Effect of avian influenza virus subtype H9N2 on the expression of complement-associated genes in chicken erythrocytes.

1. The H9N2 subtype avian influenza virus can infect both chickens and humans. Previous studies have reported a role for erythrocytes in immunity. However, the role of H9N2 against chicken erythrocytes and the presence of complement-related genes in erythrocytes has not been studied. This research investigated the effect of H9N2 on complement-associated gene expression in chicken erythrocytes.2. The expression of complement-associated genes (C1s, C1q, C2, C3, C3ar1, C4, C4a, C5, C5ar1, C7, CD93 and CFD) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative Real-Time PCR (qRT-PCR) was used to analyse the differential expression of complement-associated genes in chicken erythrocytes at 0 h, 2 h, 6 h and 10 h after the interaction between H9N2 virus and chicken erythrocytes in vitro and 3, 7 and 14 d after H9N2 virus nasal infection of chicks.3. Expression levels of C1q, C4, C1s, C2, C3, C5, C7 and CD93 were significantly up-regulated at 2 h and significantly down-regulated at 10 h. Gene expression levels of C1q, C3ar1, C4a, CFD and C5ar1 were seen to be different at each time point. The expression levels of C1q, C4, C1s, C2, C3, C5, C7, CFD, C3ar1, C4a and C5ar1 were significantly up-regulated at 7 d and the gene expression of levels of C3, CD93 and C5ar1 were seen to be different at each time point.4. The results confirmed that all the complement-associated genes were expressed in chicken erythrocytes and showed the H9N2 virus interaction with chicken erythrocytes and subsequent regulation of chicken erythrocyte complement-associated genes expression. This study reported, for the first time, the relationship between H9N2 and complement system of chicken erythrocytes, which will provide a foundation for further research into the prevention and control of H9N2 infection.

NF-кB pathway genes expression in chicken erythrocytes infected with avian influenza virus subtype H9N2.

1. Chicken erythrocytes in blood vessels are the most abundant circulating cells, which participate in the host's immune responses. The transcription factor nuclear factor-kappa B (NF-κB) plays a vital role in the inflammatory response following viral infections. However, the expression of the NF-κB pathway, and other immune-related genes in chicken erythrocytes infected with low pathogenic avian influenza virus (LPAIV H9N2), has not been extensively studied.2. The following study determined the interaction of LPAIV H9N2 with chicken erythrocytes using indirect immunofluorescence microscopy. This was followed by investigating myeloid differentiation primary response 88 (MyD88), C-C motif chemokine ligand 5 (CCL5), melanoma differentiation-associated protein 5 (MDA5), the inhibitor of nuclear factor-kappa B kinase subunit epsilon (IKBKE), NF-κB inhibitor alpha (NFKBIA), NF-κB inhibitor epsilon (NFKBIE), interferon-alpha (IFN-α), colony-stimulating factor 3 (CSF3) and tumour necrosis factor receptor-associated factor 6 (TRAF6) by mRNA expression using quantitative real-time PCR (qRT-PCR) at four different time intervals (0, 2, 6 and 10 h).3. There was a significant interaction between erythrocytes and LPAIV H9N2 virus. Furthermore, the mRNA expression of the NF-κB pathway and other immune-related genes were significantly up-regulated at 2 h post-infection in infected chicken erythrocytes, except for TRAF6, which were significantly downregulated. While at 0 h post-infection, IFN-α and CSF3 were significantly upregulated, whereas NFKBIA was significantly downregulated. Further expression of MDA5, CCL5 and NFKBIA was upregulated, while TRAF6 was downregulated at 6 h post-infection. In infected erythrocytes, expression of MyD88, CCL5 and IKBKE was upregulated. However, IFN-α and TRAF6 were downregulated at 10 h post-infection.4. These results give initial evidence that the NF-κB pathway, and other genes related to immunity, in chicken erythrocytes may contribute to LPAIV subtype H9N2 and induce host immune responses.

[NUSAP1 promotes lung cancer progression by activating AKT/mTOR signaling pathway].

