Development of a multiplex qRT-PCR assay for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine Deltacoronavirus.

Currently, porcine coronaviruses are prevalent in pigs, and due to the outbreak of COVID-19, porcine coronaviruses have become a research hotspot. porcine epidemic diarrhea virus (PEDV), Transmissible Gastroenteritis Virus (TGEV), and Porcine Deltacoronavirus (PDCoV) mentioned in this study mainly cause diarrhea in pigs. These viruses cause significant economic losses and pose a potential public health threat. In this study, specific primers and probes were designed according to the M gene of PEDV, the S gene of TGEV, and the M gene of PDCoV, respectively, and TaqMan probe-based multiplex real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was developed for the simultaneous detection of PEDV, TGEV, and PDCoV. This method has high sensitivity and specificity, and the detection limit of each virus can reach 2.95 × 100 copies/µl. An assay of 160 clinical samples from pigs with diarrhea showed that the positive rates of PEDV, TGEV, and PDCoV were 38.13, 1.88, and 5.00%; the coinfection rates of PEDV+TGEV, PEDV+PDCoV, TGEV+PDCoV, PEDV+TGEV+PDCoV were 1.25, 1.25, 0, 0.63%, respectively. The positive coincidence rates of the multiplex qRT-PCR and single-reaction qRT-PCR were 100%. This method is of great significance for clinical monitoring of the porcine enteric diarrhea virus and helps reduce the loss of the breeding industry and control the spread of the disease.

Dihydroartemisinin alleviates oxidative stress in bleomycin-induced pulmonary fibrosis.

AIMS: Dihydroartemisinin has been shown to inhibit the development of pulmonary fibrosis in rats, but its mechanism has yet to be elucidated. This study aimed to determine the mechanisms of dihydroartemisinin in bleomycin-induced pulmonary fibrosis in a rat model. MAIN METHODS: Morphological changes and collagen deposition were analyzed via hematoxylin-eosin staining and Masson staining and the expression of biotic-factor-related oxidative stress in lung tissues was assayed with standard assay kits. The expressions of α-SMA, E-cadherin, and Nrf2/HO-1 were detected by Western blot and RT-PCR, and the cell morphology and proliferation of cultured type II alveolar epithelial cells (AECs) were assessed via microscopy and immunocytochemical assay. KEY FINDINGS: Dihydroartemisinin treatment significantly decreased the level of oxidative stress and collagen synthesis and inhibited AECs differentiation in bleomycin-induced pulmonary fibrosis compared to the control group (P < 0.001). SIGNIFICANCE: Our results indicated that dihydroartemisinin might decrease oxidative damage to attenuate lung injury and fibrosis.

MiR-212-5p Suppresses the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer by Targeting Prrx2.

BACKGROUND/AIMS: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Our study investigated the functional role of miR-212-5p in TNBC. METHODS: Realtime PCR was used to quantify miR-212-5p expression levels in 30 paired TNBC samples and adjacent normal tissues. Wound healing and Transwell assays were used to evaluate the effects of miR-212-5p expression on the invasiveness of TNBC cells. Luciferase reporter and Western blot assays were used to verify whether the mRNA encoding Prrx2 is a major target of miR-212-5p. RESULTS: MiR-212-5p was downregulated in TNBC, and its expression levels were related to tumor size, lymph node status and vascular invasion in breast cancer. We also observed that the miR-212-5p expression level was significantly correlated with a better prognosis in TNBC. Ectopic expression of miR-212-5p induced upregulation of E-cadherin expression and downregulation of vimentin expression. The expression of miR212-5p also suppressed the migration and invasion capacity of mesenchymal-like cancer cells accompanied by a morphological shift towards the epithelial phenotype. Moreover, our study observed that miR-212-5p overexpression significantly suppressed Prrx2 by targeting its 3'-untranslated region (3'-UTR) region, and Prrx2 overexpression partially abrogated miR-212-5p-mediated suppression. CONCLUSIONS: Our study demonstrated that miR-212-5p inhibits TNBC from acquiring the EMT phenotype by downregulating Prrx2, thereby inhibiting cell migration and invasion during cancer progression.

Deletion of ssnA Attenuates the Pathogenicity of Streptococcus suis and Confers Protection against Serovar 2 Strain Challenge.

