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OBJECTIVE@#To detect the mutation site in a pedigree affected with autosomal dominant polycystic kidney disease (ADPKD) and verify its impact on the protein function.@*METHODS@#Peripheral blood samples were collected from the proband and his pedigree members for the extraction of genomic DNA. Mutational analysis was performed on the proband through whole-exome sequencing. Suspected variant was verified by Sanger sequencing. A series of molecular methods including PCR amplification, restriction enzyme digestion, ligation and transformation were also used to construct wild-type and mutant eukaryotic expression vectors of the PKD2 gene, which were transfected into HEK293T and HeLa cells for the observation of protein expression and cell localization.@*RESULTS@#The proband was found to harbor a c.2051dupA (p. Tyr684Ter) frame shift mutation of the PKD2 gene, which caused repeat of the 2051st nucleotide of its cDNA sequence and a truncated protein. Immunofluorescence experiment showed that the localization of the mutant protein within the cell was altered compared with the wild-type, which may be due to deletion of the C-terminus of the PKD2 gene.@*CONCLUSION@#The c.2051dupA (p. Tyr684Ter) mutation of the PKD2 gene probably underlay the pathogenesis of ADPKD in this pedigree.
Subject(s)
Female , Humans , Male , DNA Mutational Analysis , Frameshift Mutation , HEK293 Cells , HeLa Cells , Pedigree , Polycystic Kidney, Autosomal Dominant/physiopathology , Protein Kinases/genetics , Protein Transport/genetics , Exome SequencingABSTRACT
OBJECTIVE@#To explore the genetic basis for a Chinese patient with amyotrophic lateral sclerosis (ALS).@*METHODS@#Peripheral blood samples were collected from the patient and his parents for the extraction of genomic DNA. Genetic variant was identified by whole exome sequencing. Candidate variant was verified by Sanger sequencing of his parents and healthy controls.@*RESULTS@#The patient was found to harbor a heterozygous c.420C>G (p.Asn140Lys) variant of the SOD1 gene. The same variant was not detected in his parents and 100 healthy controls. The variant has not been included in HGMD, dbSNP and other databases.@*CONCLUSION@#The c.420C>G variant of the SOD1 gene may underlie the ALS in this patient. Above finding has enriched the spectrum of SOD1 gene variants.
Subject(s)
Humans , Amyotrophic Lateral Sclerosis/genetics , China , Heterozygote , Superoxide Dismutase-1/genetics , Exome SequencingABSTRACT
OBJECTIVE@#To analyze the pathogenic variant of preaxial polydactyly in a Chinese Han pedigree and identify the cause of polydactyly.@*METHODS@#The peripheral blood DNA of the proband and her parents was extracted. The polydactyly-related genes were detected by trio whole exome sequencing, and the suspected pathogenic gene was screened out. Sanger sequencing was applied to other members of the pedigree.@*RESULTS@#The results of gene sequencing showed that the LMBR1 gene had a heterozygous variant of c.423+4909(IVS5)C>T in 6 patients of the pedigree. The same variant was not detected in family members with normal phenotype. Based on the ACMG guidelines, c.423+4909(IVS5)C>T of the LMBR1 gene was predicted to be pathogenic (PM1+PM2+PP1-S(PS)+PP4+PP5).@*CONCLUSION@#The heterozygous C>T variant at position 4909 of intron 5 of the LMBR1 gene probably underlies the disease in this pedigree.
Subject(s)
Female , Humans , China , Mutation , Pedigree , Polydactyly/genetics , Thumb , Exome SequencingABSTRACT
AIM: To investigate the expression of miR-186-5p in colon cancer and the effect on colon cancer, and provide a new therapeutic target for the treatment and prognosis of colon cancer. METHODS: Serum from eligible patients were collected and divided into five groups (n=20) including the healthy group and clinical stages I, II, III and IV. The miRNAs in patients' serum were extracted by miRVana microRNA isolation kit. The differences of miR-186-5p expression in patients' serum of colon cancer at different stages were detected with real-time quantitative PCR. The full-length gene of miR-186-5p was cloned into pENTR/D-Topo vector to construct cell lines with high expression of miRNA. 5×103 cells were seeded in 96-well plates. The expression of mir-186-5p was decreased by using miR-186-5p inhibitor. The proliferation of cells was detected with cell proliferation ELISA kit after 24 hours of incubation with BrdU and absorbance was measured at 370 and 492 nm. The effect of miR-186-5p on the sensitivity of colon cancer cell lines to chemotherapeutic drugs was detected by MTT assay. RESULTS: The expression of miR-186-5p was down-regulated in the serum of patients with colon cancer compared to normal group. Expression of miR-186-5p in serum of patients with stage I, II, III and IV was significantly decreased (P<0.01) compared with the normal group. Colon cells were transfected with a plasmid integrating the miR-186-5p gene, and miR-186-5p was highly expressed in the HCT116 and SW480 cell lines (P<0.01). The expression of mir-186-5p was decreased by mir-186-5p inhibitor (hsa-miR-186-5p inhibitor) in HCT116 and SW480 cells. The proliferation rate of HCT116 and SW480 cells with high expression of miR-186-5p was lower than control group (P<0.01). After treating with mir-186-5p inhibitor, the cell proliferation rate was higher than that of the control group (P<0.01). After treatment with different concentrations cisplatin for 24 hours, the mortality of HCT116 and SW480 cells with high expression of mir-186-5p was higher than that of the control group (P<0.01). The mortality of the cells with low expression of mir-186-5p was lower than that of the control group (P<0.01). The protein level of PLK1 was decreased after transfecting with miR-186-5p plasmid and increased after stimulating with miR-186-5p inhibitor in HCT116 and SW480 cells. CONCLUSION: The expression of miR-186-5p is decreased in colon cancer patients. Overexpression of miR-186-5p can inhibit the proliferation of colon cancer cells and reduce the drug resistance of colon cancer cells; inhibition of miR-186-5p expression can increase the proliferation and drug resistance of colon cancer cells.
