Objective To observe the space-time patterns of damaged outer hair cells(OHCs) in rat cochlea at the early stage after exposure to impulse noise. Methods Wistar rats were exposed to 100 emissions of impulse noise (3 seconds interval between each emission) at 154 dB SPL. Four times (10 min, 30 min, 3h and 6h) after the noise exposure, the animals were sacrificed and the organs of Corti were processed for detection of OHC death modes. The apoptotic and necrotic OHCs were distinguished by propidium iodide (PI), a fluorescent probe specifically labeling the nuclear DNA. The specimens were examined under a fluorescence microscope for assessment of OHC damage. Results Nuclear chromatin began to shrink as the chromatin condensed around the nuclear periphery. The peripheral chromatin ring condensed into discrete mass. Chromatin masses appeared to bleb off from the nuclear surface, forming apoptotic bodies at 10 min after the noise exposure. There were a few swollen nuclei appeared 30 min after the noise exposure. Loss of OHC nuclei could be seen 3 h after the noise exposure. The cochlear lesion expanded to contain a large number of missing OHCs and seriously shrunken nuclei at 6 h after the noise exposure. Conclusions The results of the study indicate that death of OHCs takes place extremely rapid after the impulse noise exposure. The apoptosis of OHCs precedes necrosis. OHC apoptosis is a quick process. Most of dead outer hair cells were eliminated 6 h after the noise exposure.

Objective To disclose the implication of nuclear transcription factor(NF)-?B and interleukin(IL-8) in the angiogenesis and prognosis of gastric cancer. Methods The expressions of NF-?B, IL-8 and microvessel density (MVD) in 41 samples of gastric cancer were detected by immunohistochemistry. Results NF-?B and IL-8 were positive in 28 samples (68.3%) and 12 samples (29.3%) respectively. NF-?B, but not IL-8, was related to MVD significantly( P =0.002), and there was significant relationship between the expressions of NF-?B and IL-8 and TNM staging( P

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.

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 study the relationship between tyrosine kinase receptor B (TrkB)expression and anoikis-suppression and invasion in OVCAR3 ovarian cancer ceils. Methods The expression of TrkB mRNA in OVCAR3 ovarian cancer cells under two culture conditions :adhesive cells and ceil-spheroids were evaluated by RT-PCR and real-time PCR.The relationship between TrkB expression and anoikis-suppression of OVCAR3 ovarian cancer ceils was examined by RNA interference (RNAi) technic,anchorage independent culture and fluorescence-activated ceil sorting analysis.The difference in invasion and metastatic ability of OVCAR3 cells under two culture conditions and with or without TrkB silenced by small interfering RNA (siRNA) was investigated by matrigel invasion assay and in vivo studies.Results The expression of TrkB mRNA was highest in OVCAR3 ovarian cancer ceils,0.0240 ~ 0.0017,compared with the other three cell lines,0.0030±0.0006,0.0027±0.0009 and 0.0087±0.0003 respectively,andthe expression in OVCAR3 multicellular spheroids was significantly higher than that in ceils under monolayer adhesive culture,0.0437±0.0021 versus 0.0240±0.0017 (P＜0.01) . TrkB mediated anoikissuppression in OVCAR3 ovarian cancer ceils.OVCAR3 multiceilular spheroids had a higher invasion ability than OVCAR3 cells under monolayer adhesive culture,and the penetrating cells of the two groups were 71.8± 0.8 and 47.7±0.8 respectively (P＜0.01 ).The metastatic ability of OVCAR3 cells was attenuated when TrkB was silenced,and the volume of the tumors developed by OVCAR3 adhesive cells and OVCAR3 adhesive cells with TrkB silenced were (16.3±4.7) mm3 and(6.0±1.4) mm3 respectively (P＜0.01).Conclusion As an anoikis-suppressur,TrkB may increase the invasion and metastasis of OVCAR3 ovarian cancer cells.

