In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.
Artemisia annua ; drug effects ; genetics ; metabolism ; Cadmium ; pharmacology ; Plant Leaves ; drug effects ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; standards ; Reference Standards

Objective To learn the stroke occurrence status and the influencing factors of the patients with type 2 diabetes,and to provide the basis for the prevention of stroke.Methods Patients with type 2 diabetes which were reported in 2011 year from the system of chronic disease monitoring information management of Zhejiang Province were selected and followed -up until October in 2014.Stroke occurrence were recorded and the influencing factors were analyzed.Results The incidence rate was 5.22％ (59/1 129),and the annual incidence was 1.56％.Fifty six cases (94.92％) were ischemic stroke.Logistic regression analysis showed that older than 65 and hypertension were risk factors for type 2 diabetes complicated with stroke.The group of 65-and 75-were associated with higher risk of stroke than 45-(OR =3.38,95％ CI:1.39-8.24;OR =7.77,2.14-28.24).Hypertensionin were the influencing factors of stroke among patients with type 2 diabetes (OR =10.92,95％ CI:5.94-20.09).Conclusion Older age and hypertension were risk factors for type 2 diabetes complicated with stroke.Therefore effective control blood pressure among older patients with type 2 diabetes should be strengthed to prevent stroke.

Objective To analyze the epidemic characteristics of hand-foot-mouth disease (HFMD) in Jiangsu province.Methods We downloaded the case-data of HFMD in Jiangsu province during 2009-2011 from the Chinese National Notifiable Infectious Disease Reporting System,and made a comprehensive analysis on the epidemiological features of it with descriptive epidemiological methods and retrospective space-time permutation scan statistics.Results A total of 285 414 cases were reported in Jiangsu,from 2009 to 2011,with an annual incidence of 122.66 per 100 000.There were 3686 severe cases in the 3 years accumulatively,accounting for 1.29％ of the total.Proportion of the cases being 5 years old or even younger was 93.64％.Scatteredly living children accounted for 64.08％ of the total cases and 78.65％ of the severe cases,respectively.The epidemics of HFMD were visible in each city of Jiangsu province,with a lowest annual incidence rate of 44.02 per 100 000 and a highest one up to 202.90 per 100 000.Regions as Suzhou,Nanjing,Wuxi had the highest incidence in the province,with cases in these three areas occupying almost 40％ of all.The numbers of severe cases in Suqian and in Yancheng cities increased by 339.22％ and 328.33％ in 2011 compared to in 2010,respectively,and the rates of increase in these two cities were much higher than those in the other regions.Two peaks of incidence were observed every year,with the highest occurring between April and June and the second occurring in November.The spatial-temporal distribution of HFMD was not random in Jiangsu province,from 2009 to 2011.Clusters for general cases in August and 7 clusters for severe cases were detected,respectively.12 359 cases of HFMD were laboratory confirmed in the said 3 years,including 10 414 common cases and 1945 severe cases.EV71 and CoxA16 accounted for 43.49％ and 37.07％ of common cases,respectively.In terms of the severe cases,the ratios were 80.82％ and 5.96％,respectively.Conclusion HFMD was highly endemic in Jiangsu province,and the situation of prevention and control for it is still grim.Scatteredly living children of 5 years old or younger were the major population at risk,and the epidemic in different regions and seasons was different.EV71 and CoxA16 were the major etiologic agents,but the etiologic constitute showed seasonal changes.

