Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective To develop GTKO (α-1,3-galactosyltransferase gene-knockout, GTKO)/hCD55 (human CD55) gene-edited xenotransplant donor pigs and verify their function. Methods In this study, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated nuclease 9), PiggyBac transposon technology and somatic cell nuclear transfer technology were used to construct GTKO/hCD55 gene-edited Diannan miniature pigs. The phenotype and function of GTKO/hCD55 pigs were analyzed by Sanger sequencing, real-time fluorescence quantitative PCR, flow cytometry, immunofluorescence, bisulfite sequencing, antigen-antibody binding assays, and complement-dependent cytotoxicity assays. Results After transfection of PX458 and PiggyBac gene editing vectors into wild-type fetal pig fibroblasts, 48 single-cell colonies were obtained through puromycin drug screening. Two single-cell colonies were selected for somatic cell nuclear transfer, resulting in two fetal pigs at 33 days of gestation. The GGTA1(α-1,3-galactosyltransferase) genotypes of fetal pig F01 were -17 bp and wild type (WT), while the GGTA1 genotypes of fetal pig F02 were -26 bp/+2 bp and -3 bp. The hCD55 mRNA expression levels of both fetal pigs were significantly higher than those of WT pigs (P＜0.01). The fetal pig F02 was selected as the donor cell source for recloning, 11 surviving piglets were obtained, all identified as GTKO/hCD55 gene-edited pigs. These pigs showed absence of α-Gal antigen expression, but weak or no expression of hCD55 was observed. Methylation analysis of the hCD55 gene's CpG island showed hypermethylation in kidney tissue lacking hCD55 expression, whereas it was not methylated or partially methylated in kidney tissue expressing hCD55. Moreover, codon optimization of the CpG island of the hCD55 gene to reduce CG content could achieve stable expression of the hCD55 gene. In addition, antigen-antibody binding experiment showed that the amount of human IgM binding to GTKO/hCD55 gene-edited pig fibroblasts was significantly lower than that of WT pigs (P＜0.01). Complement-dependent cytotoxicity experiment showed that the survival rate of fibroblasts in GTKO/hCD55 pigs was significantly higher than that in WT pigs (P＜0.01). Conclusion This study demonstrates the successful generation of GTKO/hCD55 gene-edited xenotransplant donor pigs. Methylation-induced gene silencing of the hCD55 gene can be effectively avoided by reducing the CG content of the CpG island through codon optimization. This study provides a reference for the development of xenotransplant donor pigs and guides subsequent research on xenotransplantation.

Objective To study the distribution characteristics of TCM constitutions in the middle-aged and elderly Mongolian population undergoing physical examinations in Inner Mongolia;To analyze the correlation with related risk factors.Methods Health information and related risk factors(including BMI index,lifestyle habits,dietary habits and exercise status)of middle-aged and elderly Mongolian people who visited the physical examination departments of Inner Mongolia Autonomous Region Traditional Chinese Medicine Hospital and Tongliao City Traditional Chinese Medicine Hospital from March 2024 to April 2024 were collected.A total of 213 Mongolian people were surveyed,including 65 people who have lived in Hohhot for more than 2 years and 148 people who have lived in Tongliao for more than 2 years.Through investigation,research and analysis,the distribution characteristics of TCM constitutions in the population and its correlation with related risk factors were observed.Results Constitution distribution:101 people(47.41%)had a moderate constitution.Among the biased constitutions,the phlegm dampness constitution had the highest number of people(64 people,30.05%),with the blood deficiency constitution having the lowest number of people(2 people,0.94%).In the investigation of related risk factors,there was a significant correlation(P<0.01)between phlegm dampness and dietary habits and reduced exercise volume.Moreover,individuals with this constitution tend to have good sleep,reduced physical activity,faster eating and a diet primarily consisting of high-calorie foods(P<0.05).Conclusion The TCM constitution of the Mongolian population in Inner Mongolia is mostly composed of moderate constitution,while the biased constitution is mostly composed of phlegm dampness constitution,which is mainly related to dietary habits and reduced exercise volume.

