Objective To explore the association between the C46T polymorphism of coagulation factor Ⅻ (FⅫ) gene and the involvement of FⅫ activity (FⅫ:C) in patients with unexplained recurrent spontaneous abortion (URSA), and to elucidate its role in the pathogenesis of URSA. Methods This study included 203 patients with URSA (URSA group) and 171 healthy women with at least one child and no history of infertility or miscarriage (control group) in the southern area of Zhejiang Province. The C 46T polymorphism of the FⅫ gene was analyzed with matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) in all subjects. The values of prothrombin time, activated partial thromboplastin time (APTT), fibrinogen, FⅫ:C and other coagulant parameters were determined. The frequency distribution of the wild-type (CC), heterozygote (CT), homozygote (TT) genotypes and C and T alleles were compared between the patients and controls. A comprehensive analysis of association was conducted between C46T genotypes and the FⅫ:C levels in URSA patients. Results The CC, CT, TT genotypes of the FⅫgene were observed in 7 (3.4%, 7/203), 83 (40.9%, 83/203) and 113 (55.7%, 113/203) patients with URSA versus 7 (4.1%, 7/171), 46 (26.9%, 46/171) and 118 (69.0%, 118/171) controls. The frequency of CT in the patients with URSA was significantly higher than that in controls, but the frequency of TT in the patients was lower than that in controls (χ2=7.939, OR=1.884, 95%CI:1.210-2.935, P<0.05). The frequencies of allele C and allele T were observed in 97 (23.9%, 97/406) and 309 (76.1%, 309/406) patients with URSA versus 60 (17.5%, 60/342) and 282 (82.5%, 282/342) controls. The distribution frequency of allele T in URSA group was lower than that in control group (χ2=4.510, OR=1.475, 95%CI:1.029-2.115, P<0.05). The FⅫ:C levels in the patients were (102±13)%in CC genotype, (78±11)%in CT genotype and (59± 9)%in TT genotype, respectively. The differences of the FⅫ:C levels between the CC and CT, CT and TT, CC and TT genotypes in the patients were significant (all P<0.05). Conclusions The low level of FⅫ:C maybe result from the T allele of the FⅫgene in URSA patients. The CT genotype might be relative to the pathogenesis of URSA in a Chinese Han female population from the southern area of Zhejiang province.

OBJECTIVETo observe the changes of the blood and urine mercury (Hg) levels and liver & kidney functions of rabbits after administration of Jiuyi Dan (calcined gypsum-Sheng Dan 9: 1) for 1 month and the recovery of rabbits after the drug withdrawal.

METHODThe rabbits were randomly divided into 2 groups: the calcined gypsum group and the Jiuyi Dan group. After 36 mg of calcined gypsum and 40 mg of Jiuyi Dan were used on the surface of wound (5 cm x 5 cm) on one side of rabbit back for 4 h, the surfaces of wound were washed by saline. The bloods were taken from the rabbit hearts before and after the drug administration for 14 and 28 days, and after the drug withdrawal for 7, 40, 71, and 92 days for determining Hg level in blood, and liver & kidney function indicators (ALT, AST, CREAT and BUN). The Hg level in urine collected from bladders was examined while rabbits were dissected after the drug withdrawal for 1, 40, 71, and 92 days.

RESULTThe Hg level in blood was significantly increased (P < 0.01) after the rabbits were administrated with drugs for 14 and 28 days and after the drug treatment was stopped for 7 and 40 days. The Hg level in urine was significantly enhanced after the drug withdrawal for 1, 40, 71 days. However, the liver & kidney indicators were not influenced.

CONCLUSIONThe Hg level in rabbit blood and urine was significantly increased after the consecutive administration of double-dose Jiuyi Dan for 1 month. However, the blood Hg level and urine Hg level recover after the drug withdrawal for 71 days and 3 months, respectively. The liver & kidney indicators do not significantly change with the dose.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Blood Urea Nitrogen ; Body Weight ; drug effects ; Creatinine ; blood ; Drugs, Chinese Herbal ; administration & dosage ; toxicity ; Female ; Kidney ; drug effects ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Mercury ; blood ; metabolism ; urine ; Rabbits ; Random Allocation ; Skin ; drug effects ; injuries ; Time Factors

