Objective To determine new delhi metallo-β-lactamase-1 (NDM-1 )gene in strains of gram-negative bacilli with de-creased sensitivity to carbapenems,and to investigate the epidemic situation of strains carrying NDM-1 gene in Guangzhou area. Methods 105 strains of gram-negative bacilli with decreased sensitivity to carbapenems isolated from 201 1 to 2014 were collected. The conserved sequences of NDM-1 gene were screened initially by using polymerase chain reaction(PCR)amplification,and posi-tive strains were confirmed by PCR amplification of the whole sequence.Then NDM-1 gene was cloned into plasmid pUCm-T and sequenced.Results The resistance rates of Enterobacteriaceae bacteria against meropenem,ertapenem and imipenem were 29.09%, 50.91% and 29.09%,respectively.All strains of Acinetobacter baumanii were resistant to meropenem and imipenem.The resist-ance rates of Pseudomonas aeruginosa against meropenem and imipenem both were 88.46%.4 strains were NDM-1 gene positive, including 1 strain of Klebsiella pneumoniae,2 strains of Escherichia coli,1 strain of Enterobacter cloacae.Successful establishment of cloning plasmid pUCm-T-NDM-1 was confirmed by using double enzyme digestion and sequencing.The sequencing results were compared with BLAST,it was showed that the sequences were exactly the same in four cloned plasmids,and sequences of NDM-1 were also exactly the same with those in domestic and foreign.Conclusion Strains of NDM-1 producing gram-negative bacilli exist in Guangzhou area,and whole sequence of NDM-1 gene carried in these strains are exactly the same with those found in foreign.

Objective To investigate the correlation between IL‐6 ,hs‐CRP and blood lipids ,blood glucose in type 2 diabetes mel‐litus(T2DM) patients complicated with coronary heart disease .Methods 64 outpatients first diagnosed T2DM complicated with coronary heart disease were selected ,56 T2DM patients and 58 health examination were as compare from 2014 January to November in my courtyard .Interleukin‐6(IL‐6) ,high sensitivity C reactive protein(hs‐CRP) and total cholesterol(TC) ,low density lipopro‐tein‐C(LDL‐C) ,blood glucose and HbA1c were detected in 3 groups of person .Results T2DM group and T2DM complicated with coronary heart disease with fasting glucose ,HbA1c ,TC and LDL‐C was significantly higher than normal group ,the difference was statistically significant(P<0 .05);The level of IL‐6 ,hs‐CRP in patients T2DM with coronary heart disease complicated was signifi‐cantly higher than that of T2DM group ,and T2DM group was higher than that of healthy group ,the differences were statistically significant(P<0 .05) .Conclusion IL‐6 and hs‐CRP can be as a specific index to predict the disease process of T2DM complicated with coronary heart disease .

Objective:Molecular mechanisms underlying compound heterozygous mutations in a patient with inherited antithrombin (AT) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018 with a one-day history of sudden syncope and limb twitching. Peripheral venous blood was collected from the proband and members of his lineages, totaling nine persons across three generations, and a family lineage survey was conducted. AT activity (AT:A) was measured using a chromogenic substrate assay, while AT antigen (AT:Ag) was detected through an immunoturbidimetric assay. Mutation sites were identified by means of Sanger sequencing of the SERPINC1 gene, and silico tools were applied to predict the mutational conservation and hydrophobicity changes. Recombinant plasmid expression vectors were constructed and transfected into HEK293T cells for in vitro overexpression studies. The recombinant AT protein was characterized using Western Blotting, ELISA, and cellular immunofluorescence assays.Results:The proband was a 21-year-old man with type Ⅰ AT deficiency. His AT:A was 33%, along with a corresponding reduction in AT:Ag. The genetic analysis revealed there was a heterozygous insertion mutation at c.318_319insT (p.Asn107*) and a heterozygous missense mutation at c.922G>T (p.Gly308Cys) in exons 2 and 5, respectively. These mutation sites were entirely conserved among the homologous species. Additionally, hydrophobicity studies showed that the p.Gly308Cys mutation will decrease the hydrophilicity of amino acid residues 307-313. The in vitro expression studies indicated a reduction of approximately 46.98%±2.94% and 41.35%±1.48% in the amount of recombinant protein AT-G308C in transfected cell lysates and culture supernatants, respectively. Treatment with the proteasome inhibitor (MG132) restored the cytoplasmic levels of AT-G308C protein to a level similar to that of wild-type protein. However, neither cell lysate nor culture supernatant demonstrated the presence of the recombinant protein AT-N107*. Conclusions:The heterozygous insertion mutation of p.Asn107* and the heterozygous missense mutation of p.Gly308Cys have been associated with reduced AT levels in proband. The p.Asn107* heterozygous insertion mutation may initiate the degradation of mRNA via nonsense mutation-mediated mechanisms, which would remove the defective transcripts, as well as the p.The Gly308Cys heterozygous missense mutation may cause the AT protein to undergo proteasome-dependent degradation by modifying the hydrophobicity of nearby residues in the cytoplasm.

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.

A 34 year old female patient was scheduled to undergo surgical resection due to a "breast nodule". Preoperative examination revealed an activated partial thromboplastin time (APTT) of 66.2 seconds, coagulation factor Ⅺ activity (FⅪ: C) of 2%, and FⅪ antigen (FⅪ: Ag) of 40.3%. The patient and family members showed no abnormal bleeding symptoms. Diagnosed as hereditary coagulation factor Ⅺ deficiency. Genetic testing revealed that the F11 gene had a heterozygous nonsense mutation in exon 10, c.1107C>A (p.Tyr351stop), and a heterozygous missense mutation in exon 13, c.1562A>G (p.Tyr503Cys). The father and son were p Heterozygous carriers of Tyr351stop mutation, while the mother and daughter are p Heterozygous carriers of Tyr503Cys mutations. The in vitro expression results showed that p The Tyr351stop mutation resulted in a significant decrease in the transcription level of F11 gene, while p The Tyr503Cys mutation has no effect on the transcription level and protein expression level of F11 gene, but it leads to a significant decrease in the level of FⅪ:C in the cell culture supernatant.