OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity. METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants. RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes. CONCLUSION The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.
Legionella pneumophila/pathogenicity* ; CRISPR-Cas Systems ; Biofilms/growth & development* ; Phenotype ; Bacterial Proteins/metabolism* ; Gene Deletion

Objective The study aims to investigate the impact of microRNA-874-3p(miR-874-3p)regulation of plakophilin 3(PKP3)on the malignant biological behavior of lung adenocarcinoma cells and its underlying mechanism.Methods Immunohistochemistry and immunocytochemistry were used to detect the expression of PKP3 in lung adenocarcinoma tissue microarray and lung adenocarcinoma cell line A549 cells respectively,and the relationship between PKP3 and clinicopathological features of lung adenocarcinoma patients was analyzed.Select lung adenocarcinoma cell line A549,the experiment was divided into A549 cell group(blank control group),miR-NC group(transfected with miR-NC)and miR-mimics group(transfected with miR-874-3p mimics),sh-NC group(control group transfected with PKP3 silencing plasmid),sh-PKP3 group(transfected with PKP3 silencing plasmid),miR+pcDNA-PKP3 group(transfected with miR-874-3P mimics+pcDNA-PKP3,rescue group)and miR+pcDNA-NC group(transfected with miR-874-3p mimics+pcDNA-NC).The proliferation,invasion,migration and apoptosis of cells in each group were detected.ENCORI database was used to predict the upstream gene of PKP3,and dual luciferase assay was used to detect the targeting relationship between miR-874-3p and PKP3.MAPK/mTOR pathway-related proteins were detected by Western blotting.Results The expression of PKP3 in lung adenocarcinoma tissue was significantly higher than that in adjacent tissues.The high expression of PKP3 was related to clinical stage,tumor size,and lymph node metastasis(P＜0.05).Compared with the human normal lung epithelial cells(BEAS-2B),the expression of PKP3 in A549 cells increased significantly,and the expression of miR-874-3p decreased(P＜0.05).Overexpression of miR-874-3p decreased the PKP3 expression level(P＜0.05).Compared with the control group,both overexpression of miR-874-3p and silenced PKP3 inhibited the cloning and invasion ability of A549 cells,caused cell cycle arrest,and decreased the expression levels of cyclin dependent kinase 4(CDK4),cyclin D1,cyclin E1 proteins in A549 cells(P＜0.05).The expressions of Bax protein and Caspase-3 protein were up-regulated(P＜0.05),and apoptosis increased.Overexpression of PKP3 could reverse the biological behavior of overexpression of miR-874-3p.Overexpression of miR-874-3p and silencing of PKP3 significantly decreased the expressions of P38 MAPK and mTOR phosphorylated proteins.Conclusion MiR-874-3p can negatively regulate PKP3 expression and inhibit the malignant biological behavior of A549 cells through MAPK/mTOR pathway.

Objective To study the stability of plate-assisted intramedullary nailing for fixing proximal third tibiafractures, compare and observe biomechanical characteristics of anterolateral or posteromedial plate-assisted intramedullary nailing after fixation of proximal third tibia fractures. Methods Eight artificial tibia of 4th-generation sawbones were divided into two groups based on location of the assisted plate, namely, anterolateral plate group and posteromedial plate group, with 4 specimens in each group. Each two locking bolts were fixed to theintramedullary nail proximally and distally, and each three bicortical screws were fixed to the plate proximally and distally. The specimens were osteotomized with a 10-mm defect which located 0. 5 cm to the proximal locking bolt of intramedullary nail or 5-6 cm distally to the knee joint line, in order to simulate an AO/ OTA 41-A2 type proximal third tibia fracture after fixation of intramedullary nail. After osteotomy was finished, axial compression test, three point bending test, cyclic loading and overstress test were conducted by mechanical testing machine. The results of axial stiffness and three-point stiffness between two groups were compared and analyzed. Results Axial compression test showed that the average axial stiffness in posteromedial plate group was lower than that in anterolateral plate group, but no significantly statistical differences were found between the two groups. Three point bending test showed that the average bending stiffness in posteromedial plate group was significantly higher than that in anterolateral plate group when stimulating either varus stress (plate located at pressure side of the fracture, t = 3. 679, P<0. 05) or valgus stress (plate located at tension side of the fracture, t = 8. 975, P<0. 05). Conclusions Plate-assisted intramedullary nailing for fixation of proximal third tibia fractures can minimize the angulation malalignment, improve the stability of nailed proximal tibial fragment and allow the early weight bearing. Both anterolateral and posteromedial plate-assisted intramedullary nail can provide satisfactory axial stability for proximal third tibia fractures, while posteromedial plate-assisted intramedullary nail shows better bending stability than anterolateral plate in countering varus or valgus stress deformity. This study provides an essential basis for clinical decision making about plate-assisted intramedullary nailing for fixing proximal third tibia fractures.