Objective:To analyze the epidemiological and etiological characteristics of dengue fever in Hangzhou in 2018.Methods:RT-PCR was used to detect the nucleic acids and analyze the serotypes of dengue viruses (DENV) in serum samples collected from dengue fever cases. Phylogenetic trees based on the E gene sequences of DENV isolated from the serum samples were then constructed and analyzed. Epidemiological characteristics of these dengue fever cases were analyzed. Results:A total of 80 cases of dengue fever were detected in Hangzhou in 2018 with 55 imported cases and 25 indigenous cases (24 caused by DENV-1 and one by DENV-3). These indigenous cases mainly occurred during late July to early October with people above 50 years old accounting for 68%. Phylogenetic analysis showed that DENV-1 strains isolated from the indigenous cases in Yuhang, Jianggan-Shangcheng and Qiantang districts all belonged to genotype Ⅰ, and were respectively closely related to the strains from Indonesia in 2015, Myanmar in 2017, Ningbo in 2018 and Hangzhou imported cases from Thailand in 2018. The indigenous DENV-3 strain belonged to genotype Ⅲ, and shared 99.5% homology with the Singapore strain in 2013.Conclusions:Imported cases accounted for a large fraction of the dengue fever cases in Hangzhou, which brought a high risk to indigenous outbreak. Due to multiple imported cases, the current epidemic presented a characteristic of multiple small-scale outbreaks.

Objective To assess the feasibility of adenoviral vectors mediate cochlear gene transfer by postau-ricular microinjection through the round window membrane in mouse. Methods Twelve 5-week old C57BL/6J mice were selected for the study: 8 were implanted with Ad-EGFP by postauricular microinjection through the round window membrane, and 4 with artificial perilymphatic fluid. On postoperative days 5 and 14, the animals were sac-rificed and the surface preparation of cochleae was observed. Results Two animals died after operation. Bright green fluorescence in the cochleae was observed in Ad- EGFP groups. Gene expression on day 14 after operation was higher than that on day 5. However, the control group was free of fluorescence. Oonclusion The postauricular route of the cochlear gene transfer in mice is simple to operate with little side-effect. The technique of transgenic delivery into the inner ear through RWM by mieroinjection is feasible and effective.

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

Objective To study the epidemiological characteristics and genetic evolution of human bocavirus ( HBoV ) infection in hospitalized children with severe acute respiratory infection ( SARI ) in Hangzhou.Methods A total of 1388 clinical specimens were collected from children with SARI admitted in Affiliated Children' s Hospital, Zhejiang University School of Medicine from January 2011 to December 2014.HBoV1-4 and other respiratory pathogens were identified by fluorescent real -time polymerase chain reaction (fRT-PCR).The VP1 gene in HBoV1 positive samples was amplified and sequenced for genetic analysis with Clustal X and MEGA 6.0.Chi-square test and Fisher exact probability were used to analyze the data.Results Eighty five HBoV positive samples were detected from 1388 samples (6.12％), among which 83 (97.65％) were HBoV1 positive and 2 (2.35％) were HBoV2 positive.The positive rates of HBoV in males and females were 6.54％and 5.35％(χ2 =0.780, P>0.05).The posititve detection rate of HBoV in all age groups was statistically significant (χ2 =47.446,P <0.01).The detection rate in children aged 6 months-1 year was highest (12.84％), in children aged >3 years was lowest (1.64％), in children aged ≤6 months and aged 1-3 years was 3.04％ and 3.33％, respectively.The detection rate of HBoV in summer was the highest (14.97％), followed by that in autumn (7.14％), spring (3.19％) and winter (1.97％) (χ2 =58.807, P<0.01).The detection rates of HBoV in 2011 to 2014 were 7.39％, 7.31％, 5.58％ and 4.72％ (χ2 =3.447, P >0.05 ).The co-infection rate with other respiratory pathogens was 62.35％.The main pathogens were human rhinovirus (33.96％), parainfluenza virus (28.30％) and respiratory syncytial virus (20.75％).The incidence of anhelation and wheezing in HBoV positive group was higher than that in HBoV negative group (χ2 =15.161 and 13.914, P <0.01). Sequence analysis of VP 1 gene showed that 44 isolates belonged to the same branch ( clade 1 ) as Swedish strain ST2, and 2 isolates HZ12-S32 and HZ12-S199 belonged to a separated branch.Conclusion HBoV is an important causative agent of hospitalized children with SARI in Hangzhou area and has high co -infection with other respiratory pathogens.Most of the strains belong to the same clade as the Swedish strain ST 2, and two strains of HZ12-S32 and HZ12-S199 are identified in a separated clade.