Identification of a novel avian coronavirus infectious bronchitis virus variant with three-nucleotide-deletion in nucleocapsid gene in China.

A novel avian infectious bronchitis virus (IBV) variant, designated as GX-NN160421, was isolated from vaccinated chicken in Guangxi, China, in 2016. Based on analysis of the S1 gene sequence, GX-NN160421 belonged to the New-type 1 (GVI-1) strain. More importantly, three consecutive nucleotides (AAC) deletions were found in the highly conserved structure gene N. The serotype of GX-NN160421 was different from those of the commonly used vaccine strains. The mortality of the GX-NN160421 strain was 3.33%, which contrasted with 50% mortality in the clinical case, but high levels of virus shedding lasted at least 21 days. In conclusion, the first novel IBV variant with three-nucleotide-deletion in the N gene was identified, and this unique variant is low virulent but with a long time of virus shedding, indicating the continuing evolution of IBV and emphasizing the importance of limiting exposure to novel IBV strains as well as extensive monitoring of new IBVs.

Construction and Immunogenicity Comparison of Three Virus-Like Particles Carrying Different Combinations of Structural Proteins of Avian Coronavirus Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4+ and CD8+ T lymphocytes than the SM-VLPs, ME-VLPs and OEV of GX-YL5. Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.

Identification of MicroRNAs as Potential Biomarkers in Ovarian Endometriosis.

Endometriosis, as a prevalent gynecological disease, is characterized by the presence of endometrial-like tissue outside the uterus, causing infertility and considerable pain and affecting the quality of life of women. The pathogenic mechanism has not been fully elucidated, and there are no effective biomarkers for endometriosis. In our study, microRNA (miRNA) expression profiling of 10 ectopic endometrial plasma from patients with ovarian endometriosis and 10 normal plasma from healthy controls was analyzed using a microarray. As a result, 114 differentially expressed miRNAs were identified. Among them, 14 miRNAs were significantly downregulated in patients with ovarian endometriosis, which matched the microarray results. The diagnostic value of the 14 downregulated miRNAs in ovarian endometriosis was evaluated by receiver operating characteristic (ROC) curve analysis, and hsa-let-7i-5p showed the highest area under the ROC curve (AUC) with a value of 0.900. The target genes of the 14 miRNAs were predicted by miRWalk2.0, and the genes that were targeted by at least 2 of the 14 miRNAs were analyzed by function enrichment. The target genes were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, such as microRNAs in cancer, bladder cancer, and endocrine resistance pathways, and the Gene Ontology (GO) terms such as nucleobase-containing compound metabolic process, cellular nitrogen compound biosynthetic process, and heterocycle metabolic process. The identified 14 differentially expressed miRNAs could be potential biomarkers and therapeutic targets for the diagnosis and treatment of endometriosis.

Establishment of the PDTX model of gynecological tumors.

OBJECTIVE: Fresh tumor tissues from patients with gynecological tumors were obtained by surgery or biopsy, and transplanted into NOD-Prkdcem26ll2rgem26Nju (NCG) mice to establish a patient-derived tumor xenograft (PDTX). MATERIALS AND METHODS: A total of 15 patients with gynecologic tumors were enrolled into the present study. Among these patients, 12 patients had epithelial fallopian tube/ovarian/peritoneal cancer, one patient had metastatic ovarian cancer, and two patients had cervical cancer. Furthermore, among these patients, three patients were treated with puncture or microscopy biopsy, six patients underwent laparoscopic surgery, and six patients underwent robotic surgery. The tumor formation latency, tumor formation rate, tumor volume, tumor invasion and metastasis of the transplanted tumor were observed, the consistency of the PDTX model tumor tissue and patient's primary tumor tissue was compared by pathological H&E staining, and pharmacodynamics testing was performed. RESULTS: Seven of 15 PDTX models were successfully established, with a success rate of 46.7%. The tumor formation time ranged within 21-130 days, with a median tumor formation time of 73 days. The PDTX model maintained the differentiation, morphological and structural characteristics of tumor cells, and the pharmacodynamic test was completed in five patients. CONCLUSION: The PDTX model is highly consistent with the pathology of the patient's tumor, and can be used as a substitute for clinical patients to guide the accurate treatment and scientific research of gynecological tumors.