Objective:The present study was aimed to evaluate the nutritional risk using nutritional risk screening 2002 (NRS2002) score and to investigate the impact of nutrition support on clinical outcome in hospitalized patients.Methods:Six hundred and ninety four hospitalized patients were recruited.NRS 2002 was applied to evaluate the nutritional risk of patients.Meanwhile,the effect of nutrition support on complication rate was evaluated between different types of patients.Results:14.0％ of patients had malnutrition and the incidence of nutritional risk was 27.5％.Patients with nutritional risk had a higher complication rate (P ＜0.01).Totally,22.0％ (153/694) patients received nutrition support,including 81.7％ patients with nutritional risk and 18.3％ patients without nutritional risk.Patients with nutritional risk benefited from nutrition support,as shown by lower complication rate and shorter length of hospital stay.In patients with nutritional risk,complication rate was lower in enteral fed patients compared to parenteral fed patients.Conclusion:With nutritional risk screening,patients' nutritional status can be evaluated and appropriate nutrition support can be performed.Compared to those without nutritional risk,patients with nutritional risk will benefit more from nutrition support,as indicated by lower complication rate and reduced length of hospital stay.

Objective: To detect potential mutations of the coagulation factor Ⅶ (F7) gene in a pedigree affected with hereditary FⅦ deficiency and explore its molecular pathogenesis. Methods: The FⅦ antigen (FⅦ∶Ag) was analyzed by an enzyme-linked immunosorbent assay (ELISA) method. Prothrombin time (PT), FⅦ activity (FⅦ∶C) and other coagulant parameters were quantified with an one-stage clotting assay. The F7 gene was amplified by PCR and sequenced. Mutational sites were confirmed by reverse sequencing. Impact of amino acid substitution was assessed using SIFT and PolyPhen-2 software. Structure of the mutant protein was analyzed using Swiss-pdb Viewer software based on the three-dimensional structure in the Protein Data Bank. Results: The propositus had prolonged PT (36.3 s), with FⅦ∶C and FⅦ∶Ag significantly reduced to 2% and 44%, respectively. Her father, mother, younger sister and daughter had slightly prolonged PT and reduced FⅦ∶C (86%-120%). The FⅦ∶Ag of her father and younger sister were also reduced. DNA sequencing revealed that the propositus has carried compound heterozygous mutations (Lys341Glu and IVS6-1G>A) of the F7 gene. Her father and younger sister were heterozygous for the IVS6-1G>A mutation, while her mother and daughter were heterozygous for the Lys341Glu mutation. Bioinformatics analysis indicated that Lys341Glu mutation may affect the stability and function of the FⅦ protein. Conclusion The Lys341Glu and IVS6-1G>A mutations probably underlie the reduced activity of FⅦ in this pedigree.

OBJECTIVE: To identify potential mutations of F11 gene in a pedigree affected with hereditary coagulation factor XI (FXI) deficiency and explore its molecular pathogenesis. METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor VIII activity (FVIIIC), coagulation factor IX activity (FIXC), coagulation factor XI activity (FXIC), coagulation factor XII activity (FXIIC) and lupus anticoagulation (LA) of the proband and eight family members were determined. FXI antigen (FXIAg) was determined by enzyme-linked immunosorbent assay (ELISA). For the proband, potential mutations in the exons, flanking introns and 5'-, 3'-untranslated regions of the F11 gene were screened by direct DNA sequencing. The results were confirmed by reverse sequencing. Suspected mutations were detected in other family members. ClustalX-2.1-win and four online bioinformatic tools (PolyPhen-2, PROVEAN, SIFT, and Mutation Taster) were used to study the conservation and possible impact of the mutations. The structure of the mutational sites was processed with Swiss-PdbViewer. RESULTS: The propositus had prolonged APTT (69.6 s), whose FXIC and FXIAg were reduced to 6.0% and 10.7%, respectively. Her mother, elder sister, one younger sister, little brother, daughter and son showed slightly prolonged APTT and moderate FXIC and FXIAg levels. Gene sequencing revealed that the propositus carried a heterozygous nonsense mutation c.738G>A (p.Trp228stop) in exon 7 and a heterozygous mutation c.1556G>C (p.Trp501Ser) in exon 13. Her mother, elder sister and daughter were heterozygous for the p.Trp228stop mutation, while one younger sister and little brother and son were heterozygous for p.Trp501Ser. Her husband and the youngest sister were of the wild type. Phylogenetic analysis suggested that Trp501 was highly conserved among all homologous species. The p.Trp501Ser was predicted to be "probably damaging","deleterious", "affect protein function" and "disease causing" corresponding to PolyPhen-2, PROVEAN, SIFT and Mutation Taster. Model analysis demonstrated that the non-polar Trp501 has two benzene rings, forming a hydrogen bond with Gln512 in the wild type. Once substituted by Ser501, the side chain may form another hydrogen bond with the benzene of His396. This may affect the normal space conformation and stability of FXI protein. CONCLUSION The compound heterozygous mutations of the F11 gene probably accounted for the low FXI concentration in this pedigree.
Factor XI ; genetics ; Factor XI Deficiency ; genetics ; Female ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree ; Phylogeny

