Objective To express recombinant Vp1 protein of chlamydiaphage φCPG1, prepare monoclonal antibody against Vp1 protein and utilize it to screen clinical isolates of Chlamydia trachomatis. Methods The Vp1 protein was obtained by prokaryotic expression, and monoclonal antibody against this protein was prepared by hybridoma technique. ELISA and Western blot were used to identify monoclonal antibodies. Then,the monoclonal antibody was prepared in quantity by injecting hybridoma cells into the abdominal cavity of BALB/C mice, and purified by using protein G affinity chromatography. Clinical isolates of Chlamydia trachomatis were screened for the chlamydiaphage by immumofluorescence assay using the prepared monoclonal antibody.Results Purified Vp1 protein was obtained. The monoclonal antibody against Vp1 protein was gained after 3times of sub-clone and consistently identified as IgG1. Three hybridoma cell strains that stably secreted monoclonal antibody were generated. Chromosome analysis of hybridoma cells showed that the mean number of chromosome was 96, most of them were telocentric and a few were submetacentric. The titer of purified monoclonal antibody was more than 1: 12 800. Twenty clinical isolates were screened by using the monoclonal antibody and no positive results were obtained. Conclusions The monoclonal antibody against Vp1 protein of chlamydiaphage φCPG1 is successfully prepared, while no chlamydiaphage is detected by immumofluorescence assay using the prepared antibody in 20 clinical isolates of Ct.

Objective To optimize immunodominant protein combinations for serological screening for Cblamydia trachomatis (Ct) infection.Methods Both serum and genital swab samples were collected from 50 patients with Ct infection confirmed by colloidal gold immunochromatographic assay (GICA),and 30 GICA-negative clients without Ct infection at a sexually transmitted disease (STD) clinic in Tianjin Medical University General Hospital.The 30 serum samples from GICA-negative clients were also negative for microimmunofluorescence (MIF) assay.Eight Ct immunodominant proteins,including Pgp3,CPAF,CT143,CT101,CT694,CT875,CT813 and IncA,were selected as antigens to detect corresponding antibodies in the serum samples by enzyme-linked immunosorbent assay (ELISA) with the Ct proteins Hsp60 and major outer membrane protein (MOMP) as references.The results of ELISA were compared with those of the traditional gold standard method MIF assay to determine the immunodominant protein combination with the highest sensitivity and specificity.Results Of the 50 serum samples from patients with Ct infection,44 were positive and 6 negative by MIF.The results of ELISA with the combination of immunodominant proteins Pgp3,CT694 and CT875 as antigens were 97.73％ (43/44) consistent to those of MIF assay.Of the 30 serum samples from GICA-negative clients,all were negative by MIF.Meanwhile,no antibody was detected against any of the immunodominant proteins Pgp3,CT694 and CT875 in any of the serum samples from GICA-negative clients.Conclusions The ELISA with the combination of immunodominant proteins Pgp3,CT694 and CT875 as antigens has good sensitivity and specificity for serological screening for Ct infection,and is simple to operate and easy to popularize.

Objective To add an open reading frame in the shuttle vector of pGFP ∷ CM for transfection of exogenous genes into Chlamydia muridarum.Methods The sequence of plasmid pGFP ∷ CM and new open reading frame (including promoter of pgp4,mCherry gene of red fluorescence protein and transcription termination sequence of Chlamydia trachomatis CT579) were amplified by polymerase chain reaction (PCR),and the products were transfected into Stellar competent cells.The recombinant plasmids were identified by PCR,enzyme digestion and sequencing.Then the recombinant plasmid was transfected into plasmid-free strain CMUT3,and the GFP-and mCherry-positive inclusions were observed under the fluorescence microscope.After the ampicillin selection and plaque purification,the purified CMUT3-pGFP-mCherry-CM was identified by indirect immunofluorecesent stain using anti-pgp3 and anti-glgA antibodies.Results The correct recombinant plasmid after sequencing identification,enzyme digestion and PCR amplification was successfully transfected into CMUT3,and the GFP-and mCherry-positive inclusions were observed.The transfected strain CMUT3-pGFP-mCherry-CM was purified after ampicillin selection and plaque purification.The expression of pgp3 and glgA protein in CMUT3-pGFP-mCherry-CM was similar to that in CMUT3-pGFP ∷ CM.Conclusion An open reading frame is successfully added in the plasmid pGFP ∷ CM,and the new plasmid can be transfected into CMUT3 and express exogenous protein,which can be used for further study on the function of single chlamydial protein.

