OBJECTIVE: To investigate the molecular mechanism for a family with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. METHODS: The proband, a 63-year-old female, was admitted to the First Affiliated Hospital of Wenzhou Medical University in August 2024 for lumbar disc herniation. Coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), and FⅫ activity (FⅫ:C), were carried out for the proband and her family members (9 individuals from three generations) using a one-stage clotting assay. The level of FⅫ antigen (FⅫ:Ag) was determined with an Enzyme-linked immunosorbent assay (ELISA). Sanger sequencing was conducted to identify potential variants in the F12 gene. Multiple in silico tools were used to predict the conservation, hydrophobicity, and structural impact of the identified variants. Recombinant expression plasmids were constructed and transiently transfected into HEK293T cells. The recombinant FⅫ protein was analyzed using Western blotting (WB) and ELISA. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: The proband showed a markedly prolonged APTT (160.3 s) and significantly decreased FⅫ:C (2%) and FⅫ:Ag (5%) levels. Analysis of the F12 gene sequence revealed a 46C/T genotype in the promoter region, a heterozygous c.1457G>A (p.Cys467Tyr) missense variant in exon 12, and a heterozygous c.1561G>A (p.Glu502Lys) missense variant in exon 13. Bioinformatic analysis showed that the p.Cys467 is highly conserved across various species, and the p.Cys467Tyr variant may affect local structural stability of the FⅫ protein. The p.Cys467Tyr variant had no effect on the transcription of the F12 gene. However, the variant has significantly decreased the FⅫ:Ag levels and FⅫ protein expression in the cell culture supernatant compared to the wild-type expression vector, while in the cell lysate, it is higher than the wild-type expression vector. In other words, the p.Cys467Tyr variant has probably caused a secretion defect of FⅫ protein. CONCLUSION The 46C/T genotype, the heterozygous p.Cys467Tyr missense variant, and the heterozygous p.Glu502Lys missense variant are associated with reduced plasma FⅫ levels in this pedigree. The p.Cys467Tyr variant, which was unreported previously, did not affect the synthesis of FⅫ but may have resulted in a secretion defect.
Humans ; Female ; Middle Aged ; Mutation, Missense ; Pedigree ; HEK293 Cells ; Male ; Factor XII/genetics* ; Adult ; Factor XII Deficiency/genetics*

Objective To explore the effect of salidroside(Sal)on endoplasmic reticulum stress and connexin 43 in rats with myocardial ischemia-reperfusion injury(MIRI).Methods SD rats were randomly divided into Sham group,MIRI group,low-does Sal(Sal-L)group and high-does Sal(Sal-H)group.The Sham group and MIRI group were intraperitoneal injec-ted with 0.9％sodium chloride solution(10 mL·kg-1·d-1),the Sal-L group and Sal-H group were intraperitoneal injected at a volume of 10 mL·kg-1 with Sal(12,36 mg·kg-1·d-1),respectively.Each group was given a corresponding intervention once a day for 3 d.The MIRI model was established 30 min after the last administration in all groups except the Sham group.The patho-logical changes of myocardial tissue were observed by Hematoxylin-eosin(HE)staining.TdT-mediated-dUTP nick end labeling(TUNEL)was used to observe the apoptosis of cardiomyocyte,the genes and proteins expression of Cx43 and endoplasmic reticu-lum stress related factors such as GRP78,Caspasel2,CHOP and so on were detected by quantitative real-time polymerase chain reaction(q-PCR)and western blot analysis.Results Compared with the MIRI group,the degree of tissue and cell injury in each Sal group was alleviated,with a decreased apoptosis rate observed in the Sal-H group(P＜0.05),the gene expression of Cx43 was up-regulated while GRP78,Caspase12,and CHOP gene expressions were down-regulated in both does groups of Sal.The protein expressions of Cx43 and GRP78 were also be up-regulated and down-regulated respectively in both dose groups of Sal,meanwhile the protein expressions of CHOP,Bax,Caspasel2 and cleaved-Caspase3 were down-regulated and the protein expres-sion of Bcl-2 was up-regulated in SAL-H group(P＜0.05).Conclusion The protective effect of salidroside on cardiomyocytes may be related to the inhibition of endoplasmic reticulum stress-induced apoptosis and the imbalance of Cx43 metabolism.

