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Objective To observe the levels of four bisphenols (bisphenol A,B,S and F) and their correlation with renal function in chronic kidney disease (CKD) patients.Methods Patients with CKD were identified according to Kidney Disease:Improving Global Outcomes (KDIGO) criteria.Sixty-three CKD patients and eleven healthy controls were enrolled.CKD patients were further classified as mild renal injury group (CKD stage 1 and 2,n=30),moderate renal injury group (CKD stage 3,n=19) and severe renal injury group (CKD stage 4 and 5,n=14).The levels of four bisphenols in serum were determined by high performance liquid chromatography (HPLC).The correlation between concentrations of four bisphenols and estimated glomerular filtration rate (eGFR) was assessed by Spearman's rank correlation analysis.The associations of four bisphenols with coronary heart disease,diabetes and hypertension in CKD patients were estimated by binary multivariate logistic regression.Results (1) Four bisphenols were not detected in serum of healthy control.In the mild renal injury group the bisphenol A and bisphenol S were not detected,and patients had 5.24 (5.24,9.38) μg/L bisphenol B and 0.74 (0.74,0.74) μg/L bisphenol F.In the moderate renal injury group bisphenol S was not detected,and patients had 2.79 (1.01,4.53) μg/L bisphenol A,5.24 (5.24,5.24) μg/L bisphenol B and 0.74 (0.74,0.74) μg/L bisphenol F.In severe renal injury group patients had 14.30 (7.97,18.17) μg/L bisphenol A,0 μg/L bisphenol B,23.73 (23.73,136.59) μg/L bisphenol S and 0.74 (0.74,1.42) μg/L bisphenol F.The levels of bisphenol A and bisphenol S in severe renal injury group were higher than those in the healthy control group,mild renal injury group and moderate renal injury group (all P ＜ 0.05).Bisphenol B and bisphenol F were not statistically different among four groups.(2) Bisphenol A and bisphenol S were negatively correlated with eGFR (r=-0.779,P ＜ 0.001;r=-0.546,P ＜ 0.001).(3) Among CKD patients,bisphenol A was correlated with diabetes (OR=4.951,95％CI 1.603-15.294,P=0.005),and bisphenol S was correlated with hypertension (OR=4.466,95％ CI 1.575-12.666,P=0.005).Conclusions CKD patients have a variety of bisphenol compounds,especially bisphenol A and bisphenol S.Bisphenol A and bisphenol S have high levels,and their exposures are correlated with renal function.
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AIM:To investigate the effect of fenbendazole (FBZ) on the proliferation of human chronic myelogenous leukemia (CML) cell line K562.METHODS:The CCK-8 assay was used to detect the effect of FBZ on viability of the K562 cells and normal peripheral blood mononuclear cells (PBMC).The cell growth was measured by the method of Trypan blue exclusion.The cell cycle was analyzed by flow cytometry.The cell cycle-related proteins were detected by Western blot.RESULTS:The growth of K562 was significantly inhibited by FBZ.However, it elicited little cytotoxic effect on PBMC.Furthermore, FBZ induced G2/M phase arrest and mitotic catastrophe in the K562 cells based on the changes of nuclear morphology, DNA content, mitotic marker analysis and the number of polykaryocytes.CONCLUSION:Fenbendazole significantly inhibits the proliferation of K562 cells and induces cell cycle arrest at G2/M phase by the regulation of cell cycle-related proteins.
