Objective To discuss the preliminary application of CRISPR-Cas9 gene editing technology in repair of antithrombin gene(SERPINC1)c.318_319insT mutation.Methods The single guide RNA(sgRNA)was designed by CRISPR online design website,and AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were constructed.The gene fragments from the wild-type gene,AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were transferred into lentiviral expression vectors,and then PCR sequencing was performed for verification.The successfully constructed lentiviral recombinant plasmids were transfected into the human embryonic kid-ney cells(HEK293T).After cell culture,HEK293T cells were lysed.The AT:Ag levels in the cell lysing reagents from wild-type gene,CRISPR-Cas9 repairsome and mutant were compared by ELISA and Western blot,respectively.The recombinant AT protein was characterized in vitro by cellular immunofluorescence assay.Results Both the AT c.318_319insT mutant and CRISPR-Cas9 repair-some were successfully constructed.The results of experiments with HEK293T cells in vitro showed that the wild-type AT:Ag in the cell lysing reagents was set as 100％,the AT:Ag of CRISPR-Cas9 repairsome was 47％,and the AT:Ag of AT c.318_319insT was 22％,which were consistent with the results of western blot and cellular immunofluorescence assay.Conclusion The cellular experiments in vitro verified that CRISPR-Cas9 gene editing technology could effectively repair the SERPINC1 c.318_319 insT mutation in situ,which might provide the experimental support for the application of CRISPR-Cas9 gene editing technology in the gene therapy of hereditary thrombotic diseases.

Objectives:This study aimed to investigate the impact of quantitative flow ratio(QFR)-guided revascularization on outcome of elderly patients with coronary artery disease(CAD)undergoing valve surgery.Methods:We retrospectively analyzed 750 consecutive patients with angiographically confirmed CAD(≥50%stenosis)who underwent valve surgery at Zhongshan Hospital,Fudan University,between January 2016 and December 2021.According to the patients'ages,they were divided into the younger group(age<70 years old,n=532)and the elderly group(age≥70 years old,n=218).Revascularization strategies were evaluated using anatomical(angiography-based)and functional(QFR-based)criteria.Anatomical complete revascularization(CR)was defined as bypass grafting for all lesions with≥70%diameter stenosis in major coronary arteries or≥50%stenosis in the left main coronary artery.Functional CR referred bypass grafting for all lesions with QFR≤0.80.Incomplete revascularization(ICR)was defined as failure to meet CR criteria.According to the anatomical and functional definitions,the younger group and the elderly group were further divided into the incomplete revascularization subgroup and the complete revascularization subgroup respectively.Major adverse cardiovascular events(MACE),including death,myocardial infarction,repeat revascularization,and stroke,were assessed as the composite endpoint.Results:Over a follow-up of(3.7±1.8)years,the overall MACE rate was 13.3%.The younger group exhibited significantly lower MACE rates than the elderly group(10.7%vs.19.7%,P=0.001).In the younger group,anatomical ICR did not increase MACE risk(HR=1.46,95%CI:0.81-2.62,P=0.164),whereas functional ICR significantly increased MACE risk(HR=2.27,95%CI:1.24-4.15,P=0.001).In the elderly group,neither anatomical ICR(HR=1.22,95%CI:0.62-2.41,P=0.540)nor functional ICR(HR=1.52,95%CI:0.78-2.96,P=0.172)was associated with increased MACE risk.Conclusions:In patients undergoing valve surgery with CAD,functional ICR correlated with adverse outcomes in the younger group,whereas neither anatomical nor functional ICR significantly affected prognosis in elderly patients.These findings suggest that a moderately conservative revascularization strategy may be more appropriate for elderly populations.

