This study explored the effect of the Mycobacterium tuberculosis(Mtb)PE/PPE family protein PE_PGRS37 on the growth of Mycobacterium smegmatis(Ms)and macrophage autophagy during Mtb infection.The pe_pgrs37 gene was amplified from Mtb genome through PCR,and the recombinant vector pAIN-PE_PGRS37 was successfully constructed through homologous recombi-nation.pAIN-PE_PGRS37 and pAIN were integrated into Ms through electroshock to construct pAIN-PGRS37/Ms and pAIN/Ms re-combinant bacteria.Western blotting indicated that the PE_PGRS37 protein was correctly expressed in pAIN-PE_PGRS37/Ms.The re-combinant bacteria were inoculated in 7H9/7H10 medium,and their colony morphology and growth curves were observed.No signifi-cant difference in colony morphology was observed between pAIN-PE_PGRS37/Ms and pAIN/Ms.The growth rate significantly in-creased between 10 and 16 h,and a plateau was reached at 26 h.After infection of U937 cells with pAIN-PE_PGRS37/Ms and pAIN/Ms,macrophage autophagy flow was detected with western blotting and immunofluorescence.In the pAIN-PE_PGRS37/Ms-infected group,compared with the pAIN/Ms-infected group,macrophage LC3-II and p62 protein expression was significantly up-regulated(P<0.001)and inhibited autophagosome and lysosome fusion.The intracellular survival of the recombinant bacteria was detected through colony counting,and pAIN-PE_PGRS37/Ms showed significantly greater survival in macrophages at 12 h,24 h,and 48 h than pAIN/Ms(P<0.05).Our results suggested that PE_PGRS37 protein promotes Mycobacterium survival in macrophages by blocking macro-phage autophagy flow,thus inhibiting macrophage autophagy.

This study explored the effect of the Mycobacterium tuberculosis(Mtb)PE/PPE family protein PE_PGRS37 on the growth of Mycobacterium smegmatis(Ms)and macrophage autophagy during Mtb infection.The pe_pgrs37 gene was amplified from Mtb genome through PCR,and the recombinant vector pAIN-PE_PGRS37 was successfully constructed through homologous recombi-nation.pAIN-PE_PGRS37 and pAIN were integrated into Ms through electroshock to construct pAIN-PGRS37/Ms and pAIN/Ms re-combinant bacteria.Western blotting indicated that the PE_PGRS37 protein was correctly expressed in pAIN-PE_PGRS37/Ms.The re-combinant bacteria were inoculated in 7H9/7H10 medium,and their colony morphology and growth curves were observed.No signifi-cant difference in colony morphology was observed between pAIN-PE_PGRS37/Ms and pAIN/Ms.The growth rate significantly in-creased between 10 and 16 h,and a plateau was reached at 26 h.After infection of U937 cells with pAIN-PE_PGRS37/Ms and pAIN/Ms,macrophage autophagy flow was detected with western blotting and immunofluorescence.In the pAIN-PE_PGRS37/Ms-infected group,compared with the pAIN/Ms-infected group,macrophage LC3-II and p62 protein expression was significantly up-regulated(P<0.001)and inhibited autophagosome and lysosome fusion.The intracellular survival of the recombinant bacteria was detected through colony counting,and pAIN-PE_PGRS37/Ms showed significantly greater survival in macrophages at 12 h,24 h,and 48 h than pAIN/Ms(P<0.05).Our results suggested that PE_PGRS37 protein promotes Mycobacterium survival in macrophages by blocking macro-phage autophagy flow,thus inhibiting macrophage autophagy.

Objective:To investigate the cognition and willingness of nursing staff to Internet plus nursing service, and analyze the related factors that affect their participation in Internet plus nursing service.Methods:From April to May 2019, 150 nurses from three hospitals in Weifang were investigated by questionnaire and key person interview. Descriptive analysis and binary logistic regression analysis were used to analyze the intention of nursing staff to participate in Internet plus nursing service. The interview mode was used to analyze the appropriate mode of Internet plus nursing service from the perspective of nursing staff.Results:142 valid questionnaires were obtained, of which 137(96.5%)indicated willingness to provide Internet plus nursing services, and 135(95.1%) realized the necessity of the service. Education, marriage, nurses′judgment on the necessity of the service and their own subjective judgment of their competence were the factors affecting nurses′ participation in the Internet plus nursing service.Conclusions:Multiple factors affect the choice of nursing staff′s behavior in Internet plus nursing service.From the perspective of nursing staff, the Internet plus nursing service mode needs joint efforts from many aspects.

