Objective:To investigate the expression of a new cytokine,thymic stromal lymphopoietin (TSLP) and its receptor TSLPR in the villi of human first trimester gestation.Methods:Villi were collected from women who had undergone an artificial abortion at 7-11 weeks of normal gestation.The trophoblast cells (Tros) were isolated and cultured.The total RNA was extracted using TRIzol reagent,from both villi and the Percoll-gradient-isolated Tros,then the DNA fragments of hTSLP and hTSLPR were amplificated by RT-PCR.Villous tissues were detected for TSLP by immunohistochemistry (IHC) and Western blot.Immunocytochemistry (ICC) was carried out on cultured trophoblast cells for TSLP/TSLPR expression.Levels of TSLP in the supernatants were detected by ELISA.Results:Normal villi and the cultured Tros transcript were found to express TSLP/TSLPR mRNA and secreted TSLP protein.In addition,TSLP receptor was also expressed on trophoblasts.Conclusion:Both TSLP and its receptor are expressed on villi and trophoblast cells,which suggests that TSLP plays an important role in maternal-fetal immuno-tolerance in human early pregnancy.

Objective To investigate the occurrence law of autophagy in trophoblast cells from preeclampsia and its underlying mechanisms.Methods Twenty cases of placenta tissues were collected from women suffered from preeclampsia and normal pregnant women respectively.Autophagosome of trophoblast cells were observed by transmission electron microscope.The expressions of LC3-Ⅱ/Ⅰ and Atg4B in placenta tissues were detected by western blot and real-time PCR.Results Compared with the control group,typical autophagosomes of trophoblast cells were observed by transmission electron microscope.The ratio of LC3-Ⅱ / Ⅰ in placenta of PE patients was increased (1.43 ± 0.23) compared with control group (0.59 ±0.12),and the expression of Atg4B was up-regulated in both mRNA [(1.73 ±0.16) folds] and protein levels (0.71 ± 0.13) compared with control group (P ＜ 0.05).Conclusions Autophagy was significantly up-regulated in trophoblast cells from patients suffered from preeclampsia.Thus,all the data suggest that autophagy might be involved in the generation of preeclampsia.

Objective To detect adenovirus from children with acute upper/lower respiratory tract infections and to investigate the genetic evolution of virus .Methods A total of 1 178 clinical specimens were collected from the Children ’ s Hospital , Zhejiang University School of Medicine during March 2011 and February 2013, including 513 throat swabs from children with acute upper respiratory tract infection and 665 nasopharyngeal aspirates from children with acute lower respiratory tract infections .Besides, 9 specimens in an outbreak of adenovirus infection during 2011 and 2014 were also collected .Adenovirus was identified by real-time fluorescent polymerase chain reaction (RT-qPCR).The hypervariable region (HVR)-7 region of hexon gene in positive samples was amplified and sequenced for typing and phylogenetic analysis .Other respiratory viruses were also detected with RT-qPCR in adenovirus positive samples .Clinical characteristics of adenovirus infection were analyzed in children with lower respiratory tract infections .Chi-square test and Fisher exact probability were used for data analysis .Results Among 1 178 samples from sporadic cases , 104 samples (8.83%) were adenovirus positive .The rates of adenovirus infection in upper respiratory tract infection group and lower respiratory tract infection group were 13.65%(70/513) and 5.11%(34/665), respectively (χ2 =26.193, P<0.05).Compared with that in lower respiratory tract infections , positive rates of adenovirus were higher in upper respiratory tract infections in children aged 0-1 year and >3 years (χ2 =6.575 and 7.334, P<0.05 or <0.01).Adenovirus infection might occur throughout the year and peaked in spring and summer .Among 54 adenovirus-positive samples from 104 sporadic cases , adenovirus types 1, 2, 3, 4, 7 and 31 were identified in 4, 6, 26, 2, 15 cases and 1 case, respectively.While among 9 adenovirus-positive samples from outbreak cases , type 4 and type 3 were identified in 1 and 8 cases, respectively .The HVR-7 region of hexon gene was highly homologous in the same type , and the sequence alignment indicated that the sequence of HVR-7 might have regional differences .Nine out of 70 children (12.86%) were co-infected with other virus in upper respiratory tract infection group , while the rate of co-infection in lower respiratory tract infection group was much higher [58.82%(20/34), χ2 =24.045, P<0.05 ].There was no significant difference in clinical manifestations between children infected with adenovirus only and those with co-infection in lower respiratory tract infection group (P>0.05), but two children with co-infection died.Conclusions Adenovirus infection is more common in upper respiratory tract infection .Adenovirus type 3 and type 7 are the most prevalent serotypes in sporadic cases , while type 3 is the most prevalent serotype in outbreak cases .