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Objective · To observe the vascular structure before autogenous arteriovenous fistula (AVF) construction in patients with end-stage renal disease (ESRD) and analyze the risk factors of the pre-existing venous neointimal hyperplasia. Methods · The 8 vein samples were screened from 20 ESRD patients at their first time of the AVF construction (non-stenosis group), and the other 8 vein samples were screened from 15 ESRD patients at their at least second time of the AVF repair operation (stenosis group). Sections were prepared and stained with hematoxylin & eosin (H-E) or Masson's trichrome for observation. The intimal thickness was measured by the cellSens software, and its correlation with patients' renal function, calcium-phosphorus metabolism, iron metabolism and inflammatory reaction in the non-stenosis group were analyzed. Results · In the non-stenosis group, there were varying degrees of intimal hyperplasia in 5 (62.5%) cases, loss of endothelial cell layer in 3 (37.5%) cases, and vascular wall replacement by collagenous with atrophy or loss of muscle layer in 5 (62.5%) cases. In the stenosis group, almost all vein samples showed general thickening of the wall and 2 (25.0%) totally lost the muscle layer. Avg It of those two groups were statistically significant (P<0.01). In the non-stenosis group, both of average I/M thickness and average I/M area were negatively related to glomerular filtration rate (GFR) (P<0.05) and positively related to serum phosphorus and calcium-phosphorus product (P<0.05). Conclusion · Some apparently normal vein of ESRD patients showed varying degrees of intimal hyperplasia before AVF construction. The intimal hyperplasia had a remarkable correlation with GFR or calcium-phosphorus metabolism. Early intervention of the intimal hyperplasia prior to AVF construction may be a new prevention and control means.
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Objective This research was designed to reveal the influence of the new rural cooperative medical system (NRCMS) on anticoagulant therapy of warfarin in atrial fibrillation (AF) patients in Gaochun district,Nanjing city.The reasons of not attending NRCMS and the relationship between NRCMS and the reasons of not correctly anticoagulant therapy were also investigated.Methods We collected outpatients and inpatients in Gaochun district in our hospital who suffered with AF and did not attend any medical insurance except NRCMS.Patients' data of whether enrolled in NRCMS,whether anticoagulated with warfarin,whether measure international normalized ratio and then confine it in therapeutic extent and fundamental data of gender,age etc.were collected by questionnaire.Then,the data were analyzed.Results A total of 1 094 patients were included in our research of which 896(81.9％) patients were enrolled in NRCMS.198 subjects did not attend NRCMS.The most common reason was not getting the message of NRCMS,which covered 104 patients (52.5％).Among the patients without counter indications of warfarin,801 patients did not be anticoagulated,of which 637 were covered by NRCMS and 164 were not insured by NRCMS,which accounted for 71.1％ and 82.8％ of their total respectively.The difference was statistically significant (x2 =11.387,P =0.001).Similar to the nonNRCMS,the most common reason of not accepted anticoagulation in patients covered by NRCMS was taking substitutions,which was hold by 42.9％ patients.All the differences of proportions of patients had recorded reasons with and without NRCMS had no statistical significance except for the proportion of patients who hold the reason of not knowing the necessity of anticoagulation,having difficulty in daily life,and compound reasons.Among the anticoagulated patients,202 cases did not restrict INR in therapeutic range,of which 170 cases were covered by NRCMS and 32 cases were not insured by NRCMS,which accounted for 65.6％ and 94.1％ of their total respectively.The difference was statistically significant (x2 =11.385,P =0.001).The most common reason of not restricting INR in patients covered by NRCMS was did not know the necessity of restricting INR,which was hold by 40.6％ patients.The proportions of patients who did not take anticoagulation with the reason of having difficulty in daily life had statistical difference,while the difference of other reasons did not meet statistical significance.Conclusion There were many patients did not attended NRCMS.NRCMS can improve the proportion of anticoagnlation therapy with or without correct ways.However,there were many patients did not treated with anticoagulation and there were many patients did not restrict INR in therapeutic rainge.Propaganda,health education and directions of health information are needed.