Objective:To observe multiple locus variable-number tandem repeat analysis (MLVA) typing of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province, and to explore the relationship between the strains and strains previous isolated from Qinghai Province. Methods:Blood samples of Himalayan marmot were collected in Qinghai-Tibet Plateau of Qinghai Province from March 2019 to October 2020. Pathogens were isolated and cultured from Brucella antibody positive samples identified by using the rose bengal test (RBT). Conventional biological methods and molecular biological methods (BCSP31-PCR and AMOS-PCR) were used for strain identification. At the same time, MLVA method was used to genotype the isolated strains, and cluster analysis was used to analyze the genetic relationships between the strains based on the genotype of 70 Brucella isolated from different hosts in Qinghai Province. Results:A total of 1 466 blood samples of Himalayan marmot were collected from Qinghai-Tibet Plateau. Two strains of Brucella were isolated and cultured from 64 RBT-positive samples, named QH2013054 and QH2013062, respectively. They were identified as Brucella ovis biotype Ⅲ by conventional and molecular biological methods. The MLVA genotyping results showed that QH2013054 and QH2013062 were different at the Bru16 locus, indicating different MLVA genotypes. Cluster analysis showed that strain QH2013054 had the same MLVA genotype as 7 strains, among which 6 strains were from 3 farmers and 3 sheep from the same family in Gonghe County, and 1 strain was from a farmer in Menyuan Hui Autonomous County. The strain QH2013062 had the same MLVA genotype as 4 strains, including 3 strains from 3 farmers in Menyuan Hui Autonomous County and 1 strain from a farmer in Tu Autonomous County of Huzhu. Conclusions:The strains of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province have the same MLVA genotype as some strains of Brucella isolated from humans and sheep in Qinghai Province. It is speculated that the host humans, sheep and Himalayan marmot in Qinghai-Tibet Plateau may have a common source of infection.

Objective:To compare the early clinical efficacy between reaming after distal tibia cortical fenestration combined with antibiotic-loaded calcium sulphate and trench grooving combined with polymethyl methacrylate (PMMA) for the treatment of chronic tibial osteomyelitis after intramedullary nail fixation.Methods:A retrospective analysis was conducted in the 20 patients who had been surgically treated for chronic tibial osteomyelitis after intramedullary nail fixation at Department of Trauma Orthopaedics, Honghui Hospital from January 2019 to January 2021. According to the surgical methods, they were divided into a reaming group and a grooving group. In the reaming group, there were 6 males and 3 females with an age of (47.6±11.4) years; in the grooving group, there were 9 males and 2 females with an age of (49.2±13.9) years. The 2 groups were compared in terms of duration of infection, operation time, intraoperative blood loss, bacterial culture results, total hospital stay, time for inflammatory indexes to return to normal, time for weight-bearing, complication rate, infection control rate, and good to excellent rate by Johner-Wruhs joint function scoring.Results:There was no significant difference in the preoperative general data between the 2 groups, showing they were comparable ( P > 0.05). All patients were followed up for 12 to 25 months after operation. There were no significant differences in the duration of infection, operation time, intraoperative blood loss, bacterial culture results, time for inflammatory indexes to return to normal, complication rate, infection control rate, or Johner-Wruhs scoring between the 2 groups ( P > 0.05). The total hospital stay [(11.7 ± 4.7) d] and weight-bearing time [(5.8 ± 1.6) weeks] for the reaming group were significantly shorter than those for the grooving group [(16.8 ± 4.6) d and (8.1 ± 2.9) weeks] ( P < 0.05). Conclusion:In the treatment of chronic tibial osteomyelitis after intramedullary nail fixation, compared with conventional trench grooving combined with PMMA, reaming after distal tibia cortical fenestration combined with antibiotic-loaded calcium sulphate can not only obtain satisfactory outcomes by infection control but also shorten the treatment cycle by allowing the patients to start early weight bearing.