Objective To evaluate inhibitory effects of the Chlamydiaphage phiCPG1 capsid protein Vp1 on Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC) and Chlamydia trachomatis (Ct) serovar E,and to provide new ideas for the treatment of Ct infection.Methods The Chlamydiaphage phiCPG1 capsid protein Vp1 was expressed in Escherichia coli BL21 transfected with the recombinant plasmid Vp1-pET30a (+),identified by Western blot analysis and purified by using dialysis bags.Bicinchonininc acid (BCA) assay was performed to determine the concentration of Vp1 protein.GPIC and Ct serovar E strains were both classified into 4 groups to be firstly incubated with Vp1 protein (Vp1 group),Tris-glycine solution (Tris group),S protein (S group) or Dulbecco's Modified Eagle Medium (DMEM,DMEM group) at room temperature for 3 hours,then were used to infect Hela cells followed by 72-hour (GPIC) or 48-hour (Ct serovar E) culture with the presence of Vp 1 protein (Vp 1 group),Tris-glycine solution (Tris group),S protein (S group) or DMEM (DMEM group).Subsequently,immunofluorescence staining was conducted to observe and count chlamydial inclusions.Results The number of GPIC inclusions was significantly different between the 4 groups after 72-hour culture (F=476.632,P＜ 0.05),and lower in the Vp1 group (5.0 ± 1.5) than in the Tris group (24 ± 1.2,P＜ 0.05),S group (25 ± 1.7,P＜ 0.05) and DMEM group (25 ± 1.5,P＜ 0.05),but insignificantly different between the latter 3 groups (P ＞ 0.05).Compared with the DMEM group,the Vp1 group showed a significant decrease of 80.2％ ± 3.99％ and 77.2％ ± 1.79％ in the number of GPIC and Ct serovar E inclusions respectively,with no significant difference in the inhibitory effect of Vp1 on GPIC versus Ct serovar E (t =2.057,P ＞ 0.05).Conclusion The phiCPG1 capsid protein Vp1 can obviously inhibit GPIC and Ct serovar E infections to a similar degree.

Objective To obtain the full length (FL ) and C‐terminal fragment of polymorphic membrane protein I (PmpI) of Chlamydia trachomatis serovar D ,and to study the immunogenicity of these proteins .Methods The target genes of PmpI‐FL and PmpI‐C were amplified by polymerase chain reaction (PCR) and inserted into the prokaryotic plasmid vector pGEX‐6P‐1 .The recombinant plasmids pGEX‐6P‐1/PmpI‐FL and pGEX‐6P‐1/PmpI‐C were separately transformed into Escherichia .coli ( E . coli) DH5αand were identified by enzyme digestion ,sequencing and PCR .After the identification ,the recombinant plasmids were separately transformed into E .coli BL21 and induced to express the proteins . The expected proteins were identified by Coomassie brilliant blue staining and Western blot ,then purified by glutathione S‐transferase (GST) MagBeads .The purified proteins were then injected into BALB/c mice to prepare the polyclonal antibodies against PmpI‐FL or PmpI‐C .Enzyme‐linked immune sorbent assay (ELISA) was used for the quantitative detection of the specific antibody .Results The lengths of cloned target genes PmpI‐FL and PmpI‐C were 2 659 bp and 1 195 bp ,respectively ,and the sequences were consistent with those of Chlamydia trachomatis serovar D in GenBank .The molecular masses of target proteins were 122 000 and 69 000 ,respectively ,which were confirmed by Coomassie brilliant blue staining and Western blot and then purified .The titers of the antibodies (anti‐PmpI‐FL and anti‐PmpI‐C) in sera of immunized mice detected by ELISA were 1∶12 800 and 1∶6 400 ,respectively .Conclusion The PmpI‐FL‐GST and PmpI‐C‐GST fusion proteins with high immunogenicity are successfully expressed and purified , which lays the foundation for further study .