Two novel skeleton sesquiterpenoids (1 and 6), along with four new iphionane-type sesquiterpenes (2-5) and six new cyperane-type sesquiterpenes (7-11), were isolated from the whole plant of Artemisia hedinii (A. hedinii). The two novel skeleton compounds (1 and 6) were derived from the decarbonization of iphionane and cyperane-type sesquiterpenes, respectively. Their structures were elucidated through a comprehensive analysis of spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and 1D and 2D nuclear magnetic resonance (NMR) spectra. The absolute configurations were determined using electronic circular dichroism (ECD) spectra, single-crystal X-ray crystallographic analyses, time-dependent density functional theory (TDDFT) ECD calculation, density functional theory (DFT) NMR calculations, and biomimetic syntheses. The biomimetic syntheses of the two novel skeletons (1 and 6) were inspired by potential biogenetic pathways, utilizing a predominant eudesmane-type sesquiterpene (A) in A. hedinii as the substrate. All compounds were evaluated in LX-2 cells for their anti-hepatic fibrosis activity. Compounds 2, 8, and 10 exhibited significant activity in downregulating the expression of α-smooth muscle actin (α-SMA), a protein involved in hepatic fibrosis.
Artemisia/chemistry* ; Sesquiterpenes/chemical synthesis* ; Molecular Structure ; Humans ; Liver Cirrhosis/genetics* ; Biomimetics ; Plant Extracts/pharmacology*

Objective To explore the effect of salidroside(Sal)on endoplasmic reticulum stress and connexin 43 in rats with myocardial ischemia-reperfusion injury(MIRI).Methods SD rats were randomly divided into Sham group,MIRI group,low-does Sal(Sal-L)group and high-does Sal(Sal-H)group.The Sham group and MIRI group were intraperitoneal injec-ted with 0.9％sodium chloride solution(10 mL·kg-1·d-1),the Sal-L group and Sal-H group were intraperitoneal injected at a volume of 10 mL·kg-1 with Sal(12,36 mg·kg-1·d-1),respectively.Each group was given a corresponding intervention once a day for 3 d.The MIRI model was established 30 min after the last administration in all groups except the Sham group.The patho-logical changes of myocardial tissue were observed by Hematoxylin-eosin(HE)staining.TdT-mediated-dUTP nick end labeling(TUNEL)was used to observe the apoptosis of cardiomyocyte,the genes and proteins expression of Cx43 and endoplasmic reticu-lum stress related factors such as GRP78,Caspasel2,CHOP and so on were detected by quantitative real-time polymerase chain reaction(q-PCR)and western blot analysis.Results Compared with the MIRI group,the degree of tissue and cell injury in each Sal group was alleviated,with a decreased apoptosis rate observed in the Sal-H group(P＜0.05),the gene expression of Cx43 was up-regulated while GRP78,Caspase12,and CHOP gene expressions were down-regulated in both does groups of Sal.The protein expressions of Cx43 and GRP78 were also be up-regulated and down-regulated respectively in both dose groups of Sal,meanwhile the protein expressions of CHOP,Bax,Caspasel2 and cleaved-Caspase3 were down-regulated and the protein expres-sion of Bcl-2 was up-regulated in SAL-H group(P＜0.05).Conclusion The protective effect of salidroside on cardiomyocytes may be related to the inhibition of endoplasmic reticulum stress-induced apoptosis and the imbalance of Cx43 metabolism.

Objective:To investigate the potential mechanism of miR-1298-5p in non-small cell lung cancer(NSCLC)to regu-late the MSH2 gene and its effect on the biological behavior of tumor cells and the tumor immune microenvironment.Methods:Bioin-formatics was used to identify the key genes and miRNA involved in NSCLC.Cell proliferation was detected by CCK-8 assay,and cell invasion and migration were detected by Transwell assay.Levels of inflammatory factors were detected by ELISA assay.Western blot was used to measure the expression of MSH2 in cells,and fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the expressions of miR-1298-5p and MSH2 gene in NSCLC cells.Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-1298-5p and MSH2.Spearman correlation analysis was performed to correlate miR-1298-5p with immune cells and immune factors in the tumor immune microenvironment.Results:The level of miR-1298-5p was down-regulated in NSCLC cells compared with normal lung tissue cells.miR-1298-5p overexpression inhibited the proliferation,migration and inva-sion of NSCLC cells.MSH2 was confirmed to be a target gene of miR-1298-5p using luciferase reporter gene assay.Furthermore,down-regulation of miR-1298-5p in NSCLC cells could be reversed by silencing MSH2.miR-1298-5p expression levels were negatively corre-lated with the levels of Treg,IL-10,and TGF-β,and positively correlated with the levels of CD3+T,CD4+T,CD8+T,NK cells,IL-2,and IFN-γ.Conclusion:miR-1298-5p negatively regulates MSH2 to inhibit the proliferation,invasion and migration of NSCLC cells and improve the tumor immune microenvironment.

Acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had caused a global pandemic since 2019, and posed a serious threat to global health security. Traditional Chinese medicine (TCM) has played an indispensable role in the battle against the epidemic. Many components originated from TCMs were found to inhibit the production of SARS-CoV-2 3C-like protease (3CLpro) and papain-like protease (PLpro), which are two promising therapeutic targets to inhibit SARS-CoV-2. This study describes a systematic investigation of the roots and rhizomes of Sophora tonkinensis, which results in the characterization of 12 new flavonoids, including seven prenylated flavanones (1-7), one prenylated flavonol (8), two prenylated chalcones (9-10), one isoflavanone (11), and one isoflavan dimer (12), together with 43 known compounds (13-55). Their structures including the absolute configurations were elucidated by comprehensive analysis of MS, 1D and 2D NMR data, and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC₅₀ values of 34.89 and 19.88 μmol·L^-1, repectively while compounds 9, 43 and 47 exhibited inhibitory effects against PLpro with IC₅₀ values of 32.67, 79.38, and 16.74 μmol·L^-1, respectively.
Flavonoids/chemistry* ; SARS-CoV-2 ; Rhizome ; COVID-19 ; Peptide Hydrolases ; Antiviral Agents/chemistry*

Objective To analyze the phenotype and genotype of inherited dysfibrinogenemia pedigree associated with a novel heterozygous and deletion mutation in the FGG gene,and to investigate its molecular mechanism.Methods The clinical data were collected from the proband found at our hospital and her family members in April 2016.The activity plasma fibrinogen(Fg:C), activated partial thromboplastin time(APTT),prothrombin time(PT), thrombin time(TT)were detected by coagulation method and the antigen plasma fibrinogen(Fg:Ag), D-Dimer(D-D), fibrinogen degradation products (FDPs)were analyzed by immunoturbidimetry method.All of the exons and exon-intron boundaries of the genes of FGA, FGB and FGG with the fibrinogen(Fg)were amplified by PCR and followed by direct sequencing.And further verification were performed by cloning sequence and non-denatured polyacrylamide gel electrophoresis and silver staining.The conservatism of mutated gene locus were analyzed by ClustalX-2.1-win.The change of the protein spatial structure and the intermolecular forces with mutation were analyzed by Pymol.Results The Fg:C of the proband was significantly reduced(0.30 g/L)and the Fg:Ag of the proband was normal(2.00 g/L).Their Fg:C were both significantly reduced and the Fg:Ag were both normal(0.42 g/L,2.09 g/L & 0.47 g/L,2.42 g/L, respectively), these were found in her mother and grandma.Genetic analysis revealed a novel heterozygous and deletion mutation with c.944 _c.952 delCCTTTGATG in exon 8 of FGG gene in the proband,predicting a heterozygous 289_291delAla,Phe,Asp mutation.The same mutations were carried by her mother and grandma, but her father and grandpa were normal.Homology analysis indicated that the Ala 289,Phe290 and Asp291 were maintained highly conservative in homogenous species.Protein model analysis found that the original hydrogen bonds were disappeared when the deletion mutation happened with the Ala 289,Phe290 and Asp291.Conclusion The heterozygous and deletion mutation with 289_291delAla,Phe,Asp in the γchain of fibrinogen were identified that could cause the rearrangement of the Fg molecular space structure and the reduction of the structure stability,so the mutation probably underly the dysfibrinogenemia in this pedigree.(Chin J Lab Med,2018, 41:305-311)

BACKGROUND: Both proximal femoral nail and hemiarthroplasty have been applied in the treatment of unstable intertrochanteric fractures in osteoporotic patients. However, which has better curative efficacy is still under discussion.OBJECTIVE: To investigate the clinical effectiveness of proximal femoral nail versus hemiarthroplasty in the treatment of unstable intertrochanteric fracture in osteoporotic patients.METHODS: Sixty-seven patients diagnosised with unstable intertrochanteric fractures and osteoporosis were selected,and were randomly divided into two groups, followed by treated with hemiarthroplasty (group 1, n=35)) and proximal femoral nail (group 2, n=32), respectively. The operation time, intraoperative blood loss, length of incision, postoperative time in bed, hospitalization time and complications were recorded and compared between two groups. Harris hip scores were used to evaluate the preoperative and postoperative hip function.RESULTS AND CONCLUSION: (1) In the group 1, the operation time, intraoperative blood loss and length of incision were more than those in the group 1, and the postoperative time in bed was less than that in the group 2 (P < 0.05).However, the healing and hospitalization time did not differ significantly between two groups (P > 0.05). (2) No significant difference was observed in the excellent and good rate in Harris hip scores between two groups at the last follow-up (P >0.05). The postoperative pain scores in the group 1 were significantly superior to those in the group 2 (P < 0.05). (3)There were 1 case of delayed union and 1 case of lateral hip appearing with pain in the group 2. At the last follow-up, all patients healed completely, and no prosthesis dislocation, loosening or infection was found. (4) To condude,hemiarthroplasty provides early functional recovery and fewer complications, but with much surgical trauma. Proximal femoral nail shows similar treatment outcomes with hemiarthroplasty; therefore, it is a kind of alternative treatment strategy for unstable intertrochanteric fractures.

