Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Massive rotator cuff tears, with mean tendon tears>5 cm or involving two or more tendons in a tear, often result in shoulder pain, stiffness, limited range of motion and impaired function, significantly impacting the patients′ quality of life. At present, massive rotator cuff tears are mainly treated surgically with joint cleaning, rotator cuff repair and patch graft repair, etc, but the problems such as tendon end retraction and high rotator tension after surgical repair lead to high postoperative retear rate. The autogenous long head of biceps tendon (LHBT), as an autograft which has the advantages of high accessibility and low cost, has been employed in the repair of massive rotator cuff tears. However, there is still controversy in the choice of specific surgical method. To this end, the authors reviewed the research progress on the application of LHBT in the repair of massive rotator cuff tears, aiming to provide reference for its clinical treatment.

Objective:To compare the efficacy of superior capsular reconstruction using a "sandwich" patch graft versus a fascia lata autograft in the treatment of irreparable massive rotator cuff tears (IMRCTs).Methods:A retrospective analysis was conducted on 50 patients with IMRCTs who underwent superior capsule reconstruction at Ningbo Medical Center Li Huili Hospital from January 2019 to May 2021. Patients were categorized into two groups based on the type of graft used: the "sandwich" group (27 patients), utilizing a "sandwich" patch graft, and the fascia lata group (23 patients), utilizing a simple fascia lata graft. The "sandwich" group consisted of 10 males and 17 females with a mean age of 65.6±5.7 years (range, 55-76 years), including 6 cases on the left shoulder and 21 cases on the right shoulder. The fascia lata group comprised 10 males and 13 females with a mean age of 65.5±4.2 years (range, 56-72 years), including 4 cases on the left shoulder and 19 cases on the right shoulder. Clinical outcomes were assessed preoperatively and at 1 week, 6 months, 1 year, and 2 years postoperatively using the American Shoulder and Elbow Surgeons (ASES) score, the University of California Los Angeles (UCLA) score, and the visual analogue scale (VAS) for pain and shoulder activity. Imaging outcomes, including acromio humeral distance (AHD) and graft status, were evaluated via radiographs or MRI.Results:The follow-up duration was 40.0±8.4 months (range, 26-54 months) for all patients. The "sandwich" group demonstrated significantly better outcomes compared to the fascia lata group. Improvements were noted in the ASES score (90.1±8.7 vs. 66.8±22.0), ASES score improvement (58.0±11.8 vs. 36.7±24.2), UCLA score (31.0±3.1 vs. 23.0±8.7), UCLA score improvement (20.1±4.5 vs. 12.7±9.2), active elevation (160.0°±21.3° vs. 124.8°±37.4°), active elevation improvement (70.2°±31.4° vs. 33.7°±42.4°), external rotation (35.0°±9.0° vs. 29.0°±6.9°), external rotation improvement (11.3°±7.3° vs. 7.2°±10.4°), AHD (6.4±1.5 mm vs. 4.4±1.0 mm), AHD improvement (3.0±1.6 mm vs. 1.5±1.0 mm), the difference was statistically significant ( P<0.05). The graft healing rate of the "sandwich" group reached 93%, which was higher than the 74% in the fascia lata group, but the difference was not statistically significant (χ 2=1.984, P=0.159). One case of subcutaneous hematoma was reported in the fascia lata group postoperatively. Additionally, two patients in the fascia lata group reported mild thigh discomfort at the last follow-up, which did not impair walking. No cases of infection, joint stiffness, or vascular or nerve damage were observed. Conclusion:Superior capsular reconstruction using a "sandwich" patch graft significantly enhances the healing rate of grafts and short-term postoperative clinical outcomes in patients with irreparable massive rotator cuff tears.