Comparison of integrated PET/MRI with PET/CT in evaluation of endometrial cancer: a retrospective analysis of 81 cases.

BACKGROUND: The objective of this study was to compare the diagnostic value of integrated PET/MRI with PET/CT for assessment of regional lymph node metastasis and deep myometrial invasion detection of endometrial cancer. METHODS: Eighty-one patients with biopsy-proven endometrial cancer underwent preoperative PET/CT (n = 37) and integrated PET/MRI (n = 44) for initial staging. The diagnostic performance of PET/CT and integrated PET/MRI for assessing the extent of the primary tumor and metastasis to the regional lymph nodes was evaluated by two experienced readers. Histopathological and follow-up imaging results were used as the gold standard. McNemar's test was employed for statistical analysis. RESULTS: Integrated PET/MRI and PET/CT both detected 100% of the primary tumors. Integrated PET/MRI proved significantly more sensitivity and specificity than PET/CT in regional lymph node metastasis detection (P = 0.015 and P < 0.001, respectively). The overall accuracy of myometrial invasion detection for PET/CT and Integrated PET/MRI was 45.9% and 81.8%, respectively. Integrated PET/MRI proved significantly more accurate than PET/CT (P < 0.001). CONCLUSION: Integrated PET/MRI, which complements the individual advantages of MRI and PET, is a valuable technique for the assessment of the lymph node metastasis and myometrial invasion in patients with endometrial cancer.

Immune protection conferred by three commonly used commercial live attenuated vaccines against the prevalent local strains of avian infectious bronchitis virus in southern China.

Live attenuated vaccines are critical in the control of avian infectious bronchitis. It is necessary to know the protection conferred by commonly used commercial live vaccines. In this study, specific pathogen-free chicks were vaccinated with the commercial live vaccines H120, 4/91 and LDT3-A. Blood samples were collected at weekly intervals for the detection of IBV-specific antibodies and quantification of CD4+ and CD8+ T lymphocytes. At 21 days post-inoculation the vaccinated birds were challenged with the IBV prevalent local strains GX-YL5, GX-GL11079 and GX-NN09032, respectively. Trachea and kidney samples were collected at 5 days post-challenge for the detection of the virus. The results showed that the H120 group exhibited medium antibody levels, the lowest percentages of CD4+, CD8+ T lymphocytes and the highest viral loads. The 4/91 group showed the lowest antibody levels, but the highest percentages of CD4+, CD8+ T lymphocytes and the lowest viral loads. The LDT3-A group showed the highest antibody levels, the medium percentages of CD4+, CD8+ T lymphocytes and the medium viral loads. The protection rates of H120, 4/91 and LDT3-A groups were 41.7-58.3%, 75.0-83.7% and 66.7-75.0%, respectively. The present study demonstrated that the vaccines H120, 4/91 and LDT3-A could stimulate the immunized chicks to produce different levels of humoral and cellular immunity to resist the infection of IBV, but couldn't provide complete protection against the prevalent local strains of IBV in southern China. Also, the vaccine 4/91 offered the best immune protection among the three vaccines.

Protection against Virulent Infectious Bronchitis Virus Challenge Conferred by a Recombinant Baculovirus Co-Expressing S1 and N Proteins.

