Objective: To establish an indirect enzyme-linked immunosorbent assay (ELISA)and to compare the efficiency of receptor binding domain (RBD) proteins in different forms for Middle East respiratory syndrome coronavirus (MERS-CoV) antibody detection. Methods: The monomeric and trimeric forms of MERS-CoV RBD were expressed in Bac-insect cells, 293T cells and ExpiCHO-S™ expression system and then purified. The purified RBD proteins were identified with native gel electrophoresis and Western blot. Then, an equal amount of each RBD protein was used as coating antigen to establish an ELISA for detecting MERS-CoV IgG titer. For comparison, the newly developed ELISA and the commercial MERS-CoV IgG antibody detection kit (Euroimmune with S1 as the coating antigen) were used to measure the MERS-CoV antibody reference panel supplied by World Health Organization (WHO). Results: The purified monomeric and trimeric MERS-CoV RBD were successfully prepared using 293T cells and ExpiCHO-S™ system. RBD antigens of different forms and from different systems could recognize MERS-CoV specific antibody without having any cross reaction with the sera from healthy adults. The in-house RBD-based ELISA had good detection consistency with the Euroimmune commercial kit. The positive samples showed higher and more concentrated values based on the RBD trimer than the monomer. Conclusions Novel indirect ELISA methods based on the monomeric and trimeric forms of RBD protein were established. The trimetric form-based ELISA achieved higher detection efficiency than the one using the monomer antigen, suggesting that it could be uses as a competent alternative to the commercial kit.

Objective To explore the feasibility of MR lymphography (MR-LG) in detecting internal mammary sentinel lymph node (SLN) for rabbit models of VX2 breast cancer.Methods Totally 75 New Zealand white rabbits were randomly divided into 2 groups to establish VX2 breast cancer (tumor group) and inflammation models (inflammation group),respectively.MR-LG examinations and lymph node dissection were performed in all the rabbits.The results of internal mammary SLN and lymphatic vessels mapping were recorded,and the influence factors were analyzed.Results The success rate of model establishment was 97.33％ (73/75).The detection rate of internal mammary SLN and lymphatic vessels was 15.94％ (11/69) and 75.36％ (52/69),respectively,there was no statistical difference between the two groups (both P＞0.05).The rabbit's weight,time of tumor growth,tumor size,number,size and status of axillary SLN,as well as presence or not of internal mammary lymph vessels detection showed no statistical difference between detection and non-detection of internal mammary SLN (all P＞0.05),while the tumor size and axillary lymph node number showed statistically significant differences between detection and not detection of internal lymphatic vessels (both P＜0.05).The accuracy,sensitivity,specificity,false negative and false positive rate of internal mammary SLN detection with MR-LG was 76.81％ (53/69),39.13％(9/23),95.65％(44/46),60.86％(14/23) and 4.35％(2/46),respectively.Conclusion MR-LG can effectively detect internal mammary SLN and lymphatic vessels for rabbit models of VX2 breast cancer.

Objective: To explore the application effect of video teaching combined with Teach-back method in rehabilitation training of patients with hemiplegia. Methods: The patients with glioma in eloquent area in department of neurosurgery were selected as the research subjects, grouped by time sequence of admission. Between July 2016 to August 2016, 30 patients were selected as control group. Between September 2016 to November 2016, 30 patients were selected as observation group. The control group using conventional department of neurosurgery postoperative routine nursing and hemiplegia rehabilitation education. The observation group was given feedback teaching method on basis of the control group. On the third days after operation, 2 weeks after operation and 4 weeks after operation respectively, the muscle strength, activities of daily living and satisfaction of two groups were evaluated. Results: The recovery of muscle strength of the lower limbs of the observation group was better than the control group at 4 weeks after operation (t=5.015, 5.320, P<0.05); At 2 weeks after operation and 4 weeks after operation, the daily living ability score of the observation group was higher than the control group (t=5.360, 6.510, P<0.05); There were statistically significant differences in the scores of daily living ability between the two groups at different time points (F=68.64, 65.07, P<0.01); The observation group had higher scores of daily living ability than the control group (t=2.315, P<0.05) ; At 2 weeks after operation and 4 weeks after operation, the patient satisfaction of the observation group was higher than the control group (t=2.042, 4.317, P<0.05) . Conclusion For glioma patients with postoperative limb movement disorders, feedback teaching method combined with rehabilitation training can promote the recovery of muscle strength, especially the lower limbs, improve the ability of daily living and the satisfaction of patients.