Objective:To analyze the effect of precipitation on road traffic injuries (RTI) in Zhejiang Province.Methods:The RTI surveillance and meteorological data from 2009 to 2022 in Zhejiang Province were collected. Based on the time-stratified case-crossover design, the precipitation of case day and control day was compared, and the distributed lag nonlinear model was applied to analyze the correlation of precipitation and RTI. Stratified analyses were conducted to analyze the effect modification of gender, age, injury location, and temperature. An attributable fraction was used to assess the burden of RTI caused by precipitation.Results:A total of 239 970 RTIs were monitored in Zhejiang Province from 2009 to 2022, averaging 46 daily cases. The distributed lag nonlinear model showed that compared with no rain, the risk of RTI increased first and then decreased with the increase of precipitation. The risk of RTI was the highest when the precipitation was 30.99 mm ( OR=1.08, 95% CI: 1.05-1.11). The adverse effects on RTI mainly occurred on the day of precipitation, and it showed insignificant or protective effects with the extension of lag days. 1.34%(95% CI: 1.31%-1.36%) of RTI could be attributed to precipitation. Stratified analysis showed that gender, age, injury location, and temperature may modify the effect of precipitation on RTI. Precipitation caused a heavier burden on RTI in subgroups aged 18-64, females, and occurring on roads and in low temperatures. Conclusions:Precipitation can increase the risk of RTI. People aged 18-64 or females are the key groups for RTI prevention, and prevention and control efforts of precipitation-related RTI should be increased in road and low-temperature environments.

Adolescent idiopathic scoliosis(AIS)is a complex three-dimensional deformity involving coronal,sagittal,and axial planes,with a prevalence that should not be overlooked.With advancements in technology and in-depth research,an increasing number of hospitals and physicians are exploring standardized diagnostic and treatment approaches for AIS.Comprehensive and in-depth understanding is required for AIS,including its etiology,screening and diagnosis,classification,assessment and examination,treatment options,exploration of current focus,and evaluation of quality of life.Such understanding ensures that the diagnostic and treatment are scientific,standardized,and timely.Based on the principles of evidence-based medicine,a consensus on the diagnosis and treatment of AIS is reached after multiple discussions among spinal surgery experts,aiming to provide reference and guidance for clinical practice.

Malignant pleural mesothelioma(MPM)is strongly associated with a history of asbestos exposure and is characterized by high malignancy,high mortality,and poor prognosis.Current treatments for MPM are limited and generally suboptimal,resulting in a median overall survival(OS)of approximately one year for MPM patients.However,advancements in treatment options,including surgery,radiotherapy,chemotherapy,immunotherapy and targeted therapy,have brought new hope to patients with MPM.For early-stage MPM patients categorized under the TNM staging system,surgical treatment is feasible and can improve survival rates and quality of life.However,there is still debate regarding the optimal surgical approach for MPM.In addition to surgery,radiotherapy plays a vital role in MPM treatment.It is often used as prophylactic treatment or for alleviating local symptoms in advanced stages.Radiotherapy can also serve as neoadjuvant or adjuvant therapy in surgical contexts.For patients experiencing local progression or isolated distant metastases after systemic treatment,radiotherapy is a viable option.The advent of advanced radiotherapy techniques,such as intensity-modulated radiotherapy(IMRT)and volumetric intensity-modulated arc therapy(VMAT),has significantly improved the precision and efficacy of radiotherapy while minimizing damage to healthy tissues.Furthermore,brachytherapy can relieve pain or act as a localized supplemental therapy.Chemotherapy remains the standard treatment for MPM.The combination of pemetrexed and platinum-based drugs is widely applied as first-line therapy and has been shown to significantly extend survival.However,commonly used second-line regimens often yield suboptimal results.In recent years,immunotherapy has developed rapidly.Dual immunotherapy with nivolumab and ipilimumab has demonstrated impressive clinical efficacy and safety.The combination of immunotherapy and chemotherapy has also notably extended patients'median survival.Multiple clinical trials have confirmed that this combination therapy benefits patients.Currently available targeted therapies for MPM primarily focus on anti-angiogenesis.Bevacizumab combined with chemotherapy has established its position as a first-line treatment.Research on ramucirumab and apatinib suggests that these drugs have certain efficacy and safety profiles.Beyond conventional treatment options,the UV1 cancer vaccine combined with dual immunotherapy offers new hope for patients.Chimeric antigen receptor T(CAR-T)cell therapy is an emerging treatment method being investigated in MPM patients,with phase Ⅰ clinical trials demonstrating good antitumor effects.Additionally,some antibody-drug conjugates are becoming therapeutic options for MPM through precise targeting.Tumor treating fields combined with chemotherapy has also shown efficacy in extending survival.Despite the increasing variety of treatment options for MPM,its diagnosis and treatment still face numerous challenges,including difficulties in early detection,treatment resistance,and a lack of large-scale evidence-based clinical studies.Future research should focus on improving early diagnosis rates,developing new treatment strategies,overcoming resistance,and advancing personalized therapy.Strengthening the integration of basic research and clinical trials will also be essential.Through multidisciplinary collaboration and continuous innovation,it is hoped that more effective and safer treatment options will become available,ultimately improving the prognosis of MPM patients.

