Objective To develop a real-time RT-PCR assay ( rRT-PCR) for efficient detection of duck hepatitis virus type 1 ( DHV-I) .Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay ( rRT-PCR) .Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates.The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China.Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.

The objective of the study was to clone avian beta-defensin (AvBD) 5 gene from pigeon bone marrow tissues and liver tissues, to express the recombinant AvBD5 protein in E. coli, and to determine its antimicrobial activity. The mRNA of duck AvBD5 was cloned from pigeon bone marrow tissues and liver tissues by RT-PCR. In addition, phylogenetic relationships between amino acid sequence of the pigeon AvBD5, AvBDs from other avian species, and some mammalian beta-defensin-5 were analyzed. The cDNA of pigeon AvBD5 was sub-cloned into pGEX-6p-1 vector to construct recombinant plasmid pGEX-pigeon AvBD5. The recombinant protein was expressed into E. coli and purified. Antimicrobial activity and physical-chemical stability of the recombinant fusion protein were measured in vitro. The complete nucleotide sequence of both cDNAs contained 201 bp nucleotides, encoding a polypeptide of 66 amino acids. Both beta-defensins have six conserved cysteines. Phylogenetic relationships were analyzed. Both pigeon AvBDs shared the highest amino acid homology (87.9% and 78.8%) with duck AvBD5. So it was named as pigeon AvBD5alpha (bone marrow) and AvBD5beta (liver). Both recombinant plasmids were transformed into E. coli BL21 and the bacteria were induced with Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). After purification, antibacterial activity of the purified was investigated. In addition, effect of ionic strength on the antibacterial activity, and hemolytic recombinant protein activity of the purified recombinant protein were investigated. A 32 kDa protein was highly expressed. Both purified recombinant pigeon AvBD5alpha and AvBD5beta exhibited extensive antimicrobial activities against 12 bacteria, including Gram-positive and Gram-negative. In high salt ions concentrations, antibacterial activity of both recombinant proteins was decreased. In addition, the hemolysis activity of recombinant protein was extremely low.
Amino Acid Sequence ; Animals ; Anti-Infective Agents ; metabolism ; pharmacology ; Avian Proteins ; biosynthesis ; genetics ; pharmacology ; Cloning, Molecular ; Columbidae ; genetics ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; beta-Defensins ; biosynthesis ; genetics ; pharmacology

The objective of the study was to clone avian beta-defensin (AvBD) 3 gene from goose tissues, express the recombinant AvBD3 protein in Escherichia coli, and determine its antimicrobial activity. The mRNA of goose AvBD3 was cloned from spleen and bursa of Fabricius of the gooses by RT-PCR. The sequence analysis showed that the genefragment of AvBD3 contained 182 bp, and encoded 60 amino acids. Homology analysis showed that goose AvBD3 shared the highest percentage of amino acid homology (100%) with chicken AvBD3. The cDNA of goose AvBD3 was sub-cloned into BamH I and Sal I sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-goose AvBD3. The recombinant plasmid was transformed into E. coli BL21 and the bacteria was induced with IPTG It was demonstrated by SDS-PAGE that a 31 kDa protein which was equal to goose AvBD3 protein in molecular weight was highly expressed. The purified recombinant goose AvBD3 exhibited extensive antimicrobial activity against twelve bacteria strains, including Gram-positive and Gram-negative investigated. At high salt ions conditions, antimicrobial activity of recombinant goose AvBD3 protein against both Staphylococcus aureus and Pasteurella multocida decreased significantly. In addition, hemolysis activity of the recombinant protein was extremely low, and the recombinant protein remained antimicrobial activity under different pH values.
Amino Acid Sequence ; Animals ; Anti-Bacterial Agents ; chemistry ; metabolism ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Geese ; genetics ; metabolism ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; beta-Defensins ; genetics ; isolation & purification ; metabolism

OBJECTIVETo explore the protective effects of rhubarb aglycone combined with urokinase (UK) thrombolysis on brain microvascular basement membrane impairment in rats with thrombus-occluded cerebral ischemia by regulating the expression of IgG, CoLIV and LN in rats brain, by which the level of injury of brain microvascular basement membrane could be detected.

METHODRats were randomly divided into sham-operated, model, thrombolysis, rhubarb aglycone and combination (rhubarb aglycone combined with thrombolysis) groups. Moreover, rats in model, thrombolysis, rhubarb aglycone and combination groups were randomly divided into 3, 6, and 9 h groups respectively. Model of thrombus-occluded cerebral ischemia was duplicated by using the combination of rats' auto-thrombus with inserting the nylon thread. Rats were administrated with thrombolysis therapy through artery at 3, 6, and 9 h after cerebral ischemia. At 24 h of administration through artery, intracranial hemorrhage ratio (ICHR) and mortality of rats were observed, and then the brain of rats was taken. In the study, expression of IgG, CoLIV and LN in rats brain were measured.

RESULTThrombolysis at 9 h of cerebral ischemia made rats mortality and BHR increase, administration of combined therapy could make them decrease. Expression of IgG level in rats brain of 9 h and 6 h model groups increased, while CoLIV and LN expression decreased significantly. In each administration 9 h group, IgG level was lower, and CoLIV and LN were higher, such changes appeared significantly in rhubarb aglycone and association groups.