Objective To discuss the preliminary application of CRISPR-Cas9 gene editing technology in repair of antithrombin gene(SERPINC1)c.318_319insT mutation.Methods The single guide RNA(sgRNA)was designed by CRISPR online design website,and AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were constructed.The gene fragments from the wild-type gene,AT c.318_319 insT mutant and CRISPR-Cas9 repairsome were transferred into lentiviral expression vectors,and then PCR sequencing was performed for verification.The successfully constructed lentiviral recombinant plasmids were transfected into the human embryonic kid-ney cells(HEK293T).After cell culture,HEK293T cells were lysed.The AT:Ag levels in the cell lysing reagents from wild-type gene,CRISPR-Cas9 repairsome and mutant were compared by ELISA and Western blot,respectively.The recombinant AT protein was characterized in vitro by cellular immunofluorescence assay.Results Both the AT c.318_319insT mutant and CRISPR-Cas9 repair-some were successfully constructed.The results of experiments with HEK293T cells in vitro showed that the wild-type AT:Ag in the cell lysing reagents was set as 100％,the AT:Ag of CRISPR-Cas9 repairsome was 47％,and the AT:Ag of AT c.318_319insT was 22％,which were consistent with the results of western blot and cellular immunofluorescence assay.Conclusion The cellular experiments in vitro verified that CRISPR-Cas9 gene editing technology could effectively repair the SERPINC1 c.318_319 insT mutation in situ,which might provide the experimental support for the application of CRISPR-Cas9 gene editing technology in the gene therapy of hereditary thrombotic diseases.

Objectives:This study aimed to investigate the impact of quantitative flow ratio(QFR)-guided revascularization on outcome of elderly patients with coronary artery disease(CAD)undergoing valve surgery.Methods:We retrospectively analyzed 750 consecutive patients with angiographically confirmed CAD(≥50%stenosis)who underwent valve surgery at Zhongshan Hospital,Fudan University,between January 2016 and December 2021.According to the patients'ages,they were divided into the younger group(age<70 years old,n=532)and the elderly group(age≥70 years old,n=218).Revascularization strategies were evaluated using anatomical(angiography-based)and functional(QFR-based)criteria.Anatomical complete revascularization(CR)was defined as bypass grafting for all lesions with≥70%diameter stenosis in major coronary arteries or≥50%stenosis in the left main coronary artery.Functional CR referred bypass grafting for all lesions with QFR≤0.80.Incomplete revascularization(ICR)was defined as failure to meet CR criteria.According to the anatomical and functional definitions,the younger group and the elderly group were further divided into the incomplete revascularization subgroup and the complete revascularization subgroup respectively.Major adverse cardiovascular events(MACE),including death,myocardial infarction,repeat revascularization,and stroke,were assessed as the composite endpoint.Results:Over a follow-up of(3.7±1.8)years,the overall MACE rate was 13.3%.The younger group exhibited significantly lower MACE rates than the elderly group(10.7%vs.19.7%,P=0.001).In the younger group,anatomical ICR did not increase MACE risk(HR=1.46,95%CI:0.81-2.62,P=0.164),whereas functional ICR significantly increased MACE risk(HR=2.27,95%CI:1.24-4.15,P=0.001).In the elderly group,neither anatomical ICR(HR=1.22,95%CI:0.62-2.41,P=0.540)nor functional ICR(HR=1.52,95%CI:0.78-2.96,P=0.172)was associated with increased MACE risk.Conclusions:In patients undergoing valve surgery with CAD,functional ICR correlated with adverse outcomes in the younger group,whereas neither anatomical nor functional ICR significantly affected prognosis in elderly patients.These findings suggest that a moderately conservative revascularization strategy may be more appropriate for elderly populations.

Objective To understand the current status of radiation diagnosis and treatment resources and their use in Taiyuan City, China, and provide data support for optimizing resource allocation and standardizing diagnosis and treatment. Methods A census-based approach was implemented using a standardized questionnaire to collect basic information on radiation diagnosis and treatment institutions in Taiyuan City. The number and use frequency of radiation diagnosis and treatment resources were calculated based on the resident population of Taiyuan City at the end of 2023. Results There were a total of 562 radiation diagnosis and treatment institutions in Taiyuan City, with 3970 workers and 1354 pieces of equipment. The numbers of radiation diagnosis and treatment institutions, workers, and equipment per 1 000 000 population were 103, 728, and 248, respectively. There were significant differences in radiation diagnosis and treatment resources among different districts and counties. There were 526 (93.59%) radiation diagnosis and treatment institutions and 25 (96.15%) tertiary hospitals in the six urban districts. All institutions offering nuclear medicine diagnosis and treatment and radiotherapy were located in the urban districts. There were substantial disparities among hospitals of different levels. The average numbers of radiation workers and equipment were in the order of tertiary hospitals (99.42, 17.15) > secondary hospitals (13.31, 4.62) > primary and unrated hospitals (1.43, 1.38). The frequency of radiation diagnosis and treatment in tertiary hospitals was 112 149 per hospital, which is 5 times that of secondary hospitals and 44 times that of primary and unrated hospitals. The use frequency of radiation diagnosis and treatment was 975.79 per 1000 population. The use frequency was 941.12 per 1000 population for X-ray diagnosis, 21.68 per 1000 population for interventional radiology, 8.70 per 1000 population for nuclear medicine diagnosis, 2.33 per 1000 population for nuclear medicine treatment, and 1.95 per 1000 population for radiotherapy. Conclusion There are abundant radiation diagnosis and treatment resources in Taiyuan City. The use frequency of radiation diagnosis and treatment has reached the level of Class II medical and health care regions, and there remains considerable room for growth and improvement. At present, the distribution of radiation diagnosis and treatment resources and services is unbalanced among different districts and counties as well as among institutions of different levels, mainly concentrated in urban districts and tertiary hospitals. The health administrative department needs to make overall plans and allocate resources scientifically.

