Objective:To investigate the molecular pathogenic mechanism of venous thrombosis caused by heterozygous missense mutations in two protein C (PROC) genes through laboratory phenotype analysis, genetic mutation analysis, and in vitro expression experiments.Methods:Two probands presented with venous thromboembolism at the First Affiliated Hospital of Wenzhou Medical University. Clinical data and blood samples were collected from the probands and their family members to evaluate the plasma protein C (PC) activity (PC∶A), PC antigen (PC∶Ag) levels, and other relevant coagulation parameters. The anticoagulant capacity was assessed using the thrombin generation test (TGT). The mutation sites of the PROC gene were identified using direct DNA sequencing. Bioinformatics software was used to analyze the conservation and pathogenicity of the mutated gene. PyMOL software was used for the analysis of the protein three-dimensional models and interactions between mutated amino acids. Wild-type and two mutant expression vectors were constructed and HEK293T cells were transiently transfected. Total cellular RNA was extracted from positively transfected cells to investigate the transcriptional levels of the mutant PROC gene. Enzyme-linked immunosorbent assay, Western blot, and cellular immunofluorescence assays were used to investigate the translation levels of the mutant PROC protein.Results:Probands 1 and 2 exhibited PC∶A levels of 35% and 40% and PC∶Ag levels of 44% and 39%, with increasing D-dimer levels to 4.42 mg/L and 0.83 mg/L, respectively. Meanwhile, other coagulation parameters revealed no significant abnormalities. TGT demonstrated impaired anticoagulant function in both proband witnesses and their familial PC carriers. Sequencing analysis revealed heterozygous missense mutations c. 833T>C (p. Leu278Pro) in proband 1 and c. 1330T>C (p. Trp444Arg) in proband 2 within exon 9 of the PROC gene. Conservation analysis revealed that Leu278 and Trp444 were highly conserved across homologous species. Pathogenicity analysis indicated that both p. Leu278Pro and p. Trp444Arg mutations are deleterious. Protein modeling analysis demonstrated that both mutations induce structural alterations in the protein. In vitro expression experiments revealed that compared with the wild-type, both p. Leu278Pro and p. Trp444Arg mutations showed no significant differences in the mRNA expression level of the PC protein. However, both mutations caused significantly lower PC∶Ag content and protein expression levels in the cell culture supernatant compared with the wild-type, whereas higher levels were observed in the cell culture lysate. This indicates the association of both mutations with the secretion function of the PC protein.Conclusion:The heterozygous missense mutations p. Leu278Pro and p. Trp444Arg in exon 9 of the PROC gene in both probands are associated with decreased PC levels.

Objective:To investigate the efficacy and safety of dupilumab in the treatment of elderly patients (≥ 60 years old) with atopic dermatitis (AD), with particular attention paid to rare adverse reactions.Methods:A single-center retrospective analysis was conducted on data collected from 281 elderly AD patients who received the standard regimen of dupilumab at the Department of Dermatology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from January 2020 to May 2024. Clinical characteristics such as gender, disease duration, skin lesion manifestations, and itch severity were analyzed. Changes in skin lesions and itch severity, as well as related adverse events were recorded during the follow-up period of 0 - 16 weeks. The efficacy and safety of dupilumab in the treatment of elderly AD patients were evaluated.Results:Among the 281 elderly AD patients, 214 were males (76.16%) and 67 were females (23.84%), with the age being 71.13 ± 7.91 years and the age at onset being 59.92 ± 15.72 years. The disease duration ( M[ IQR]) was 5.00 (13.00) years. After standard-regimen dupilumab treatment, the improvement rates of clinical outcome indicators SCORing Atopic Dermatitis (SCORAD) and pruritus numerical rating scale (NRS) scores gradually increased. At week 16, the improvement rates of SCORAD and NRS scores ( M[ IQR]) reached the maxima of 72.37% (23.89%) and 75.00% (29.72%), respectively. The overall incidence of adverse events was relatively low, with only 16 patients (6.05%) reporting adverse events. Common adverse reactions such as conjunctivitis (2 cases, 0.71%) and facial erythema (1 case, 0.36%) were mild and well-tolerated. Phenotype switching occurred in 10 cases (3.56%) . Conclusion:Dupilumab was an effective and safe treatment for elderly AD, but phenotype switching may occur.

