Induction of COX-2 by feline calicivirus via activation of the MEK1-ERK1/2 pathway, and attenuation of feline lung inflammation and injury by MEK1 inhibitor AZD6244 (selumetinib).

Feline calicivirus (FCV) is an important and highly prevalent pathogen of cats that causes acute infectious respiratory disease. Here it is shown in vitro that FCV induces the production of cyclooxygenase-2 (COX-2) through the MEK1-ERK1/2 signaling pathway. Screening of FCV proteins revealed that FCV non-structural protein VPg enhanced COX-2 mRNA expression and protein production in CRFK cells in a concentration-dependent manner. Regions 24-54aa and 84-111aa in FCV VPg were essential for up-regulation. In vivo, COX-2 and IL-6 production caused by FCV infection of kittens was significantly suppressed by the MEK1 inhibitor AZD6244 (selumetinib) and lung inflammation and injury were practically eliminated, with body temperature being returned to normal. AZD6244 may therefore find application as an effective therapeutic agent for the treatment of FCV infection.

An enhanced anti-tumor effect of apoptin-cecropin B on human hepatoma cells by using bacterial magnetic particle gene delivery system.

The gene therapy of cancer, due to the limit of its efficiency and safety, has not been widely used in clinical. Recently, bacterial magnetic particles (BMPs), which are membrane-bound nanocrystals found in magnetotactic bacteria, have been exploited as a new gene delivery system. However, its application on gene therapy remains to be explored. In our previous study, we found that a combination of cecropin B (ABPs) and apoptin (VP3) could serve as an effective gene therapeutic agent. Thus, in this study, we used BMPs to deliver the co-expression plasmid of these two gene, namely pVAX1-VA, and evaluated its therapeutic effect on human hepatocellular carcinoma (HepG2). Our results showed that BMPs significantly improved the efficiency of gene transfection (almost 3-fold than Lipofectamine 2000 at 48 h, P < .001), which led to stronger apoptosis (in a peak almost 2-fold than Lipofectamine 2000-pVAX1-VA, P < .01) and growth inhibition of HepG2 cells. More importantly, compared with Lipofectamine 2000-pVAX1-VA group, BMP-pVAX1-VA strikingly inhibited tumor growth (0.60 ±â€¯0.09 g vs. 0.88 ±â€¯0.11 g, P < .05) in nude mouse tumor models and increased the tumor-infiltrating lymphocytes considerably without apparent cytotoxicity. These findings suggest that BMPs could be an attractive gene delivery system for gene therapy and provide a potential available treatment for human hepatocellular carcinoma and maybe some other kinds of tumors.

Combination of specific single chain antibody variable fragment and siRNA has a synergistic inhibitory effect on the propagation of avian influenza virus H5N1 in chicken cells.

BACKGROUND: The avian influenza virus (AIV) causes frequent disease with high morbidity and mortality. RNA interference (RNAi) has been shown to provide an effective antiviral defense in animals, and several studies have focused on harnessing small interfering RNAs (siRNAs) to inhibit viral infections. In addition, single chain variable fragments (scFvs) contain the complete antigen binding site, and specific scFvs can bind to and neutralize viruses. RESULTS: Fourteen positive scFvs were selected by the yeast two-hybrid system. Using molecular docking technology, we selected the three highest affinity scFvs for further functional validation. Results of indirect ELISA and IFA showed that all three scFvs could bind to FJ13 strain and had neutralizing activity, decreasing the viral infectivity markedly. Chicken fibroblastic DF-1 cells were transfected with scFvs in combination with siRNA-NP604 (an siRNA of anti-AIV NP protein previously reported). Following infection with FJ13 virus, copy numbers of the virus were significantly reduced from 12 h to at least 60 h post-infection compared to that achieved in cells transfected with scFv or siRNA-NP604 separately. CONCLUSIONS: A novel combination of antiviral siRNAs expressed in chicken cells and chicken antibody single-chain variable fragments (scFvs) secreted from the cells has a synergistic inhibitory effect on the avian influenza viral proliferation in vitro. Intracellular application of scFvs and anti-viral siRNA may provide a new approach to influenza prevention and treatment.

The first evidence of shaking mink syndrome-astrovirus associated encephalitis in farmed minks, China.

A novel neurological disorder, shaking mink syndrome (SMS), emerged in Denmark and Sweden in 2000. SMS has seldom been reported in China, but the causative agent has not been detected in the country. SMS outbreaks occurred in multiple provinces in 2020. A total of 44 brain samples from minks associated with SMS were collected from Heilongjiang, Liaoning and Shandong provinces of which 28 samples (63.3%) were SMS-astrovirus (SMS-AstV)-positive by reverse transcription PCR. Histopathological examination revealed non-suppurative encephalitis in three minks. Moreover, the complete coding region sequences (CDSs, 6559 bp) of a sample collected from a 2-month-old mink (termed SMS-AstV-H1, GSA accession No. SAMC816786) were amplified by PCR and Sanger sequencing. The complete CDS and open reading frame 2 sequences of SMS-AstV-H1 were 94.3% and 96.4% identical to an SMS-AstV strain (GenBank accession number: GU985458). Phylogenetically, SMS-AstV-H1 was closely related to an SMS-AstV strain (GU985458). Based on the above results, we describe SMS-AstV-associated encephalitis in farmed minks in China. Future studies need to focus on epidemiology, virus isolation and potential interspecies transmission of SMS-AstV.

[Cloning and characterization of the promoter region of Musca domestica yolk protein-1 gene].

