Animals ; Cardiomyopathy, Dilated ; Disease Models, Animal ; Mice ; Mice, Transgenic

Suitable pretreatment of biological samples can truly reflect the role of law of the measured components played in the body and will provide experimental evidence for the studies on metabolic process, material basis of efficacy, mechanism of action, pharmacology, toxicology and the others. Biological samples include blood, urine, hair, tears, etc. There are also many samples processing methods, such as the direct protein precipitation, liquid-liquid extraction and solid phase extraction and so on. These methods could be used alone or combined.
Animals ; Body Fluids ; chemistry ; Chemical Precipitation ; Chemistry Techniques, Analytical ; methods ; Humans ; Liquid-Liquid Extraction ; Proteins ; isolation & purification ; Solid Phase Extraction

Objective To observe the nuclear translocation of transcription factor NF-κB and IRF-3 in TLR4 silenced EVC304 cells infected by HTNV and to provide new information for anti-HTNV innate immunity and its signal transduction. Methods TLR4~- cells and TLR4~+ cells were infected by HTNV 76-118, respectively. The cells stimulated by LPS were selected as positive control groups, and the cells without stimulation were selected as negative control groups. After 6 hours, indirect immunofluorescence assay(IFA) was used to detect the nuclear translocation of NF-κB and IRF-3. Results The transcription factor NF-κB and IRF-3 transfered into nuclear 6 hours after stimulated by HTNV 76-118. Conclusion TLR4 may mediate the nuclear translocation of transcription factor NF-κB and IRF-3 in HTNV infected human umbilical vein endothelial cells.

Objective To analyse MMACHC mutations for 45 pedigrees with combined methylmalonic aciduria and homocyctinuria by Sanger sequencing, and to discuss the utility of prenatal genetic diagnosis for these pedigrees.Method Peripheral blood was collected from 45 probands and their parents from 2012-2015 in Genetic Counselling Clinic of the First Affiliated Hospital of Zhengzhou University, and the DNA were extracted from the blood.Then the coding sequence of MMACHC gene was amplified by PCR, and the PCR products were further sequenced to detect mutations for each pedigree.For 12 families, chorionic villus sampling was performed on the pregnant women to make prenatal genetic diagnosis.Result There were 14 distinct mutations detected in the 45 pedigrees, and the most frequent mutations are c.609G>A(W203X),c.658-660delAAG(K220del)and c.80A>G (Q27A).Two of those mutations have not been reported before:one is a splicing site mutation c.81+1G>A;while the other is a missense mutation c.665A>G,p.Y222C.Most mutations were found in exon 4.Among the 12 pedigrees who received prenatal diagnosis, 2 fetuses were normal, 7 fetuses were carriers of heterozygous mutation, and the other 3 fetuses were patients with compound heterozygous mutation or homozygous mutation.The couples whose fetuses were normal or carriers continued the gestation, while the couples whose fetuses were patients decided to terminate the pregnancy.After delivery, the outcome of the fetuses was the same as the prenatal diagnose results.Conclusion Two novel mutations of MMACHC were identified and prenatal genetic diagnosis helps to avoid the delivery of combined methylmalonic aciduria and homocyctinuria patients.

SARS-CoV is a newly discovery pathogen causing severe acute respiratory problems.It has been established that the S protein in this pathogen plays an important rule in the adsorption and penetration of SARS-CoV into the host cell by interaction with the ACE2 receptor.To determinant which functional motif of the S protein was involved in the interaction with ACE2,seven truncated S proteins deleted from the N or C terminal were obtained by an E.coli expression system and purified by column chromatography to homogeneity.Each truncated S protein was fixed on to the well of an ELISA plate and an interaction was initiated with the ACE2 protein.The adsorption were quantified by ELISA,and the results indicated that amino acids from 388 to 496 of the S protein was responsible for the interaction with the ACE2 receptor,and the interaction could be completely disrupted by an antibody specific to these amino acids.Deletions adjacent to this domain did not appear to have a significant impact on the interaction with ACE2,suggesting that the S protein of SARS-CoV could be developed as a vaccine to prevent the spread of SARS-CoV.