Streptococcus suis serotype 2 (SS2) is a major porcine and human pathogen which causes arthritis, meningitis, and septicemia. Streptococcus suis nuclease A (SsnA) is a recently discovered deoxyribonuclease (DNase), which has been demonstrated to contribute to escape killing in neutrophil extracellular traps (NETs). To further determine the effects of ssnA on virulence, the ssnA deletion mutant (ΔssnA) and its complemented strain (C-ΔssnA) were constructed. The ability of ΔssnA mutant to interact with human laryngeal epithelial cell (Hep-2) was evaluated and it exhibited dramatically decreased ability to adhere to and invade Hep-2 cells. This mutation was found to exhibit significant attenuation of virulence when evaluated in CD1 mice, suggesting ssnA plays a critical role in the pathogenesis of SS2. Finally, we found that immunization with the ΔssnA mutant triggered both antibody responses and cell-mediated immunity, and conferred 80% protection against virulent SS2 challenge in mice. Taken together, our results suggest that ΔssnA represents an attractive candidate for designing an attenuated live vaccine against SS2.

Chinese medicinal plants for advanced endometriosis after conservative surgery: a prospective, multi-center and controlled trial.

The trial was to explore the effects of Chinese medicinal plants (CMP) treatment on the advanced endometriosis (stage III-IV) after conservative surgery. A prospective, multi-center and controlled trial was conducted from June 2012 to September 2013. Sixty-five post-operative women with advanced endometriosis (stage III-IV) after conservative surgery were included in the trial. They had undergone laparoscopic surgical excision of the endometriosis lesions and the diagnosis of endometriosis was confirmed by pathological examination. The patients received either CMP treatment or goserelin acetate sustained-release depot treatment (as comparison) according to the willingness of the patients. In the post-treatment follow-up visit at 6 and 12 months, the patients were respectively undergone ultrasonic and gynecological examinations. The serum levels of cancer antigen 125 (CA-125) and interleukin 18 (IL-18) were also detected in the post-treatment follow-up visit at 12 months. We found that in the post-treatment follow-up visit at 6 months, the recurrence rate of CMP group and comparison group was 1/31 (3.23%) and 1/34 (2.94%), respectively. In the post-treatment follow-up visit at 12 months, the recurrence rate of CMP group and comparison group was 5/31 (16.13%) and 6/34 (17.65%), respectively. There were no significant differences between the two groups (P>0.05). The serum levels of CA-125 and IL-18 significantly decreased in both of the two groups (P<0.05) and no marked differences existed between them on the serum levels of IL-18 (P>0.05). The serum CA-125 levels of CMP group were significantly lower than those of the comparison group (P<0.05). No adverse effect was reported in both of the two groups during the research and the follow-up period. It concluded that CMP showed promise in preventing the recurrence of stage III-IV endometriosis after conservative surgery, although the conclusion is somewhat limited due to the small size of the trial.

The extracts of Pacific oyster (Crassostrea gigas) alleviate ovarian functional disorders of female rats with exposure to bisphenol a through decreasing FSHR expression in ovarian tissues.

BACKGROUND: Bisphenol-A (BPA) is one of the widespread industrial compounds, which has adverse effects on animal and human health. The study was aimed to explore the effects of Crassostrea gigas extracts (CGE) in alleviating ovarian functional disorders of female rats with exposure to BPA and the underlying possible mechanism. MATERIALS AND METHODS: Eighteen four-week-old female Sprague-Dawley (SD) rats were randomly divided into BPA group (50mg/kg BPA), BPA+CGE group (50mg/kg BPA+50mg/kg CGE), and control group (equivalent dosage of vehicle) with 6 rats in each group. After a 6-week treatment ended, the serum levels of estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH) were measured by using commercial standard assay kits. The expression levels of FSH receptor (FSHR) in the rat ovarian tissues were respectively detected by immunohistochemistry and Real-time PCR. RESULTS: CGE treatment markedly increased E2 levels and decreased FSH levels in the serum (P<0.05), however, the alterations of serum LH levels were not significant (P>0.05). The protein and mRNA expression levels of FSHR were the lowest in the ovaries of control rats and the highest in BPA rats (P<0.05). CGE treatment markedly decreased the expression levels of FSHR in the ovarian tissues (P<0.05). CONCLUSIONS: Crassostrea gigas successfully alleviates ovarian functional disorders of female rats with exposure to BPA partly through decreasing FSHR expression levels in the ovarian tissues.