BACKGROUND/AIMS: Early colorectal (CR) neoplasm can be cured by endoscopic submucosal dissection (ESD), but clinical experience and factors associated with complications from ESD for CR neoplasms in China have not been reported. METHODS: Seventy-eight cases of early CR neoplasm treated with endoscopic resection performed between December 2012 and December 2013 at Beijing Military General Hospital were included. Factors associated with ESD complications and procedure times were evaluated. RESULTS: The en bloc resection rate was 88.5% (69/78), tumor size was 32.1+/-10.7 mm, and procedure time was 71.8+/-49.5 minutes. The major complication was perforation, which occurred in 8.97% of the ESD procedures. Multivariate logistic regression analysis indicated that only tumor size (p=0.022) was associated with ESD perforation. Tumor size (p<0.001) and the non-lifting sign (p=0.017) were independent factors for procedure time, and procedure time (p=0.016) was a key factor for en bloc resection. After a median 10 months (range, 4 to 16) of follow-up, no patients had local recurrence. CONCLUSIONS: This study indicated that ESD is an applicable method for large early CR neoplasm in the colon and rectum. Tumor size and the non-lifting sign might be considerable factors for increased complication rate and procedural time of ESD.
China* ; Colon ; Colorectal Neoplasms* ; Follow-Up Studies ; Hospitals, General ; Humans ; Logistic Models ; Military Personnel ; Rectum ; Recurrence

OBJECTIVETo evaluate the activity and genotype of thiopurine methyltransferase (TPMT) and the concentration of thioguanine nucleotides (TGNs) as parameters for individualizing mercaptopurine (6-MP) therapy.

METHODSErythrocyte TPMT activity was measured by means of radiochemical assay. Sequence specific primer (SSP) PCR and restriction fragment length polymorphism (RFLP) were used to determine the mutations in TPMT genomic DNAs. Erythrocyte TGNs concentration in acute lymphoblastic leukemia (ALL) patients after 6-MP treatment was detected by high-performance liquid chromatography (HPLC).

RESULTSThe erythrocyte TPMT activity in Han population was 16.6 +/- 4.5U/ml pRBCs (man 16.8 +/- 5.0 U/ml pRBCs, woman 16.5 +/- 4.4 U/ml pRBCs), the activity in 8.1% of the samples was lower than 10 U/ml pRBCs. There was no difference for TPMT activity by gender, age, and between healthy donors and ALL patients. For TPMT genotypes, there were 5 cases of TPMT * 2, 4 TPMT * 3A, 6 TPMT * 3B, 10 TPMT * 3C and 5 unknown in 30 subjects who had low TPMT activity. In children with ALL, the TGNs level did show a negative correlation with TPMT activity at diagnosis and 7 approximately 14 days after 6-MP therapy and with WBC count 14 days after the determination of TGNs.

CONCLUSIONDetection of TPMT activity before 6-MP therapy and TGNs level during 6-MP therapy may be helpful for preventing side effects from antipurine metabolic drug overdose, and individualizing 6-MP chemotherapy in children with ALL.

Adolescent ; Adult ; Aged ; Antimetabolites, Antineoplastic ; therapeutic use ; Case-Control Studies ; Child ; Child, Preschool ; Chromatography, High Pressure Liquid ; Erythrocytes ; metabolism ; Female ; Genotype ; Humans ; Male ; Mercaptopurine ; therapeutic use ; Methyltransferases ; genetics ; metabolism ; Middle Aged ; Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; metabolism ; Purine Nucleotides ; metabolism ; Time Factors

OBJECTIVETo construct a gene knock-out mutant of response regulator named RevS in Streptococcus suis serotype 2 virulent strain 05ZYH33, and to investigate the effects of its deletion on the biological characters of this pathogen and the pathogenesis to mice and piglets.

METHODSRecombinant gene knock-out vector consisting of Spc(r) cassette was constructed and flanking was constructed consisting of Spc(r) cassette with flanking homology regions to the RevS genes while the isogenic RevS-deficient mutant was screened by allelic replacement. The effects of RevS deletion on the basic biological characters of 05ZYH33 including growth stability, colonial morphology, haemolysis, Gram staining, growth curve and protein expression were examined in vitro. The mice and piglets were infected with 10(8) CFU wild virulent and mutant isolates.

RESULTSPCR analysis confirmed that the coding genes of RevS were replaced completely by Spc(r) cassette and the basic biological characters of 05ZYH33 did not undergo any apparent change. Balb/c mice infection assay indicated that RevS play a role in the pathogenesis of Streptococcus suis infections, while no remarkable difference was observed in the piglets' pathogenesis infection rates between mutant isolates deltaA05ZYH33 and wild-type isolates 05ZYH33.