Objective:To analyze the recovery of olfactory function in patients diagnosed with chronic rhinosinusitis with nasal polyps (CRSwNP) after endoscopic sinus surgery and to identify factors influencing recovery to provide a theoretical foundation for taking effective measures.Methods:This was a retrospective analysis included 277 CRSwNP patients with olfactory dysfunction who underwent endoscopic sinus surgery at the First Affiliated Hospital of Chongqing Medical University from October 2021 to September 2023. This study included 189 males and 88 females, with a median age of 46 years (range: 18-84 years). Routine laboratory tests, sinus CT Lund-Mackay score, modified sinus CT score, endoscopic polyp score, and nasal endoscopic Lund-Kennedy score were included for preoperative assessments. Eosinophil counts were obtained from nasal polyp tissues during surgery. The University of Pennsylvania smell identification test (UPSIT) was administered before surgery and 6 months after surgery to evaluate olfactory function. Based on results of postoperative olfactory test, patients were divided into two groups: the group with improved olfactory function and without improvement of olfactory function. Chi-square test and multivariate Logistic regression were used to analyze relevant factors affecting postoperative olfactory function of CRSwNP patients, and the prediction model was constructed to verify its consistency and to analyze its prediction efficiency.Results:Of the 277 patients, 155 (56%) showed improved olfactory function and 122 (44%) did not improve after surgery. Multivariate Logistic regression analysis identified following independent factors associated with improved postoperative olfactory function: concurrent allergic rhinitis ( OR=2.34), long duration of olfactory dysfunction ( OR=1.13), higher total score of CT olfactory zone ( OR=1.26), higher Lund-Kennedy score ( OR=1.23), presence of olfactory cleft polyps ( OR=4.72), higher tissue eosinophil count ( OR=1.01) and high IL-6 levels ( OR=1.51). Conversely, a higher endoscopic polyp score ( OR=0.74) was associated with a lower likelihood of olfactory improvement. The nomogram model, validated using receiver operating characteristic (ROC) analysis, demonstrated good clinical efficacy with an area under the curve (AUC) of 0.80 (95%CI: 0.748-0.852). The calibration curve showed an absolute error of 0.021, indicating good consistency and predictive accuracy. Conclusions:Factors such as a medical history of allergic rhinitis, duration of olfactory dysfunction, total score of sinus CT olfactory zone, endoscopic Lund-Kennedy score, olfactory cleft polyps, tissue eosinophil count, IL-6 level and endoscopic polyp score independently influence postoperative olfactory function of CRSwNP patients. The nomogram model based on these independent factors has good clinical efficacy, which can be used to predict the postoperative olfactory function in CRSwNP patients with olfactory dysfunction.