Objective To study the implementation effects of China′s contracted service policy for family physicians. Methods Systematic evaluation method was used to extract, describe and analyze the literature information of the research on the implementation effect of family physicians contracted service policy. Results A total of 80 papers of four types were rounded up, including 47 on the effect of health management on patients with chronic diseases, 11 on the effect of health management on the elderly, 15 on the effect on the first diagnosis in the community, and 7 on the effect on the control of medical expenses.The research is mostly distributed in the developed areas in the east. The family physicians contracted service promotes the health management effect of patients with chronic diseases and the elderlies, improves the first visit ratio of residents at their community, and effectively controls the medical expenses.Existing research shows that such a service has achieved initial success.However, the research also identified such common problems as the shortage and low competence of family physicians, low quality, resource integration and inadequate policy publicity. Conclusions The contracted service policy in China has begun to play the role of " health gatekeeper" and " cost gatekeeper" to some extent.It is suggested to strengthen the training of general practitioners, establish and perfect incentive mechanism, and improve the construction of information platform, while the implementation effect of contracted services for family physicians deserve further study in a broader scope, deeper level and design specifications.

Objective Grey red-backed voles (Myodes rufocanus) are agile, fierce and hard to catch, thus, it is difficult to judge their gender by external appearance, especially for the juvenile voles. Therefore, it may cause difficulties to their allocation and later breeding in laboratories. The aim of this paper is to establish a rapid, simple and accurate method for gender identification of grey red-backed voles. Methods Fresh hair follicles were taken from 6 adult male voles, 3 adult females and 14 4-week-old juvenile voles, 5 male and 5 female 9-week-old Wistar rats, and 5 male and 3 female 6-week-old BALB/c mice. The genomic DNA was extracted using Chelex-100 resin and the zinc-finger Y/X gene (ZFY/ZFX) and the gene of sex-determining region of the Y (SRY) chromosome were amplified by PCR, and a double PCR amplification method was established. Results The ZFY/ZFX gene and SRY gene were simultaneously amplified from the male voles, while only the ZFY/ZFX gene was amplified from the females. The gender of all 23 voles, 10 Wistar rats and 8 BALB/c mice were correctly identified with this method, and the PCR results were consistent with the phenotypic and autopsy results. Conclusions Using fresh hair follicles as experimental materials for gender identification of grey redbacked voles can alleviate shock and damage to the animals. The established double PCR amplification method is accurate, simple, rapid, and deserves to be used for gender identification of grey red-backed voles.

Objective To establish a breeding method ofMyodes rufocanus in the laboratory,collect their growth and reproduction data,and provide a basis for carrying out the experimental animalization.Methods Wild Myodes rufocanus caught in the Moranbong woodland were brought back to the laboratory.They were bred artificially in a large hard wall rodent negative pressure isolator.Their growth and reproduction data were recorded for evaluating the results of breeding.Results The Myodes rufocanus were successfully bred in the laboratory.The pregnancy rate was 54.55%.The average pregnancy length was 20.4 days(8 to 22 days).During one breeding period,they gave birth 2.9 times on average.The maximum number of births was 7 times,far more than the number tested under field conditions.The average litter size was 4.3±1.22.The highest litter number of a single nest was 8.The weaning rate of pups was 94.8%.The growth and development of pups were good.Conclusions The breeding method for Myodes rufocanus is established.The growth and reproduction data are tested too.The results of our study laid a foundation for the experimental animalization of Myodes rufocanus.

Objective The aim of this study was to investigate the polymorphism of SLA class II genes in Canadian SPF Yorkshire and Landrace pigs.Methods Blood samples were obtained from 15 SPF Yorkshire and 22 Landrace pigs for isolation of peripheral blood mononuclear cells respectively, and the DQB1, DRB1 and DQA genes were amplified by PCR after reverse transcription.SLA class II genes were obtained by analyzing the direct and cloning result.The polymorphism of alleles was analyzed using the DNAsp 5.0 software.Results A total of 25 alleles were identified at three genes, including eight DQB1, ten DRB1 and seven DQA, and three alleles were submitted the complete sequences for the first time.The official allele names were assigned as SLA-DQB1*0212 (KU754590), SLA-DQB1*0203 (KU754591) and DRB1*06:07(KU754601) by the SLA Nomenclature Committee.Three novel DQA alleles were discovered.Five of the 15 amino acids, one of the 16 amino acids and 11 of the 19 amino acids, which bind processing antigens, showed well conserved among the alleles of DQB1, DRB1 and DQA genes in the SPF Yorkshire and Landrace pigs, respectively.Neighbor-joining tree showed that the three genes were divided into two clusters, respectively.There was a close relationship between SPF Yorkshire and Landrace pigs and foreign Yucatan miniature pigs, and it showed no obvious genetic distance with other pigs.Conclusions A total of 25 SLA class II alleles have been identified successfully in this study, and there are more abundant polymorphism for them.There is a widely distribution for SLA class II alleles identified in this study in other pig breeds.It is critical for the eventual future use of SPF Yorkshire and Landrace pigs as classical laboratory animal models.