Objective To observe the improved effect of propofol on vascular hyporeactivity in septic rats and its underlying mechanism.Methods A total of 96 SD rats(12 weeks old,both genders,weighing 200～220 g)were randomly divided into sham group(n=16),sepsis group(n=16,cecal ligation and puncture),propofol group(n=16),propofol+ROCK inhibitor Y-27632 group(n=16),propofol+PKCαinhibitor GO6976 group(n=16),propofol+IP3 inhibitor 2-APB group(n=8)and propofol+gap junction inhibitor metoclopramide sodium(Movens)group(n=8).In vitro vascular ring reactivity and vascular calcium sensitivity were measured to observe the improved effects of propofol on vascular hyporeactivity in septic rats and its relationships with RhoA/ROCK,PKCα,IP3 and cell gap junction.Results Determination of in vitro vascular ring and calcium sensitivity showed that the contractile reactivity to norepinephrine(NE)and to calcium sensitivity were significantly decreased in the arterial rings isolated from the septic rats compared with those from the sham group,with the dose-response curve shifting to the right,and most significant decrease by 51.42％in the superior mesenteric artery(SMA,P＜0.05).Propofol treatment significantly improved the hyporeactivity and calcium sensitivity of the vessels isolated from the septic rats,especially those of the femoral artery with a recovery rate of 89.57％(P＜0.05).In comparison with the propofol group,the dose-response curves of the propofol+Y-27632 group and the propofol+GO6976 group were shifting to right,indicating that Y-27632 and GO6976 could significantly inhibit the amelioration of propofol on calcium sensitivity of SMA in severely septic rats with an inhibitory rate of 146.95％and 88.63％(P＜0.05),respectively.Isolated vascular reactivity measurement demonstrated that Y-27632 and Movens treatment significantly antagonized the ameliorated role of propofol on hyporeactivity of blood vessels from the septic rats with an inhibitory rate of 40.79％and 169.90％(P＜0.05),separately,while no such effect was observed in the propofol+GO6976 and propofol+2-APB groups.Conclusion Propofol treatment can significantly improve vascular hyporeactivity of septic rats,which may attribute to the increase of vascular calcium sensitivity through RhoA/ROCK pathway.

OBJECTIVES: Surgical robot system has broken the limitation of traditional surgery and shown excellent performance in surgery, and has been widely used in minimally invasive treatment in most areas of surgery. This study aims to verify the basic performance of the domestic surgical robot system and the safety and effectiveness of the integrated bipolar electrocoagulation and ultrasonic knife. METHODS: The basic performance of the domestic surgical robot system was evaluated by completing the square knot and surgical knot, vertical and horizontal perforation and right ring perforation and suture, as well as picking up beans. Compared with laparoscopy, the safety and effectiveness of the domestic surgical robot after integrated interconnection bipolar electrocoagulation and ultrasonic scalpel were evaluated by detecting the vascular closure performance and the degree of histopathological damage in animals. RESULTS: Compared with freehand knotting, domestic robot knotting speed and circumference were slightly worse, but better than laparoscopic knotting. There was no statistical significance in the tension difference of the surgical knots among the 3 methods (P>0.05), but the tension of the square knots made by the freehand and the domestic surgical robot was greater than that of the laparoscopy (P<0.05). The space required for both the left and right forceps heads of knots was smaller than that of laparoscopy (P<0.001), which successfully completed the 4 quadrant suture tasks, and the time of picking up beans was significantly less than that of laparoscopy (P<0.05). There was no significant difference in the temperature of the liver tissue after the bipolar electrocoagulation between the interconnected domestic surgical robot and the laparoscopy (P>0.05), and the acute thermal injury was observed under the light microscope. The temperature of the liver tissue treated by the domestic robotic ultrasound knife was higher than that of the laparoscopic ultrasound knife (P<0.05). CONCLUSIONS Domestic surgical robots are obviously superior to laparoscopy in suturing, knotting, and moving objects, and domestic surgical robots' interconnect bipolar electrocoagulation and ultrasonic knife have achieved success in animal experiments, and hemostasis is considered to be safe and effective.
Animals ; Robotics ; Laparoscopy/methods* ; Ultrasonography