Objective To investigate the effect of postoperative reduction quality in femoral neck fracture internal fixation on mechanical properties of the femoral head from the perspective of trabecular bone biomechanics.Methods From patients who underwent hip replacement surgery for femoral neck fractures,a total of 26 femoral head slice specimens were obtained.The central axis of the primary compressive trabeculae was defined as the 0° group,with the intersection point of the primary compressive trabeculae and the femoral calcar serving as the center.By rotating the specimens to simulate different reduction angles,the cut femoral head slice specimens were randomly divided into five groups:-10°,-5°,0°,5°,and 10°,representing femoral heads with varying reduction qualities.The specimens were subjected to single compression load tests and fatigue load tests.The load was set from 70 N to 1 400 N,at a frequency of 1 Hz,with 10 000 cycles.Axial stiffness,displacement,and the number of collapse cycles were measured,to compare the biomechanical properties of femoral head specimens under different reduction qualities.Results There were differences in the axial stiffness,displacement,and number of collapse cycles among the femoral head specimens in different groups.Under 800 N load,the axial stiffness of 0° group was significantly greater than that of±10° groups(P＜0.05).The axial stiffness of 0° group was also greater than that of the±5° groups,but the differences were not statistically significant(P＞0.05).The axial stiffness of±5° groups was greater than that of±10° groups(P＜0.05).0° group had a lower displacement than±5° groups and±10° groups.However,the differences in displacement between 0° group and±5° groups were not statistically significant(P＞0.05),while the differences between the 0° group and±10° groups were statistically significant(P＜0.05).The differences in displacement between±5° groups and±10° groups were also statistically significant(P＜0.05).0° group had a significantly higher number of collapse cycles than±10° groups(P＜0.05).The number of collapse cycles in 0° group was also higher than that in±5° groups,but the differences were not statistically significant(P＞0.05).The number of collapse cycles in±5° groups was significantly higher than that±10° groups(P＜0.05).Conclusions The quality of reduction after internal fixation of femoral neck fractures significantly affects the biomechanical properties of the femoral head.This study provides a scientific basis for optimizing treatment and postoperative management,aiming to improve clinical outcomes and patients' quality of life.

Objective:To analyze the effect of precipitation on road traffic injuries (RTI) in Zhejiang Province.Methods:The RTI surveillance and meteorological data from 2009 to 2022 in Zhejiang Province were collected. Based on the time-stratified case-crossover design, the precipitation of case day and control day was compared, and the distributed lag nonlinear model was applied to analyze the correlation of precipitation and RTI. Stratified analyses were conducted to analyze the effect modification of gender, age, injury location, and temperature. An attributable fraction was used to assess the burden of RTI caused by precipitation.Results:A total of 239 970 RTIs were monitored in Zhejiang Province from 2009 to 2022, averaging 46 daily cases. The distributed lag nonlinear model showed that compared with no rain, the risk of RTI increased first and then decreased with the increase of precipitation. The risk of RTI was the highest when the precipitation was 30.99 mm ( OR=1.08, 95% CI: 1.05-1.11). The adverse effects on RTI mainly occurred on the day of precipitation, and it showed insignificant or protective effects with the extension of lag days. 1.34%(95% CI: 1.31%-1.36%) of RTI could be attributed to precipitation. Stratified analysis showed that gender, age, injury location, and temperature may modify the effect of precipitation on RTI. Precipitation caused a heavier burden on RTI in subgroups aged 18-64, females, and occurring on roads and in low temperatures. Conclusions:Precipitation can increase the risk of RTI. People aged 18-64 or females are the key groups for RTI prevention, and prevention and control efforts of precipitation-related RTI should be increased in road and low-temperature environments.