Objective To investigate the clinical efficacy of the combined therapy of promoting blood circulation and removing blood stasis and anisodamine hydrobromide in the treatment of sepsis-induced coagulopathy(SIC).Methods A total of 102 SIC patients treated in our hospital from Sep-tember 2021 to September 2024 were selected as study subjects.They were divided into control group(n=51)and experimental group(n=51)according to different treatment methods.The control group received conventional treatment,while the experimental group received an additional combined therapy of promoting blood circulation and removing blood stasis and anisodamine hydrobromide on the basis of the control group.Coagulation function and thrombotic risk were assessed in both groups before treatment and at 24,48,and 72 h after treatment.Clinical efficacy and treatment safety were com-pared,and lactate clearance rate was measured before treatment and at 2,6,and 24 h after treat-ment.Results After treatment,the levels of thrombin time(TT),prothrombin time(PT),acti-vated partial thromboplastin time(APTT),and D-dimer(D-D)decreased,while the platelet(PLT)count and fibrinogen(FIB)levels increased compared with treatment before,with all differ-ences being statistically significant(P＜0.05).At 24 hours post-treatment,the TT,PT,and D-D levels in the experimental group were lower than those in the control group(P＜0.05),whereas no significant differences were observed in the remaining indicators(P＞0.05).At 48 hours post-treat-ment,the TT,PT,APTT,and D-D levels in the experimental group were lower than those in the control group,while the PLT level was higher(P＜0.05).At 72 hours post-treatment,the TT,PT,and APTT levels in the experimental group were lower than those in the control group,whereas the PLT and FIB levels were higher(P＜0.05).The levels of the four new thrombotic indicators,namely thrombomodulin(TM),thrombin-antithrombin complex(TAT),plasmin-α2-antiplasmin complex(PIC),and tissue plasminogen activator-plasminogen activator inhibitor complex(t-PAIC),decreased with prolonged treatment duration in both groups,with differences in different time points being statistically significant(P＜0.05).At 24 hours post-treatment,the TM and TAT levels in the experimental group were lower than those in the control group,with both differences being statistical-ly significant(P＜0.05).At 48 hours post-treatment,the TM,PIC,and t-PAIC levels in the ex-perimental group were lower than those in the control group(P＜0.05).At 72 hours post-treat-ment,the levels of four indicators thrombosis in the experimental group were lower than those in the control group,but only the differences in TM and PIC levels were statistically significant(P＜0.05).The thromboelastography indicators,including R value,K value,and maximum amplitude(MA)decreased,while α angle increased with prolonged treatment duration in both groups(P＜0.05).At 24 and 48 hours post-treatment,the R value in the experimental group was lower than that in the control group,with both differences being statistically significant(P＜0.05).At 72 hours post-treatment,the R value,and MA in the experimental group were lower than those in the control group(P＜0.05).The Sequential Organ Failure Assessment(SOFA)score,SIC score,and Acute Physiology and Chronic Health Evaluation Ⅱ score decreased post-treatment in both groups compared with pretreatment levels,and were lower in the experimental group than in the con-trol group(P＜0.05).The lactate clearance rate increased with prolonged treatment duration in both groups(P＜0.05).At 2,6,and 24 hours post-treatment,the lactate clearance rate in the ex-perimental group was higher than that in the control group(P＜0.05).The 28-day survival rate was 100.00％in the experimental group,which was higher than 92.16％in the control group(P＜0.05).Conclusion The combined therapy of promoting blood circulation and removing blood sta-sis and anisodamine hydrobromide has good clinical efficacy in the treatment of SIC.It can improve patients' coagulation function,reduce thrombotic risk,and has high treatment safety.