Objective To compare aortic root anatomical characteristics between severe aortic valve stenosis (AS) and aortic regurgitation (AR) patients, and to provide useful information for transcatheter aortic valve replacement (TAVR) device designs and procedural techniques for treatment of AR. Methods Consecutive patients admitted between April 2014 to May 2016 with severe AS or AR and planned to undergo transcatheter aortic valve replacement were included. There were a total of 57 AR and 113 AS patients. All patients underwent multi-detector computed tomographic imaging and echocardiography examinations. Results The mean aortic annulus diameter in AR patients was slightly but significantly larger than AS patients[ (26.4±3.7) mm vs. (25.2±2.9) mm, P=0.001]. The mean diameters of the ascending aorta[ (38.3±6.9) mm vs. (33.9±6.7) mm, P<0.001]and Valsalva sinus[ (38.9±6.9) mm vs. (32.7±4.5) mm, P<0.001] in AR patients were larger than in AS patients. The left coronary ostia height was of no significant difference between the 2 groups [ (12.5±3.7) mm vs. (13.4±3.2) mm, P=0.08] and the right coronary ostia height was higher in the AR group than in the AS group [ (17.5±5.0) mm vs. (15.3±3.3) mm, P=0.001]. Conclusions The anatomical aortic root data from patients with AS or AR in the present study may provide useful information for transcatheter aortic valve replacement device designs and procedural techniques for treatment of AR.

BACKGROUND: Cell-free stem cell therapy has been an issue of concern, but there is no conclusion on how to extract high-quality exosomes. OBJECTIVE: To extract exosomes from human umbilical cord mesenchymal stem cells by using three different methods, and then to screen the optimal method. METHODS: Exosomes were extracted from human umbilical cord mesenchymal stem cells by using the Total Exosome Isolation test kit, Exo Quick test kit and differential ultracentrifugation method, respectively. Then, transmission electron microscopy was used for morphological observations, BCA was utilized to quantify the protein, and western blot assay was applied to detect surface markers CD9, CD81 and CD63. RESULTS AND CONCLUSION: Extraction of exosomes was completed by all the three methods, and round or oval membranous vesicles were observed under the transmission electron microscope. The protein content and purity of exosomes was highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group, and there were significant differences among the three groups (P < 0.05). Under the same protein concentration, surface specific markers, CD81, CD63 and CD9, were expressed highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group. The operating time was significantly lower in the Exobiology Quick kit group compared with the other two groups (P < 0.05). To conclude, despite a longer operating time, the differential ultracentrifugation method is a rational method to extract enough exosomes with relative high purity.

Objective To explore the association between Toll-like receptors(TLR) gene polymorphisms and the primary immune response level to Hepatitis B Vaccine in Han children in Guangxi. Methods A total of 513 Han children aged 8-9 months were collected from the department of pediatrics in the Maternal and Child Hospital of Guangxi Zhuang Autonomous Region and Nanning Maternal and Child Health Hospital from 2014 to 2016. Peripheral venous blood of each study object was collected to detect HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc and HBV DNA. The polymorphisms of 10 sites of TLR gene were detected by SNPscanTM multiple SNP typing techniques. The association between allele, genotype of TLR gene and anti-HBs levels were analyzed by non-conditional logistic regression. Results The genetic polymorphism of TLR3 gene rs13126816 was related to immune response after primary Hepatitis B immunization in Han children in Guangxi (OR=1.79，95% CI: 1.11-2.89, P=0.018). The anti-HBs level of children with A/A genotype［238.04(519.75) mIU/L］and G/A genotype［347.96(619.68) mIU/L］were significantly lower than those with G/G genotype［489.08(854.76) mIU/L］, and the differences were statistically significant (all P<0.05). Anti-HBs level of children carrying allele A ［317.20(608.72) mIU/L］was significantly lower than those carrying allele G［457.01(852.66) mIU/L］, and the difference was statistically significant (Z=-3.055， P<0.05). The rest of the TLR genes were not related to the immune response of Hepatitis B Vaccine (all P>0.05). Conclusions The allele A of TLR3 gene rs13126816 may be the influencing factor for the low response of primary immune response to Hepatitis B Vaccine in Han children.

