Objective To investigate the clinical application of multiplex allele-specific PCR assays for simultaneous detection of the mitochondrial 12S rRNA A1555G and C1494T mutations associated with aminoglycoside-induced hearing impairment.Methods Three standard plasmids of different genotypes (wild-type, A1555G mutant and C1494T mutant) were constructed for templates and allele-specific primers aiming directly at wild-type and mutant of mitochondrial DNA nt1555 and nt1494 were designed for developing a multiplex allele-specific PCR technique to detect the A1555G and C1494T mutations.Then the method was applied to clinical screening of 138 non-syndromic hearing loss subjects and confirmed by DNA sequencing.Results Multiplex allele-specific PCR was successfully applied to the detection of A1555G and C1494T mutations in a cohort of 138 Han Chinese genetically unrelated hearing-loss subjects.Finally, 11(7.97%) unrelated affected subjects harbored the A1555G and C1494T mutations in the 12S rRNA gene(10 cases for A1555G and 1 cases for C1494T), which was well consistent with results of DNA sequencing [7.97%(11/138), Kappa=1.000, P＜0.01].Conclusion This study indicates that the multiplex allele-specific PCR assay is useful, convenient and reliable in the detection of the A1555G and C1494T mutations, which could identify the subjects at risk and effectively prevent of aminoglycoside-induced hearing loss.

Objective To observe the change of interleukin-8(IL-8) during perioperative period, and to define whether the increase of IL-8 in response to cardiac surgery is related to the presence of a certain allele in a functional polymorphism. To explore the relationship between postoperative inflammation and clinical outcome. Methods One hundred and forty-five patients undergoing selective off-pump coronary artery bypass (OPCAB) for the first time were enrolled. The IL-8 (-251A >T) polymorphisms were analyzed by using polymerase chain reaction (PCR) and gene sequencing. The plasma levels of cytokine, troponin T (TnT). creatine kinase-MB (CK-MB) and creatinine (Cr) were measured before and 4, 24 and 72 hours after operation by suspension array system. Results After surgery, the IL-8 concentration increased and reached the highest level at 4 hours after surgery [18.0 (8.4, 37.1) ng/L, P = 0.000], and then it decreased to the preoperative level at 3 days after surgery. Four hours after surgery, the patients with IL-8-251 AA homozygous genotype had higher concentration of IL-8 C33.1 (16.6, 49.5) ng/L, P =0.0353. They had higher TnT and CK-MB levels than patients homozygous for AT and TT genotype 4 hours after surgery [TnT:0.53 (0.43, 4.92) ng/ml, P = 0.037; CK-MB: 41.5 (28.8, 65.5) U/L, P=0.025], and patients homozygous for AT genotype had higher Cr level 24 hours after surgery C93.1 (76.4, 121.5) μmol/L, P = 0. 021]. The patients who underwent ventilation for more than 1 day or post-operative hospital stay for more than 14 days had higher IL-8 levels (P=0.036, 0.038). IL-8-251AA genotype was an independent risk factor for patients undergoing ventilation for more than 1 day (OR=11.80, 95% CI: 1.87-74.48) and post-operative hospital stay for more than 14 days (OR=38.00, 95% CI:4.15-347. 87) . Conclusions OPCAB results in postoperative inflammatory response. IL-8-251AA genotype is associated with longer mechanical ventilation and hospital staying. Genetic background might alter the extent of inflammatory response and relate to postoperative prognosis. 、

Objective The surgery time for patients with infective endocarditis (IE) has been transformed.It has been supported that,for the patients with surgical indications,the surgery time should be as early as possible to improve the clinical outcome.The purpose of the research is to identify whether the patients with IE could get further benefit from early surgery.Methods Between June 1996 and July 2011,135 IE patients'data has been collected retrospectively,all of whom were verified through the modified Duke categories.The patients were devided into group A( the new therapeutic schedule group after 2008 ) and group B( the traditional therapeutic schedule group before 2008 ) by the year of 2008.The end points of observation were death associated with IE,cardiac failure,embolism,and re-infection.The comparison between the groups was by means of non-parameter rank and inspection test,variance analysis,t test,chi-square test,fisher exact test.The outcome comparison between the groups was via the Kaplan-Meier survival analysis.Results There were no significant differences in baseline data between the groups.No survival differences could be observed via the Kaplan-Meier analysis( Log Rank P =0.189).During the following-up visit,compared with the patients in group B,the mortality in group A is lower(9.4％ vs.23.0％,P=0.016),the incidence of heart failu re was less in group A (5.4％ vs.26.2％,P ＜0.001 ).No differences could be found in re-infection between the two groups(0 vs.4.9％,P =0.112 ). More patients in group A underwent surgery (67.6％ vs.32.8％,P ＜0.001 ).Conclusion The new therapeutic sehedule of IE coull reduce the mortality rate and promote the cardiae funetion.The incidence of re-infeetion didn't increase.

Objective:To explore the effect of peer support system on social anxiety and resilience among new graduate nurses. Methods:Totally 36 new graduate nurses entry in 2014 who's Interaction Anxiousness Scale (IAS) score were ＞43 were selected as control group. Totally 39 new graduate nurses entry in 2015 who's IAS score were ＞ 43 were as experimental group. The control group accepted routine standardized training. The experimental group received peer support, which consisted on conduct group intervention and twining intervention (6 months). IAS and Connor-Davidson Resilience Scale(CD-RISC) were used to investigate the nurses'social anxiety and resilienceat 3 months and 6 months after intervention. The scores were analyzed by using repeated ANVOA Results: Repeated measures analysis of variance of IAS showed that, there were significant differences on the IAS scores for interaction between measure time and group processing (P ＜0.001). The differences between the two groups in the main effects of interaction and time on the total score of CD-Rescan its three dimensions score were all statistically significant (Ps ＜ 0.001). Separate analyses showed that at baseline, there was no significant difference between two groups in all variables (Ps ＞0.05). After 3 months and 6 months of intervention, the IAS scores were lower in the intervention group than in the control group (Ps ＜0.001), the CD-RISC total scores and the three dimensions scores were higher in the intervention group than in the control group (Ps ＜0.05). Conclusion: It suggests that the peer support system could improve the social anxiety and resilience of new graduate nurses.