Objective To explore the the detection of MMP-2,-7,-9,and-12 enzymatic activity using the CEACAM1-derived fluorescent peptide substrate Site 84,investigating the application of substrate Site 84 to distinguishing between MMP-2 and MMP-9 in the gelatinase spectrum of MMPs.Methods The fluorescent enzymatic method was employed to observe the detection of MMP-2,-7,-9,and-12 enzymatic activity using substrate Site 84;further observations were made on the sensitivity and specificity of substrate Site 84 to enzymatic activity of MMP-2 and MMP-9 within the gelatinase spectrum;the kinetic parameters(Km and Kcat)of the enzymatic reaction between substrate Site 84 and MMP-2 were obtained.Results Using Site 84 as a substrate,enzymic kinetics curves for MMP-12,-7,-2 were obtained,but no enzymatic activity curve for MMP-9 was observed.Furthermore,Site 84 specifically detected the enzymatic activity of MMP-2 within the gelatinase spectrum,capable of detecting low concentration(0.6 μM)of MMP-2 enzymatic activity,but no obvious enzymatic reaction was observed for high concentration(6 μM)of MMP-9;the kinetics parameters for the enzymatic reaction between Site 84 and MMP-2 were Km=315 μM,Kcat/Km=2 565/MS.Conclusion The CEACAM1-derived substrate Site 84 serves as a novel fluorescent peptide substrate,enabling the acquisition of enzymatic activity curves for MMP-12,-7 and-2,and specifically detecting the enzymatic activity of MMP-2 within the MMP gelatinase spectrum.

Objective:To investigate the efficacy and safety of dupilumab in the treatment of elderly patients (≥ 60 years old) with atopic dermatitis (AD), with particular attention paid to rare adverse reactions.Methods:A single-center retrospective analysis was conducted on data collected from 281 elderly AD patients who received the standard regimen of dupilumab at the Department of Dermatology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from January 2020 to May 2024. Clinical characteristics such as gender, disease duration, skin lesion manifestations, and itch severity were analyzed. Changes in skin lesions and itch severity, as well as related adverse events were recorded during the follow-up period of 0 - 16 weeks. The efficacy and safety of dupilumab in the treatment of elderly AD patients were evaluated.Results:Among the 281 elderly AD patients, 214 were males (76.16%) and 67 were females (23.84%), with the age being 71.13 ± 7.91 years and the age at onset being 59.92 ± 15.72 years. The disease duration ( M[ IQR]) was 5.00 (13.00) years. After standard-regimen dupilumab treatment, the improvement rates of clinical outcome indicators SCORing Atopic Dermatitis (SCORAD) and pruritus numerical rating scale (NRS) scores gradually increased. At week 16, the improvement rates of SCORAD and NRS scores ( M[ IQR]) reached the maxima of 72.37% (23.89%) and 75.00% (29.72%), respectively. The overall incidence of adverse events was relatively low, with only 16 patients (6.05%) reporting adverse events. Common adverse reactions such as conjunctivitis (2 cases, 0.71%) and facial erythema (1 case, 0.36%) were mild and well-tolerated. Phenotype switching occurred in 10 cases (3.56%) . Conclusion:Dupilumab was an effective and safe treatment for elderly AD, but phenotype switching may occur.