BACKGROUND:Core decompression and tantalum rod implantation after core decompression are common methods to repair early and middle stages of necrosis of femoral head, can effectively control and even reverse the progress of necrosis of the femoral head. Comparison of mechanical support and curative effect of femoral head after operation deserves further investigation. OBJECTIVE:To explore the effect of core decompression on mechanical pulp femoral head support by using the finite element analysis and the advantages of tantalum implant treatment in the repair of avascular necrosis of the femoral head. METHODS: The right femur of healthy adults was chosen as the research object, and CT scanning was conducted to get the images of cross-sections. The images were then inputted into computer to get contour of femur and rebuild three-dimensional model. Distal end of femur was completely fixed, the angle of the top of femoral head and the femoral shaft was 25°, and 570 N pressure on the femoral head was applied according to the three-dimensional space distribution of femur force under physiological state. Three-dimensional finite element models were calculated to get the colapse values in different necrotic areas of the femoral head before and after different repair methods. RESULTS AND CONCLUSION:After core decompression, colapse values were apparently increased, especialy in the weight-bearing area. With increased range of necrosis, colapse values also increased. After core decompression, colapse values decreased obviously after porous tantalum rod implantation. Although core decompression could remove dead bone, decompression itself further reduced the mechanical properties of the femoral head and changed the original femoral head support. On the basis of core decompression, porous tantalum rod provided safe and effective mechanical support for femoral head and subchondral bone plate, could effectively prevent colapse and provide conditions for the restoration of bone tissue.

OBJECTIVETo identify potential mutations and explore the molecular mechanism underlying combined inherited coagulation factors VII(FVII) and X(FX) deficiency for a family featuring consanguineous marriage between maternal cousins.

METHODSProthrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FVII activity (FVII:C), FX activity (FX:C), FVII antigen (FVII:Ag), FX antigen (FX:Ag) and other coagulant parameters of the proband and 5 family members were measured. Potential mutations in exons, exon-intron boundaries and 5', 3' untranslated sequences of F7 and F10 genes were screened by polymerase chain reaction and direct sequencing. Suspected mutations were confirmed by sequencing the opposite strand.

RESULTSPT and APTT of the proband were obviously prolonged to become 76.4 s and 60.2 s, respectively. FVII:C, FVII:Ag,FX:C and FX:Ag of the proband were obviously reduced to become 4%, 6%, 6% and 33%, respectively. Both PT and APTT of her grandmother, father, mother and daughter were slightly prolonged, which have measured 16.4 s, 15.8 s,16.9 s, 16.5 s, and 44.0 s, 42.1 s, 41.1 s, 43.5 s, respectively. And their FVII:C (34%, 39%, 31%, 40%, respectively), FX:C (50%, 58%, 47%, 42%, respectively) and FX:Ag (51%, 54%, 58%, 47%, respectively) were slightly reduced, while FVII:Ag was in the normal range. The coagulant parameters of her younger brother were within normal range. Two homozygous mutations, g.11267C to T in exon 8 of F7 gene, which resulted in an Arg277Cys substitution, and g.28139G to T in exon 8 of F10 gene which led to a Val384Phe substitution, were identified in the proband. The proband's grandmother, parents and daughter were heterozygous for both Arg277Cys and Val384Phe mutationss. Wild-type alleles of both F7 and F10 genes were also found in the younger brother.

CONCLUSIONA homozygous Arg277Cys mutation and a Val384Phe mutation have been respectively identified in the F7 and F10 genes, which can explain the low levels of FVII and FX in this family. The former has been inherited from the consanguineous parents.

Adult ; Aged ; Consanguinity ; Factor VII Deficiency ; genetics ; Factor X Deficiency ; genetics ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Phenotype