Avian infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis, which results in considerable economic losses. It is imperative to develop safe and efficient candidate vaccines to control IBV infection. In the current study, recombinant baculoviruses co-expressing the S1 and N proteins and mono-expressing S1 or N proteins of the GX-YL5 strain of IBV were constructed and prepared into subunit vaccines rHBM-S1-N, rHBM-S1 and rHBM-N. The levels of immune protection of these subunit vaccines were evaluated by inoculating specific pathogen-free (SPF) chickens at 14 days of age, giving them a booster with the same dose 14 days later and challenging them with a virulent GX-YL5 strain of IBV 14 days post-booster (dpb). The commercial vaccine strain H120 was used as a control. The IBV-specific antibody levels, as well as the percentages of CD4+ and CD8+ T lymphocytes, were detected within 28 days post-vaccination (dpv). The morbidity, mortality and re-isolation of the virus from the tracheas and kidneys of challenged birds were evaluated at five days post-challenge (dpc). The results showed that the IBV-specific antibody levels and the percentages of CD4+ and CD8+ T lymphocytes were higher in the rHBM-S1-N vaccinated birds compared to birds vaccinated with the rHBM-S1 and rHBM-N vaccines. At 5 dpc, the mortality, morbidity and virus re-isolation rate of the birds vaccinated with the rHBM-S1-N vaccine were slightly higher than those vaccinated with the H120 control vaccine but were lower than those vaccinated with the rHBM-S1 and rHBM-N vaccines. The present study demonstrated that the protection of the recombinant baculovirus co-expressing S1 and N proteins was better than that of recombinant baculoviruses mono-expressing the S1 or N protein. Thus, the recombinant baculovirus co-expressing S1 and N proteins could serve as a potential IBV vaccine and this demonstrates that the bivalent subunit vaccine including the S1 and N proteins might be a strategy for the development of an IBV subunit vaccine.

[Efficacy of gamma-irradiated adipose-derived stem cells for treatment of thin endometrium in rats].

OBJECTIVE: Transplantation of adipose-derived stem cells (ADSCs) is associated with potential risks of late complications including tumorigenesis due to the active proliferation of the cells. We aimed to test the effect of transplantation of ADSCs with suppressed proliferation by gamma irradiation in the treatment of thin endometrium in rats. METHODS: ADSCs were isolated from female SD rats and identified by detecting the surface antigens with flow cytometry. After exposure to gamma irradiation at 0, 5 Gy and 10 Gy, the cells were examined for changes in colony-forming ability. Twenty-four female rats with chemically induced thin endometrium were randomized into 4 equal groups and at 6-8 h after modeling, the rats received intrauterine injection of non-irradiated ADSCs (group I), 5 Gy irradiated ADSCs (group II), 10 Gy irradiated ADSCs (group III), or PBS only (group IV). Endometrial pathology was analyzed with HE staining in these rats in the third estrus phase following the cell transplantation. RESULTS: The ADSCs showed a complete loss of proliferative capacity after exposure to 10 Gy irradiation. After the cell transplantation, the endometrium thickness was thicker in group I and II than in group IV (P<0.01), but there was no significant difference between groups III and IV. CONCLUSIONS: Gamma irradiation impairs the proliferative capacity of ADSCs in vitro. Exposure to 10 Gy irradiation causes a total loss of proliferation capacity of the ADSCs, which have no therapeutic potential; 5 Gy irradiation causes partial loss of proliferation capacity of the cells, which still retain the activity to promote endometrial cell regeneration.

ARHI overexpression induces epithelial ovarian cancer cell apoptosis and excessive autophagy.

OBJECTIVE: ARHI is a maternally imprinted tumor suppressor gene that is responsible for initiating programmed cell death and inhibiting cancer cell growth. However, the influence of ARHI on epithelial ovarian cancer cell death and the underlying mechanisms behind how ARHI regulates cancer cells still require further studies. METHODS: Epithelial ovarian cancer cells TOV112D and ES-2 were used in this in vitro study. Cell proliferation, apoptosis, and autophagy activities were compared in TOV112D and ES-2 cells transfected with ARHI vectors or control vectors. Bcl-2 siRNA was transfected into TOV112D cells to investigate the roles of Bcl-2 played in regulating apoptosis and autophagy. RESULTS: ARHI expression was reduced in TOV112D and ES-2 cells compared with normal epithelial ovarian cells (NOE095 and HOSEpiC). Overexpressed ARHI inhibited cancer cell proliferation, whereas induced forced cell apoptosis and excessive formation of autophagosomes inhibited promoted cell death. Furthermore, we found that Bcl-2 expression moderately declined in response to ARHI overexpressing in ES-2 and TOV112D cells; meanwhile, more apoptotic cells and higher LC3 level presented after silence of Bcl-2 in TOV112D cells. Reduced Bcl-2-Beclin 1 complex were observed in ARHI overexpressing cells. Moreover, modulation of ARHI to Bcl-2 expression could be ascribed partially to the activation of PI3k/AKT pathway. The addition of LY294002 enabled to suppress Bcl-2 expression and cell proliferation. CONCLUSIONS: The silence of ARHI expression in vitro seems to accelerate the malignant transformation of healthy ovarian cells by restraining apoptosis and autophagy. The overexpressed ARHI in TOV112D cancer cells suppresses the activation of PI3K/AKT and reduces the expression of Bcl-2, leading to enhanced cell apoptosis and autophagic cancer cell death.

Molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern china.

To gain comprehensive genetic information of circulating avian coronavirus infectious bronchitis virus (IBV) isolates in China, analysis of the phylogenetic tree, entropy of the amino acid sequences, and the positive selection as well as computational recombinations of S1, M and N genes of 23 IBV isolates was conducted in the present study. The phylogenetic trees based on the S1, M and N genes exhibited considerably different topology and the CK/CH/LSC/99I-type isolates were the predominant IBVs based on the phylogenetic analysis of S1 gene. Results of entropy of amino acid sequences revealed that the S1 gene had the largest variation; the M gene had less variation than the N gene. Positive selections were detected in not only S1 but also M and N gene proteins. In addition, five S1 gene recombinants between vaccine strain 4/91 and CK/CH/LSC/99I-type field isolate were confirmed. In conclusion, multiple IBV genotypes co-circulated; genetic diversity and positive selections existed in S1, M and N genes; 4/91 vaccine recombinants emerged in China. Our results show that field IBVs in China are continuing to evolve and vaccine strains may have an important role in the appearance of new IBV strains via recombination. In addition, the present study indicates that IBV evolution is driven by both generations of genetic diversity and selection.

STAT3 acetylation-induced promoter methylation is associated with downregulation of the ARHI tumor-suppressor gene in ovarian cancer.

ARHI is a Ras-related imprinted tumor-suppressor gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of ovarian cancer cells, and promoter methylation is considered to be associated with its loss of expression. however, the underlying mechanisms are not well understood. Thus, the present study aimed to investigate the specific functions of ARHI and its methylation in ovarian cancer cell proliferation. Furthermore, we examined the possible role of acetylated STAT3 in modulating the expression of ARHI and its methylation. In accordance with the majority of previous studies, reduced ARHI expression was found in epithelial ovarian cancer tissues and cancer cell lines as indicated by immunohistochemistry and RT-PCR. In addition, CpG islands I and II within ARHI promoter regions were partially methylated or hypermethylated in cancer cell lines (SKOV-3 and HO-8910) as analyzed by pyrosequencing assays, resulting in enhanced proliferation of the cancer cells. This proliferation was reversed by the administration of 5-aza-2'-deoxycytidine. Subsequently, we demonstrated that STAT3 acetylation was increased in HO-8910 cells, and the methylation status of CpG I was altered in response to the acetylation of STAT3 using western blotting. Finally, chromatin immunoprecipitation (ChIP) and IP analysis indicated that acetylated STAT3 bound to the ARHI promoter and recruited DNA methyltransferase 1 for genetic modification. In conclusion, acetylated STAT3-induced promoter gene methylation accounts for the loss of ARHI expression and cancer cell proliferation.

Continuous evolution of avian infectious bronchitis virus resulting in different variants co-circulating in Southern China.

Sixty field strains of avian infectious bronchitis virus (IBV) were isolated from chicken flocks in different regions of Guangxi from 1985 to 2012. Phylogenetic analysis of S1 subunit glycoprotein genes revealed that field isolates from 2009-2011 mostly belonged to the LX4 type, while those from 1985-2008 belonged to the HN08 type, and a few others belonged to the 4/91 type, the TW type and the Mass type. In addition, it is noteworthy that no obvious regional differences were found among these 60 strains isolated from six regions in Guangxi, while there was a high degree of sequence identity among the isolates in the same period of time.