Objective:To investigate the molecular pathogenic mechanism of venous thrombosis caused by heterozygous missense mutations in two protein C (PROC) genes through laboratory phenotype analysis, genetic mutation analysis, and in vitro expression experiments.Methods:Two probands presented with venous thromboembolism at the First Affiliated Hospital of Wenzhou Medical University. Clinical data and blood samples were collected from the probands and their family members to evaluate the plasma protein C (PC) activity (PC∶A), PC antigen (PC∶Ag) levels, and other relevant coagulation parameters. The anticoagulant capacity was assessed using the thrombin generation test (TGT). The mutation sites of the PROC gene were identified using direct DNA sequencing. Bioinformatics software was used to analyze the conservation and pathogenicity of the mutated gene. PyMOL software was used for the analysis of the protein three-dimensional models and interactions between mutated amino acids. Wild-type and two mutant expression vectors were constructed and HEK293T cells were transiently transfected. Total cellular RNA was extracted from positively transfected cells to investigate the transcriptional levels of the mutant PROC gene. Enzyme-linked immunosorbent assay, Western blot, and cellular immunofluorescence assays were used to investigate the translation levels of the mutant PROC protein.Results:Probands 1 and 2 exhibited PC∶A levels of 35% and 40% and PC∶Ag levels of 44% and 39%, with increasing D-dimer levels to 4.42 mg/L and 0.83 mg/L, respectively. Meanwhile, other coagulation parameters revealed no significant abnormalities. TGT demonstrated impaired anticoagulant function in both proband witnesses and their familial PC carriers. Sequencing analysis revealed heterozygous missense mutations c. 833T>C (p. Leu278Pro) in proband 1 and c. 1330T>C (p. Trp444Arg) in proband 2 within exon 9 of the PROC gene. Conservation analysis revealed that Leu278 and Trp444 were highly conserved across homologous species. Pathogenicity analysis indicated that both p. Leu278Pro and p. Trp444Arg mutations are deleterious. Protein modeling analysis demonstrated that both mutations induce structural alterations in the protein. In vitro expression experiments revealed that compared with the wild-type, both p. Leu278Pro and p. Trp444Arg mutations showed no significant differences in the mRNA expression level of the PC protein. However, both mutations caused significantly lower PC∶Ag content and protein expression levels in the cell culture supernatant compared with the wild-type, whereas higher levels were observed in the cell culture lysate. This indicates the association of both mutations with the secretion function of the PC protein.Conclusion:The heterozygous missense mutations p. Leu278Pro and p. Trp444Arg in exon 9 of the PROC gene in both probands are associated with decreased PC levels.

Objective:To investigate the effectiveness and safety of ultrasound-guided endovascular therapy for autogenous arteriovenous fistula (AVF) thrombosis.Methods:It was a single-center retrospective cohort study. Data of patients undergoing ultrasound-guided intravascular therapy due to AVF thrombosis in the First Hospital of Hebei Medical University from August 2018 to June 2021 were analyzed. According to different surgical procedures, the patients were divided into two groups. Patients treated with percutaneous transluminal angioplasty (PTA) + drilling thrombectomy were in group A, and patients treated with PTA only were in group B. After 1 year of follow-up, the surgical technique success rate, primary patency rate, secondary patency rate and complications were compared between the two groups.Results:A total of 152 patients were enrolled, including 74 in group A and 78 in group B. There were no significant differences in gender, age, proportion of patients with diabetes and hypertension, and thrombosis time of AVF between the two groups (all P>0.05). Compared with group B, the diameter and length of thrombus in group A were larger [13.0(9.0, 16.0) mm vs. 6.0(5.0, 6.5) mm, Z=-9.362, P<0.001; 12(8, 15) cm vs. 3(3, 4) cm, Z=-10.061, P<0.001], and the establishment time of AVF was longer [5(2, 7) years vs. 2(1, 5) years, Z=-2.698, P=0.007]. Among the overall patients, the success rate of surgery was 96.7% (147/152), and the success rate of surgery was 95.9% (71/74) in group A and 97.4% (76/78) in group B respectively, with no statistical difference ( χ2=0.004, P=0.952). Kaplan-Meier survival analysis showed that, overall, the primary patency rate at 3rd, 6th and 12th month after operation was 87.1%, 71.4% and 56.6%, and the secondary patency rate was 97.1%, 96.4% and 94.1%, respectively. The primary patency rate of group A at 3rd, 6th and 12th month was 82.4%, 66.7% and 53.6%, and the secondary patency rate was 95.7%, 94.2% and 89.7%, respectively. The primary patency rate of group B at 3rd, 6th and 12th month was 91.5%, 73.2% and 59.7%, and the secondary patency rate was 98.6%, 98.6% and 98.5%, respectively. There was no significant difference in the primary and secondary patency rate between group A and group B at 3rd, 6th and 12th month (all P>0.05). The duration of operation in group A was longer than that in group B [2.0(1.9, 2.0) h vs. 2.0(1.0, 2.0) h, Z=-5.181, P<0.001], but no serious complications occurred in both groups. Conclusion:The two surgical methods are effective, safe and reliable in the treatment of AVF thrombosis, and have high clinical application value.