Objective: To investigate the inhibitory effects of nucleolar and spindle associated protein 1 (NUSAP1) on lung cancer and the related mechanisms. Methods: A549 cells were transfected with NUSAP1 siRNA, the cell proliferation, migration and invasion, and apoptosis were detected by CCK8, Transwell and flow cytometry, respectively. Western blot was used to detect the expressions of apoptosis and AKT/mTOR signal pathway related proteins. Results: Compared with the negative control group, the proliferation [(0.610±0.058) vs (1.724±0.067), P<0.05], migration [(178.267±14.780) vs (272.464±36.232), P<0.05] and invasion [(73.527±6.617) vs (120.585±13.235), P<0.05] of NUSAP1 deleted A549 cells were significantly inhibited, while the apoptosis [(3.572±0.214)% vs (11.358±1.047)%, P<0.05] was significantly increased. The expressions of apoptosis related protein Bax and active-caspase 3 were increased (P<0.05), while the expressions of anti-apoptosis protein Bcl-2 and proliferation related protein P70, the phosphorylation levels of AKT and mTOR were reduced in NUSAP1 knockdown cells (P<0.05). Conclusion: NUSAP1 knockdown can inhibit the proliferation, migration and invasion, and promote the apoptosis of tumor cells through suppressing AKT/mTOR signaling pathway in lung cancer cells.

[Clinical application and efficacy of TIPS combined with AngioJet mechanical thrombectomy for liver cirrhosis with extensive portal vein thrombosis].

Objective: To assess the safety and efficacy of transjugular intrahepatic portosystemic shunt (TIPS) combined with AngioJet mechanical thrombectomy for liver cirrhosis with extensive portal vein thrombosis. Methods: From March 2018 to April 2019, a total of 11 patients with liver cirrhosis and extensive portal vein thrombosis were treated by TIPS combined with AngioJet mechanical thrombectomy, including 6 males and 5 females, with the age of 37-71 (46±9) years old, 3 cases of Child-Pugh grade A, 8 cases of grade B and 0 cases of grade C. The intraoperative immediate thrombus clearance rate, perioperative complication rate, postoperative thrombus recurrence rate, rebleeding rate, the incidence of hepatic encephalopathy and the rate of stent patency of all cases were collected and analyzed. Results: All the patients were treated successfully. The immediate complete thrombus clearance (grade Ⅲ) rate of portal vein trunk was 9/11, and grade Ⅱ was 2/11, The average dose of urokinase was 30-60 (40±5) ten thousand U, slight puncture point bleeding occurred in 3 cases, and recurrence of PVT in portal vein trunk occurred in 1 case with Ⅱ grade clearance rate after operation, rebleeding occurred in 1 case, hepatic encephalopathy occurred in 2 cases, the primary patency rate of stents was 9 cases. Conclusion: TIPS combined with AngioJet mechanical thrombectomy can treat the liver cirrhosis with extensive portal vein thrombosis effectively and safely, and postoperative portal vein patency rate and intrahepatic shunt patency rate are high.

MicroRNA-889 promotes cell proliferation in colorectal cancer by targeting DAB2IP.

OBJECTIVE: Colorectal cancer (CRC) remains one of the most frequent lethal malignant tumors worldwide. The correlation between miR-889 expression and CRC progression has not been well identified in the recent literature. Here, we aim to detect the role and mechanism of miR-889 in CRC. PATIENTS AND METHODS: First, miRNA RT-PCR (Real Time-Polymerase Chain Reaction) was performed to determine miR-889 expression in CRC tissues and cells. The proliferative capacity of cells transfected with miR-889 mimics, miR-889 inhibitor or NC was measured by CCK-8 (cell counting kit-8), colony formation and EdU (5-Ethynyl-2'-deoxyuridine) assays. The online bioinformatics sites were chosen to predict possible downstream regulatory genes of miR-899. The dual-luciferase report assay was conducted to verify the relation between DAB2IP (DAB2 interacting protein) and miR-899. The expression changes of DAB2IP were assessed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. RESULTS: MiR-889 was upregulated in CRC tissues and CRC cells, and upregulated miR-889 was confirmed to promote cell growth in vitro. Dual-luciferase reporter, qRT-PCR, and Western blot assays suggested that DAB2IP might be regulated by miR-889. The effects of miR-889 on proliferation could be abolished by DAB2IP through confirmatory experiments. By directly targeting DAB2IP, miR-889 served as a vital part in accelerating CRC cell proliferation. CONCLUSIONS: Our current study substantiated that miR-889 might participate in controlling CRC proliferation by regulating DAB2IP, which provides potential and prospective therapeutic target for CRC.

FABP4 accelerates glioblastoma cell growth and metastasis through Wnt10b signalling.

