Mechanical ventilation (MV) with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses,resulting in ventilator-induced lung injury (VILI).The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear.VILI-related genes such as cysteine-rich angiogenic inducer 61 (Cyr61)have been demonstrated to play a detrimental role in the aggressive ventilation strategies.In the present study,we investigated the involvement of Cyr61 in the VIM and the underlying mechanism.A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting,respectively.Additionally,after exposure ofA549 cells to cyclic stretch for 5 min to 1 h,the expression levels of nuclear factor kappaB (NF-κB) and IL-8 were detected by ELISA and Western blotting.Thereafter,Cyr61 expression was depressed in A549 cells with the siRNA pGenesill.1-Cyr61-3 before the cyclic stretch,and IL-8 secretion and the activation of NF-κB pathways were probed by ELISA and Western blotting,respectively.Moreover,a NF-κB inhibitor (PDTC) and an activator (TNF) were used before mechanical stretch.Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8,respectively.The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells.Additionally,cyclic stretch also mobilized NF-κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells.The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch.Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA.It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells.These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

Objective To construct the small interfering RNA (siRNA) targeting heparanase gene and its expressing vector,and to observe its interference effect on the expression of heparanase gene and inhibitory effect on the invasive potential of human malignant melanoma A375 cells.Methods Three siRNAs were designed.The recombinant plasmid pRNATU6.1/heparanase-siRNA was designed and constructed. A375 cells were cultured,and transfected with pRNATU6.1/heparanase-siRNA.The cells treated with lipo- fectamine or Opti-MEM served as the controls.Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein in these treated A375 cells.The in vitro invasive potential of treated A375 cells was assessed by Matrigel gel assay.Results The siRNA targeting heparanase gene was successfully cloned to the eukaryotic expressing vector pRNATU6.1.The expression levels of both heparanase RNA and protein decreased significantly in siRNA-transfected A375 cells than those in the control cells.The in vitro invasive potential of siRNA-transfected cells was also signifi- cantly inhibited as compared with that of the control cells (P

This study was purposed to investigate the intercellular cell adhesion molecule-1 (ICAM-1) gene K469E (A/G) (rs5498) and K56M (A/T) (rs5491) single nucleotide polymorphisms (SNP) and soluble ICAM-1 (sICAM-1) levels in plasma in three Chinese populations of Yugur, Tibetan and Han nationalities, to analyze comparatively the genotypes and allele frequencies distribution in different ethnic groups, and to explore the effects of ICAM-1 K469E and K56M polymorphism and sICAM-1 levels in plasma. EDTA-anticoagulant venous blood from Yugur(327 cases), Tibetan (400 cases) and Han (126 cases) people was collected, the DNA was extracted by using whole blood genomic DNA extraction kit, DNA SNP were analyzed by PCR-RFLP, genotype was judged by gel scan imaging system after agarose gel electrophoresis, the gene sequence was determined and the distribution of ICAM-1 genotypes and allele frequencies were compared among different ethnic groups, besides, the group representativeness was tested via the Hardy-Weinberg genetic equilibrium. Finally, the human sICAM-1 plasma levels were detected by using human ICAM-1 ELISA kit. The results showed that DNA sequencing result was consistent with PCR-RFLP analysis. In Yugur, Tibetan and Han nationalities, the KK, KE and EE three genotypes at ICAM-1 K469E gene locus were detected, the genotype distribution was not statistically significantly different, while the K, E allele frequency distribution was statistically significantly different (P < 0.05). Both of genotype and allele frequency distribution between Yugur, Tibetan and Han nationalities were statistically significantly different (P < 0.05). In K56M site only KK, KM two genotypes were detected, but the MM genotype was not detected in the three ethnic groups; the difference of two genotypes and K, M allele frequencies between Yugur and Han population was statistically significantly different (P < 0.05). Among three ethnic groups, the sex ratio and age distribution of K469E, K56M genotypes and allele frequencies of ICAM-1 gene were not significantly different, and distribution was in accordance with Hardy-Weinberg genetic equilibrium (P > 0.05). The plasma sICAM-1 level at ICAM-1 K469E allele locus in K individuals [(253 ± 122), (185 ± 97) µg/L] was higher than that at non-K allele [(145 ± 110) µg/L, P < 0.01]; the plasma sICAM-1 level of ICAM-1 K56M sites with KK genotype [(253 ± 122) µg/L] was higher than that of the KM genotypes [(168 ± 103) µg/L, P < 0.01]. In Yugur and Tibetan groups, the plasma sICAM-1 levels [(224 ± 80), (214 ± 111) µg/L] were higher than that in the Han group [(175 ± 125)µg/L, P < 0.05]. Pairwise comparison indicated that the plasma sICAM-1 levels between Yugur and Han group were statistically significantly different (P < 0.01), that was significantly different between Tibetan and Han group (P < 0.05). It is concluded that in Yugur, Tibetan and Han population, the genotypes and gene frequencies of two amino acid sites K469E and K56M in ICAM-1 were KK/KE-type, KK-type and K allele, moreover, the ratio of them in Yugur and Tibetan group was higher than that in Han, while there is not significant difference in sex ratio and age distribution, therefore, ICAM-1 genotype and allele frequency distribution in this study had ethnic representativeness. ICAM-1 gene K469E and K56M polymorphisms were likely to affect the plasma sICAM-1 expression level. K469E gene K allele may be a genetic risk factor, while K56M gene M allele a may be genetic protective factor for some diseases.
Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Intercellular Adhesion Molecule-1 ; blood ; genetics ; Male ; Middle Aged ; Plasma ; metabolism ; Polymorphism, Genetic ; Young Adult