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Objective To explore the mechanism ofHuanshuai Oral Liquid in the process of hypoxia-microvascular waste-renal interstitial fibrosis in mice with atherosclerosis renal artery stenosis.Methods The atherosclerosis renal artery stenosis model was established on Apoe-/- mice by high fat feed and uninephrectomy. After 2 weeks of renal ablation, model mice were randomly divided into TCM group (Huanshuai Oral Liquid) and model group. C57BL/6J mice were taken as the blank group. The mice of TCM group receivedHuanshuai Oral Liquid 0.01 mL/(g?d) for gavage, while the blank group and the model group received normal saline 0.01 mL/(g?d) for gavage. Renal function, atherosclerosis and renal interstitial fibrosis were assessed in the 12th week after taking medicine via immunohistochemical method to detect the expressions of the collagen typeⅣ, alpha smooth muscle actin (α-SMA) andⅧ factor related antigen (vWF), and via RT-PCR to detect the expressions of HIF-1α and TGF-β.ResultsCompared with the blank group, the model group suffered severe renal artery stenosis and renal fibrosis, and the expressions of HIF-1α, TGF-β, collagen typeⅣ andα-SMA significantly increased (P<0.05), while the expression of vWF decreased (P<0.05). Compared with the model group, the expressions of HIF-1α, TGF-β, collagen typeⅣ andα-SMA group, the expressions of HIF-1α, TGF-β, collagen typeⅣ andα-SMA significantly decreased in the TCM group (P<0.05), while the expression of vWF significantly increased (P<0.05).ConclusionHuanshuai Oral Liquid could significantly improve the renal function and the renal tubular-interstitial fibrosis. The mechanism might be associated with regulating the expressions of HIF-1α and TGF-β.
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ObjectiveTo reproduce a clinically relevant two-hit model of sepsis complicated by pneumonia and to explore the correlation between two-hit and immune state.Methods Eighty-one male Sprague-Dawley ( SD ) rats were divided into groups according to the random number table. Forty-five male rats were assigned respectively to sepsis-alone group, pneumonia 4 days and 7 days after sepsis groups, respectively. Survival rate of each group was observed. Another group of 36 male rats were divided into normal control group, sepsis-alone for 1, 4 and 7 days groups, and sepsis complicated by pneumonia for 4 days and 7 days after sepsis groups, each group consisted of 6 rats. Cecal ligation and puncture (CLP) was done in rats, andStreptococcus pneumoniae suspension (bacteria count 1×1010 cfu/mL) was injected via the nose on the 4th day or 7th day after CLP. Rats were sacrificed at corresponding time points, and 1 day after challenge ofStreptococcus pneumoniae on the 4 days or 7 days post CLP for the collection of blood and tissue samples to make bacterial count of the blood, splenocyte count, biochemical indices, cytokines concentration, pathological changes in spleen and apoptotic cells.Results① Compared with the rats of sepsis-alone group, the rats in pneumonia 4 days after CLP group had poor survival rate (4 vs. 11,χ2 = 6.533,P = 0.011), while no difference was found between pneumonia 7 days after CLP group and sepsis-alone group (9 vs. 11,χ2 = 0.600,P = 0.439).② The blood bacterial count and all the biochemical indexes were sharply increased on 1 day post-CLP in the rats of sepsis-alone group, and then they gradually lowered. Compared with the rats of 1 day post-CLP, the proportion of splenocytes were decreased on the 4th day post-CLP [dendritic cells (DC): (0.69±0.09)% vs. (0.87±0.31)%, CD4+T cells: (21.05±2.89)% vs. (24.84±4.59)%, CD8+ T cells: (10.62±1.79)% vs. (13.40±1.31)%, allP 0.05). The same trend of changes, with slight reduction in splenocytes and biochemical indices were found between the groups of sepsis followed by pneumonia and sepsis-alone, but no significant difference was found. The level of HMGB1 in the 4-day group of sepsis with complication of pneumonia was further decreased compared with sepsis-alone group (μg/L:1.17±0.74 vs. 1.76±0.71,P 0.05).Conclusions The mortality of this two-hit model with complication of pneumonia 4 days after CLP was significantly higher than that of single sepsis model. The ability of bacteria clearance was decreased, and immunocyte apoptosis was exacerbated. These findings may be with the result of the occurrence of immunoparalysis in the mid stage of sepsis. The two-hit model reproduced on 7 days after CLP might suggest reconstruction of host immune function, and maybe associated with the recovery of immune response.