Objective:To study the influence of anticoagulation timing on incidence of perioperative deep venous thrombosis (DVT) in elderly patients with hip fracture.Methods:A retrospective analysis was made of the 179 elderly patients with hip fracture who had been admitted to Department of Orthopedics and Traumaology, Hong-Hui Hospital from July 2017 to December 2018. They were 78 males and 101 females, aged from 62 to 91 years (mean, 79.5 years). There were 79 femoral neck fractures and 100 intertrochanteric fractures, 109 of which were treated by internal fixation and 70 by hip replacement. The patients were divided into 3 groups depending on the timing of anticoagulation after injury. In group 1 of 74 cases, anticoagulation started <24 h after injury; in group 2 of 36 cases, anticoagulation started 24 to 48 h after injury; in group 3 of 69 cases, anticoagulation started >48 h after injury. Anticoagulation continued until 12 h before surgery in all patients but was resumed 8 to 12 h after surgery. The 3 groups were compared in incidence of perioperative DVT.Results:The 3 groups were comparable due to insignificant differences between them in their pre-operative general data ( P>0.05). DVT occurred perioperatively in 84 patients, yielding an incidence of 46.9% (84/179). The incidences of perioperative DVT were 27.0% (20/74), 47.2% (17/36) and 68.1% (47/69) in groups 1, 2 and 3, respectively, showing significant differences ( χ2=24.206, P<0.001), between any 2 groups ( P<0.05). Conclusion:Since the earlier anticoagulation starts after injury the lower incidence of perioperative DVT in elderly patients with hip fracture, early standardized prophylactic anticoagulation after injury can effectively reduce incidence of perioperative DVT.

Objective:To evaluate short-term clinical efficacy of femoral neck system (FNS) for treatment of femoral neck fractures in young and middle-aged patients.Methods:A retrospective analysis was conducted of the 70 middle-aged and young patients who had been surgically treated for femoral neck fractures at Department of Trauma Orthopaedics, Honghui Hospital from January to November 2020. Of them, 32 cases were fixated by FNS; they were 16 males and 16 females, with an age of (49.4±11.0) years, including 10 cases of type Ⅱ, 12 cases of type Ⅲ and 10 cases of type Ⅳ by the Garden classification. The other 38 patients were fixated by cannulated compression screws (CCS); they were 19 males and 19 females, with an age of (48.8±10.1) years, including 12 cases of type Ⅱ, 15 cases of type Ⅲ and 11 cases of type Ⅳ by the Garden classification. The 2 groups were compared in terms of operation time, intraoperative blood loss, fracture reduction, fracture union time, weight-bearing time, complications, Barthel index at 3 months after surgery, and hip function at 6 months after surgery.Results:There was no statistically significant difference in preoperative general information or follow-up time between the 2 groups, showing comparability between groups ( P>0.05). There was no significant difference in operation time, intraoperative blood loss or fracture reduction quality between the 2 groups ( P>0.05). In the FNS group, weight-bearing time [(11.4±3.4) weeks] and fracture healing time [(3.1±0.9) months] were significantly shorter than those in the CCS group [(16.4±3.9) weeks and (3.6±0.9) months], rate of complications (12.5%, 4/32) was significantly lower than that in the CCS group (34.2%, 13/38), Barthel index at 3 months after operation (98.1±2.8) and Harris hip score at 6 months after operation (96.8±4.0) were significantly higher than those in the CCS group (93.8±4.1 and 93.6±6.7) ( P<0.05). Conclusion:In the treatment of femoral neck fractures in young and middle-aged patients, compared with CCS fixation, FNS fixation can obtain better short-term curative effects, due to its advantages of shorter bone union and weight-bearing time, a decreased rate of complications and early functional recovery of daily activities.

Objective:To report our experience in the admission and perioperative management of 88 patients with lower extremity fracture in a mildly affected area in the epidemic of COVID-19.Methods:A retrospective analysis was conducted of the 88 patients with 97 lower extremity fractures who had been admitted to Department of Orthopedic Trauma, Honghui Hospital Affiliated to Xi'an Jiaotong University College of Medicine from 23rd January, 2020 to 22nd February, 2020. They were 43 males and 45 females, aged from 15 to 95 years (average, 65.5 years). The patients underwent screening for COVID-19 infection before admission. Their fractures were located at the femoral neck in 33 cases and at the femoral trochanter in 26. Open reduction and internal fixation was performed for 29 cases, internal fixation with proximal femoral nail anti-rotation (PFNA) for 25 and hip replacement for 28. The time from injury to admission, time from admission to surgery, operation time, fracture reduction, hospital stay, and perioperative deep venous thrombosis (DVT) of lower limbs were recorded. COVID-19 infection was observed in the medical staff and patients as well.Results:All the 88 patients were COVID-19 negative in the screening before admission. The time from injury to admission averaged 4.5 days, the time from admission to surgery 3.7 days and hospital stay 6.6 days. The prostheses were all well located in the 28 patients undergoing hip replacement. The rate of functional and anatomic reduction was 94.2%(65/69) in the 69 patients undergoing internal fixation. Peri-operatively, DVT occurred in 25 cases (28.4%). High temperature was observed in 7 patients within 3 days after operation, which was diagnosed as absorption fever. No medical staff or patients were infected by COVID-19.Conclusion:In the epidemic of COVID-19, orthopedic surgeons in a medical institute in a mildly affected area can still provide effective and safe medical services for fracture patients and reduce nosocomial infection, as long as they comprehend the diagnosis and treatment guidelines for the epidemic, strictly screen the patients accordingly, stick to operative indications, protect against possible infection cautiously, and carry out the procedures in a standard manner.