Objective:To summarize the clinical experiences of managing patients with novel coronavirus(2019-nCoV) infection after kidney transplantation.Methods:Clinical data were retrospectively analyzed for two patients with 2019-nCoV infection after renal transplantation in January 2020. Case 1 was a 48-year-old male with CMV pneumonia secondary to 2019-nCoV infection at 4 months post-transplantation. CT imaging showed multiple patchy ground-glass opacities of both lungs. Case 2 was a 59-year-old male who screened positive for 2019-nCoV nucleic acid due to fever at 9 days post-transplantation and he showed no clinical manifestations of pneumonia. After a definite diagnosis, case 1 was transferred to a designated hospital for isolation. Treatment regimens: cefoperazone sulbactam sodium plus linezolid for anti-infection, gamma globulin for enhancing immunity, methylprednisolone for controlling inflammatory responses and antiviral regimens of arbidol tablets plus lopina-velitonavir tablets. Case 2 was isolated in a single room. The treatment plan included cefoperazone sulbactam sodium for anti-infection, gamma globulin for enhancing immunity, arbidol for antiviral therapy and other symptomatic measures.Results:During a follow-up period of 3 weeks, case 1 recovered with renal dysfunction, nucleic acid test of nasopharyngeal swab turned negative and pulmonary imaging improved. Case 2 showed no obvious clinical symptoms and nucleic acid test of nasopharyngeal swab turned negative thrice.Conclusions:Renal transplant recipients should take precautions to avoid exposure to high-risk environments. A definite diagnosis should be made on the basis of clinical manifestations and results of nucleic acid test and pulmonary imaging. Currently there is no effective antiviral agent and symptomatic treatment is a major option.

Objective:To explore the clinical efficacy of Kinesio taping combined with extracorporeal shock wave and the plantar pressure evaluation in the treatment of plantar fasciitis. Method:A total of 67 patients with unilateral plantar fasciitis were randomly divided into the control group(ESWT group)and the experimental group(KT combined group).The patients in the two groups were given the same health education and extracorporeal shock wave treatment(ESWT),and the experimental group was treated with Kinesio taping(KT).The pain and functional activity were evaluated by pain visual analogue scale and AOFAS ankle and hind-foot function scale;The insole plantar pressure measuring system was used to mea-sure the peak pressure values of each plantar regions.The clinical efficacy and plantar pressure of the affected side were compared before treatment,the 3rd week of treatment and 5th week of treatment. Result:There was no significant difference in various indcators of the measurements between the two groups before treatment(P＞0.05).The score of pain rating,functional activity scale score and plantar pressure of pa-tients in both groups were significantly improved at the 3rd week and the 5th week of treatment(P＜0.05),com-pared with those before treatment.At the 3rd week of treatment,there was no significant difference between the two groups in pain degree and functional activity scale score(P＞0.05),but there was statistical difference in plantar pressure analysis(P＜0.05).The peak force weight ratio of the medial heel in the KT combined group was significantly greater than that in the control group.At the 5th week of treatment,there were statisti-cally significant differences between the two groups in the score of pain rating,functional activity scale score and plantar pressure(P＜0.05).The KT combined treatment group was significantly better than the control group in the pain improvement and functional activity,and the weight bearing of the middle foot and hind foot was also significantly higher than the control group. Conclusion:Compared with the simple extracorporeal shock wave therapy,the combination of KT therapy can better relieve pain,improve the function of hind foot,correct abnormal foot weight bearing,and improve the gait of patients.

