Objective To establish a tandem affinity purification(TAP) system of innate immune-regulatory protein PKR and analyze PKR function, for the future screen and identification of novel PKR-interaction proteins. Methods PKR gene was amplified by PCR, and then cloned into a mammalian expression vector pcTAP-A. Recombinant pcTAP-PKR was transfected into PKR knock-down(PKRkd) HeLa cells by LipofectAMINE 2000,and the PKR overexpressed HeLa cells were harvested for mitogen-activated protein kinases(MAPK) activation analysis. Cell extracts of PKR overexpressed cells were purified using TAP kit and examined by Western blot. Results Cal modulin resin(CBP) and streptavidin resin(SBP) tagged PKR was detected in PKRkd HeLa cells as early as 24 h upon transfection with pcTAP-PKR, and its expression decreased at later time points. The overexpression of PKR was autophosphorylated, and thus involved in the regulation of MAPK actviation. After small-scale TAP kit purification, PKR protein was detectable by Western blot. Conclusion We have successfully established a TAP system that over-expresses functional PKR, providing a useful tool for the future study on the identification of PKR interacting proteins.

Objective To clone and express Dengue virus nonstructural protein 4A (NS4A) gene and express in eukaryotic cells.Then,to isolate and purify and isolate cellular proteins interacted with NS4A.Methods With specific primers,NS4A gene fragment tagged with FLAG and HA (FLAG-NS4A-HA) was amplified by PCR and cloned into an expression vector,pSG5 vector.Recombinant plasmid was transfected into A549 cells by LipofectAMINETM2000.Transient expression of FLAG-NS4A-HA was detected by Western blot.The NS4A interacting proteins were isolated and purified by tandem affinity purification (TAP) system using HA and FLAG antibodies,and then assayed by silver stained SDS-PAGE.Results Dengue virus NS4A gene tagged with FLAG and HA was successfully constructed into pSG5 vector and expressed in A.549 cells.Silver stained SDS-PAGE showed that the expressed NS4A and two potential interacting proteins that interact with NS4A were isolated after TAP purification and SDS-PAGE.Conclusion Cellular proteins that potentially interacted with Dengue virus NS4A were successfully purified and isolated,which provided a basis for further research.

Objective: To investigate the cognition of medical staffs in grass-roots hospitals to clinical pharmacy practice and clini-cal pharmacists to provide some references for the further development of clinical pharmacy service. Methods: Totally 625 clinicians and nurses from 4 grass-roots hospitals were surveyed using a questionnaires, and the results were statistically analyzed. Results: The clinicians and nurses had an approval attitude towards clinical pharmacy practice and clinical pharmacists with small difference (overall average score was 3. 73±0. 86 and 3. 70±0. 90, respectively). They showed the highest cognition to clinical pharmacy practice, and the lowest cognition to the development, theory and regulation of clinical pharmacy and clinical pharmacists. Conclusion: It is necessary for grass-roots hospitals to carry out some comprehensive measures to improve clinical pharmacists' professional quality and general ca-pacity, promote the consummation of rules and regulations, and enhance cognition of medical staffs to clinical pharmacy practice and clinical pharmacists so as to promote the development of clinical pharmacy practice, which is helpful in training better clinical pharma-cists.

Objective:To explore the prognostic value of lymphocyte subsets in adult hemophagocytic syndrome (HPS).Methods:A total of 172 adult HPS patients diagnosed in 8 medical centers from January 2013 to August 2020 were selected for the study, of whom 87 were male (50.6%, 87/172), and 85 were female (49.4%, 85/172), with 68 survivors and 104 deaths. The clinical data were summarized, and variables such as lymphocyte subsets, immunoglobulin characteristics and fibrinogen were retrospectively analyzed, and the correlation between the mentioned variables and patient prognosis was analyzed. The optimal cut-off values of continuous variables were calculated by MaxStat, and the prognostic factors of HPS patients were screened based on the Cox proportional hazard regression model.Results:The median age of HPS patients was 56 (42, 66) years old, and the 5-year cumulative survival rate was 37.4% (37.4/100). The median age, platelet and albumin were 48 (27, 63) years, 84×10 9/L and 32.3 g/L in the survival group, and 59 years, 45.5×10 9/L, and 27.3 g/L in the death group, respectively. The differences between the two groups was statistically significant ( Z=?3.368, P=0.001; Z=?3.156, P=0.002; Z=?3.431, P=0.001). Patients with differentiated cluster 8+(CD8+)<11.1%, CD3+<64.9%, CD4+>51%, and CD4/CD8 ratio>2.18 had poor prognosis (χ 2=7.498, P=0.023; χ 2=4.169, P=0.041; χ 2=4.316, P=0.038; χ 2=9.372, P=0.002). Multivariable analysis showed that CD4/CD8 ratio, age, fibrinogen and hemoglobin were independent prognostic factors in HPS patients ( HR=2.435, P=0.027; HR=5.790, P<0.001; HR=0.432, P=0.018; HR=0.427, P=0.018). Conclusion:Peripheral blood lymphocyte subsets can be used to evaluate the prognosis of patients with HPS; CD4/CD8 ratio, age, fibrinogen, and hemoglobin are independent prognostic factors in HPS patients.