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Clinical decision support system (CDSS) has been developed and integrated into clinical nursing system by building up rules and knowledge base through literature retrieval, expert argumentation, and situation analysis. With such algorithms as reasoning algorithm, case study, artificial intelligence and big data analysis, three modules have been constructed as follows. The first is an Intelligent Knowledge-based Decision Module composed of 15 document-driven knowledge-based decision-makers and 5 task-driven knowledge-based decision-makers. The second is an Intelligent Quality Control Recording Module which has achieved intelligent quality control of nursing records through Feedforward Control and Feedback Control. The third is an Intelligent Human-Computer Interaction Module which consists of real-time static interactions through e-board and dynamic reminders/alarms triggered by the system. CDSS, since in use, has reduced the incidence of adverse nursing events and problems of quality control in nursing records, and improved satisfaction of patients.
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Objective To observe the effect of acupuncture at the muscle region of meridians plus tuina on facial nerve function in peripheral facial paralysis. Method Sixty patients with peripheral facial paralysis (recovery stage) were randomized into a treatment group (intervened by acupuncture at the muscle region of meridians plus tuina) and a control group (intervened by conventional acupuncture), 30 cases in each group. Result The facial nerve electromyography (EMG) showed that the action potential latency and amplitude were changed significantly after the treatment in both groups (P<0.05), indicating that the two methods were both effective in recovering facial nerve function, while acupuncture at the muscle region of meridians plus tuina produced a more significant treatment effect compared to conventional acupuncture. In the treatment group, same treatment with different intervention times produced significantly different effects on the Toronto facial grading system (TFGS) (P<0.05), indicating that the earlier the intervention, the more significant the therapeutic efficacy. Conclusion Acupuncture at the muscle region of meridians plus tuina can produce a more significant effect in recovering facial nerve function in peripheral facial paralysis compared to conventional acupuncture.
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Objective To investigate the role of CC chemokine ligand 20 (CCL20) and CC chemokine receptor 6 (CCR6) in the pathogenesis of acute necrotizing pancreatitis (ANP). Methods 48 SD rats were randomly divided into two groups: control group and ANP group. The ANP model was induced by retrograde infusion of 4 % sodium taurocholate into the biliary and pancreatic duct in SD rats. The same amount of saline was injected in the control group. The rats were sacrificed at 1, 3, 6, 12 h, the serum amylase levels and the pathological score of the pancreas were measured. The expressions of CCL20 and CCR6 mRNA and protein in pancreas were detected by immunohistochemistry and semi-quantitative RT-PCR,respectively. Results The levels of serum amylase and the histological score of ANP group were significantly higher than those of control group (P ＜ 0.01 ). The expression of pancreatic CCL20 mRNA and protein was increased in a time-dependant manner ( P ＜ 0.05 ). The expression of pancreatic CCR6 mRNA at 6h was significantly higher than that of control group (0.88 ± 0.05 vs 0. 23 ± 0.09, P ＜ 0.01 ). The expression of pancreatic CCR6 mRNA at 12h was decreased when compared with that of 6h group, but it was still higher than that of control group (0.37 ± 0. 10 vs 0. 15 ± 0.07, P ＜ 0.05 ), the change of CCR6 protein was consistent with that of CCR6 mRNA. Conclusions CCL20 and CCR6 may play an important role in the pathogenesis of ANP.