BACKGROUND:Kinesio taping is often used for the treatment of various sports injuries.The methods of foot and ankle sports taping are complex and diverse.Among them,Fascia taping is applicable to a wider range of people and can be used for different foot posture types,but it still lacks of practical verification,and its specific biomechanical role is not clear. OBJECTIVE:To observe the changes in plantar pressure characteristics of subjects with different foot positions during walking and jogging after Fascia taping. METHODS:Thirty-seven young healthy subjects were recruited from the Yantai campus of Binzhou Medical University to conduct the test.They were scored according to the foot posture index-six items version,and were divided into the supination foot group,the neutral foot group,and the pronation foot group.The static foot morphological indexes(including navicular drop,arch height index,arch height flexibility-longitudinal arch,and arch height flexibility-transverse arch)and the pressure-time integral of each foot zone during walking and jogging were collected and calculated respectively before and after Kinesio taping.The specific biomechanical mechanism of Fascia taping was analyzed. RESULTS AND CONCLUSION:(1)General data:There was no statistical difference among the three groups of subjects in general data,such as gender,height,and body mass index(P>0.05).Before taping,there was a significant difference in the foot morphological indexes and the areas of the outer front foot,midfoot,and hindfoot between different foot posture groups(P<0.01).(2)Static foot morphological indexes:After taping,there was no statistically significant difference between the groups in navicular drop,arch height flexibility-longitudinal arch,and arch height flexibility-transverse arch(P>0.05),while there was still a significant difference between the groups in the arch height index(P<0.05).In the supination foot group,the arch height index increased slightly,but there was no significant difference before and after taping(P>0.05).In the pronation foot group,the navicular drop and arch height flexibility-longitudinal arch was significantly reduced,and the arch height index was increased.There was a significant difference before and after taping(P<0.05).(3)The index of plantar pressure during walking:After taping,there was no significant difference between the three groups in the area of lateral forefoot and medial midfoot(P>0.05).In the pronation foot group,the lateral load of the forefoot increased after taping(P<0.05).In the supination position group,the load of the lateral forefoot and midfoot regions increased significantly(P<0.05),while the difference in the rear foot region was not significant(P>0.05).(4)The index of plantar pressure during jogging:After taping,there was no statistically significant difference between groups in the lateral forefoot(P>0.05).In the pronation foot group,the load of the medial forefoot increased significantly(P<0.05).In the supination position group,the load of the lateral forefoot,the middle foot and the rear foot region increased significantly(P<0.05).(5)The results showed that the Fascia taping was suitable for different foot postures.It could not only correct the static foot structure of subjects with different foot postures,but also regulate the abnormal plantar pressure distribution during the dynamic activities of walking and jogging,and the load of the midfoot,forefoot,and hindfoot in the supination and pronation posture tended to normal foot posture load level.

Objective To certify that Chlamydia can spread from the genital tract to the gastrointestinal tract for long-lasting colonization.Methods Totally,120 female C57BL/6J mice aged 5-6 weeks were divided into 4 experimental groups to be inoculated with purified Chlamydia muridarum (C.muridarum) elementary bodies in the vagina (n =35),gastric area (n =30),anus and rectum (n =30),retro-orbital venous plexus (n =5) respectively.Moreover,corresponding negative groups inoculated with sucrose phosphate glutamate buffer (n =5) were set up for each experimental group.On days 3,7,and every 7 days,vaginal and rectal discharges were collected with swabs from the mice,and the number of live C muridarum orgnisms in exfoliated cells infected with C muridarum in the swabs was determined.Indirect immunofluorescence assay and quantitative PCR (qPCR) were performed to determine the number of live chlamydial organisms and the copy number of chlamydial genomes in the mouse genital tract (vagina,uterus,oviduct and ovary),gastrointestinal tract (stomach,small intestine,cecum,colon,rectum)and parenteral tissues (heart,liver,spleen,lung,kidney) on days 7,14,28,56 and 105 after the inoculation.The number of live chlamydial organisms and copy number of chlamydial genomes were transformed logarithmically with a base of 10.The degree of hydrosalpinx and inflammation in the genital tract,and histopathological changes of the gastrointestinal tract were observed.The infectivity and virulence of C.muridarum in the genital tract and gastrointestinal tract were evaluated in the intragastric inoculation group and intra-anal and intrarectal inoculation group on days 28 and 56 after the inoculation.Blood samples were obtained from the mouse caudal vein in the retro-orbital venous plexus inoculation group on days 3,5,7,10 and 14 after the inoculation,the number of live chlamydial organisms and the copy number of chlamydial genomes in the blood samples were determined,and chlamydial infectivity in the genital tract and gastrointestinal tract was evaluated on day 56.Results On day 7 after the inoculation in the vagina,both C.muridarum live organisms and genomes were detected in the genital tract,gastrointestinal tract and parenteral tissues of all the mice.The largest common logarithm of the number of C.muridarum inclusion forming units (IFU) was observed in the vagina (6.26 ± 0.56),with the common logarithm of the copy number of chlamydial genomes in the vagina being 7.30 ± 0.23,and the common logarithms of the number of Chlamydia IFU and genomic copy were 2.60 ± 1.95 and 4.87 ± 0.09 respectively in the rectum.On day 28,no live Chlamydia was detected in the heart,lung or other parenteral tissues,while live Chlamydia could be found in the genital tract and gastrointestinal tract.The common logarithms of the number of Chlamydia IFU and genomic copy were 3.47 ± 1.06 and 5.80 ± 1.49 respectively in the vagina,and 4.00 ±0.35 and 5.14 ± 0.81 respectively in the rectum.On day 56,live Chlamydia could only be detected in the gastrointestinal tract.On day 105,live Chlamydia and its genomes could be still detected in the gastrointestinal tract,and the common logarithms of the number of Chlamydia IFU and genomic copy could be up to 2.60 ± 0.65 and 4.29 ± 0.57 respectively in the rectum.On days 28 and 56 after the inoculation,both live Chlamydia and its genomes could be detected in the gastrointestinal tract of all the mice in the intragastric inoculation group and intra-anal and intrarectal inoculation group.Chlamydia could survive in the blood for about 14 days in the retro-orbital venous plexus inoculation group,and live Chlamydia was detected in anal-rectal swabs in all the mice on day 14.On day 56 after the intravaginal inoculation with C.muridarum,severe hydrosalpinx,chronic inflammation and oviduct dilation occurred in the genital tract of 5 mice,but there was no obvious infiltration of inflammatory cells in the gastrointestinal tract,and inflammatory pathological changes were not observed in the gastrointestinal tract of mice after inoculation with Chlamydia through other routes either.Conclusion The infection with Chlamydia in the genital tract can lead to systemic dissemination,and Chlamydia can be spread to the gastrointestinal tract,and colonize and survive in the gastrointestinal tract for a long time.