Objective To investigate the genetic variation and molecular evolution of human bo-cavirus 1 (HBoV1) strains isolated during 2009 to 2014 in Hangzhou, China.Methods Throat swab sam-ples were collected from children with acute respiratory tract infections in the Children′s Hospital Affiliated to the Zhejiang University School of Medicine from 2009 to 2014.Real-time PCR was performed for the detec-tion of HBoV1 strains.Fifteen HBoV1 strains with high virus load were screened out for the amplification and sequencing of complete genomes.The complete genomes were submitted to GenBank for further analysis with bioinformatics software.Results A total of 48 nucleotide mutations were detected in the complete genomes of 15 HBoV1 strains, resulting in 11 amino acid mutations with 5 of them located in the active region of phospholipase A2 ( PLA2) .The 15 HBoV1 isolates along with 16 HBoV1 strains in GenBank were classified into three clusters as indicated by the phylogenetic analysis based on their complete coding sequences.All of the 15 strains were belonged to clusterⅠ, the representative strain of which was the Sweden prototype strain ST2.The phylogenetic trees constructed using genes encoding the capsid proteins VP1 and VP2 were highly similar to those based on the complete coding sequences.The estimated mean evolutionary rate of HBoV1 with regard to the complete coding sequence was 3.03×10-4(95%HPD, 2.14×10-4-3.92×10-4 ) substitu-tions per site per year.With regard to each gene, the NS1 gene was considered to the most conserved gene while the NP1 gene showed the highest substitution rate.The dN/dS ratios (ω) of the four genes were all less than 1, indicating that all of them were under negative selection.Moreover, the VP2 gene was under the strongest negative selection, while the NP1 gene was under the weakest negative selection.Conclusion All of the HBoV1 isolates circulating in Hangzhou province during 2009 to 2014 were belonged to ST2 genotype with a relatively high mutation in the area of PLA2.Despite the complete genome was conservative, its evo-lutionary rate was high.Among the four genes, the NP1 gene showed the highest substitution rate.All of the four genes were under negative selection, of which the VP2 gene was under the strongest negative selection.

Objective To evaluate the value of real-time ultrasonic elastography in the differential diagnosis of rectal tumors.Methods One hundred patients (30 cases of adenoma and 70 cases of adenocarcinoma) with rectal tumors proved by pathology and 70 normal subjects referred to the First Affiliated Hospital of China Medical University from April 2012 to October 2013 were included in this prospective cohort. All patients underwent real-time ultrasonic elastography. The tumour tissue and reference tissue were chosen for strain ratio measurements. Only tumor tissue was selected for the tumor sample area. Tissue recognized as normal rectal wall and perirectal tissue was selected as the reference sample area. At the same time the elasticity score were calculated. Receiver operating characteristic (ROC) curves of the elasticity score and strain ratio method were used to find the cut-off point of adenoma and adenocarcinoma. The corresponding sensitivity, accuracy and specifi city were calculated. One-way ANOVA was used to comparestrain ratio value among healthy control group, retal adenoma group and rectal adenocarcinoma group and among patients with different preoperative stages of adenocarcinoma group. LSD-t test was used to compare strain ratio value between two groups.Results ROC curve showed that the best cut-off value of elasticity score in diagnosis of rectal tumor was 3, the sensitivity, specificity and accuracy were 85.1%, 73.1% and 82.0%, and the area under ROC curve was 0.780. The best cut-off value of strain ratio in diagnosis of rectal tumour was 2.34, the sensitivity, specifi city and accuracy in diagnosis were 91.4%, 83.3% and 89.0%, and the area under ROC curve was 0.945. Strain ratio of healthy control group, rectal adenoma group and rectal adenocarcinoma group was 0.74±0.44, 1.75±0.58 and 7.48±6.80. There was signifi cantly statistical difference among three groups in strain ratio. Compared with the strain ratio of healthy control group and rectal adenoma group, that of rectal adenocarcinoma group was higher (t=-8.26, P=0.000; t=-6.98,P=0.000). Compared with the strain ratio of healthy control group, that of rectal adenoma group was higher (t=-8.53,P=0.000). Strain ratio of patients with preoperative pathological stages T1, T2, T3 and T4 rectal carcinoma was 4.91±3.60, 7.07±7.23, 8.64±2.62 and 8.58±9.95 and there was no significantly statistical difference among patients with different preoperative stages of adenocarcinoma group (F=0.86,P=0.47).Conclusion The real-time ultrasonic elastography is a promising modalityfor the differential diagnosis of rectal tumors.

