Objective To explore the association between fibrinogen gene polymorphism(rs1049636) and serum γ′ fibrinogen level and ischemic stroke (IS) .Methods 421 IS patients and 421 age‐and gender‐ matched healthy controls ,including 283 males and 138 females ,were recruited in this assay .The plasma γ′fibrinogen concentration was measured by enzyme‐linked immunosor‐bent assay (ELISA) .Fibrinogen gene polymorphism(rs1049636) were genotyped by using PCR‐LDR assay .Results γ′fibrinogen concentrations in IS patients[(159 .4 ± 97 .4)U/dL] were significantly higher than that in control group[(114 .2 ± 73 .0)U/dL] with statistically significant difference(P<0 .001) .Single nucleotide polymorphism(SNP) analysis showed that rs1049636 C allele was significantly associated withγ′fibrinogen level ,but not associated with increased risk of IS(P=0 .077) .Conclusion An associ‐ation between increasedγ′fibrinogen level and IS existed in Chinese Han population .However ,no association between rs1049636 C allele and IS risk was observed in our study .

Objective:To explore the clinical significance of combined detection of the promoter methylation of plasma free Septin9, SDC2 and BCAT1 genes in peripheral blood for the diagnosis of colorectal cancer. Methods The data of patients admitted to the Department of Gastroenterology, Shanghai East Hospital Affiliated to Tongji University from January to September 2019 were retrospectively analyzed. They were divided into colorectal cancer group (62 cases of colon cancer, 59 cases of rectal cancer), precancerous lesions group (77 cases of colorectal adenoma, 5 cases of high-grade intraepithelial neoplasia), interference group (61 cases of colorectal cancer and advanced adenoma negative but suffered other intestinal lesions, 17 cases of non-colorectal cancer) and healthy group (94 cases). The methylation status of three genes (Septin9, SDC2 and BCAT1) in peripheral blood plasma was detected simultaneously by fluorescence PCR. The relationship between the positive rate of three genes detected jointly and the clinic pathological characteristics of colorectal cancer was analyzed and compared with serum carcinoembryonic antigen (CEA) positive rate. The colorectal cancer group was divided into stage Ⅰ, Ⅱ, Ⅲ and Ⅳ according to TNM stage, and the colorectal cancer group was analyzed and counted by grade. The diagnostic efficiency of detection methods was analyzed by receiver operating characteristic (ROC) curve, and the area under ROC curve (AUC) was compared.Results:The positive rate of combined detection of SDC2 and BCAT1 gene methylation was higher than other three groups (χ 2 =237.246, P<0.001). The positive rate of combined detection of plasma Septin9, SDC2 and BCAT1 gene methylation was higher than CEA in colorectal cancer group ( P<0.001). The positive rates of the combined detection of plasma Septin9, SDC2 and BCAT1 gene methylation in stage Ⅰ-Ⅳ of colorectal cancer group were 73%(16/22), 87%(34/39), 86%(30/35) and 96%(24/25), respectively. Compared with CEA group, the positive rate of combined detection of plasma Septin9, SDC2 and BCAT1 gene methylation in stage Ⅰ-Ⅲ of colorectal cancer group was higher than serum CEA ( P<0.001), but the positive rate of stage Ⅳ was not statistically significant compared with CEA group ( P>0.05). ROC curve analysis showed that the AUC of Septin9, SDC2 and BCAT1 was 0.857(95% CI 0.810-0.903),0.819(95% CI 0.768-0.871)and 0.862(95% CI 0.816-0.909), respectively. The AUC of combined detection of three gene methylations was 0.889 (95% CI 0.846-0.933), and the AUC of combined detection with serum CEA was 0.913 (95% CI 0.874-0.951). There was no significant difference in the positive rate of combined detection of Septin9, SDC2 and BCAT1 gene methylation among different gender, age and cancerous site of colon cancer patients (all P>0.05). Conclusion:The combined detection of the promoter methylation of plasma free Septin9, SDC2 and BCAT1 genes in peripheral blood plasma is helpful for the early diagnosis of colorectal cancer. The positive rate in stage Ⅰ-Ⅲ of colorectal cancer group is higher than serum CEA. The combined diagnosis of the three genes can improve the diagnostic efficiency.

BACKGROUNDBoth p16(INK4) and p21(Waf1) are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16(INK4) could be activated by p21(Waf1) through transcriptional factor Sp1 in HeLa cells. This study was undertaken to determine the effects of p16(INK4) on the expression and functions of p21(Waf1).

METHODSHuman diploid fibroblast 2BS cells were stably transfected with sense (2BS/p16(INK4)), antisense p16(INK4) (2BS/asp16(INK4)) or empty vector (2BS/neo). Then they were assayed by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence activated cell sorting (FACS) and Western blot.

RESULTS2BS/p16(INK4) cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21(Waf1) protein levels increased twofold in the 2BS/p16(INK4) cells, but not decreased in the 2BS/asp16(INK4) cells. p21(Waf1) mRNA levels were not affected in neither 2BS/p16(INK4) nor 2BS/asp16(INK4) cells.