The aim of this study was to investigate the similarities and differences of A1381T (rs216311) and -1793G/C (rs7966230) single nucleotide polymorphisms (SNP) in Chinese Yugur, Tibetan, and Han nationalities and their influence on plasma vWF concentration in order to explore the sensitivity of these 3 nationalities to vWF-related diseases. Peripheral venous blood was obtained from 322 Yugur, 399 Tibetan, and 120 Han healthy people. The DNA were then extracted. vWF gene A1381T and -1793G/C polymorphisms were analyzed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequenced when it was necessary. The vWF:Ag level in plasma was determined by ELISA. The results showed that the genotype distribution of vWF gene at both A1381T and -1793G/C loci in Yugur, Tibetan and Han nationalities was different with statistically significance (P < 0.05). GG genotype of A1381T locus accounted for 69.9% in Yugur nationality, which was much higher than 56.6% and 53.3% in Tibetan and Han nationalities respectively(P < 0.01); AA genotype of A1381T locus expressed a low level of vWF in plasma. For the -1793G/C locus, the proportion of CG genotype in Yugur was much higher than that in Han, CC genotype expressed a high level of vWF in plasma. The plasma vWF levels with different nationalities and the polymorphism of vWF gene were significantly different. It is concluded that the polymorphisms of vWF gene at both A1381T and -1793G/C loci in Yugur, Tibetan and Han are significantly different; the polymorphism of vWF gene influences the plasma vWF level; the plasma vWF levels in Yugur and Tibetan are significantly higher than that in Han, which may be associated with the living environment and habits.
Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; China ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Plasma ; chemistry ; Polymorphism, Genetic ; Young Adult ; von Willebrand Factor ; genetics ; metabolism

OBJECTIVEIn order to understand the epidemiological and virologic characteristics of coronavirus HKU1 infection in hospitalized children with acute respiratory tract infection (ARTI) in Changsha.

METHODS1165 nasopharyngeal aspirates (NPA) specimens were collected from hospitalized children with ARTI between September 2007 and August 2008 in Changsha. Specimens were screened for pol gene of coronavirus HKU1 by polymerase chain reaction. All positive amplification products were confirmed by sequencing and compared with those in GenBank.

RESULTSCoronavirus HKU1 were detected in 12 patients (1.03%) out of the 1165 children. The patients were from 8 days to 3 years. The most common clinical diagnosis was bronchopneumonia(83.33%). Similarity of coronavirus HKU1 with those published in the GenBank at nucleotide levels was 98.18% - 100%.

CONCLUSIONCoronavirus HKU1 may be important pathogens in children with acute lower respiratory tract infection. Coronavirus HKU1 infections are common in children under 3 years old. There is no significant difference in the infectious rate between the boys and the girls. The peak of its prevalence is in spring and winter. A single genetic lineage of Coronavirus HKU1 was revealed in human subjects in Changsha.

Acute Disease ; Child, Hospitalized ; Child, Preschool ; China ; Coronavirus ; classification ; genetics ; isolation & purification ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Phylogeny ; Polymerase Chain Reaction ; Respiratory Tract Infections ; virology

OBJECTIVETo explore the viral etiology of acute low respiratory tract infection (ALRTI) among hospitalized children in Changsha of Hunan Province of China.

METHODSNasopharyngeal aspirates were collected from 1165 hospitalized children with ALRTI in Changsha from September 2007 to August 2008. Respiratory syncytin virus (RSV), human rhinovirus (HRV), influenza virus A (IFVA), influenza virus B (IFVB), parainfluenza 1-3 (PIV 1-3), human metapneumovirus (hMPV), human coronaviruses NL63 (HCoV-NL63), and human coronaviruses HKU1 (HCoV-HKU1) were detected by reverse transcription polymerase chain reaction (RT-PCR). Adenovirus (ADV) and human bocavirus (HBoV) were detected by standard polymerase chain reaction (PCR). WU polyomaviruses (WUPyV) and KI polyomaviruses(KIPyV) were detected by nested PCR. The positive samples further underwent genetic sequencing.