BACKGROUND: Management of gastric leak after sleeve gastrectomy (SG) is challenging due to its unpredictable outcomes. We aimed to summarize the characteristics of SG leaks and analyze interventions and corresponding outcomes in a real-world setting. METHODS: To retrospectively review of 15,721 SG procedures from 2010 to 2020 based on a national registry. A cumulative sum analysis was used to identify a fitting curve of gastric leak rate. The Kaplan-Meier method and log-rank tests were performed to calculate and compare the probabilities of relevant outcomes. The logistic regression analysis was conducted to determine the predictors of acute leaks. RESULTS: A total of 78 cases of SG leaks were collected with an incidence of 0.5% (78/15,721) from this registry (6 patients who had the primary SG in non-participating centers). After accumulating 260 cases in a bariatric surgery center, the leak rate decreased to a stably low value of under 1.17%. The significant differences presented in sex, waist circumference, and the proportion of hypoproteinemia and type 2 diabetes at baseline between patients with SG leak and the whole registry population ( P = 0.005, = 0.026, <0.001, and = 0.001, respectively). Moreover, 83.1% (59/71) of the leakage was near the esophagogastric junction region. Leakage healed in 64 (88.9%, 64/72) patients. The median healing time of acute and non-acute leaks was 5.93 months and 8.12 months, respectively. Acute leak (38/72, 52.8%) was the predominant type with a cumulative reoperation rate >50%, whereas the cumulative healing probability in the patients who required surgical treatment was significantly lower than those requring non-surgical treatment ( P = 0.013). Precise dissection in the His angle area was independently associated with a lower acute leak rate, whereas preservation ≥2 cm distance from the His angle area was an independent risk factor. CONCLUSIONS Male sex, elevated waist circumference, hypoproteinaemia, and type 2 diabetes are risk factors of gastric leaks after SG. Optimizing surgical techniques, including precise dissection of His angle area and preservation of smaller gastric fundus, should be suggested to prevent acute leaks.
Humans ; Male ; Retrospective Studies ; Diabetes Mellitus, Type 2/complications* ; Obesity, Morbid ; Anastomotic Leak/epidemiology* ; Gastrectomy/methods* ; Reoperation/methods* ; Registries ; Laparoscopy/methods* ; Treatment Outcome

Objective　To analyze the composition, population density, seasonal trends, and insecticide resistance monitoring results of vector mosquitoes in Liaocheng City from 2020 to 2022, providing a reference basis for further mosquito control measures. Methods　According to the "Implementation Plan for National Vector Biomonitoring", adult mosquito monitoring was carried out using mosquito traps and double-layer stacked nets at two national level monitoring points in Dong’e and Chiping. The Bretu Index method was used for monitoring throughout the city, and the immersion method was used for monitoring mosquito insecticide resistance. The monitoring period for adult mosquitoes was from March to November, and for juvenile mosquitoes was from May to October. Results　From 2020 to 2022, a total of 816 mosquito traps were deployed, capturing 4 717 adult mosquitoes with an average density of 5.78 mosquitoes per trap-night. Among them, 4 232 were Culex pipiens pallens, accounting for 89.72% of the total mosquito population, followed by 15 were Anopheles sinensis, accounting for 0.32%; 10 Aedes albopictus, accounting for 0.21%; 460 other mosquito species, accounting for 9.75%. Mosquito density was higher in rural areas compared to urban areas, with the highest density found in livestock sheds, followed by parks, hospitals, farms, and residential areas. Adult mosquito was first observed in May, with the peak activity occurring from June to August. The double-layer net method captures only Aedes albopictus, with an average net trapping index of 2.99 mosquitoes/(net·h), and their activity was concentrated from June to September. The average Brayton Index (BI) from May to October was 15.95, with a peak occurring in July and gradually decreasing after August. Insecticide resistance monitoring found that Culex pipiens pallens in Liaocheng City exhibited low resistance to permethrin, deltamethrin, dithion, and beta cypermethrin, with beta cypermethrin having the highest resistance. Conclusion　The dominant mosquito species in Liaocheng City is Culex pipiens pallens, followed by Aedes albopictus. The peak activity of adult and juvenile mosquitoes is concentrated between July and September, showing a distinct seasonal trend of growth and decline, posing a risk of dengue fever outbreaks. Culex pipiens pallens exhibits resistance to some insecticides, and timely measures should be taken to prevent the outbreak of mosquito-borne infectious diseases.