CONCLUSIONp16(INK4) may play an important role in the regulation of cellular senescence by modulating the p21(Waf1) protein level via the posttranscriptional mechanism.

Cell Cycle ; Cells, Cultured ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Cyclin-Dependent Kinase Inhibitor p21 ; physiology ; Fibroblasts ; metabolism ; Humans ; Transcription, Genetic

OBJECTIVEGas chromatography (GC) was used to investigate the cellular fatty acid (CFA) composition of 141 Acinetobacter baumannii and 32 A. calcoaceticus isolates from different locations in China and to find chemical markers to differentiate these two closely related bacteria.

METHODSWhole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation, and extraction for GC analysis, followed by a standardized Microbial Identification System (MIS) analysis.

RESULTSAll A. baumannii and A. calcoaceticus strains contained some major fatty acids, namely, 18:1 ω9c, 16:0, Sum In Feature 3, 12:0, 17:1ω8c, 3-OH-12:0, 17:0, Sum In Feature 2, 2-OH-12:0, and 18:0 compounds. Although most of the total CFAs are similar between A. baumannii and A. calcoaceticus strains, the ratios of two pairs of CFAs, i.e., Sum In Feature 3/18:1 ω9c versus 16:0/18:1 ω9c and Sum In Feature 3/18:1 ω9c versus unknown 12.484/18:1 ω9c fatty acids, could differentiate these two closely related bacteria. A. baumannii could be easily classified into two subgroups by plotting some ratios such as Sum In Feature 3/16:0 versus 17:0 and Sum In Feature 3/2-OH-12:0 versus 17:0 fatty acids.

CONCLUSIONThe ratios of some CFAs could be used as chemical markers to distinguish A. baumannii from A. calcoaceticus.

Acinetobacter baumannii ; classification ; cytology ; metabolism ; Acinetobacter calcoaceticus ; classification ; cytology ; metabolism ; Biomarkers ; metabolism ; Fatty Acids ; metabolism ; Species Specificity

Objective To analyze the similarities and differences of the clinical manifestations between the children with upper airway resistance syndrome (UARS) and obstructive sleep apnea-hypopnea syndrome (OSAHS), and to explore the clinical features and characteristics of sleep respiratory parameters. Methods Using the double-blind method, all children were diagnosed as UARS or OSAHS through the polysomnography test and the results of all children were analyzed by a sleep technician and an otolaryngologist. Another ENT doctor recorded their clinical and physical examination in detail. Results Polysomnography showed that the apnea-hypopnea index (AHI) and the lowest oxygen in 253 children with OSAHS were 3.60 [ 2. 00 ;7. 55 ] times/h and 0. 90 [ 0. 85 ;0. 91 ], and were 0. 90 [ 0. 50; 1.10 ] times/h and 0. 95 [ 0. 92 ;0. 96 ] in 102 children with UARS, the difference of the two groups by rank test was statistically significant. The proportion of UARS and OSAHS was more common during preschool period than during school-age period. The chief complaint in two groups was sleep snoring, and the main symptoms were sleep restless, attention deficit/hyperactivity and breath with mouth open. The incidence rate of above symptoms were as follows, 94. 1% ,72. 5% ,62. 7% and 37. 3% in children with UARS, 92. 9% ,78. 7% ,57. 7% and 45. 5% in children with OSAHS. The difference was not significant by chi-square test (P >0. 05). Tonsil and adenoid hypertrophy were also observed in the two groups, the difference was not significant ( x2 = 0. 27, P =0. 87). However, the children with OSAHS were more apt to have the sleep apnea than with UARS, the difference was statistically significant (x2 =34.07,P<0.001). Conclusions The clinical manifestations of two groups are similar, the difference between UARS and OSAHS can not be determined by the patient's clinical performance. Sleep apnea can be more easily observed in children with OSAHS than that in UARS, the final diagnosis is based on polysomnography.

Objective To explore the correlation between periodic limb movement index (PLMI) and the apnea-hypopnea index (AHI), apnea index (AI), hypopnea index (HI) and lowest oxygen saturation (LSaO_2) in sixty-four children with sleep-disordered breathing(SDB).Methods Between March 2008 and May 2009, sixty-four children suspected of OSAHS underwent overnight polysomnogram monitoring in our medicine sleep center.OSAHS was diagnosed according to the general criterion.Sixty-four children were divided into two groups.Thirty children were diagnosed as OSAHS and 34 children were diagnosed as primary snoring (PS, 32 children) or upper airway resistance syndrome (UARS, 2 children).The difference of PLMI and periodic limb movement index during sleep associated with arousals (PLMl-arousal) were compared between the two groups.Besides this, the correlation between PLMI, periodic limb movement index during sleep associated with arousals and AHI, AI, HI and LSaO_2 were also analyzed in all SDB children.Furthermore, all SDB children were divided into two groups according to PLMI (< 5 events/h vs ≥5 events/h).AHI, AI, HI, LSaO_2 and sleep structure were compared between the two groups.Results ①The difference of PLMI and PLMI-arousal between the children with OSAHS and children with other SDB types(PS and UARS) were not significant (z value, - 1.279, - 1.490; P value, 0.201,0.136, respectively).② The increased sleep stage I was significant as being compared between the two groups (<5 events/h vs ≥5 events/h, t = -2.16, P <0.05).However, other sleep stages and sleep efficiency were not significantly different (P value, all > 0.05).③ The difference of HI, AI, AHI, arousals index (ArI) and LSaO_2 were not significant between the two groups(<5 events/h vs ≥5 events/h, P value, all > 0.05).④ PLMI and PLMI-arousal were not correlated with AHI, HI, AI, AHI and LSaO_2 (Spearman rank correlation analysis).Conclusions PLMS may be independent of SDB and PLMS had a little influce on sleep structure.

BACKGROUNDPromoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay.

METHODSHuman embryonic lung fibroblast cells (2BS), HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined various factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments.

RESULTSThe sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein (EGFP). Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity.

CONCLUSIONSTo detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

Cells, Cultured ; Genes, Reporter ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Luciferases ; genetics ; Plasmids ; Promoter Regions, Genetic ; Transfection

BACKGROUNDThe accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro.

METHODSThe effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS).

RESULTSAminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD) and increased cumulative population doublings by at least 17 - 21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 micromol/L H(2)O(2) for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 micromol/L H(2)O(2) had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells.

CONCLUSIONAminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, improvement of DNA repair ability and the slowdown of telomere shortening.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cellular Senescence ; drug effects ; DNA Damage ; DNA Repair ; Diploidy ; Dose-Response Relationship, Drug ; Female ; Fibroblasts ; drug effects ; Glycation End Products, Advanced ; analysis ; Guanidines ; pharmacology ; Humans ; Hydrogen Peroxide ; toxicity ; Telomere

BACKGROUNDAstragali Radix, the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1, 3] dioxolan-2, 6'-spirane-5', 6', 7', 8'-tetrahydro-indolizine-3'-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2 strongly delay replicative senescence of human fetal lung diploid fibroblasts (2BS). In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence.

METHODSThe effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants.

RESULTSThere was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings (PDs) grown from PD28 in 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of p16 was observed. The expression level of p21 increased with cell ageing. Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 micromol/L H2O2 for 5 minutes return to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 (1.0 micromol/L ) or HDTIC-2 (10 micromol/L ) for 1 hour.

CONCLUSIONSExpression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16(INK4a)/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.

Antioxidants ; pharmacology ; Astragalus Plant ; chemistry ; Cells, Cultured ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; analysis ; genetics ; Dioxolanes ; pharmacology ; Female ; Fibroblasts ; chemistry ; drug effects ; metabolism ; Humans ; Indolizines ; pharmacology ; Plant Roots ; chemistry ; RNA, Messenger ; analysis

Objective To analyse the clinical features of children with obstructive sleep apneahypopnea syndrome(OSAHS),accompanying with risk factors.Methods The clinic data of 19 patients treated in the Department of Otorhinolaryngology of Guangzhou Children's Hospital between January 2005 to January 2008 were investigated retrospectively.Among them,5 were＜2 years old,6 with craniofacial deformity:small mandible and(or)mandibular retrusion(5 cases),transverse facial cleft(1 case),Down's syndrome(2 cases),cerebral palsy(2 cases),chronic bronchitis(3 cases)and mucopolysaccharidoses(1 case).Nineteen patients with symptoms of snoring, mouth breathing,were diagnosised as OSAHS by polysemnography(PSG)and treated by tonsillectomy and(or)adenoidectomy in hospital.All patients were closely followed-up.Results Fourteen patients underwent PSG 6 months to 1 year after operation,11 patients recovered,the median [percentiles 25;percentiles 75]apnea-hypopnea index(AHI)decreased from the pre-operative 22.5[16.5;24.3]times/h to 2.0[1.5;4.3]times/h,and the lowest oxygen saturation(LSaO2)before operation was 0.63,and was higher than 0.92 after operation,1 case accompanying with chronic bronchitis,the pulmonary hypertension was improved after operation.One case with Down's syndrome wag not significantly impmved,preoperative AHI and LSaO2 was 22.4 times/h and 0.67,and after operation was 14.2 and 0.84;2 cases accepted adenoidectomy only,snoring,mouth breathing reappeared 3 mornths after operation.pre-operative PSG results showed AHI 24.6 times/h and 26.6 times/h,LSaO2 was 0.69 and 0.73,after operation the AHI was 10.6 times/h and 8.5 times/h,LSaO2 was 0.90 and 0.88,the symptoms disappeared after adenotonsillectomy.Five cases did not have PSG because they lived far away in the other cities,their pre-operative PSG showed AHI 16.4 to 26.2 times/h,LSaO2 was 0.65 to 0.76.One year after operation,these patients were followed-up by telephone,4 children were significantly improved,1 case with mandibular symptoms showed no improvement.Conclusions For OSAHS children accompanying with risk factors,if they have adenoid and tonsil hypertrophy,adenotonsillectomy is the major treatment.Because of the existence of risk factors,perioperative risk increased,even the failure of operation.so these patients must be comprehensively assessed before operation.Satisfied results Can be achieved by close observation after operation and management of complications as soon as possible.