RESULTSAmong the 1165 nasopharyngeal aspirates, viruses were detected in 871 samples (74.76%), among which RSV (27.03%) was the most common virus, followed by HRV (17.33%), PIV3 (13.73%), HBoV (8.67%) and hMPV (6.52%). The overall positive rate of viral detection showed no significant differences between males and females (X2=2.241, P=0.134), whereas the positive rates of PIV3, hMPV, and HBoV in males were higher than in females. The positive rate of viral detection showed significant differences among different age groups (X2=10.934, P=0.027), and the highest positive rate was noted in the age group of 6 months to 1 year. Furthermore, the overall positive rate of viral detection showed a significant difference in term of seasonal distribution, with a peak prevalence in winter.

CONCLUSIONSVirues predominate in the etiology of pediatric ALRTI in Changsha, and RSV, HRV and PIV3 are the main viruses for ALRTI. HBoV and hMPV have become increasingly important. Viral infection-associated ALRTI shows a prevail in the age group of 6 months to 1 year as well as in winter.

Adolescent ; Age Distribution ; Child ; Child, Hospitalized ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Nasopharynx ; virology ; Respiratory Tract Infections ; etiology ; virology ; Seasons ; Sex Distribution ; Viruses ; isolation & purification

OBJECTIVETo establish an inductively coupled plasma mass spectrometry(ICP-MS) method for determination of 30 trace elements including As, Ba, Be, Bi, Ni, Cd, Co, Cr, Cs, Cu, Ga, Mn, Pb, Sr, Tl, V, Ge, Mo, Nb, Ti, W, Te, Se, Zr, In, Sb, Hg, Ce, La, and Sm in human blood.

METHODThe blood samples were analyzed by ICP-MS after diluted 1/10 with 0.01% Triton-X-100 and 0.5% nitric acid solution. Y, Rh and Lu were selected as internal standard in order to correct the matrix interference of Cr, As, Se, and Hg by a hex pole-based collision-reaction cell. Other elements were determined with standard method. The limits of detection, precision and accuracy of the method were evaluated. The accuracy was validated by the determination of the whole blood reference material.

RESULTSAll the 30 trace elements have good linearity in their determination range, with the correlation coefficient > 0.9999. The limits of detection of the 30 trace elements were in the range of 1.19 - 2.15 µg/L and the intra-precision and inter-precision (relative standard deviation, RSD) were less than 14.3% (except Hg RSD < 21.2%, and Ni RSD < 15.4%). The spiked recovery for all elements fell within 59.3% - 119.2%. Among the 13 whole blood reference materials, V, Cr, Mn, Co, Ni, Cu, As, Se, Cd, Te, and Pb (1.45, 1.19, 18.40, 0.18, 1.57, 591.00, 2.97, 61.00, 0.35, 1.86, and 9.70 µg/L respectively) fell within the acceptable range and the detection results of Hg (0.59 µg/L) and Mo (1.59 µg/L) were slightly beyond the range.

CONCLUSIONThis method was simple, fast and effective. It can be used to monitor the multi-elementary concentration in human blood.

Humans ; Limit of Detection ; Mass Spectrometry ; methods ; Metalloids ; blood ; Metals ; blood ; Trace Elements ; blood

INTRODUCTIONKnowledge of stroke symptoms is associated with seeking medical attention early, and knowledge of risk factors is an essential factor in stroke prevention. In this study, we evaluated the level of stroke literacy in Singapore.

MATERIALS AND METHODSA cross-sectional study of Singapore citizens and permanent residents aged 21 years and above was conducted in a public housing estate. Participants were randomly sampled using multi-stage stratified sampling. Assessment of awareness of stroke symptoms and risk factors was performed using open-ended questions. In total, 687 respondents were recruited, with a response rate of 69.7%.

RESULTSOverall, 52.4% of respondents identified the brain as the source of pathology, and 47.6% could cite at least 1 of the 3 FAST symptoms (facial droop, arm weakness and speech difficulty), while 40% could name 2 or more of 7 established risk factors for stroke (high blood pressure, high cholesterol, cigarette smoking, diabetes mellitus, older age, previous heart attacks and stroke). Respondents at higher risk of stroke (older individuals and those with stroke risk factors) did not have greater awareness of stroke symptoms and risk factors. The majority of respondents reported they would seek immediate medical care if they experienced stroke symptoms. Only 59.4% knew the emergency ambulance service telephone number.

CONCLUSIONIn a sample of Singaporean adults residing in a public housing estate, we found evidence of poor stroke literacy, highlighting the need for comprehensive population-based education efforts. There is a role for opportunistic education among those at higher risk of stroke.