A partial Musca domestica genomic library was constructed. It was consisted of 1.2 x 10(5) recombinants with insert length ranging from 10 kb to 23 kb(15 kb average). High molecular weight genomic DNA with more than 50 kb size was extracted from the larva hatched 36 h and digested with unfrequently cutting restriction enzyme Bcl I. DNA fragments of 10 approximately 23 kb were recovered by agarose gel electrophoresis and ligated with EMBL3 BamH I Arms CIPase treated. Then the products of ligation were packed in vitro using packing protein. The cloning efficiency of the genomic library was 5 x 10(4) pfu/mL. The genomic library was screened by hybridization using a probe of a 768 bp partial cDNA fragment of Musca domestica yolk protein 1 (mdYP1) gene obtained by PCR and the probe was labeled with Digoxigen. A positive plaque was chosed and purified by in situ hybridization. A genomic DNA fragment about 4.0 kb mdYP1 was isolated from purified positive plaque by southern blotting analysis. Sequence analysis revealed that mdYP1 genomic gene was composed of 5'-upstream region about 1.7 kb with typical CAAT/TATA box. The promoter of the mdYP1 gene was characterized by examining the ability of 5'-upstream fragments to regulate expression of green fluorescent protein (GFP) in Musca domestica larva. Four fragments of the promoter region, P1 (+296/+7), P2 (+684/+7), P3(+1165/+7) and P4 (+1616/+7) ,were obtained by PCR specific amplification using template of recombinant A-lambdaNA containing mdYP1 gene sequence. Then the four fragments were respectively subcloned into pCMV-GFP reporter vector deleted CMV promoter. All the fragments showed no promoter activity when the four recombinant vectors were transfected into Sf9 and BHK -2 cells respectively, but three of them, P2, P3 and P4, showed significant promoter activity when they were respectively introduced into Musca domestica larva by electroporation. The two fragments, P5 (+684/+302) and P6 (+165/+302), obtained by digesting P2 and P3 with Spe I and Hind III, were also subcloned into pGFP vector, and they showed no promoter activity in Sf9 cells, BHK -21 cells and Musca domestica larva. The results demonstrated that the core promoter spanned 302bp and contained a CCAAT box and a TATA box upstream translation initiation codon (ATG), but itself had no transcriptional activity, and that regulatory promoters or enhancers and other cis-elements presented from +302 to +1616 were necessary to maintain the specific expression.

Distribution and expression of phosphorylated histone H3 during porcine oocyte maturation.

Phosphorylation modification of core histones is correlated well with diverse chromatin-based cell activities. However, its distribution pattern and primary roles during mammalian oocyte meiosis are still in dispute. In this study, by performing immunofluorescence and Western blotting, spatial distribution and temporal expression of phosphorylated serine 10 or 28 on histone H3 during porcine oocyte meiotic maturation were examined and distinct subcellular distribution patterns between them were presented. Low expression of phosphorylated H3/ser10 was detected in germinal vesicle. Importantly, following gradual dephosphorylation from germinal vesicle (GV) to late germinal vesicle (L-GV) stage, a transient phosphorylation at the periphery of condensed chromatin was re-established at early germinal vesicle breakdown (E-GVBD) stage, and then the dramatically increased signals covered whole chromosomes from pre-metaphase I (Pre-MI) to metaphase II (MII). Similarly, hypophosphorylation of serine 28 on histone H3 was also monitored from GV to E-GVBD, indicating dephosphorylation of histone H3 maybe involved in the regulation of meiotic resumption. Moreover, the rim staining on the chromosomes and high levels of H3/ser28 phosphorylation were observed in Pre-MI, MI, and MII stage oocytes. Based on above results, such stage-dependent dynamics of phosphorylation of H3/ser 10 and 28 may play specific roles during mammalian oocyte maturation.

Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA.

We established a rapid procedure for obtaining transgenic mice by directly injecting an enhanced green fluorescent protein (EGFP)-expressing plasmid (pIRES-EGFP) into the ovaries of fertile mice. The frequency of transgenic mouse production was determined by pair-mating, and by polymerase chain reaction (PCR) and sequence analysis of DNA taken from the tails of the offspring. The mice that received the EGFP gene transmitted it to their offspring (F(1)). Genetic and PCR analyses of F(1) progeny confirmed that the inserted EGFP was stably inherited. Of six female F(1) mice, all were able to pass the foreign DNA on to the next generation (F(2)). In situ hybridization using paraffin-embedded sections of ovarian and testicular tissues from the F(1) and F(2) progeny showed that the introduced gene was expressed in the gonads of the animals. The chromosomal location of the injected DNA was determined by fluorescence in situ hybridization, and the frequency of multiple site versus single site insertions is 85.71% (18/21) analyzed by FISH. We anticipate great progress in murine genetic engineering using this technique.

[Study on screening differentially expressed genes in mice livers by silver staining DD-PCR].

AIM: To screen swimming-fatigue related genes in mice and lay theoretic basis for researching the molecular mechanism of fatigue. METHODS: 30 male BALB/c mice (20 +/- 2g) were divided into control group, dipping in water group and swimming-fatigue group respectively. After fatigue for swimming in swimming-fatigue group, with control group and dipping in water group, liver tissues in mice were collected. With improved silver staining mRNA differential display method, the differentially expressed genes in mice livers were screened and evaluated by reversed Northern blot. The positive segments were analyzed homology by BLAST. RESULTS: 7 of DD-ESTs were gained. Two of them only expressed in swimming-fatigue group, two down-regulated expressed, and three up-regulated. One of them was a novel gene and was accepted by GenBank, AY615302. CONCLUSION: Seven DD-ESTs in swimming-fatigue mice were gained by silver staining mRNA differential display method.