OBJECTIVE: To detect potential mutations of the coagulation factor Ⅶ (F7) gene in a pedigree affected with hereditary FⅦ deficiency and explore its molecular pathogenesis. METHODS: The FⅦ antigen (FⅦ:Ag) was analyzed by an enzyme-linked immunosorbent assay (ELISA) method. Prothrombin time (PT), FⅦ activity (FⅦ:C) and other coagulant parameters were quantified with an one-stage clotting assay. The F7 gene was amplified by PCR and sequenced. Mutational sites were confirmed by reverse sequencing. Impact of amino acid substitution was assessed using SIFT and PolyPhen-2 software. Structure of the mutant protein was analyzed using Swiss-pdb Viewer software based on the three-dimensional structure in the Protein Data Bank. RESULTS: The propositus had prolonged PT (36.3 s), with FⅦ:C and FⅦ:Ag significantly reduced to 2% and 44%, respectively. Her father, mother, younger sister and daughter had slightly prolonged PT and reduced FⅦ:C (86%-120%). The FⅦ:Ag of her father and younger sister were also reduced. DNA sequencing revealed that the propositus has carried compound heterozygous mutations (Lys341Glu and IVS6-1G>A) of the F7 gene. Her father and younger sister were heterozygous for the IVS6-1G>A mutation, while her mother and daughter were heterozygous for the Lys341Glu mutation. Bioinformatics analysis indicated that Lys341Glu mutation may affect the stability and function of the FⅦ protein. CONCLUSION The Lys341Glu and IVS6-1G>A mutations probably underlie the reduced activity of FⅦ in this pedigree.
Factor VII ; genetics ; Factor VII Deficiency ; genetics ; Female ; Genetic Testing ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree

OBJECTIVE: To explore the molecular basis for a Chinese pedigree affected with hereditary coagulation factor VII (FVII) deficiency. METHODS: The coding regions of F7 gene were amplified by PCR and sequenced. Suspected variants were confirmed by reverse sequencing and validated in other members from the pedigree. Pathogenicity of the variants was analyzed with multiple bioinformatic tools. RESULTS: Genetic analysis revealed that the proband has carried compound heterozygous c.985T>C (p.Ser329Pro) and c.1091G>A (p.Arg364Gln) variants in exon 8 of the F7 gene. Her mother, brother and son were heterozygous for c.985T>C (p.Ser329Pro), while her father was heterozygous for c.1091G>A (p.Arg364Gln). Phylogenetic analysis suggested that both p.Ser329 and p.Arg364 are highly conserved among homologous species. Online bioinformatic software predicted both variants to be deleterious. Protein model analysis suggested that the Pro329 side chain may form a new hydrogen bond with Leu333. The Pro benzene ring may clash with Glu325 in the p.Ser329Pro variant model. The p.Arg364Gln variant have two additional hydrogen bonds compared with wild type Arg364. Both variants may lead to alteration of the protein structure. CONCLUSION The p.Ser329Pro and p.Arg364Gln variants in exon 8 of the F7 gene probably account for the reduced FVII in this pedigree.