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To detect cadmium ions, we constructed a specific microbial sensor and screened detecting cassettes and different fluorescence proteins. Blue fluorescence protein mTagBFP2 was selected as a reporter and a double-promoters model was used in the construction of the fusion reporter vector Pmer::merR-m-Pmer::mTagBFP2-pMD19-T. The reporter vector was then transformed into Escherichia coli MC4100 wild type strain. The medium, incubation time, initial density for induction, and the optimal detection range were determined. The specificity of the biosensor was also checked. The biosensor responded specifically to cadmium irons with low background, and the linear concentration range detection ranged from 0.1 to 75 μmol/L at the initial OD600 = 0.1 with 2 h incubation in IHMM medium. Thus we successfully constructed a specific biosensor to detect cadmium irons and provided useful strategies for development and optimization of microbial sensors to detect heavy metals.
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Biosensing Techniques , Cadmium , Escherichia coli , Genetic Vectors , Promoter Regions, GeneticABSTRACT
Objective To assess the relationship between mitochondrial DNA (mtDNA) ND2 gene C5178A polymorphism and complications of cardio-cerebral-vascular in patients with type 2 diabetes mellitus (T2DM).Methods This is a case-control study.448 unrelated patients with T2DM were collected from Zhejiang Provincial People's Hospital from 2010 to 2011,including 274 males and 174 females.Direct nucleotide sequencing analysis was used to screen mtDNA ND2 gene C5178A genotyping in ）patients.Meanwhile,detailed clinical and laboratory information for all of study subjects were collected.Body mass index (BMI),blood pressure,blood lipid,blood glucose and incidence rate of cerebral infarction were compared between 5178C patients and 5178A patients.Furthermore,according to the genotyping results,we 2analyzed whether these differences exist in patients with different gender by using t test or x2 test.Results 348 out of 448 patients with T2DM were C carriers and the remaining patients were A carriers.There're significant differences between T2DM patients with 5178A and T2DM patients with 5178C on systolic pressure (124.6 mm Hg ± 9.0 mm Hg vs 127.8 mm Hg ± 10.7 mm Hg,t =2.700,P =0.007)and HDL (1.3 mmol/L ± 0.2 mmol/L vs 1.2 mmol/L ± 0.3 mmol/L,t =2.968,P =0.003).Moreover,the incidence of cerebral infarction in T2DM patients with 5178A (8.0％,8/100) was much lower than that with 5178C (21.0％,73/348 ; x2 =8.832,P =0.003).No statistical gender difference was found in the distribution of C5178A (P ＞ 0.05).Our results also revealed that the female T2DM patients with 5178A had a lower serum triglyceride (1.5 mmol/L ±0.8 mmol/L; t =2.601,P =0.011) and lower systolic pressure (123.6 mm Hg±6.6 mm Hg; t =2.887,P =0.004) than that with 5178C (1.8 mmol/L ± 1.0 mmol/L and 128.0 mm Hg ± 9.0 mm Hg,respectively).Furthermore,cerebral infarction was more common in female T2DM patients with 5178C (21.3％,29/136; x2 =5.232,P =0.022) than that with 5178A (5.3％,2/38).Similarly,male T2DM patients with 5178A had a much lower incidence rate of cerebral infarction (9.7％,6/62; x2 =3.946,P =0.047) than that with 5178C (20.7％,44/212).In contrary,the serum concentration of HDL was higher in male T2DM patients with 5178A (1.4 mmol/L ±0.2 mmol/L;t=3.511,P =0.001) than that with 5178C (1.2 mmol/L±0.3 mmol/L).Conclusions The polymorphism site mtDNA C5178A correlates with cerebral-cardiovascular complications in patients with type 2 diabetes mellitus.mtDNA 5178A allele may protect T2DM patients from developing cerebral-cardiovascular diseases through regulation of blood pressure and lipid metabolism.