We investigated the genetic diversity and evolution of the M gene of human influenza A viruses in Hangzhou (Zhejiang province, China) from 2009 to 2013, including subtypes of A(H1N1) pdm09 strains and seasonal A(H3N2) strains. Subtypes of analyzed viruses were identified by cell culture and real-time reverse transcription-polymerase chain reaction, followed by cloning, sequencing and phylogenetic analyses of the M gene. Assessment of 5675 throat swabs revealed a positive rate for the influenza virus of 20.46%, and 827 cases were diagnosed as. infections due to influenza A viruses. Seventy-six influenza-A strains were selected randomly from nine stages during six phases of a virus epidemic. Sequences of the M gene showed high homology among six epidemics with identities of amino-acid sequences of 98.98-100%. All strains contained the adamantine-resistant mutation S31N in its M2 protein. Two of the A(H1N1)pdm09 strains had double mutants of V27A/S31N or V271/S31N. One of the seasonal A(H3N2) viruses had another form of double-mutant R45H/S31N. Evolutionary rate of the M gene was much lower than that of the HA gene and NA gene. Compared with A(H3N2) strains, higher positive pressure on the M1 and M2 proteins of A(H1N1) pdm09 viruses was observed. Separate analyses of M1 and M2 proteins revealed very different selection pressures. Knowledge of the genetic diversity and evolution of the M gene of human influenza-A viruses will be valuable for the control and prevention of diseases.
Amino Acid Substitution ; China ; epidemiology ; Evolution, Molecular ; Genetic Variation ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Phylogeny ; Selection, Genetic ; Viral Matrix Proteins ; genetics ; Viral Proteins ; chemistry ; genetics

Objective To establish the tissue culture system of Cynanchum otophylum in genetic transformation. Methods Asepsis seedling was set up by using different explant parts, disinfectors, and disinfecting time. The callus was induced, breeded, and differentiated by using different media, different hormone categories and combinations, and different hormone concentrations of 2,4-D and KT. Results It was the best method that asepsis seeding was built up by using seed as the explant and using 10% NaClO to treat the seed for 20—30 min or using 0.2% HgCl2 to treat the stem for 3 min. The seed, root, stem, and leaf of C. otophylum can form callus easily. The 2,4-D was important in forming callus. C. otophylum was more sensitivity to KT. The effect was better when KT was used to induce callus or adventitious buds. MS+2,4-D 1.0 mg/L+KT 1.0 mg/L was the better medium to induce callus to form. The callus cant be induced to form adventitous buds without reference to using seed or root, stem, and leaf as the explant, without reference to using hormone 6-BA or KT, ZT, 2ip. Gentamicin 100 ?g/mL is the optimum pressure in genetic transformation of C. otophylum. Conclusion The seed or stem is the ideal material bombarded by particle gun that is induced to form callus for 14 d in the media MS+2, 4-D 1.0 mg/L+KT 1.0 mg/L and without forming adventitious buds.