Adult ; Aged ; Cross-Sectional Studies ; Female ; Health Literacy ; Humans ; Male ; Middle Aged ; Public Housing ; Risk Factors ; Singapore ; Socioeconomic Factors ; Stroke ; Surveys and Questionnaires ; Young Adult

The aim of current study was to detect intron 22 inversion of factor VIII gene in severe hemophilia A (HA) patients and screen the carriers of the gene inversion. Fifty-five cases of severe HA were involved and factor VIII gene inversion was detected and identified by long distance-PCR (LD-PCR) and 0.6% agarose gel electrophoresis. The 11 kb and 12 kb bands indicate the factor VIII gene inversion and non-inversion, respectively. Occurring of both 11 kb and 12 kb bands indicates a carrier of the inversion. The results showed that factor VIII gene inversion existed in 22 out of 55 cases, which accounted for about 40% of total detected patients. Five carriers of factor VIII gene inversion were diagnosed from the members in 15 families. In conclusion, LD-PCR assay is a simple, rapid and accurate method for detection of factor VIII gene inversion, and this approach is helpful in screening, carrier testing, and prenatal diagnosis of severe hemophilia A.
Adolescent ; Adult ; Antigens ; analysis ; Child ; Child, Preschool ; Chromosome Inversion ; Factor VIII ; genetics ; Hemophilia A ; blood ; genetics ; Humans ; Infant ; Male ; Polymerase Chain Reaction ; methods ; von Willebrand Factor ; immunology

AIM:To investigate the mechanism of angiotensinⅡ(AngⅡ)/angiotensinⅡ type 1 receptor (AT1R)pathway activating protein phosphatase 2A(PP2A)which leads to down-regulation endothelial nitric oxide syn-thase(eNOS)phosphorylation level in mesenteric arteries of rats.METHODS: METHODS: The mesenteric arteries of adult male SD rats(weighing 160~180 g;n=90)were isolated under aseptic conditions.Firstly,to determine the effect of angiotensinⅡdown-regulated eNOS(Ser1177)phosphorylation level,the mesenteric arteries were randomly divided into normal control(control)group and AngⅡgroup.The mesenteric arteries in AngⅡgroup were incubated with AngⅡat 1×10 -7mol/L,1×10 -6mol/L and 1×10 -5mol/L for 6 h,12 h and 24 h,respectively.Secondly,to investigate the mo-lecular mechanism by which angiotensinⅡ activated PP2A leading to down-regulation eNOS(Ser1177)phosphorylation level,the mesenteric arteries were randomly divided into control group, AngⅡ group and candesartan(CAN; a specific AT1R blocker)+AngⅡgroup.The mesenteric arteries were pretreated with 1×10 -5mol /L CAN for 1 h,then incubated with 1×10 -7mol/L AngⅡfor 12 h in CAN+AngⅡgroup.The protein levels of eNOS,p-eNOS(Ser1177),PP2Ac,p-PP2Ac(Tyr307)and protein phosphatase 2A inhibitor 2(IPP2A2 )in the arteries were determined by Western blot.The ac-tivity of PP2A in the arteries was detected by PP2A activity kit.RESULTS:Compared with the control group,the protein level of p-eNOS(Ser1177)in the mesenteric arteries was decreased after incubated with AngⅡfor 6 h,12 h and 24 h(P<0.05).The decreasing tendency of p-eNOS(Ser1177)showed concentration-dependently,especially in 12 h and 24 h groups.The expression of eNOS protein showed no significant difference in each group.Compared with the control group, the mesenteric arteries of the rats were incubated with AngⅡ at 1×10-7mol/L for 12 h in vitro, the protein levels of p-eNOS(Ser1177)were down-regulated(P<0.05); pretreatment with CAN significantly increased the protein level of p-eNOS(Ser1177)(P<0.05);the protein levels of eNOS showed no significant difference in each group.Compared with the control group,the protein levels of p-PP2Ac(Tyr307)and IPP2A2 were decreased after the mesenteric arteries were trea-ted with AngⅡat 1×10 -7mol/L for 12 h(P<0.05).Candesartan pretreatment restored the protein levels of p-PP2Ac (Tyr307)and IPP2A2 (P<0.05),however the expression of PP2Ac protein showed no significant difference in each group. Compared with the control group,the activity of PP2A was increased in the mesenteric arteries incubated with AngⅡat 1× 10-7mol/L for 12 h(P<0.05).Candesarten pretreatment inhibited the activity of PP 2A significantly(P<0.05).CON-CLUSION:AngⅡincreases PP2A activity via AT1R pathway,thus leading to down-regulation eNOS(Ser1177)phospho-rylation level in mesenteric arteries.The molecular mechanism of PP2A activation may be associated with decreasing the protein levels of p-PP2Ac(Tyr307)and IPP2A2.