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In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.
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Animals , Cricetinae , Adiponectin , Genetics , Bioreactors , CHO Cells , Cell Culture Techniques , Methods , Cricetulus , Receptors, Tumor Necrosis Factor, Type II , Genetics , Recombinant Fusion Proteins , GeneticsABSTRACT
Objective To prepare hepatocyte growth factor(HGF) recombinant protein and confirm its activity preliminarily according to building HGF gene prokaryotic expression vector and transforming into E.coli.Methods Clone HGF inserted into the vector pET-26b(+) to construct prokaryotic expression vector pET-26b(+)-HGF and transform into E.coli Rosseta(DE3).The transformed bacteria induced by IPTG was purified through Ni-NTA resin affinity chromatography frozen-drying after renaturation.Results HGF gene recombinant prokaryotic expression vector pET-26b(+)-HGF was constructed successfully.E.coli Rosseta(DE3) which was transformed into pET-26b(+)-HGF expresses the target protein as the form of inclusion bodies,accounting for 38％ of the total bacterial proteins,and confirmed by Western blot.HGF protein which was purified by Ni-NTA resin affinity chromatography,has a purity of about 95％,and can promote proliferation,migration,and inhibition of apoptosis for human non-small cell lung cancer cell line A549 cells after interaction.Conclusion HGF gene recombinant prokaryotic expression vector pET-26b (+)-HGF was constructed and expressed in transformed E.coli Rosseta(DE3) successfully.They resumed their recombinant HGF protein structure after purification and renaturation,and had biological activity confirmed by in vitro studies.
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Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting (HRM) and study its preliminary clinical application.Methods Two common mutations [ IVS-2-654 ( C ＞ T ) and -28 ( A ＞ G ) ]of β-thalassemia in Wenzhou city population were selected.The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls.A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity,sensitivity and repeatability were methodologically evaluated.One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children's Hospital of Wenzhou Medical College were enrolled into this study.The genomic DNA was extracted from whole blood cells and detected by HRM method.The results were compared with the direct sequencing data.Results HRM method could detect the mutations [ IVS-2-654( C ＞ T) and -28 ( A ＞ G ) ]of β-thalassemia and the results did not show any non-specific amplified fragments.All within-run and between-run coefficients of variation for different DNA types' Tm were smaller than 0.1％.And minimum 103 copies of DNA of each assay and 10％ mutation could be determined by this method.One hundred and seventeen patients with clinically suspected β-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing.There were 45 IVS-2-654 ( C ＞ T)heterozygous mutation and 9-28 ( A ＞ G)heterozygous mutation and none homozygous mutation.Conclusion The method of rapid identification of β-thalassemia gene mutations based on HRM analysis is successfully established,which is a convenient,rapid,specific,sensitive and accurate technique for screening gene mutations in β-thalassemia as well as a general technical platform to identify other gene mutations.
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AIM: To explore the molecular mechanism of asthenospermia(AST) by preliminary screening of nucleotide sequences from the ND3 and ND4L genes of mitochondrial DNA(mtDNA). METHODS: Samples from 50 AST patients and 42 age-matched normal controls were collected according to the WHO criteria. Density gradient centrifugation was applied to separate spermatozoa with different vigor. The ND3 and ND4L genes of mtDNA were amplified and sequenced directly from the extracted genomic DNA from AST patients and normal controls. The sequences were compared with revised Cambridge Reference Sequence(rCRS) to analyze the variants. RESULTS: A total of 22 nucleotide variations were found in ND3 and ND4L genes of mtDNA in asthenospermia group and control group. G10320A, A10398G and T10609C were missense mutations, while A10157G and A10313C were the reported for the first time in this study. Haplotype N in patients with AST(33/50) was higher than that in control group(14/42, P<0.05), and haplotype R9 in patients with AST(15/50) was also higher than that in control group(4/42, P<0.05) through genetic testing of ND3 gene. Rates of sperm progressive motility of haplotype F1, F2 and R9 were significantly lower than those of haplotype M and M rest. Two haplotype differences, haplotype M and N, were found in the same AST patient's spermatozoas which had different vigor. Haplotype M had stronger vigor, while haplotype N had lower vigor. By sequencing ND3 gene of mtDNA from 50 AST patients, we detected G10310A heteroplasmic mutation in 2 specimens of asthenospermia with poor and moderate motility spermatozoa, respectively. No mutation occurred in good motility spermatozoa. CONCLUSION: Haplotype of mitochondrial may have some correlation with sperm motility. The nt10398G-10400T polymorphisms may have benefit for sperm motility, whereas the mutation in nt10310A may impair sperm motility.