Objective:To analyze losses and gains (L&G) of basic medical institutions induced by the Essen-tial Medicines Policy.Methods: Choosing some poverty-stricken county in western China as sample area to conduct field research,using 2009 as baseline year,to calculate L&G and L&G ratio of basic medical institutions caused by adjustments of drug policy,medical services prices, and government subsidies from 2009—2015. Results: Medical facilities have gained after the implementation of the Essential Medicines Policy as a whole. Gains were on an upward trend from 2009—2015,and L&G ratio increased from -2.15% in 2009 to 47.70% in 2015. For medical facilities at different levels, their gains attributed to different causes. Gains for medical facilities at village and town levels mainly attributed to government subsidies;gains for medical facilities at county level mainly attributed to adjustment of medical services prices. Conclusions:Implementation of the Essential Medicines Policy has helped adjust composi-tion of losses and gains of medical facilities. Moving forward,functions and development of medical facilities should be strengthened with a focus on adjusting medical services prices for medical facilities at town level.

Objective: To investigate the effect of 1q21 amplification (1q) on the therapeutic response and prognosis of bortezomib(Btz) in the treatment of newly diagnosed multiple myeloma (MM) patients. Methods: A total of 180 newly diagnosed MM were included for analyses of clinical characteristics, cytogenetics, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS), retrospectively. Gene expression profiling (GEP) was analyzed using publicly available R2 platform. Results: ① In 180 patients, 1q was found in 51.1% cases. Of them, 174 patients had complete follow-up data, including 88 cases with 1q and 86 without 1q (non-1q). ②Incidence of 1q was positively associated with percentage of IGH rearrangement (72.2%, P=0.017) and 1p deletion (1p) (27.8%, P=0.040). ③ The median PFS was 15.0 and 20.3 months for the 1q group and non-1q group, and the median OS was 29.4 and 44.0 months, respectively. Both PFS and OS of 1q group was significantly shorter than those of the non-1q group (P=0.029 and 0.038, respectively). Multivariate analysis further revealed that 1q was an independent prognostic factor for both PFS (HR=1.910, 95% CI 1.105-3.303, P=0.020) and OS (HR=2.353, 95% CI 1.090-5.078, P=0.029). ④ In 91 evaluable cases with 1q, very good partial remission (VGPR) rate was higher after treatment with Btz than those without Btz (62.1% vs 40.0%, P=0.032). Of note, the patients with 1q who received auto-HSCT after induction with Btz had significantly longer PFS than those without auto-HSCT (19 months vs 13 months, P=0.048). ⑤GEP analysis revealed that 1q21 amplification predominantly up-regulated expression of >50% genes within 1q21 region, and also altered expression of 28% genes in chromosome 1 and 10% genes in whole genome, particularly related to DNA repair and cell cycle. Conclusions: 1q is an independent adverse prognostic factor in patients with newly diagnosed MM. It is often associated with 1p deletion and IGH rearrangement. Patients with 1q respond well to Btz-based regimen, but they fail to gain long-term benefit from this treatment itself. However, auto-HSCT following Btz induction might improve survival of patients with 1q, suggesting a potential strategy to treat this high-risk subset of MM. GEP analysis warrants further attention in understanding the mechanisms underlying the high-risk of 1q.
Bortezomib/therapeutic use* ; Chromosome Aberrations ; Humans ; Multiple Myeloma/drug therapy* ; Prognosis ; Retrospective Studies