CONCLUSIONBrain microvascular basement membrane impairment could be caused by the therapy of delayed thrombolysis, which made the mortality and BHR increase. Rhubarb aglycone combined with the therapy of thrombolysis could perform the protective effects on brain microvascular basement membrane and then decrease the ICHR and mortality caused by thrombolysis after cerebral ischemia.

Animals ; Basement Membrane ; blood supply ; drug effects ; Brain Ischemia ; drug therapy ; mortality ; Disease Models, Animal ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Intracranial Thrombosis ; drug therapy ; mortality ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rheum ; chemistry ; Thrombolytic Therapy ; Urokinase-Type Plasminogen Activator ; therapeutic use

Amid ongoing reforms in the healthcare system and the pursuit of high-quality development in public hospi-tals,the significance of party building in leading the standardization of hospital party branches has become increasingly promi-nent.Taking a university's affiliated hospital as an example,this study comprehensively analyzes the current situation of Party building on the standardized construction of party branches within university-affiliated public hospitals using the SWOT method.Meanwhile,this paper proposes targeted strategies by assessing the strengths,weaknesses,opportunities,and challenges of party building leadership.These strategies are intended to refine the framework for the role of Party building in advancing the standard-ized construction of Party branches in university-affiliated public hospitals.

OBJECTIVES: To investigate whether necrostatin-1 (Nec-1) can protect islet cells from the damage induced by TNF-α. METHODS: After isolation and purification, the neonatal porcine islet cell clusters (NICCs) were divided into 3 groups (islets 10 000 IEQ/group): a Nec-1 group (Nec-1+TNF-α was added to the culture medium), a TNF-α group (TNF-α was added to the culture medium), and a control group (pure medium). The number of cells was observed after 48 h of co-culture. The cell death was evaluated by AO/EB staining. Insulin secretion and DNA of islets were detected by chemiluminescence and nucleic acid quantitative analysis. RT-PCR assay was used to examine the mRNA expressions of insulin gene, glueogan gene and somatostatin gene. Flow cytometry analysis was used to detect the viability of B cells. RESULTS: The number of islets in Nec-1 group, TNF-α group and the control group were (8 425±2 187), (4 325±778), and (7 122±1 558) IEQ, respectively. Compared to the other two groups, the number of dead cells in TNF-α group was greatly increased. The insulin/DNA values in the Nec-1 group, TNF-α group and blank control group were (13.21±3.15), (2.47±0.45), and (7.44±0.97) mIU/mg, respectively. Compared to the TNF-α group and the control group, the mRNA relative expression levels of insulin gene (6.73±1.07), glucagon gene (10.13±1.98), somatostatin gene (8.57±1.11) were significantly increased in the Nec-1 group (all <0.05), the rate of live cells (97.32±1.87)% and live B cells (90.86±3.68)% were increased significantly in the Nec-1 group (all <0.05). CONCLUSIONS TNF-α can induce neonatal porcine islet cells damage, which is attenuated in the presence of Nec-1. Nec-1 can increase the content of endocrine cells in NICCs.
Animals ; Imidazoles ; Indoles ; Insulin ; Islets of Langerhans ; Swine ; Tumor Necrosis Factor-alpha ; genetics

Objective: To investigate the epidemiologic features of respiratory viral etiology in hospitalized children with acute respiratory tract infection (ARTI) in Shijiazhuang. Methods: A total of 28 512 cases of hospitalized children with clinical diagnosis of ARTI in Children′ s Hospital of Hebei Province from 2014 to 2017 were recruited into this study. One nasopharyngeal swab was collected from each patient. Immunofluorescence assay was used to detect seven kinds of respiratory viruses, including respiratory syncytial virus (RSV), parainfluenza virus (PIV) type 1-3, influenza virus type A, B (FluA, FluB) and adenovirus (ADV). Results: At least one viral pathogen was identified in each of 9 263 out of 28 512 patients and the overall positive rate was 32.5%. Of 9 263 virus-positive patients, 9 070 (97.9%) had mono-infection. The most frequently detected virus was RSV, followed by PIV-3 and FluA. The positive rates of RSV and PIV-1 showed annually decreasing tendency, meanwhile the positive rate of FluA increased in the nearly two years. The detection rate of ADV and PIV-1 increased every other year. There was a significant difference in the positive rate among different years (P<0.05). The overall positive rate decreased along with the age increased (linear by linear association χ²=1191.289, P<0.05). The detection rates of RSV and PIV-3 were the highest in groups of <1 year old and 1-3 years old and decreased along with the age increased. The preschool children were more susceptible to developing FluA, FluB and ADV related diseases. There was a significant difference in the positive rate among different age groups (P<0.05). The viral distribution was uneven in different seasons, and the infection peaked in winter, the difference was statistically significant (P<0.05). The epidemic seasons of RSV and FluA were winter, and FluB infection was epidemic in winter and spring. The positive rates of PIV-1 and PIV-2 were most common in summer and autumn. PIV-3 was usually prevalent in spring and summer and ADV was prevalent sporadically. Conclusions RSV is the most common pathogen in hospitalized children with clinical diagnosis of ARTI during 2014-2017 and the positive rate of which showed an annually decreasing tendency. The positive rate of FluA increased in the nearly two years. Children in infancy are susceptible to the seven common respiratory viruses and winter is the epidemic season for these viruses.