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To produce TEM-116 extended-spectrum beta-lactamase (ESBL) from recombinant bacteria in a cost-effective way, we purified and renatured the recombinant TEM-116 ESBL from the inclusion bodies by Ni(2+)-NTA affinity and gel filtration chromatography through subcloning the bla(TEM-116) into expression vector pET28a(+), transforming into Escherichia coli BL21(DE3) and inducing with IPTG. We characterized the purified protein that had the molecular weight of 30 kDa and specific activity of 476 IU/mg. The recombinant TEM-116 ESBL showed higher efficiency in eliminating penicillin and cephalosporin in vitro and in vivo. Specifically, the recombinant TEM-116 ESBL could eliminate 7000 mg penicillin G (PG) when used at 10.0 IU in 1 L fermentation medium. When used at 320.0 IU, it could also degrade a mix of PG, ampicillin and cefazolin each at 200 mg in 1 L of urine. In milk, 1.0-2.5 IU of the recombinant enzyme could remove 80 U/L of PG. The recombinant enzyme was fully active at the temperature ranged from 4 degrees C to 37 degrees C. Furthermore, the recombinant enzyme used at 2.0x10(4)-2.3x10(4) IU/(kg bw) (body weight) eliminated 8.0x10(4)-9.1x10(4) microg/(kg bw) PG in mouse models in vivo. The recombinant TEM-116 ESBL has the potential as a tool enzyme in food and environmental protection to eliminate harmful residues of antibiotics.
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Animals , Mice , Cephalosporins , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Penicillins , Recombinant Proteins , Genetics , Pharmacology , beta-Lactamases , GeneticsABSTRACT
Objective To predict the CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains, which were prevailing in China. Methods Total of 236 plasma samples were from the 3rd National HIV Molecular Epidemic Survey (NMES3). All the subjects were infected with HIV-1 CRF07_BC viruses. The tat exon 1 region was amplified by reverse transcription reaction and nested polymerase chain reaction (nested-PCR), then the PCR products were sequenced. The distribution of CTL epitopes of this region were predicted by on-line software BIMAS HLA Peptide Binding Predictions and statistics software. Results To-tal of 236 CRF07_BC strains were from 16 provinces, mainly in intravenous drug asers(58.9%)and then sex(25.0%). It was showed that there were 12 CTL epitopes of 236 Tat exon 1 region of CRF07_BC strains mainly located in proline-rich region, cysteine-rich region and core-region. Those epitopes were banded by 5 HLA presenting molecules in genotype(A * 2501 ,A * 2902, B * 15,B * 5301 and Cw * 1203) and 6 HLA presenting molecules in serotype (B53, B58 ,B57 ,A3 ,A68 and Cw12). The frequency of single amino acid substitution was more than 50% in 7 CTL epitopes. Conclusion The CTL epitopes in Tat exon 1 of CRF07 _BC strains were located in different functional regions, and there were some amino acid variations in them.