ObjectiveTo identify the biomechanical deviations of patients with adolescent idiopathic scoliosis (AIS) during static standing and level walking. MethodsCross-sectional, observational studies comparing biomechanical characteristics between individuals with AIS and healthy controls were searched in the databases of PubMed, Web of Science, EMBASE, Cochrane Library, CNKI, CBM, Wanfang data and VIP. The retrieval time was from the establishment of the database to August, 2021. Quality assessment and data extraction were performed independently by two reviewers. The data were analyzed with RevMan 5.4. ResultsA total of 23 studies were finally included, with a total of 1 092 subjects. The overall methodological quality was moderate to high level. During static standing, the sway area (ES = 0.69, 95%CI 0.35 to 1.02, P < 0.001), anteroposterior transfer speed (ES = 0.59, 95%CI 0.29 to 0.89, P < 0.001) and mediolateral transfer speed (ES = 0.47, 95%CI 0.12 to 0.83, P < 0.05) of plantar pressure center were more in AIS group than in the control group, however, there was no significant difference in the inclination of pelvic frontal plane (ES = 0.10, 95%CI -0.16 to 0.36, P > 0.05). During walking, the sagittal range of motion of knee (ES = -0.59, 95%CI -1.00 to -0.17, P = 0.005) and ankle (ES = -0.55, 95%CI -0.96 to -0.14, P = 0.008) were less in AIS group than in the control group, and the duration of EMG activity of the gluteus medius (ES = 1.21, 95%CI 0.23 to 2.18, P = 0.02), the quadratus lumborum (ES = 1.13, 95%CI 0.68 to 1.57, P < 0.001) and the erector spinae (ES = 1.20, 95%CI 0.75 to 1.65, P < 0.001) was longer in AIS group than in the control group, while no significant difference was observed in the step speed (ES = -0.12, 95%CI -0.45 to 0.21, P = 0.49). Compared with the control group, the step length of the spinal concave side (ES = -0.37, 95%CI -0.72 to -0.02, P = 0.04) was shorter, but the overall step length (ES = -0.22, 95%CI -0.76 to 0.31, P = 0.42) was not significantly different in AIS group. ConclusionCompared with the healthy controls, some characteristic biomechanical changes were exhibited in the AIS patients, including spatiotemporal characteristics, kinematic and mechanical characteristics, and electromyographic signal characteristics.