Objective:To analyze the risk factors for postoperative one-year mortality in elderly patients with femoral intertrochanteric fracture following multidisciplinary treatment (MDT) by intramedullary nailing.Methods:The clinical data were retrospectively analyzed of the 158 elderly patients with femoral intertrochanteric fracture who had undergone MDT by proximal femoral intramedullary nailing between January 2018 and August 2020 at Department of Orthopedics, Trauma Center, Zhongda Hospital Affiliated to Southeast University. There were 41 males and 117 females with an average age of 82.5 years (from 65 to 95 years). By the modified Evans classification, there were 15 cases of type Ⅰ, 16 cases of type Ⅱ, 35 cases of type Ⅲ, 81 cases of type Ⅳ, and 11 cases of type Ⅴ. The one-year mortality was documented in the patients after surgery. To screen for risk factors, univariate analysis was conducted of gender, age, body mass index (BMI), modified Evans classification of fractures, time from injury to operation, American Society of Anesthesiologists (ASA) classification, Charlson comorbidity index (CCI) and comorbidities, as well as preoperative hemoglobin (Hb), serum albumin (ALB) and total lymphocyte count (TLC). The factors with P<0.05 were included in the multivariate logistic regression model analysis to determine the risk factors. Results:A total of 13 patients died within one year after surgery, yielding a mortality of 8.2% (13/158). Univariate analysis showed significant differences in age, body mass index, modified Evans classification of fractures, CCI and Hb between the surviving and dead patients ( P<0.05). Multivariate logistic regression analysis showed that age >85 years ( OR=0.122, 95% CI: 0.018 to 0.834, P=0.032), BMI>23.9 kg/m 2 ( OR=0.083, 95% CI: 0.013 to 0.510, P=0.007), CCI≥3 points ( OR=0.051, 95% CI: 0.090 to 0.275, P=0.001) and preoperative Hb<90 g/L ( OR=4.733, 95% CI: 1.036 to 21.624, P=0.045) were the independent risk factors for postoperative one-year mortality in the elderly patients with intertrochanteric fracture following MDT by proximal femoral intramedullary nailing. Conclusions:After MDT by proximal femoral intramedullary nailing of femoral intertrochanteric fractures, the geriatric patients with an age >85 years, BMI>23.9 kg/m 2, CCI≥3 points and Hb<90 g/L are likely to die. Therefore, special care should be taken for them.

Spontaneous intracranial hypotension can frequently result in several complications including subdural hygroma, subdural hematoma and cerebral venous thrombosis, but coma rarely. A case of spontaneous intracranial hypotension presented with orthostatic headaches was described. He experienced somnolence, disorientation, incontinence, and then coma, though received conservative treatment. Brain imaging demonstrated acute-on-chronic subdural hematoma, magnetic resonance myelography using heavily T 2-weighted fast spin-echo pulse sequences showed spinal longitudinal extradural collection, and magnetic resonance myelography with intrathecal gadolinium revealed cerebrospinal fluid leak at the level of T 6, T 7. The patient recovered consciousness after surgical evacuation of the hematoma, and the headache disappeared after a targeted epidural blood patch. The hematoma resolved 2 months later and the patient kept free from headache during follow-up.