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Objective To clone the gene encoding protein of EspA and Stx2B from EHEC OI57:H7 by DNA recombinant technology, construct prokaryotic expression vector pET-28a ( + )-espAstx2B, express fusion protein of EspA-Stx2B and to analyze the biological and immunological characteristics of the fusion protein. Methods the sequence encoding the protein of EspA and Stx2B was amplified by PCR from the enterohemorrhagic Escherichia coli strain. The amplified products were connected with linker by recombinant technology and cloned into pET-28a( + ) vector. The vector was then transferred to the host cells E. Coli BL21 strain (DE3). Following, the protein expression was induced by IPTG. The expression quantities and style of fusion protein was then determined by SDS-PAGE. Its immunoreactivity was analyzed by Western blot. Finally, BALB/c mice were injected with the preliminarily purified recombination protein EspA-Stx2B, then oral challenged these mice with EHEC O157-SMR2 and counteracted toxic substances with O157 ultrasonic supernatant. Results The determination of the sequence encoding of the espA-stx2B fusion gene has 100% of consistency with the sequence from GenBank Sakai strain and contrivable linker. This fusion protein EspA-Stx2B was expressed as inclusion body formation and the percentage is approximately 40%. Western blot suggested the fusion protein has excellent immunoreactivity. Titer of antiserum of the mice to EspA-Stx2B increased evidently. EspA-Stx2B could not decrease bacterial number attached to the intestinal tract of mice based on fecal shedding of Oi57 in mice. In the test of death of BAI,B/c causing by conteracting toxic substances with O157 ultrasonic supernatant, immunoprotection of EspA-Stx2B rate was 66.7%. Conclusion A recombinant plasmid that has high performance on expression of EspA-Stx2B prorein was successfully constructed in present study, and the fusion protein has excellent immunoreactivity and immunogenicity. EspA-Stx2B could not decrease bacteria] number attached to the intestinal tract of mice based on fecal shedding of O157 in mice, but evidently decrease the mortality rate of the mice. The antiEspA and anti-Stx2B had immunoprotection effect by different means. These results may provide the foundation for the further development on EHEC O157:H7 double subunit vaccine.
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Objective To investigate the suppression effects of Tripotolide (TL) on the pancreatic cancer xenograft models and angiogenesis. Methods The growth suppression effect of TL on SW1990 was determined using cell count kit (CCK-8), apoptotic cells induced by TL were examined by morphology and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The inhibitory effects of TL on the growth of tumor xenografts and tumor microvascular density (MVD) were investigated. ResultsTL inhibited the growth and proliferation of SW1990 cells in a concentration-dependent and time-dependent manner. The inhibition ratios of cells treated at 160 mg/ml TL for 24 h was 50. 6%, the apoptotic rate increased from 9.6% in the control group to 45.1% (P <0.01 ). The inhibition rate of cancer xenograft growth was 89.9% when TL was intratumorally injected at the dose of 0.5 mg/kg. The expression of VEGF in tumor tissue decreased while MVD also decreased from 36.25±8.64 to 9.87±3.34 (P <0.01 ). ConclusionsTL induced prominent growth inhibition and apoptosis in human pancreatic cancer cell lines. TL.can attenuate the growth of pancreatic caner xenografts through its effect on antiangiogenesis.
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Objective To investigate immunoprophylactic potential of genetic engineering vaccines of enterohaemorrhagie Escherichia coli O157:H7 in BALB/c mice after immunization with these vaccines. Methods Sixty BALB/c mice (3 weeks old) were randomized averagely into 5 groups. Group 1-3 were im-munized respectively with IntiminC300, Stx2B and HIyAN436, group 4 with a combination of these three vaccines, and group 5 with PBS. Each mouse was immunized with vaccine(100 μg)and Al(OH)3 adjuvant (100 μg) for 3 times. After 7 d of the second and third immunization, serum of each mouse was collected and the different antibodies were detected. After 10 d of the last immunization, all mice were given drinking water containing streptomycin for 3 d before and following oral challenge with O157:H7 (109 CFU), and treated with clinical, microbiological and pathological examination. Results The three vaccines elicited high titer antiserum, and some mice were died after infection with O157. The livability of group 1-4 was re-spectively 73%, 64%, 36% and 91%. And these vaccines depressed fecal and colon shedding with O157. Condusion IntiminC300, Stx2B and HIyAN436 have certain protective efficacy for infection of O157, and combined immunization was more effective than single vaccine.
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<p><b>OBJECTIVE</b>To study the effect of transcatheter arterial chemoembolization (TACE) combined with beta-ultrasound guided portal vein embolization (PVE) through fine-needle liver puncture for hepatocellular carcinoma.</p><p><b>METHODS</b>209 patients with primary hepatocellular carcinoma were divided into TACE group (104 patients) and TACE + PVE group (105 patients).</p><p><b>RESULTS</b>The response rates (CR + PR) were 37.5% in TACE group and 57.2% in TACE + PVE group (P < 0.01). Tumor thrombi became lessened or resolved in the portal vein with incidences of 22.2% in TACE group and 68.8% in TACE + PVE group (P < 0.01). The 1-, 2- and 3-year survival rates were 65.1%, 36.3% and 20.5% in TACE group and 95.6%, 59.6% and 39.1% in TACE + PVE group (P < 0.05).</p><p><b>CONCLUSION</b>The effect of TACE combined with PVE is much more effective than TACE alone for hepatocellular carcinoma. Beta-ultrasound guided portal vein embolization through fine-needle liver puncture is effective, easy, safe and should be widely practiced.</p>