OBJECTIVE: Glioblastomas are one of the most dangerous types of malignancies because of their metastatic capacity and challenge to treat with chemotherapy or radiotherapy. Hence, detailed research to explain the molecular mechanisms of glioblastoma metastasis is crucial to improve glioblastoma treatment. PATIENTS AND METHODS: The expression level of fatty acid-binding protein 4 (FABP4) in glioblastoma cell lines and tissues was detected by Western blotting and quantitative Real-time polymerase chain reaction (qRT-PCR) assays. Proliferation assay, colon formation analysis and transwell/migration assay were performed to detect the relationship between FABP4 and malignant behaviors of glioblastoma cells in vitro. Subcutaneous xenograft and intravenous metastasis models were used to determine the role of FABP4 in vitro. Rescue assays were conducted to confirm the contribution of Wingless-Type MMTV Integration Site Family, Member 10B (Wnt10b) to the progression of glioblastoma cells regulated by FABP4. RESULTS: Glioblastoma cells exhibited a higher level of FABP4 expression than control cells, and down-regulation of FABP4 suppressed tumor cell growth and metastasis in vitro and in vivo. Wnt10b, as a regulator gene of FABP4, restored the effects of FABP4 down-regulation in glioblastoma cells. CONCLUSIONS: We provided substantive evidence that FABP4 is a growth and metastasis promoter in vivo and revealed that it functions in part through Wnt10b, which suggests that FABP4 might act as a probable target to block glioblastoma metastasis.

Mifepristone alleviates cerebral ischemia-reperfusion injury in rats by stimulating PPAR γ.

OBJECTIVE: To investigate the changes of peroxisome proliferator-activated receptor gamma (PPAR γ) in focal cerebral ischemia-reperfusion injury, and to explore the effect and mechanism of mifepristone on the cerebral ischemia-reperfusion injury. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats were selected, and the middle cerebral artery occlusion and reperfusion (MCAO/R) rat model was constructed using the longa's suture-occluded method. The sham operation group was not inserted with occlusion sutures. All experimental rats were divided into four groups: the sham operation group (SHA group), the MCAO/R model group (MCR group), the mifepristone intervention group (MIF group) (3 mg/kg, intragastric administration), and the mifepristone + bisphenol A diglycidyl ether (BADGE) intervention group (MIF+BAD group) [3 mg/kg mifepristone (intragastric administration) + 30 mg/kg BADGE (intraperitoneal injection)]. The long's scoring method (5 grades) was applied for scoring after reperfusion, at the time when the animals woke up, and at 48 h after awaking before execution, respectively. 48 h after the model was successfully established, triphenyl tetrazolium chloride (TTC) staining was performed to calculate the volume of cerebral infarction, and Nissl staining was conducted to observe the cranial nerve tissue morphology. Meanwhile, immune-histochemical staining was used to detect PPAR γ. Moreover, the protein expression levels of PPAR γ, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitor of metalloproteinase 1 (TIMP-1) were examined by Western blotting (WB). RESULTS: Mifepristone could significantly enhance the neurological function after cerebral ischemia-reperfusion injury, reduce the volume of cerebral infarction, and improve the morphology of nerve tissues in rats. The expression of PPAR γ in the brain tissues of rats after cerebral ischemia-reperfusion injury markedly declined, whereas mifepristone could remarkably increase the protein expression of PPAR γ. After mifepristone intervention, the protein levels of TNF-α, IL-1ß, IL-6, MMP-2, and MMP-9 in the infarcted brain tissues of rats were markedly decreased, while the expression of the TIMP-1 protein was increased. When combined with BADGE, the effect of mifepristone was partially offset. CONCLUSIONS: Mifepristone acts as a PPAR γ agonist, and relieves cerebral ischemia-reperfusion injury by restoring the balance between MMPs and TIMPs and inhibiting inflammatory cytokines.

[FAT1 inhibits cell proliferation of esophageal squamous cell carcinoma through regulating the expression of CDK4/CDK6/CCND1 complex].

Objective: To explore the expression of FAT1 in esophageal squamous cell carcinoma (ESCC) tissues, and its effect on cell proliferation. Methods: The expression levels of FAT1 protein in human ESCC tissues and matched adjacent normal tissues were determined by immunohistochemistry (IHC). Lentivirus based knockdown of FAT1 was carried out in YSE2 and Colo680N cell lines and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assays was performed to examine the effect of FAT1 on the proliferation of these ESCC cells. Colony formation assay was used to detect the colony formation ability. Flow cytometry was performed to analyze the cell cycle and apoptosis. The expression levels of cell cycle markers in FAT1 knock out ESCC cell lines were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot. Results: The relative expression of FAT1 in ESCC tissues was 66.97±21.53, significantly lower than 78.13±16.76 of adjacent normal tissues(P<0.05). Knockdown of FAT1 promoted cell proliferation and colony formation. In YSE2 cell, the division time in negative control (NC) group was (1 570±51) min, significantly longer than (1 356±31) min in shFAT1 group. In Colo680N cell, division time in NC group was (1 532±53) min, significantly longer than (1 290±30) min in shFAT1 group (P<0.05). Knockdown of FAT1 promoted G1-to S-phase transition and resulted in the upregulation of CDK4/CDK6/CCND1. Conclusion: FAT1 inhibits the proliferation and G1-to S-phase transition of ESCC cells through regulating the protein expression of CDK4/CDK6/CCND1 complex.