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Objective To analysis the β-globin gene mutation in β-thalassemia in the population of Wenzhou natives,and identify the major mutation in Wenzhou and further provide valuable information for genetic counseling,prenatal diagnosis and prevention programs in this region.Methods Patients with β-thalassemia were diagnosed and the genomics DNA were extracted from whole blood cells and amplified with PCR,sequenced and compared to the standard sequence.Some mutations were further identified by subcloned.Results 44 of 66 patients were diagnosed β-Thalassemia,9 mutations were found in the 44 sporadic patients with the sequence analysis,2 of which were known polymorphisms(exonl 59,IVS-2-665),3 belonged to the common mutations in Chinese(IVS-2-654,CD_(41/42)-TTCT and TATA box nt-28),2 were scarce abnormalities(CD_(47),CD_(66))and 2 novel variants(-24T→C,CD_(26A)→G,same sense mutation,unreported).Conclusion The mutations of β-globin gene in Han Chinese in Wenzhou are complex (9 mutations found in all),the rare and novel mutations are identified,which provide the valuable information for genetic counseling in Wenzhou.
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Objective To develop and evaluate an aptamer based biosensor (aptasensor) for rapid colorimetric detection of enteropathogenic Escherichia coli (EPEC). Method The aptasensor was fabricated by modifying the truncated LPS-binding aptamer on the surface of nanoscale polydiacetylene vesicles using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between EPEC and aptamer at the interface of the vesicle led to blue-red transition of polydiacetylene which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Transmission electron microscopy (TEM) was used to confirm the specific interactions between EPEC and polydiacetylene vesicles. Result Truncated aptamer showed the similar LPS-binding activity. The aptasensor could detect the target bacteria in a range of 105-108 colony-forming units (CFU)/ml within less than 30 minutes and its specificity was 100% for detection of EPEC O111. The sensor reproducibiliry obtained at 106 CFU/ml was 6. 08% R. S. D. The results of TEM confirmed that the specific interactions between EPEC and polydiacetylene vesicles. Conclusion A new aptasensor was developed successfully for rapid colorimetric detection of EPEC.
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Objective To prenatally diagnose HA fetus with different clinical backgrounds. Methods Genetic tests were performed on 15 gravidas subjected for prenatal diagnosis of HA and different methods were employed for diagnosis according to the gestational weeks and clinical data. Amniotic fluid were taken from pregnant women within 23 gestational weeks for direct genotyping and indirect linkage analysis, since these women had probands with clear-cut mutations. Cordocentesis was performed for linkage analysis in pregnant women over 23 gestational weeks with probands whose types of mutation were unknown, while the FⅧ activity tests were carried out simultaneously. For the pregnant women over 23 gestational weeks without proband, cordocentesis was operated for measurement of FⅧ activity and karyotyping, but carriers of hemophilia A could not be detected in these cases. The introns 22 and 1 inversion of F8 gene were identified by long distance-polymerase chain reaction. Nucleotide sequencing was employed if the gene inversion could not be found and linkage analysis of 7 polymorphic markers, including DXS1108, F8Civs13, INTRON22,DXS1073,DXS9901, DXS15, DXS8069 and sex site (Amelo) were applied eventually. Identification of maternal blood contamination must be done before the tests. Results Fifteen samples were identified without maternal blood contamination. Five fetuses were diagnosed with hemophilia A. Meanwhile there were three pregnant women whose cord blood FⅧ activities were less than 1%. One of them was accompanied by trisomy 21; another had inversion mutation in introns 22 of F8 gene; the remaining one was identified with missense mutation in exon 23 (p. Arg2182Cys) of F8 gene. Conclusions Diverse methods should be applied in prenatal diagnosis of hemophilia A with different clinical backgrounds, for the sake of birth defects prevention.