OBJECTIVETo study the relationship between mitochondrial DNA (mtDNA) mutations and hypertension.

METHODSClinical data of two pedigrees with maternally transmitted hypertension was collected. Whole mtDNA sequence was analyzed.

RESULTSThe family members on the maternal side presented with various levels of hypertension, with the onset age ranging from 44 to 55 years old. Analysis of the mtDNA sequence of the two families members showed all patients have carried a matrilineal 4329C> G mutation of the tRNA(Ile) and tRNA(Gln) genes. The same mutation was not found in 366 healthy controls. The 4329C site of mtDNA is highly conserved across species, and has been associated with the fidelity of amino acid accept arm of the tRNAs, as well as functionality and stability in the formation of tRNAs.

CONCLUSIONThe 4329C> G point mutation in tRNA(Ile) and tRNA(Gln) probably has contributed to the pathogenesis of hypertension, possibly in association with other modifying factors.

Adult ; Base Sequence ; DNA Mutational Analysis ; DNA, Mitochondrial ; chemistry ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Hypertension ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Pedigree ; Point Mutation ; RNA, Transfer, Gln ; genetics ; RNA, Transfer, Ile ; genetics ; Sequence Homology, Amino Acid

OBJECTIVETo identify secondary mutations associated with deafness in a Chinese family affected with deafness.

METHODSThe family has been subjected to clinical and molecular analyses, in addition with measurement of reactive oxygen species and doubling time after establishment of immortalized lymphocyte cell lines.

RESULTSThe results showed that the hearing loss level and audiometric configuration were discrepant among the family members with maternally transmitted hearing loss. The penetrance of hearing loss in this family was respectively 66.7% and 44.4% when aminoglycoside-induced hearing loss was included or excluded. Analysis of whole mitochondrial genome has found 33 variants as previously reported polymorphisms, except for a 12s rRNA A1555G mutation and a tRNA(Thr)T15943C mutation. Haplotype evolutionary tree has verified that this family belonged to East-Asian haplogroup F. 15943 position was located on the T-stem of the tRNA(Thr), which has destroyed the extremely conserved T-A base pair when T changed to C at this position. However, functional experiments indicated that the population doubling time in special galactose and glucose were longer, whilst the level of reactive oxygen species has increased. Compared with the control cell line groups and a family only carrying the 12s rRNA A1555G mutation, all of the three groups belonged to the same haplogroup.

CONCLUSIONMitochondrial tRNA(Thr)T15943C mutation may act as a potential modifying factor and interact with 12s rRNA A1555G mutation, and thereby enhance the penetrance and expression of deafness.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; China ; DNA, Mitochondrial ; genetics ; Deafness ; genetics ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Pedigree ; Phenotype ; Point Mutation ; RNA, Ribosomal ; genetics ; RNA, Transfer, Thr ; genetics ; Young Adult

Objective To establish a stable strain of H9c2 cardiomyocytes overexpressing Cx40 and preliminarily investigate the effect of lentiviral vector-mediated Cx40 protein overexpression on the proliferation of H9c2 cells and its related mechanisms. Methods The Cx40 gene fragment was cloned from H9c2 cells by PCR and linked with lentivirus vector pLVX-IRES-Puro to obtain the recombinant plasmid pLVX-Flag-Cx40. Recombinant lentiviral particles carrying Flag-Cx40 were obtained by cotransfection with packaging plasmids into HEK293T cells. A stable expression strain (H9c2-Flag-Cx40 cell) was screened from infected H9c2 cells by purinomycin. The expression of Cx40 protein was detected by Western blot analysis, and the effect of Cx40 on H9c2 cells proliferation was determined by CCK-8 assay; cell cycle changes were measured by flow cytometry; the expression of the cell cycle protein cyclin D1 was detected by qRT-PCR and Western blot analysis. Co-immunoprecipitation (Co-IP) immunoprecipitation and Western blot analysis were used to identify the binding of Cx40 and Yes associated protein (YAP) in H9c2 cells; cytoplasmic and cytosolic proteins were isolated to detect the effect of Cx40 on the localization of YAP using Western blot analysis. Results Sequencing results showed that the recombinant pLVX-Flag-Cx40 expression vector was successfully established. A stable transfected cell line containing recombinant Flag-Cx40 lentivirus (H9c2-Flag-Cx40 cell) was successfully constructed from H9c2 cells. Compared with the control group, overexpression of Cx40 significantly reduced the proliferation of H9c2 cells, arrested the cell cycle at G0/G1 and reduced cyclin D1 expression. A significant increase in YAP expression was observed in the cytoplasm of the H9c2-Flag-Cx40 stable cell line, while the expression in the nucleus was significantly reduced. Cx40 bound to YAP in the cytoplasm and prevented it from entering the nucleus to play the role of transcriptional coactivation. Conclusion Overexpression of Cx40 induces cell-cycle arrest at G0/G1 phase and inhibits the proliferation in H9c2 cells.
Rats ; Humans ; Animals ; Cyclin D1/genetics* ; Transfection ; Myocytes, Cardiac ; HEK293 Cells ; Cell Cycle Checkpoints/genetics* ; Cell Proliferation/genetics* ; Lentivirus/genetics* ; Genetic Vectors/genetics* ; Gap Junction alpha-5 Protein