CONCLUSIONThe mutant of Streptococcus suis 05ZYH33 response regulator was successfully constructed, while the mutation did not obviously affect the bacterial biological characters, while the knock-out mutant of RevS was shown to be attenuated in pathogenesis to mice and piglets.

Animals ; Bacterial Proteins ; genetics ; Gene Knockout Techniques ; methods ; Mice ; Mice, Inbred BALB C ; Models, Genetic ; Polymerase Chain Reaction ; Streptococcal Infections ; microbiology ; Streptococcus suis ; genetics ; pathogenicity

OBJECTIVETo determine the prevalence of Streptococcus suis and major pathogenic serotypes in middle part of Jiangsu province.

METHODSTonsillar specimens from 303 slaughtered pigs aged 6 to 8 months were investigated for the presence of Streptococcus suis and major pathogenic serotypes by polymerase chain reaction (PCR) method. Bacteriological examination compared with molecular genetics identification for three Streptococcus suis isolates were also done.

RESULTSThe overall carrier rate of Streptococcus suis was up to 88.0%, with the percentages of serotype 1(14), 2(1/2), 7 and 9 were 9.6%, 8.5%, 11.3% and 29.5% respectively in 2005. While in 2006, the prevalence of Streptococcus suis was 82.5%, with capsular types 1 (14), 2 (1/2), 7 and 9 were accounted for 17.6%, 2.4%, 25.8% and 20.0% of all the specimens. All the three isolates belonged to Streptococcus suis serotype 2,named 2a, 2f and 14e, which exhibiting the virulent phenotype cps2+/gdh+/mrp-/lepf-/sly-/fbps+/orf2+/89k-, cps2+/lgdh+/mrp-/epf-/sly-/fbps-/orf2-/89k- and cps2+/gdh+/mrp-/epf-/sly-/fbps/orf2-/ respectively. These isolates were all susceptible to amoxicillin, ampicillin, penicillin and resistant to amikacin and tetraycline. Clinical signs were not noted in BALB/c mice and rabbit.

CONCLUSIONPrevalence of the Streptococcus suis among the healthy herds in the areas was very high, with various capsule types of Streptococcus suis involved in the same herds, and the virulent phenotype of these 3 isolates were very different from those prevalent Streptococcus suis serotype 2 virulent isolates frequently discovered from the epidemic areas.

Amikacin ; therapeutic use ; Amoxicillin ; therapeutic use ; Ampicillin ; therapeutic use ; Animals ; China ; epidemiology ; Mice ; Mice, Inbred BALB C ; Molecular Epidemiology ; methods ; Penicillins ; therapeutic use ; Polymerase Chain Reaction ; Streptococcal Infections ; drug therapy ; epidemiology ; microbiology ; Streptococcus suis ; classification ; drug effects ; genetics ; pathogenicity ; Tetracycline ; therapeutic use ; Virulence

OBJECTIVEIt was noticed that coxsackievirus A16 (CA16) and enterovirus 71 (EV71) were two major etiological agents of hand, foot and mouth disease (HFMD) in children. Recently there were several large outbreaks of HFMD in the Asia-Pacific region, and there was a propensity to cause severe complications or death in children under 5 years of age. The severe forms were associated with EV71 infection. Although epidemics of HFMD have been reported in the mainland of China, few reports about EV71 as the pathogen of HFMD epidemics are available. The present study was conducted to investigate the causal agent of an HFMD epidemic in children in Shanghai from April to June of 2002.

METHODSTotally 102 specimens (including vesicle fluid, stool and throat swabs) were collected from 72 patients with HFMD. The specimens were inoculated into Vero and/or RD cells. At first all the isolates were respectively neutralized by the RIVM pools of enterovirus antiserum, the type-specific antisera to EV71 or to CA16. Secondly all untyped isolates were tested by RT-PCR assay with two specific primer pairs for VP1 genes of EV71 and CA16 respectively. The EV71 and CA16 were identified depending on the size of PCR products. Sequence analyses of VP1 genes of 9 virus strains were performed by the laboratory of China CDC.