Objective To analyse phenotype and genetic variation of two congenital hypodysfibrinogenemia(Fg)caused by compound heterozygous variants and preliminary investigate their molecular pathogenic mechanisms.Metheds The proband A and B and their family members(a total of 19 members in 3 generations)who visited the First Hospital of Wenzhou Medical University on 4 May 2023 and 20 May 2023 for"parkinson's disease"and"pre-bilateral eyelid excision"were enrolled for the study.Prothrombin time(TT)and fibrinogen(Fg)activity were measured by coagulation assay and Fg antigen(Fg∶Ag)was measured by immunoturbidimetric assay for the two family members,and Fg aggregation assay was catalysed using human thrombin.FGG gene was amplified by PCR and se-quenced directly.The variant sites were analysed using Chromas software.Multiple sequence comparison was performed by ClustalX-2.1-win software.Pathogenicity analysis of the variant sites was performed using bioinformatics software.The analysis for FGG protein model was performed using PyMOL software.Results Phenotypic results showed TT of proband A and B extended to 27.5 s and 26.1 s,and plasma Fg activity reduced to 0.6 g/L and＜0.5 g/L,respectively.Genetic sequencing identified heterozygous c.1129+62_65delAATA on intron 8 of FGG gene in the both probands,resulting in the formation of aberrant amino acids at p.γGly377-Gly388 and an early ter-mination codon at p.γTyr389 site.A heterozygous missense variant c.103C＞A(p.AαArg35Ser)was found in exon 2 of the FGA gene of proband A,and a heterozygous missense variant c.569A＞G(p.BβAsn190Ser)was found in exon 4 of the FGB gene of proband B.Compared to the control group,the both probands showed significant decreases in peak and rate of Fg aggregation.Multiple sequence comparison analyses showed that all the three variant sites were conserved.Three bioinformatics software predicted both the missense variants were pathogenic.Protein modelling analysis showed that the number of hydrogen bonds in p.γGly377-Gly388 variant region was altered,resulting in steric hinderance.Conclusion All the two types of compound heterozygous variants,i.e.,c.1129+62_65delAATA and p.AαArg35Ser,c.1129+62_65delAATA and p.BβAsn190Ser,have been reported for the first time in Chi-na and worldwide to date,and the three variants may be related to the reduced Fg level and function in the two pedigree.

Objective To investigate the effect of miR-486-5P on ferroptosis in H9c2 cardiomyocytes after hypoxia/reoxygenation(H/R),and to analyze its mechanism.Methods Using H9c2 cardiomyocytes as the research object,a H/R injury model was established using cobalt chloride(CoCl2)and fresh culture medium.The cells were divided into control group,H/R group,H/R+miR-486-5P mimic NC group,H/R+miR-486-5P mimic group,H/R+miR-486-5P inhibitor NC group and H/R+miR-486-5P inhibitor group.The relative expression level of miR-486-5P was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).The cell viability was detected by CCK-8 method.The activities or levels of lactate dehydrogen-ase(LDH),glutathione(GSH),Fe2+and malondialdehyde(MDA)were detected by colorimetric method.The levels of reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were detected by DCFH-DA fluorescent probe and JC-1 assay,respectively.Western blot was used to detect the levels of AkT/mTOR signaling pathway proteins and ferroptosis related protein solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4)and acyl-coa synthetase long chain family member 4(ACSL4).Results Compared with the control group,the level of miR-486-5P and cell viability in the H/R group de-creased significantly(P＜0.05),while LDH activity,MDA,Fe2+level,ROS level and ACSL4 protein level in-creased significantly(P＜0.05).The GSH,MMP,SLC7A11 and GPX4 levels and p-Akt/Akt and p-mTOR/mTOR ratios were significantly decreased(P＜0.05).After H/R treatment,compared with the H/R+miR-486-5P mimic NC group,the cell viability of the H/R+miR-486-5P mimic group was significantly increased(P＜0.05).The LDH activity,MDA,Fe2+level,ROS level and ACSL4 protein level were significantly de-creased(P＜0.05),while GSH,MMP,SLC7A11 and GPX4 levels and p-Akt/Akt and p-mTOR/mTOR ratios were significantly increased(P＜0.05).Compared with the H/R+miR-486-5P inhibitor NC group,the trend of the above indicators in the H/R+miR-486-5P inhibitor group was opposite.Conclusion miR-486-5P allevi-ates hypoxia/reoxygenation-induced ferroptosis in H9c2 cells by regulating Akt/mTOR signaling pathway,and thus alleviates hypoxia/reoxygenation induced cardiomyocyte injury.