Objective The abnormal ankle-brachial index ( ABI) is associated with the incidence of cardiocerebral vascular diseases, but little is known about its relationship with cerebral microbleeds (CMB).This study aimed to investigate the correlation be-tween ABI≤0.9 and different distribution patterns of CMB . Methods We enrolled 187 patients with acute lacunar infarction , inclu-ding 115 non-CMB cases and 72 CMB cases (20 strictly lobar, 24 strictly deep, and 28 lobar and deep).We analyzed the differences between the two groups and the association of abnormal ABI with the occurrence and distribution of CMB by logistic regression analysis . Results ABI≤0.9 was found in 57 (30.5%) of the patients, with a significantly higher incidence rate in the CMB group than in the non-CMB group (43.1%vs 22.6%, P=0.003).The level of ABI was negatively correlated with the number of CMBs (r=-0.211, P=0.006).Multivariate logistic regression analysis after adjusted for confounders indicated that ABI ≤0.9 was significantly associated with the presence of CMB (OR=2.363;95%CI:1.181-4.729), deep CMB (OR=3.434;95%CI:1.283-9.187), and lobar and deep CMB ( OR=2.837;95%CI:1.098-7.333) in patients with ischemic cerebrovascular disease . Conclusion Decreased ABI is a risk factor of CMB, particularly deep CMB, in patients with ischemic stroke.

Objective To investigate the risk factors for reduced renal function in patients with ischemic stroke.Methods The medical records of patients with ischemic stroke were analyzed retrospectively.They were divided into normal renal function group and reduced renalfunction group.Reduced renal function was defined as estimated glomerular filtration rate (eGFR) ＜60 ml/(min·1.73 m2).Multivariate logistic regression analysis was used to identify the risk factors for reduced renal function in patients with ischemic stroke.Results A total of 805 patients with ischemic stroke were enrolled in the study.8.8％ of patients had a reduced renal function.There was no significant differences in the proportion of patients with mild and moderate neurological deficit between the reduced renal function group and the normal renal function group (all P ＞ 0.05),however,the proportion of patients with severe neurological deficit was significantly higher than that in the normal renal function group (8.4％vs.2.6％,x2 =5.573,P =0.017).The proportion of small artery occlusion in the reduced renal function group was sigaificantly higher than that in the normal renal function group (66.2％ vs.46.5％,x2 =9.962,P =0.002),and the proportion of large artery atherosclerosis was significantly lower than that in the normal renal function group (19.7％ vs.43.5％,x2 =15.045,P =0.000).Multivariate logistic regression analysis indicated that old age (odds ratio [ OR] 3.301,95％ confidence interval [ CI],1.575 to 6.918; P=0.002) was the most important independent risk factor for reduced renal function,then was female (OR,2.291,95％ CI 1.355to 3.872; P=0.002) and hyperlipidemia (OR,2.527,95％ CI 1.095 to 5.831; P=0.030).Conclusions Reduced renal function in patients with ischemic stroke is strongly associated with old age,female,and hyperlipidemia.

OBJECTIVETo determine the level of genetic variation of human parainfluenza virus type 3 (HPIV-3), and to describe infection and co-infection characteristics of HPIV-3 in children.

METHODSSingle respiratory samples from 856 pediatric patients with acute respiratory tract infection (ARI) in Hangzhou were collected from December 2009 to March 2013. All samples were screened for HPIV-3 by real-time RT-PCR and followed by HN sequencing and phylogenetic analysis. In all RSV positive specimens, we screened for the other pathogens, and co-infection characteristics were evaluated.

RESULTSA total of 9.6% of 856 samples were positive for HPIV-3, the nucleotide among the strains ranged from 96.9% to 100%. All Hangzhou strains were placed in C3 subgroup based on HN gene analysis. 49% (n=41) of all HPIV-3-positive children with ARI were found to be co-infected with at least one of the other pathogen. The highest co-infection rate of HPIV-3 was with HRV (n=17). Children in the younger groups (≤12 months old) were significantly more prone to be co-infected with other pathogen (χ(2)=4.78, P=0.029). Pneumonia infection rate was significantly higher in the mono-infection group than the co-infection group (χ(2)=3.92, P=0.048).

CONCLUSIONHPIV-3 was an important pathogen in children with ARI in Hangzhou. HN gene variation rate was low, but showed a more local pattern. The co-infections with other respiratory viruses were popular. Except for pneumonia, no significant differences in other clinical presentation between the HPIV-3 mono-infection and co-infection groups were observed.

Child ; China ; epidemiology ; Genetic Variation ; Humans ; Parainfluenza Virus 3, Human ; Phylogeny ; Real-Time Polymerase Chain Reaction ; Respiratory Tract Infections ; epidemiology ; virology ; Respirovirus Infections ; epidemiology