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Objective To investigate the application value for predicting human cytomegalovirus(HCMV) infection with viral load in urinary epithehal cell (EC).Methods Peripheral blood and urine specimens from 82 infants with HCMV latent infection and 84 infants with HCMV active infection were collected respectively.Plasma HCMV DNA load and the levels of HCMV lgM/IgG antibody were detected by real-time fluorescence quantitative polymernse chain reaction (FQ-PCR) and chemiluminescence immunsassay.HCMV pp65 antigen in peripheral blood polymorphonuclear leukocytes (PMNLs) was detected by indirect immunofluorescence assay.The urinary EC count and HCMV DNA load were detected by UF-100 automated urine sediment analyzer and FQ-PCR,respectively.HCMV DNA load in urinary EC was calculated accordingly.At the same time,the sensitivity and specificity for diagnosis of active HCMV infection were evaluated by receiver operating characteristic curve (ROC).Results The positivity of HCMV DNA in urinary EC was 94.58% (157/166),which was the highest among the urinary EC from 166 cases of HCMV infection.HCMV DNA load ranged from 5.67×102to 1.31×107 copies/103 EC There was no significantly statistical difference among urine specimens from different periods of time(P>0.05).HCMV DNA load in active infection group [5.13±0.99(copies/103EC,lg)]is significantly higher than that in latent infection group [3.92±0.82 (copies/103 EC,lg),t = 8.52,P < 0.01];According to ROC curve analysis,its sensitivity and specificity were 71.4% and 75.2% respectively when cut-off value was 4.55.The virus load was significantly decreased in urinary EC in post-treatment infants as compared with pre-trestment(t=5.44,P<0.01).Conclusion Detection of HCMV DNA load in the urinary EC is a cost-effective method and can be used to predict HCMV active infection in infants and monitor treatment of HCMV infection.
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We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
Subject(s)
Humans , Bacterial Proteins , Genetics , Catalytic Domain , Cysteine Endopeptidases , Genetics , Endopeptidases , Genetics , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Green Fluorescent Proteins , Genetics , Luminescent Proteins , Genetics , Recombinant Fusion Proteins , Genetics , SUMO-1 Protein , GeneticsABSTRACT
Objective To construct quantitative standard for quantification of hepatocyte growth factor (HGF) mRNA and establish its real-time fluorescence quantitative(FQ)-PCR assay to estimate its clinical relevance in lymphoma.Methods Recombinant plasmid Was constructed with target cDNA obtained from isolated total RNA by RT-PCR After PCR products were identified and purified,recombined plasmids were quantitated and then acted as quantitative standard.A new real time FQ-PCR analysis system Was established with the second pair of primers and the probe after amplification condition and the concentrations of components were optimized.HGF mRNA expressions in 47 lymohoma cases[11 Hodgkin disease(HD) cases,36 non-Hodgkin lyphoma(NHL)cases.among these patients,36 patients in remission while 11 patients without remission ] were analyzed quantitatively,and its specificity and sensitivity for lymphoma diagnosis were evaluated by receiptor operation character(ROC)curve method.Results HGF mRNA quantitative standard was constructed successfully.and its real time FO.PCR analysis system Was established combined with hot.start PCR and down.touch PCR technique. According to slope of standard curve (-3.513)and correlation cofficient(0.999),amplification efficiency of the system was 92.6%.Coefficient variation of intra-assay,intra-day and inter-day-assay were 2.1%,4.0% and 6.8%,respectively.Sensitivity of FQ-PCR Was 2 eopies/μl.Expressions of HGF mRNA in lymphoma group Was higher than that in control group(6.425±2.172 and 0.317±0.192,respectively,t=15.883,P<0.001),and its expressions in remission group was lower than no remission group(6.157±1.712 and 7.59l ±1.184,respectively,t=2.768,P<0.05).However,there Was not difference of HGF mRNA level between group HD and group NHL(P>0.05).According to ROC analysis,its sensitivity and specificity were 93.6% and 100% when cutoff value for lymphoma clinical diagnosis Was 3.136.Conclusion HGF mRNA'8 quantitative standard and its real time F9-PCR analysis system have been successfully constructed,and it can be used for quantitative detection of its mRNA expression in lymphoma.