Objective To investigate the effect of postoperative reduction quality in femoral neck fracture internal fixation on mechanical properties of the femoral head from the perspective of trabecular bone biomechanics.Methods From patients who underwent hip replacement surgery for femoral neck fractures,a total of 26 femoral head slice specimens were obtained.The central axis of the primary compressive trabeculae was defined as the 0° group,with the intersection point of the primary compressive trabeculae and the femoral calcar serving as the center.By rotating the specimens to simulate different reduction angles,the cut femoral head slice specimens were randomly divided into five groups:-10°,-5°,0°,5°,and 10°,representing femoral heads with varying reduction qualities.The specimens were subjected to single compression load tests and fatigue load tests.The load was set from 70 N to 1 400 N,at a frequency of 1 Hz,with 10 000 cycles.Axial stiffness,displacement,and the number of collapse cycles were measured,to compare the biomechanical properties of femoral head specimens under different reduction qualities.Results There were differences in the axial stiffness,displacement,and number of collapse cycles among the femoral head specimens in different groups.Under 800 N load,the axial stiffness of 0° group was significantly greater than that of±10° groups(P＜0.05).The axial stiffness of 0° group was also greater than that of the±5° groups,but the differences were not statistically significant(P＞0.05).The axial stiffness of±5° groups was greater than that of±10° groups(P＜0.05).0° group had a lower displacement than±5° groups and±10° groups.However,the differences in displacement between 0° group and±5° groups were not statistically significant(P＞0.05),while the differences between the 0° group and±10° groups were statistically significant(P＜0.05).The differences in displacement between±5° groups and±10° groups were also statistically significant(P＜0.05).0° group had a significantly higher number of collapse cycles than±10° groups(P＜0.05).The number of collapse cycles in 0° group was also higher than that in±5° groups,but the differences were not statistically significant(P＞0.05).The number of collapse cycles in±5° groups was significantly higher than that±10° groups(P＜0.05).Conclusions The quality of reduction after internal fixation of femoral neck fractures significantly affects the biomechanical properties of the femoral head.This study provides a scientific basis for optimizing treatment and postoperative management,aiming to improve clinical outcomes and patients' quality of life.

Malignant pleural mesothelioma(MPM)is strongly associated with a history of asbestos exposure and is characterized by high malignancy,high mortality,and poor prognosis.Current treatments for MPM are limited and generally suboptimal,resulting in a median overall survival(OS)of approximately one year for MPM patients.However,advancements in treatment options,including surgery,radiotherapy,chemotherapy,immunotherapy and targeted therapy,have brought new hope to patients with MPM.For early-stage MPM patients categorized under the TNM staging system,surgical treatment is feasible and can improve survival rates and quality of life.However,there is still debate regarding the optimal surgical approach for MPM.In addition to surgery,radiotherapy plays a vital role in MPM treatment.It is often used as prophylactic treatment or for alleviating local symptoms in advanced stages.Radiotherapy can also serve as neoadjuvant or adjuvant therapy in surgical contexts.For patients experiencing local progression or isolated distant metastases after systemic treatment,radiotherapy is a viable option.The advent of advanced radiotherapy techniques,such as intensity-modulated radiotherapy(IMRT)and volumetric intensity-modulated arc therapy(VMAT),has significantly improved the precision and efficacy of radiotherapy while minimizing damage to healthy tissues.Furthermore,brachytherapy can relieve pain or act as a localized supplemental therapy.Chemotherapy remains the standard treatment for MPM.The combination of pemetrexed and platinum-based drugs is widely applied as first-line therapy and has been shown to significantly extend survival.However,commonly used second-line regimens often yield suboptimal results.In recent years,immunotherapy has developed rapidly.Dual immunotherapy with nivolumab and ipilimumab has demonstrated impressive clinical efficacy and safety.The combination of immunotherapy and chemotherapy has also notably extended patients'median survival.Multiple clinical trials have confirmed that this combination therapy benefits patients.Currently available targeted therapies for MPM primarily focus on anti-angiogenesis.Bevacizumab combined with chemotherapy has established its position as a first-line treatment.Research on ramucirumab and apatinib suggests that these drugs have certain efficacy and safety profiles.Beyond conventional treatment options,the UV1 cancer vaccine combined with dual immunotherapy offers new hope for patients.Chimeric antigen receptor T(CAR-T)cell therapy is an emerging treatment method being investigated in MPM patients,with phase Ⅰ clinical trials demonstrating good antitumor effects.Additionally,some antibody-drug conjugates are becoming therapeutic options for MPM through precise targeting.Tumor treating fields combined with chemotherapy has also shown efficacy in extending survival.Despite the increasing variety of treatment options for MPM,its diagnosis and treatment still face numerous challenges,including difficulties in early detection,treatment resistance,and a lack of large-scale evidence-based clinical studies.Future research should focus on improving early diagnosis rates,developing new treatment strategies,overcoming resistance,and advancing personalized therapy.Strengthening the integration of basic research and clinical trials will also be essential.Through multidisciplinary collaboration and continuous innovation,it is hoped that more effective and safer treatment options will become available,ultimately improving the prognosis of MPM patients.