Objective To analyse phenotype and genetic variation of two congenital hypodysfibrinogenemia(Fg)caused by compound heterozygous variants and preliminary investigate their molecular pathogenic mechanisms.Metheds The proband A and B and their family members(a total of 19 members in 3 generations)who visited the First Hospital of Wenzhou Medical University on 4 May 2023 and 20 May 2023 for"parkinson's disease"and"pre-bilateral eyelid excision"were enrolled for the study.Prothrombin time(TT)and fibrinogen(Fg)activity were measured by coagulation assay and Fg antigen(Fg∶Ag)was measured by immunoturbidimetric assay for the two family members,and Fg aggregation assay was catalysed using human thrombin.FGG gene was amplified by PCR and se-quenced directly.The variant sites were analysed using Chromas software.Multiple sequence comparison was performed by ClustalX-2.1-win software.Pathogenicity analysis of the variant sites was performed using bioinformatics software.The analysis for FGG protein model was performed using PyMOL software.Results Phenotypic results showed TT of proband A and B extended to 27.5 s and 26.1 s,and plasma Fg activity reduced to 0.6 g/L and＜0.5 g/L,respectively.Genetic sequencing identified heterozygous c.1129+62_65delAATA on intron 8 of FGG gene in the both probands,resulting in the formation of aberrant amino acids at p.γGly377-Gly388 and an early ter-mination codon at p.γTyr389 site.A heterozygous missense variant c.103C＞A(p.AαArg35Ser)was found in exon 2 of the FGA gene of proband A,and a heterozygous missense variant c.569A＞G(p.BβAsn190Ser)was found in exon 4 of the FGB gene of proband B.Compared to the control group,the both probands showed significant decreases in peak and rate of Fg aggregation.Multiple sequence comparison analyses showed that all the three variant sites were conserved.Three bioinformatics software predicted both the missense variants were pathogenic.Protein modelling analysis showed that the number of hydrogen bonds in p.γGly377-Gly388 variant region was altered,resulting in steric hinderance.Conclusion All the two types of compound heterozygous variants,i.e.,c.1129+62_65delAATA and p.AαArg35Ser,c.1129+62_65delAATA and p.BβAsn190Ser,have been reported for the first time in Chi-na and worldwide to date,and the three variants may be related to the reduced Fg level and function in the two pedigree.

Objective:To analyze the recovery of olfactory function in patients diagnosed with chronic rhinosinusitis with nasal polyps (CRSwNP) after endoscopic sinus surgery and to identify factors influencing recovery to provide a theoretical foundation for taking effective measures.Methods:This was a retrospective analysis included 277 CRSwNP patients with olfactory dysfunction who underwent endoscopic sinus surgery at the First Affiliated Hospital of Chongqing Medical University from October 2021 to September 2023. This study included 189 males and 88 females, with a median age of 46 years (range: 18-84 years). Routine laboratory tests, sinus CT Lund-Mackay score, modified sinus CT score, endoscopic polyp score, and nasal endoscopic Lund-Kennedy score were included for preoperative assessments. Eosinophil counts were obtained from nasal polyp tissues during surgery. The University of Pennsylvania smell identification test (UPSIT) was administered before surgery and 6 months after surgery to evaluate olfactory function. Based on results of postoperative olfactory test, patients were divided into two groups: the group with improved olfactory function and without improvement of olfactory function. Chi-square test and multivariate Logistic regression were used to analyze relevant factors affecting postoperative olfactory function of CRSwNP patients, and the prediction model was constructed to verify its consistency and to analyze its prediction efficiency.Results:Of the 277 patients, 155 (56%) showed improved olfactory function and 122 (44%) did not improve after surgery. Multivariate Logistic regression analysis identified following independent factors associated with improved postoperative olfactory function: concurrent allergic rhinitis ( OR=2.34), long duration of olfactory dysfunction ( OR=1.13), higher total score of CT olfactory zone ( OR=1.26), higher Lund-Kennedy score ( OR=1.23), presence of olfactory cleft polyps ( OR=4.72), higher tissue eosinophil count ( OR=1.01) and high IL-6 levels ( OR=1.51). Conversely, a higher endoscopic polyp score ( OR=0.74) was associated with a lower likelihood of olfactory improvement. The nomogram model, validated using receiver operating characteristic (ROC) analysis, demonstrated good clinical efficacy with an area under the curve (AUC) of 0.80 (95%CI: 0.748-0.852). The calibration curve showed an absolute error of 0.021, indicating good consistency and predictive accuracy. Conclusions:Factors such as a medical history of allergic rhinitis, duration of olfactory dysfunction, total score of sinus CT olfactory zone, endoscopic Lund-Kennedy score, olfactory cleft polyps, tissue eosinophil count, IL-6 level and endoscopic polyp score independently influence postoperative olfactory function of CRSwNP patients. The nomogram model based on these independent factors has good clinical efficacy, which can be used to predict the postoperative olfactory function in CRSwNP patients with olfactory dysfunction.