[Function and mechanism of zinc finger protein ZNF50 in inhibiting the growth of esophageal squamous cell carcinoma cells].

Objective: To investigate the function and mechanism of zinc finger protein 750 (ZNF750) in esophageal squamous cell carcinoma (ESCC). Methods: Xenograft in nude mice was applied to detect the tumorigenesis of ZNF750-depleted ESCC cells. Western blot was performed to observe the expression of downstream target protein of ZNF750 in ESCC cell lines and xenograft tumor tissues in which ZNF750 was knocked down. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay was used to determine the proliferation of ZNF750 stably depleted cells after restoration of its target protein. Results: The tumor weight of blank control, negative control and ZNF750 knockdown groups was 137±26 mg, 161±31 mg and 463±89 mg, respectively, with a statistically significant difference (P<0.01). The expressions of Kruppel-like factor 4 (KLF4) in ZNF750-depleted ESCC cells and its derived tumor tissue xenograft in nude mice were significantly down-regulated. Restoration of KLF4 in ZNF750 stably depleted cells significantly inhibited the cell proliferation (P<0.01). Conclusions: ZNF750 may be a new tumor suppressor in the tumorigenesis of ESCC, and the inhibition of cell proliferation induced by ZNF750 may be partially through the regulation of KLF4 expression.

Diversity of endophytic fungi of Myricaria laxiflora grown under pre- and post-flooding conditions.

Myricaria laxiflora is distributed along the riverbanks of the Yangtze River valley. The Three Gorges Dam has dramatically changed the habitat of M. laxiflora, which has evolved to develop increased resistance to flooding stress. In order to elucidate the relationship between plant endophytic fungi and flooding stress, we isolated and taxonomically characterized the endophytic fungi of M. laxiflora. One hundred and sixty-three fungi were isolated from healthy stems, leaves and roots of M. laxiflora grown under pre- and post-flooding conditions. Culture and isolation were carried out under aerobic and anaerobic conditions. Based on internal transcribed spacer sequence analysis and morphological characteristics, the isolates exhibited abundant biodiversity; they were classified into 5 subphyla, 7 classes, 12 orders, 17 families, and 26 genera. Dominant endophytes varied between pre- and post-flooding plants, among different plant tissues, and between aerobic and anaerobic culture conditions. Aspergillus and Alternaria accounted for more than 55% of all isolates. Although the number of isolates from post-flooding plants was greater, endophytes from pre-flooding plants were more diverse and abundant. Endophytes were distributed preferentially in particular tissues; this affinity was constrained by both the host habitat and the oxygen availability of the host.

[Animal model and multiple trait BLUP applied in poultry genetic evaluation].

Best linear unbiased prediction (BLUP) is a powerful method to estimate genetic values of animals, and is widely applied in many animal species but poultry. Beijing White Leghorn nested data in 1986-1987 with 777 individuals were analysed by animal model and multiple trait BLUP. Two traits (40-week egg production and 36-week egg weight) and two fixed effects (house-pen effect and hatching batch effect) were considered. The way to calculate a large set of mixed model equations in micro-computer was studied. Only non-zero elements of the coefficient matrix of MME were stored on a disk. The iteration process was reduced by block iteration. It also simplified the multiple trait BLUP method, the dimension of equations is only 1/q (q is the number of traits) of regular method. So it saved a lot of compute time and cost, and BLUP became applicable in poultry. The significance of using BLUP in poultry are: (1) Eliminating some fixed effects; (2) Reducing the estimation error for unbalanced data; (3) Estimating breeding values of progeny, so we can shorten the generation interval; (4) It can estimate breeding values of individuals without records from the relatives' information; (5) The breeding value of sires and dams can be estimated from their progeny records, used for family selection; (6) Due to genetic and environmental correlation between traits and all relatives information were considered, it can increase the selection accuracy.