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 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 explore the effectiveness and feasibility of the machine learning models based on radiomics in the diagnosis of pituitary prolactin macroadenoma.Methods:Totally 122 histologically proven pituitary macroadenoma patients, including 70 cases of pituitary prolactin macroadenoma (PPM) and 52 cases of non-pituitary prolactin macroadenoma (NPPM), were retrospectively recruited. The differences of age, sex, serum prolactin value, bleeding, cystic degeneration and Knosp classification were compared between PPM and NPPM. The pre-processing, delineation of the region of interest and feature extraction of the preoperative axial contrast-enhanced T 1WI image were performed in the 3Dslicer software. The optimal feature set were selected by least absolute shrinkage and selection operator. All patients were randomly divided into the training group ( n=85) and the test group ( n=37) at a ratio of 7∶3. The models were established in the training group by logistic regression and support vector machine (SVM), and then verified by the test group. ROC curves were drawn respectively, and specificity, sensitivity, accuracy and area under the ROC curve (AUC) were calculated. Results:The age [(38±12) years vs . (43±11) years], gender ratio (male/female 50 cases/20 cases vs . 14 cases/38 cases) and prolactin value [366.00 (117.75, 1 156.25)μg/L vs . 47.25 (32.68, 62.40) μg/L] of patients with PPM and NPPM were statistically different ( P<0.05). The AUC values of logistic regression and SVM in the training group were 0.936 and 0.946, and the AUC values of the test group were 0.768 and 0.774, respectively. The diagnostic accuracy of logistic regression and SVM in the training group were 88.2% and 91.8%, and the accuracy of the test group were 73.0% and 77.8%. Conclusion:The machine learning models based on the radiomics can predict the pituitary prolactin macroadenoma well with a high accuracy.

Objective:To study the clinical value of color Doppler ultrasonography in the diagnosis of persistent sciatic vein(PSV).Methods:A retrospective study was performed on 17 patients who were diagnosed with PSV by color Doppler ultrasound in the Second Hospital of Shandong University and the Shandong Provincial Hospital Affiliated to Shandong First Medical University from May 2010 to December 2021. Their sonographic features were analyzed, summarized and classified.Results:In all the 17 cases, the sciatic vein showed a vein adjacent to the sciatic nerve in the pelvis or back of the thigh. According to anatomy, persistent sciatic vein could be divided into three types: complete PSV, upper PSV and lower PSV. There were 7 cases of complete PSV, 2 cases of upper PSV and 8 cases of lower PSV. Femoral vein dysplasia was found in 11 of 17 patients with PSV. In addition to 1 case of bilateral PSV, the diameter of the femoral vein on the affected side was (0.36±0.19)cm in 16 cases, and the diameter of femoral vein at the corresponding position on the healthy side was (0.61±0.11)cm, there was significant difference between the two groups ( P<0.001). Conclusions:Color Doppler ultrasonography is the effective imaging method for diagnosis of the PSV.

Posterior cruciate ligament (PCL) injury is common in sports medicine. Arthroscopic reconstruction of PCL has become a routine procedure to stabilize the knee joint after PCL injury. The location of tibial tunnel during operation is crucial to a successful surgery. This article reviews the current studies on transtibial PCL reconstruction from the aspects of the anatomy related to the tibial tunnel, the anteromedial and anterolateral tibial tunnels, the maximum angle and optimal angle of tibial tunnel, and the anatomical and non-anatomical tibial tunnels, hoping to provide helpful references for the treatment of PCL injury.

Objcetive: To explore the treatment of long segment defect of tibia by using tensor fascia lata combined with iliac flap or deep circumflex iliac pedicle iliac flap. Methods: From February 2012 to August 2017, The People′s Hospital of Zun Yi City Bo Zhou District treated 16 patients who had long segment defect of tibia.There were 11 males and 5 females, age from 22 to 58 years old, the average age was 42 years old. Iliac flap grafting with tensor fascia lata combined with iliac flap or deep circumflex iliac pedicle was used to treat the defect of long segment of tibia. There were 4 cases with simple tibial defect and 12 cases with skin defect. The longest tibial defect was 5-8 cm. Results: In this study, four patients used iliac flaps with deep circumflex iliac pedicle, the area of flaps ranged from 2.5 cm×5.0 cm to 5.0 cm×10.0 cm, while the area of iliac flaps ranged from 5.0 cm×2.5 cm to 8.0 cm×4.0 cm. Twelve patients used grafting with tensor fascia lata combined with iliac flap, the area of flaps ranged from 5.0 cm×12.0 cm to 12.0 cm×23.0 cm, while the area of iliac flaps ranged from 7.0 cm×2.0 cm to 8.0 cm×4.0 cm. All 16 cases of bone flap were survived, fracture healing, without surgical complications. The average follow-up period was 1.5 years, the flaps had good appearance in 10 cases and was slightly bloated in 6 cases; the ankle had normal motion in 14 cases and had poor dorsal extension in 2 cases. X-ray films showed that the bone flap repaired the bone defects and reached bone healing. Conclusions Vascularized tensor fascia lata combined with iliac flap or deep circumflex iliac pedicle iliac flap grafts increase local blood supply and accelerate the process of fracture healing.