[Serum interleukin 18 and 33 levels and its clinical significance in asthma patients].

OBJECTIVE: To study the levels of serum interleukin (IL)-18 and IL-33 in asthmatic patients pre-and post-treatment with glucocorticoids during the acute exacerbation period and therefore to investigate the role of IL-18 and IL-33 in the pathogenesis of bronchial asthma. METHODS: Thirty cases of moderate to severe asthma admitted in the Second Affiliated Hospital of Dalian Medical University from June, 2009 to October, 2010 were chosen as the asthma group, and 23 healthy people who underwent routine physical examination were chosen as the control group. The subjects in the 2 groups were all nonsmokers. All the patients were treated with a glucocorticoid (methylprednisolone 40 - 80 mg/d) for 3 to 5 days and combined with aerosol inhalation of budesonide for 1 to 2 weeks as adjuvant therapy. The serum levels of IL-18 and IL-33 were detected for the asthma group before and after the treatment and for the control group. Other data including immunoglobulin E (IgE), the number of eosinophil cells (EOS) and the pulmonary function (FEV(1)%pre) were measured as well. The differences of IL-18 and IL-33 levels between the asthma group before and after treatment and the control group were analyzed, and the correlation between IL-18, IL-33 and IgE, EOS and FEV(1)%pre were analyzed as well by SPSS software. RESULTS: Compared with the healthy control group [(158 ± 45) ng/L and (143 ± 32) ng/L], the levels of IL-18 and IL-33 in the asthma pre-treatment group were significantly increased [(300 ± 181) ng/L and (208 ± 95) ng/L, t = 3.67 and 3.51, all P < 0.05)]; and the IgE level and EOS count showed a similar change (t = 5.76 and 5.24, all P < 0.05). After systemic glucocorticoid treatment and local aerosol inhalation, the levels of IL-18 and IL-33 [(183 ± 83) ng/L and (148 ± 77) ng/L] were decreased significantly in the asthma patients [t = 5.42 and 12.09, all P < 0.05], and the IgE level and EOS count also showed a same trend as well (t = 11.87 and 4.56, all P < 0.05), whereas FEV(1)%pred [(54 ± 14)%] increased significantly [(81 ± 16)%, t = -7.81, P < 0.05]. The Pearson correlative analysis showed that there were positive correlations between the levels of IL-18, IL-33 and IgE and EOS, respectively (all P < 0.05), and there were negative correlations between IL-18, IL-33 levels and FEV(1)%pre, respectively (all P < 0.05). CONCLUSIONS: Our results suggest that IL-18 and IL-33 participate in the pathogenesis of asthma, and may play stimulation role in the process of the disease.

Expression of Elf-1 and survivin in non-small cell lung cancer and their relationship to intratumoral microvessel density.

BACKGROUND AND OBJECTIVE: The expression of transcription factor Elf-1 and inhibitor of apoptosis survivin in non-small cell lung cancer (NSCLC) is correlated with the angiogenic factor vascular endothelial growth factor (VEGF), and are both factors affecting the cell cycle. This study investigated the expression of Elf-1, survivin, and intratumoral microvessel density (iMVD) assessed by monoclonal antibody CD105 in NSCLC, and explored their correlations with clinicopathologic features and angiogenesis of NSCLC. METHODS: PowerVision(TM)-9000 immunohistochemistry was used to evaluate the expression of Elf-1, survivin, and CD105 in tissue microarrays containing 60 specimens of NSCLC and 9 specimens of normal tissue. Western blot analysis was used to evaluate the protein levels of Elf-1 and survivin in 17 specimens of NSCLC and 5 specimens of normal tissue. RESULTS: Elf-1 and survivin were detected in 1 of the 9 normal tissues. The positive rates of Elf-1 and survivin in NSCLC were 70.0% and 65.0%, respectively. The expression levels of both Elf-1 and survivin were significantly related to tumor differentiation, lymphatic metastasis, clinical stage, and postoperative survival time (P < 0.05). Overexpression of both were related to poor prognosis: the survival rates were significantly lower in patients with positive expression than in those with negative expression (P < 0.01). Elf-1 expression was positively correlated with survivin expression (r = 0.769, P < 0.01). Elf-1 and survivin expressions were positively correlated with iMVD (r = 0.446, P < 0.01; r = 0.435, P < 0.01). CONCLUSIONS: The expression of Elf-1 and survivin in NSCLC is related to differentiation, lymphatic metastasis, clinical stage, and prognosis, and both are positively correlated with iMVD. Detection their combined expression can help to predict the malignant behavior of NSCLC. Blocking the activity of Elf-1 and survivin may be a new way to inhibit angiogenesis in NSCLC.