Objective:To investigate the effects of surgical revision for humeral shaft nonunion on health-related quality of life.Methods:The data of 62 patients were studied retrospectively who had been hospitalized at Department of Orthopaedics and Traumatology, Hong-Hui Hospital for humeral shaft nonunion from March 2013 to September 2018. They were 43 males and 19 females, aged from 20 to 73 years (average, 42.3 years). Their nonunions belonged to the atrophic type in 19 cases, to the ischemic type in 14 cases and to the hypertrophic type in 29 cases. Their demographic and clinical data, imaging manifestations and treatment methods were recorded and analyzed. The 12-item short form health survey (SF-12) and brief pain inventory (BPI) were used to evaluate their health-related quality of life and the Mayo elbow performance score (MEPS) was used to evaluate their elbow function.Results:Upon admission, their physical component summary (PCS) scored 24.3±5.2, mental component summary (MCS) 26.3±3.8, brief pain inventory- severity (BPI-S) 6.5±1.9, and brief pain inventory-interference (BPI-I) 6.7±2.5. At 1-year follow-up, their PCS averaged 43.6±8.1, MCS 34.7±4.4, BPI-S 4.9±1.2, and BPI-I 4.4±1.4. There were statistically significant differences between preoperation and postoperation in the above 4 groups of indicators ( P<0.05). Their MEPS at the last follow-up revealed a 95.2% rate of excellent elbow function (59/62). By comparison with the literature data, their postoperative PCS scores were not significantly different from those for the patients with Barrentt＇s esophagus, primary hypertension, chronic obstructive pulmonary disease and silicosis ( P>0.05). There was no significant difference either in the postoperative MCS scores between them and stroke patients ( P>0.05). Conclusions:As a destructive chronic disease, humeral shaft nonunion has negative effects on physical and mental health and quality of life of the patients. Although the pain effect on the limb can be alleviated by surgical revision, the entire treatment may cause permanent psychological trauma to the patients.

Objective:To explore the clinical efficacy of proximal femoral anatomical locking plate and autogenous iliac graft for failed primary internal fixation in treatment of femoral intertrochanteric fracture.Methods:A retrospective analysis was conducted of the 29 patients with femoral intertrochanteric fracture who had been treated after failed primary internal fixation at Department of Orthopaedics and Trauma, Xi'an Honghui Hospital between January 2014 and March 2017. They were 17 men and 12 women, aged from 35 to 83 years (mean, 68.7 years). Their primary internal fixation involved dynamic hip screw in 12 cases, proximal femoral locking plate in 13 cases, and intramedullary nail in 4 cases. The causes for their internal fixation failure included head cutting in 8 cases, fracture nonunion in 10 cases, plate or screw breakage in 6 cases, intramedullary nail breakage in 3 cases, and hip varus in 2 cases. Their revision surgery was performed with anatomical proximal femoral locking plate and autogenous iliac bone graft. Their fracture union time, and visual analogue scale (VAS), hip Harris score, SF-36 health survey scale and complications at the final follow-ups were recorded.Results:All the 29 patients were followed up for 12 to 24 months (18 months on average). Bony union was eventually achieved in all the 29 patients after an average time of 4.5 months (from 3 to 7 months). There were no such complications as nonunion, re-fracture or internal fixation failure. The VAS pain score at the final follow-up(4.6±1.6) was significantly lower than that before surgery(7.1±2.1), and the Harris hip score(85.2±8.2) and SF-36 score(75.9±15.5) at the final follow-up were significantly higher than those before surgery (48.0±12.7 and 48.7±18.8) (all P<0.05). According to their hip Harris scores at the final follow-ups, the therapeutic efficacy was rated as excellent in 9 cases, as good in 15 cases and as poor in one, yielding an excellent and good rate of 82.8%. Conclusion:For patients with femoral intertrochanteric fracture whose primary internal fixation has failed, especially those with fine femoral head and neck and hip joint, proximal femoral anatomic locking plate and autogenous iliac bone graft can result in satisfactory clinical efficacy.