Objective To investigate the effects of hydrogen sulfide on sepsis-induced cardiac dysfunction in rats.Methods Male SD rats were randomly ( random number) divided into 4 groups:control group ( Ⅰ group),sepsis group ( Ⅱ group),sepsis + NaHS group (Ⅲ group),sepsis + PAG group (Ⅳ group). Cecal ligation and puncture technique was used in Ⅱ, Ⅲ, Ⅳ group. Hemodynamic was observed,and H2S synthesis,CSE (cystathionine-r-lyase) mRNA,MPO activity and the level of TNF-α,IL-1β were determined.The morphological changes and infiltration of inflammatory cells in myocardium were also observed.Results Compared with Ⅰ group,H2S synthesis,the levels of TNF-α,IL-1 β,the activity of MPO increased ( P ＜ 0.05 or P ＜ 0.01 ),and the expression of CSE-mRNA increased,and blood pressure of rats decreased significantly in Ⅱ group.Compared with Ⅱ group,H2S synthesis,the levels of TNF-α,IL-1β and the activity of MPO increased (P ＜ 0.05),the expression of CSE mRNA did not change noticeably ( P ＞ 0.05),and blood pressure of rats decreased more significantly in Ⅲ group.Compared with Ⅱ group, H2S level decreased,the levels of TNF-α,IL-1β and the activity of MPO decreased significantly,the expression of CSE mRNA decreased and blood pressure of rats decreased in Ⅵ group (P ＜0.05).Histopathological changes of myocardium were aggravated in the following severity order: Ⅰ ＜ Ⅳ ＜＜ Ⅲ.Conclusions CSE/H2S system of the myocardium was upregulated in sepsis rats.Hydrogen sulfide could raise the levels of MPO,TNF-α,IL-1β,aggravating myocardial injury. Contrarily,the inhibitor of H2S could counteract it.

Objective To investigate the changes of the expression of HIF-1α (hypoxia induciblefactor-1a) mRNA in myocardium of rats after cardiopulmonary resuscitation (CPR) and the intervention effects of exogenous hydrogen sulfide on it. Method Forty five male SD rats were randomly divided into three groups: control group ( n = 15 ), cardiopulmonary resuscitation group ( CPR group, n = 15 ) and NaHS + CPR group (n = 15 ) . Rats of control group were anesthetized and intubated without asphyxia and cardiac arrest. The rats of CPR group and NaHS + CPR group were operated to induce cardiac arrest by asphyxiation. In the rats of NaHS + CPR group, NaHS in dose of 50 ug/kg was administrated via the femoral venous line 1 minute before CPR. Hemodynamic was monitored continuously. The expression of HIF - lα mRNA in myocardium of rats in each group was determined by using RT-PCR and the activity of myeloperoxidase (MPO) in the myocardium of rats in each group was assayed by using a patent reagent box 6 h after CPR. The histopathological changes of myocardium were also observed. The t- test was used for statistical analysis. Results There was no statistically significant difference in hemodynamic changes between CPR group and CPR + NaHS group ( P ＞ 0. 05 ) . When compared with the control group, the activity of MPO and the expression of HIF-1α mRNA in CPR group and CPR + NaHS group were both increased, and those increased in CPR + NaHS group was more significant (P ＜ 0. 05) . When compared with CPR group, the expression of HIF-1α mRNA in CPR + NaHS group was higher, however, the activity of MPO in CPR + NaHS group was lower ( P ＜ 0. 05) . There were various histopathological changes of myocardium of rats found in CPR group and CPR + NaHS group, and the damage of myocardium of rats in CPR group was more obvious than that in CPR + NaHS group. Conclusions The expression of HIF-1α mRNA in myocardium of rats was increased after CPR. Exogenous hydrogen sulfide can protect myocardium cells from resuscitation-perfusion injury, and the protection is associated with increase in expression of HIF 1 αmRNA.

To investigate the interaction and involvement of sodium hydrosulfide (NaHS), a H(2)S donor, on hippocampus of rats suffering from sepsis-associated encephalopathy, rats were subjected to cecal ligation and puncture (CLP)-induced sepsis. Adult male Sprague-Dawley rats were randomly divided into four groups: Sham group, CLP group, CLP+NaHS group and CLP+aminooxyacetic acid (AOAA, an inhibitor of H(2)S formation) group. The four groups were observed at 3, 6, 9, 12 h after treatment. We examined hippocampal H(2)S synthesis and the expression of cystathionine-β-synthetase (CBS), a major enzyme involved in the H(2)S synthesis in hippocampus. CBS expression was detected by reverse transcription polymerase chain reaction (RT-PCR). The concentrations of inflammatory cytokines (TNF-α, IL-1β) were determined in hippocampus by using enzyme-linked immunosorbent assay (ELISA). Neuronal damage was studied by histological examination of hippocampus. In CLP group, H(2)S synthesis was significantly increased in hippocampus compared with sham group and it peaked 3 h after CLP (P<0.05). Sepsis also resulted in a significantly upregulated CBS mRNA in hippocampus. The levels of TNF-α and IL-1β in the hippocampus were substantially elevated at each time point of measurement (P<0.05), and they also reached a peak value at about 3 h. Administration of NaHS significantly aggravated sepsis-associated hippocampus inflammation, as evidenced by TNF-α and IL-1β activity and histological changes in hippocampus. In septic rats pretreated with AOAA, sepsis-associated hippocampus inflammation was reduced. It is concluded that the rats subjected to sepsis may suffer from brain injury and elevated pro-inflammatory cytokines are responsible for the process. Furthermore, administration of H(2)S can increase injurious effects and treatment with AOAA can protect the brain from injury.