[Expression and significance of Elf-1 and vascular endothelial growth factor in non-small cell lung cancer].

BACKGROUND AND OBJECTIVE: The roles of vascular endothelial growth factor (VEGF) in tumor angiogenesis is related with Ets family. Elf-1, a member of Ets family, has seldom been studied. This study aimed to investigate the expression of Elf-1 and VEGF in non-small cell lung cancer (NSCLC), and explore their correlations to clinicopathologic features of NSCLC. METHODS: Tissue microarray containing 69 specimens of NSCLC and six specimens of normal lung tissues was constructed. The expression of Elf-1 and VEGF was detected by PowerVision-9000 immunohistochemistry. RESULTS: Elf-1 and VEGF were not detected in all normal tissues; the positive rates of Elf-1 and VEGF were 72.46% and 63.77% in NSCLC, respectively. The expression levels of both Elf-1 and VEGF were significantly related with tumor differentiation, lymphatic metastasis, clinical stage, and postoperative survival time (all P < 0.01). Overexpression of them was related with poor prognosis: the survival rates were significantly lower in positive patients than in negative patients (both P < 0.01). Elf-1 expression was positively correlated to VEGF expression (r = 0.702, P < 0.01). CONCLUSIONS: The expression of Elf-1 and VEGF in NSCLC is related to differentiation, lymphatic metastasis, clinical stage and prognosis. Detecting their expression in combination can help to predict the malignant behavior of NSCLC.

Effects of Yiweining Recipe on expressions of metalloproteinase-2 and cyclooxygenase-2 mRNAs in ectopic endometrium of rats with endometriosis.

OBJECTIVE: To explore the effects of Yiweining Recipe (YWNR), a compound Chinese herbal medicine, on expressions of metalloproteinase-2 (MMP-2) and cyclooxygenase-2 (COX-2) mRNAs in rats with endometriosis (EM). METHODS: Operational self-transplantation was applied in establishing the rat models. Detection of MMP-2 and COX-2 mRNAs was conducted with hybridization in situ. RESULTS: There were significant differences in the expressions of MMP-2 and COX-2 mRNAs between the untreated group and the high-dose YWNR-treated group. YWNR could reduce the expressions of MMP-2 and COX-2 mRNAs. CONCLUSION: YWNR can treat EM through reducing the positive expressions of MMP-2 and COX-2 mRNAs.

Comparative study on the efficacy of Yiweining and Gestrinone for post-operational treatment of stage III endometriosis.

OBJECTIVE: To observe the clinical efficacy and safety of Yiweining (YWN) and gestrinone (GT) in treating post-operational patients of stage III endometriosis (EM-III). METHODS: Fifty-two patients of EM-III after operation were randomly assigned into three groups, the YWN group (20 patients) was treated through oral intake of YWN 200 ml, twice a day; the GT group (19 patients) treated with gestrinone 2.5 mg, twice every week, with the medication starting from the 7th post-operational day and lasting for 6 months. The control group (13 patients) was untreated. Six months was one therapeutic course, and follow-up study was carried out in the 6 - 30 months after the end of the medication. RESULTS: The recurrence rate in the YWN group and the GT group were 5.0% and 5.3% respectively, showing insignificant difference between the two groups, but they were lower than that in the control group (30.7%, P < 0.05). Besides, the adverse reaction rate in the YWN group was lower than that in the GT group (10.0% vs 31.6%, P < 0.05). CONCLUSION: Application of YWN to prevent the post-operational recurrence of endometriosis is effective and safe, and its efficacy is similar to that of GT.