Objective:To investigate the molecular pathogenesis of a newly discovered gene mutation in a family with hereditary coagulation factor Ⅺ(FⅪ) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in September 2021 due to "calculus of intrahepatic duct". The patient had no symptoms of spontaneous bleeding.The clinical data and blood samples of the proband and her family members (10 persons in 3 generations) were collected.The activated partial thromboplastin time (APTT) and FⅪ activity (FⅪ:C) were performed by the one-stage clotting assay. FⅪ antigen (FⅪ:Ag) were detected by enzyme linked immunosorbent assay (ELISA). Genomic DNA extracted from peripheral blood cells of subjects was used as template to analyze F11 gene mutation by DNA direct sequencing. Bioinformatics software was used to analyze the effects of mutations on protein structure and function. Wild-type and mutant FⅪ protein expression vectors were constructed and transient transfected into HEK293T cells. The total RNA was extracted from positive transfected cells and then reversely transcribed into cDNA. The mRNA expression level of F11 gene in transfected cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The content of FⅪ:Ag and the expression of FⅪ protein in transfected cell lysates and culture supernatant were detected by ELISA and western blot.Results:The APTT of the proband was significantly prolonged to 107.9s (reference range 29.0-43.0s), while FⅪ:C and FⅪ:Ag were significantly decreased to 2% (reference range 84%-122%) and 5% (reference range 76%-127%), respectively. Gene sequencing analysis indicated that the proband had c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation in exon 6 and 13 of the F11 gene, respectively. Bioinformatics analysis showed that the amino acids at site 161 of FⅪ protein were threonine (Thr) in the matrix composed of five different species, indicating that Thr161 site was highly conserved among homologous genes in different species. p.Thr161Met heterozygous mutation affected the stability of local intermolecular structure of FⅪ protein. In vitro expression experiments of p.Thr161Met mutation showed that FⅪ protein had a normal synthesis in the cells but secretion dysfunction.Conclusions:c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation were mainly responsible for the decrease of FⅪ in this family. p.Thr161Met mutation was first reported in the world and did not affect the normal synthesis of FⅪ protein, but caused secretion dysfunction.

Objective:To explore the molecular pathogenesis of hereditary protein C (PC) deficiency due to a p. Gly86Asp variant of the PROC gene through in vitro expression experiment.Methods:Wild type and Gly86Asp mutant expression plasmids of PC were constructed and respectively transfected into HEK 293FT cells. Total RNA was extracted from the transfected cells, and the expression of PROC gene was determined by quantitative real-time PCR (qRT-PCR). PC antigen (PC: Ag) in the supernatant of cell culture and cell lysate was determined by enzyme-linked immunosorbent assay (ELISA), and the level of PC protein was detected by Western blotting. Results:qRT-PCR has detected no significant difference in the transcription level of wild-type and mutant-type PC. Compared with the wild type, the level of mutant PC: Ag in the supernatant and cell lysate were 81.3%±2.6% and 110.0%±2.8%, respectively. No difference was detected in the molecular weight between the wild-type and mutant-type PC by Western blotting. The PC content of mutant type was higher than wild-type in cell lysate, while the opposite was found with the cell culture supernatant.Conclusion:The impaired secretion by mutant PC may be the molecular mechanism of PC deficiency caused by the p.

Objective To investigate the effects of chronic intermittent hypoxia(CIH)and reoxygenation on insulin resistance(IR)and expressions of miR-27a-3p/PPARγ/IRS1/PI3K/AKT in rat skeletal muscle.Methods GEO database was used for screening the differentially expressed miRNAs in CIH,and their target genes were subjected to GO and KEGG enrichment analysis followed by construction of the miRNA-mRNA-pathway regulatory network using Cytoscape.In the animal experiment,48 male SD rats were randomly divided into normoxia group and CIH group(8 weeks of CIH followed by 4 weeks of normoxic recovery).Blood and skeletal muscle samples were collected at baseline,8 weeks,and 12 weeks to evaluate the changes in fasting blood glucose(FBG)and fasting insulin(FINS)levels and muscular pathology.RT-qPCR and Western blotting were used to detect the changes in the expressions of miR-27a-3p,PPARγ,GLUT4,IRS1,p-IRS1,PI3K,p-AKT and AKT in the muscular tissues.Results No muscular miRNA datasets for CIH were available in GEO database,from which only a kidney-related dataset(GSE202480)was obtained,based on which a total of 165 differentially expressed miRNAs were identified.GO/KEGG analysis suggested that these miRNAs were involved in muscular regulation and insulin signaling.The miRNA-mRNA-pathway network highlighted miR-27a-3p as a crucial regulator in the PPAR and PI3K/AKT pathway.In the animal experiment,the rats subjected to CIH for 8 weeks showed significantly increased FBG,FINS,HOMA-IR,and PPARγ levels,loose muscle fiber arrangement,decreased cross-sectional area of the muscle fibers,and lowered expressions of miR-27a-3p,p-IRS1/IRS1,PI3K,and p-AKT/AKT in the skeletal muscles.Conclusion CIH increases IR,causes skeletal muscle pathology,downregulates miR-27a-3p expression,upregulates PPARγ expression,and inhibits IRS1/PI3K/AKT insulin signaling in the skeletal muscles of rats,and these changes can be reversed by reoxygenation.MiR-27a-3p may participate in CIH-induced IR by modulating the PPAR γ/IRS1/PI3K/AKT signaling pathway.