RESULTSViruses were isolated from 91 specimens from 67 patients. Serotyping by neutralization failed for all the isolates. But the RT-PCR results indicated that the viruses isolated from 78 specimens from 58 patients were identified as positive for CA16 and the isolates from 13 specimens from 9 patients were identified as positive for EV71, the ratio between CA16 and EV71 was 6.4:1. The results of sequence analyses were consistent with those of PCR assay. Two EV71 strains isolated in this study belonged to a new lineage (C4) within genogroup C. One patient with EV71-associated HFMD had a complication of encephalitis with convulsion, shock, coma and dyspnea.

CONCLUSIONCA16 and EV71 were the primary causes of HFMD during the epidemic. It was the first report of EV71-associated severe encephalitis occurred in patients with HFMD in Shanghai.

Animals ; Cercopithecus aethiops ; Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; diagnosis ; epidemiology ; Disease Outbreaks ; Enterovirus ; isolation & purification ; Enterovirus A, Human ; isolation & purification ; Female ; Genes, Viral ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Vero Cells

OBJECTIVETo explore the pathogenesis of tumors by blocking the normal differentiation process of stem cells.

METHODSBone marrow mesenchymal stem cells (BMSCs) from rats were isolated, cultured and purified by whole bone marrow adherence method. The rat BMSCs were induced to differentiate into adipocytes with dexamethasone, insulin and indomethacin. Blockage of the differentiation process was induced by 3-methylcholanthrene (3-MC).

RESULTSThe differentiation experiment showed that at 30 days after the induction, oil red O staining-positive cells occurred with increased intracytolasmic lipid droplets, characteristic for adipocytes. The differentiation blockage experiment showed that at 30 days after induction, the deposits of oil red O staining-cytoplasmic lipid droplets was significantly reduced, indicating that the blocked cells were adipocytes, but not fully differentiated. Morphological identification showed that cell contact inhibition disappeared, abnormal cell nuclei, increased number of micronucleus aberration and karyotype abnormalities, indicating that malignant transformation of the stem cells occurred after the differentiation blockage.

CONCLUSIONSThe results of this study show a blockage of the differentiation of that stem cells at the intermediate phase, and a tendency of malignant transformation of the stem cells. The results of our study provide new evidence that cancer stem cells may be originated by suppression of stem cell differentiation.

Adipocytes ; cytology ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cell Transformation, Neoplastic ; Cells, Cultured ; Dexamethasone ; pharmacology ; Drug Combinations ; Female ; Indomethacin ; pharmacology ; Insulin ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Methylcholanthrene ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo compare the efficacy between subtotal colectomy with antiperistaltic cecoproctostomy and total colectomy with ileorectal anastomosis (TAC-IRA) for patients with severe refractory slow transit constipation(STC).

METHODSDuring 1999 to 2002, TAC-IRA was the preferred procedure for 20 STC patients in our department. From 2003 to 2005, 17 STC patients underwent subtotal colectomy plus antiperistaltic cecoproctostomy. Clinical data of the two groups were collected and compared retrospectively.

RESULTSThere were no significant differences in basic preoperative clinical data between the two groups. During the follow-up period, the time of daily defecation in the antiperistaltic cecoproctostomy group was less than that of TAC-IRA group (2.4+/-0.9 vs 3.4+/-0.8, P=0.0014), meanwhile the Wexner continence score was significantly lower in the antiperistaltic cecoproctostomy group (4.3+/-1.8 vs 5.8+/-1.9, P=0.0223). Barium enema after subtotal colectomy showed that residual ascending colon and cecum presented a sign of "reservoir".

CONCLUSIONSubtotal colectomy with antiperistaltic cecoproctostomy is a better method for appropriately selected patients with STC than TAC-IRA.

Adult ; Aged ; Anastomosis, Surgical ; Cecum ; surgery ; Colectomy ; methods ; Colon ; surgery ; Constipation ; surgery ; Female ; Humans ; Male ; Middle Aged ; Rectum ; surgery ; Retrospective Studies