Objective To study the value of CT lymphography (CT-LG) in evaluating axillary sentinel lymph nodes (SLN) of rabbit with breast cancer using 3D and curved planar reformation (CPR).Methods CT-LG examinations were completed on the VX2 rabbit models bearing breast cancer established by tumor tissue mass suspension injection.All raw data were transferred to a postprocessing workstation, then the 3D and CPR were used to show lymphatic vessel and lymph nodes.On the CT-LG images, one or several lymph nodes displayed firstly in the lymphatic drainage pathway was defined as the SLN which was then confirmed by the biopsy.Results (1) 20 rabbits bearing tumor underwnet CT-LG examinations, and both SLNs and draining lymphatic vessels were detected using 3D and CPR techniques.70% (14/20) of experimental rabbits had fine images, and 16 SLNs and 7 non-SLNs were demonstrated on 14 rabbits on CT-LG images with good quality.(2) 8 continuous lymphatic vessels displayed well, exhibiting a homogeneous and continuous tube with uniform contrast-medium filling on CPR images.Most lymphatic vessels demonstrated well, and the 14 vessels with local interrupt or fuzzy on 3D images showed continuous tubular contrast-filling with heterogeneous high-density on the CPR images from the injection sites to axillary SLN.Among 6 rabbits with poor images, 3 rabbits showed the SLN but without lymphatic vessels on 3D images, 2 showed only initial segment of lymphatic vessel,and 1 had no lymphatic vessel and SLN.(3) A total 24 SLNs were obtained by SLNB,including 18 metastatic SLNs and 6 negative ones.On CT-LG images, there were statistical differences between the metastatic and negative SLNs in the maximum diameter and filling defect (P=0.041 and P=0.001, respectively).However, the short diameter, shape and margin of SLNs had no statistical differences between the two groups (all P>0.05).Conclusion On CT-LG examinations, 3D and CPR images may show axillary SLNs and lymphatic vessels effectively,and CPR can demonstrate more details than 3D.The maximum diameter and fillingdefect of SLN are helpful for distinguishing metastatic and negative SLNs.

Cognitive dysfunction,as a common symptom among patients with chronic fatigue syndrome (CFS) and patients with fibromyalgia(FM),impacts on life quality,occupation and study of these patients.However,the neural correlates to the cognitive impairment are unknown.Event related potentials,which reflect the information processing objectively and constantly,provide possibility for taking a insight into and estimating the dysfunction.By summarizing and analyzing studies in event related potentials about chronic fatigue syndrome,fibromyalgia,we found that CFS patients were characterized with prolonged latency of N200 and P300 accompanied by decreased P300 amplitude when they performed on Oddball paradigm,fibromyalgia patients were characterized with lower P300 amplitude when they concentrated on Oddball task,meanwhile,fibromyalgia patients also showed decreased P100/N100,P200,P300,LPC in emotional word decision task and somatic pictures decision task.It's suggests that the cognitive dysfunction in CFS is mainly caused by slowed speed of information identification and classification,whereas in FM it's dysregulation in attention control system results in the cognitive dysfunction.Limitations in current studies and prospects on researches about cognitive dysfunction in CFS for future were also discussed.

OBJECTIVETo evaluate the validity and reliability of Diabetes Self-management Knowledge, Attitude, and Behavior Assessment Scale (DSKAB).

METHODSWe selected 460 patients with diabetes in the community, used the scale which was after two rounds of the Delphi method and pilot study. Investigators surveyed the patients by the way of face to face. by draw lots, we selected 25 community diabetes randomly for repeating investigations after one week. The validity analyses included face validity, content validity, construct validity and discriminant validity. The reliability analyses included Cronbach's α coefficient, θ coefficient, Ω coefficient, split-half reliability and test-retest reliability.