Objective:To analyze the types of genetic variants and clinical characteristics of three Chinese pedigrees affected with Hereditary coagulation factor Ⅶ (FⅦ) deficiency.Methods:Three pedigrees who had visited the First Affiliated Hospital of Wenzhou Medical University between December 2021 and October 2022 were selected as the study subjects. Prothrombin time (PT), activated partial thromboplastin time (APTT) and FⅦ activity (FⅦ: C) were measured in the three probands and their pedigree members. All exons and their flanking sequences were analyzed by direct sequencing, and candidate variants were verified by reverse sequencing. The corresponding variant loci in the family members were also analyzed. ClustalX-2.1-win was used to analyze the conservation of the variant loci. Varcards and Spcards online software was used to predict the pathogenicity of the variants. Pymol software was used to analyze the changes in protein structure and molecular forces.Results:Three cases of hereditary FⅦ deficiency were found to have decreased FⅦ: C, prolonged PT and normal APTT. Genetic analysis identified a total of four genetic variants, and all three probands had harbored compound heterozygous variants of the F7 gene, including p. Cys389Gly and p. His408Gln in proband 1, p. Cys389Gly and IVS6+ 1G>T in proband 2, and IVS6+ 1G>T and IVS1a+ 5G>A in proband 3. Conservation analysis showed that both the p. Cys389 and p. His408 loci are highly conserved among orthologous species. Analysis with Varcards and Spcards software showed that these variants were pathogenic. Protein modeling analysis showed that the p. Cys389Gly and p. His408Gln variants may result in altered protein structures and changes in hydrogen bonds. Conclusion:The clinical manifestations of the three FⅦ-deficient probands may be attributed to the compound heterozygous variants of p. Cys389Gly/p.His408Gln, p. Cys389Gly/ⅠⅤS6+ 1G>T and ⅠⅤS6+ 1G>T/ⅠⅤS1a+ 5G>A of the F7 gene. The combination of the three compound heterozygous variants was unreported previously.

Objective To explore the mediating effect of fatigue between social support and depression in hospitalized patients with ischemic stroke,so as to provide reference for improving post-stroke depression.Methods A total of 142 hospitalized patients with ischemic stroke were enrolled and investigation was conducted with self-rating depression scale(SDS),social support rating scale(SSRS),and fatigue severity scale(FSS).Spearman's correlation analysis was used to analyze the correlation between the variables.A model for the mediating effect between the variables was established by using the AMOS 23.0 software to analyze the mediating effect of fatigue between social support and depression.The Bootstrap method was used to test the significance of intermediary effect.Results The scores for SDS,SSRS,and FSS in the hospitalized ischemic stroke patients enrolled were 48.96±9.09,31.34±8.35,and 30.70±13.99,respectively.Spearman's correlation analysis showed that depression was positively correlated with fatigue and negatively correlated with social support.In addition,fatigue was negatively correlated with social support.Analysis of the mediating effect model established that fatigue played a mediating role between social support and depression in patients with ischemic stroke,with the mediating effect value being-0.170 and the mediating effect accounting for 90.0％of the total effect.Conclusion The effect of social support on depression in hospitalized patients with ischemic stroke is mainly achieved by affecting the sense of fatigue.Health workers should pay attention to the severity of fatigue of patients and reduce their sense of fatigue as much as possible,which will help enhance social support for the patients and reduce their depression.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.