BACKGROUND:Increased homocysteine level induces apoptosis of human umbilical vein endothelial cells,but the mechanism remains unclear. OBJECTIVE:To investigate the role of hsa-circ-0001360 in human umbilical vein endothelial cell apoptosis induced by homocysteine. METHODS:In vitro cultured human umbilical vein endothelial cells were divided into control group,homocysteine group,interference control group,interference control + homocysteine group,hsa-circ-0001360 interference group,hsa-circ-0001360 + homocysteine interference group,overexpression control group,overexpression control + homocysteine group,hsa-circ-0001360 overexpression group and hsa-circ-0001360 + homocysteine overexpression group.All groups were treated with 100 μmol/L homocysteine.After 72 hours of intervention,the expressions of apoptosis-related proteins Bax,Bcl-2,and Caspase-3 were detected by western blot assay.The apoptotic rate was detected by flow cytometry.Quantitative real-time PCR was used to detect the expression of hsa-circ-0001360. RESULTS AND CONCLUSION:(1)Compared with the control group,the expression of Caspase-3 and Bax was significantly increased(P<0.01),and the expression of Bcl-2 was significantly decreased(P<0.01),and the apoptotic rate was significantly increased(P<0.01)in the homocysteine group.(2)Compared with control group,the expression of hsa-circ-0001360 was significantly increased in the homocysteine group(P<0.01).(3)The expression of hsa-circ-0001360 was significantly higher in the cytoplasm than that in the nucleus(P<0.01).(4)Compared with the interference control C group and interference control + homocysteine group,the expressions of Caspase-3 and Bax were significantly decreased(P<0.01),while the expression of Bcl-2 was significantly increased(P<0.01);the apoptotic rate was significantly decreased(P<0.01)in sh-hsa-circ-0001360 interference group and sh-hsa-circ-0001360 + homocysteine interference group.(5)Compared with overexpression control group and overexpression control + homocysteine group,the expressions of Caspase-3 and Bax were significantly increased(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01);the apoptotic rate was significantly increased(P<0.01)in the hsa-circ-0001360 overexpression group and the hsa-circ-0001360 + homocysteine overexpression group.(6)In conclusion,hsa-circ-0001360 can promote the apoptosis of human umbilical vein endothelial cells induced by homocysteine.

Objective: To explore the biological effects of electromagnetic pulse (EMP) with different high intensities on condylar cartilage in rats. Methods: SD rats were randomly divided into a sham group (Sham) and an irradiation group (EMP1: 500 kV/m, 10 Hz; EMP2: 270 kV/m, 10 Hz). Then, they were sacrificed at 1 h, 3 h, 12 h, 24 h and 3 d after irradiation. The degree of cartilage degeneration was evaluated by HE, safranine O-fast green, type Ⅱ collagen immunohistochemistry and TUNEL staining. Immunohistochemistry and western blot were performed to detect the expression of the matrix degradation factors: matrix metalloproteinase-13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5) and the apoptosis key factor cleaved-cysteinyl aspartate specific proteinase (cleaved-Caspase3) in condylar cartilage. Results : HE staining showed that, compared with the Sham group, a small amount of exfoliation was found on the fibrous surface layer of the cartilage after irradiation in the EMP1 and EMP2 groups. Compared with the Sham group, the percentage of safranine O-fast green-positive area decreased significantly at 12 h and 24 h (both P<0.01) in the EMP1 group and 12 h and 24 h in the EMP2 group (both P<0.05); the percentage of type Ⅱ collagen-positive area decreased significantly at 3 h and 12 h (P<0.05, P<0.001) in the EMP1 group. In addition, the number of TUNEL-positive apoptotic cells increased significantly at 1 h, 3 h, 12 h, and 24 h in the EMP1 group and 1 h, 3 h, and 12 h in the EMP2 group (P<0.05). Moreover, at different timepoints (except at 3 d) in the EMP1 group and EMP2 group, the percentage of MMP-13, ADAMTS-5- and cleaved Caspase3-positive chondrocytes and their protein levels in condylar cartilage increased significantly after irradiation (P<0.05). Conclusion EMP with a certain degree of high-intensity can induce early transient damage to condylar cartilage. This effect is dose-and time-dependent.