RESULTSThis study distributed a total of 460 questionnaires, reclaimed 442, qualified 432. The score of the scale was 254.59 ± 28.90, the scores of the knowledge, attitude, behavior sub-scales were 82.44 ± 11.24, 63.53 ± 5.77 and 108.61 ± 17.55, respectively. It had excellent face validity and content validity. The correlation coefficient was from 0.71 to 0.91 among three sub-scales and the scale, P<0.001. The common factor cumulative variance contribution rate of the scale and three sub-scales was from 57.28% to 67.19%, which achieved more than 50% of the approved standard, there was 25 common factors, 91 items of the total 98 items held factor loading ≥0.40 in its relevant common factor, it had good construct validity. The scores of high group and low group in three sub-scales were: knowledge (91.12 ± 3.62) and (69.96 ± 11.20), attitude (68.75 ± 4.51) and (58.79 ± 4.87), behavior (129.38 ± 8.53) and (89.65 ± 11.34),mean scores of three sub-scales were apparently different, which compared between high score group and low score group, the t value were - 19.45, -16.24 and -30.29, respectively, P<0.001, and it had good discriminant validity. The Cronbach's α coefficient of the scale and three sub-scales was from 0.79 to 0.93, the θ coefficient was from 0.86 to 0.95, the Ω coefficient was from 0.90 to 0.98, split-half reliability was from 0.89 to 0.95.Test-retest reliability of the scale was 0.51;the three sub-scales was from 0.46 to 0.52, P<0.05.

CONCLUSIONThe validity and reliability of the Diabetes Self-management Knowledge, Attitude, and Behavior Assessment Scale are excellent, which is a suitable instrument to evaluate the self-management for patients with diabetes.

Objective To prepare and screen out monoclonal antibodies against the receptor bind-ing domain (RBD) of Middle East respiratory syndrome coronavirus ( MERS-CoV) spike ( S) protein in mice. Methods The RBD of MERS-CoV S protein expressed in the insect-baculovirus system was purified and then used to immunize the female BALB/ c mice. The spleen cells collected from the mice were fused with myeloma Sp2 / 0 cells. The positive hybridoma cells were obtained by using limited dilution method. Enzyme-linked immunosorbent assay ( ELISA), Western blot assay and neutralization test based on the MERS-CoV pseudovirus were performed for further screening and identification. Results Twelve strains of hybridoma cells that produced the monoclonal antibodies against RBD of MERS-CoV S protein were screened out. All of the 12 monoclonal antibodies (McAbs) could have specific reaction with the RBD of MERS-CoV S protein as indicated by the results of ELISA. Of the 12 McAbs, two were identified as the immunoglobulin M (IgM) isotype and the rest were IgG1 isotype by using double antibodies sandwich ELISA. Four McAbs including 1F1, 2E4, 3C3 and 3E6 were identified as having neutralizing activity by the neutralization test based on MERS-CoV pseudovirus. Results of the Western blot assay showed that the four McAbs (1F1, 2E4, 3C3 and 3E6) could have specific reaction with the RBD of MERS-CoV S protein, but no cross-reac-tion with that of SARS-CoV S protein. Conclusion Twelve mouse-derived McAbs against the RBD of MERS-CoV S protein were obtained. The prepared hybridoma cells showed the characteristics of high speci-ficity and stability in antibody secretion. Four out of the 12 McAbs were proved to have neutralizing activity.

Objective To establish an inactivated viral particle-based ELISA for the detection of antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) in serum samples collected from a MERS-CoV associated case. Methods Serum samples were collected from 10 newborns and 40 healthy adults. A viral particle-based ELISA was established by using the inactivated MERS-CoV virions as antigen. The levels of IgM and IgG antibodies in the serum samples were detected by the established ELISA and the cut-off values for positive detection were determined. Then the inactivated MERS-CoV virion-based ELISA was used to detect the antibodies against MERS-CoV in 5 serum samples collected from the first im-ported MERS case in China. Results The cut-off values of IgM and IgG antibodies in serum samples for ELISA were determined to be A450 readings of 0. 32 and 0. 42, respectively. The titers of IgM and IgG anti-bodies in serum samples collected at early admission to hospital from the first imported MERS case in China were both 1 ︰ 40. Seroconversion occurred 2 weeks after his admission to hospital with the titers of IgM and IgG reaching to 1 ︰ 320. Conclusion The inactivated MERS-CoV virion-based ELISA was established successfully and could be used for the detection of serum antibodies (IgG and IgM) in MERS associated cases.

Objective To investigate the feasibility of using recombinant infectious clones of hu-man coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids ( pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12. 9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12. 9 genes with the reporter gene en-coding the green fluorescent protein ( GFP ) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the res-cued virus strains were characterized by immunofluorescence assay ( IFA) and Western blot ( WB) assay af-ter transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12. 9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12. 9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12. 9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was ob-tained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infec-ted with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43 FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors.