Objective:To retrospectively analyze the molecular epidemiological features and genetic recombination of coxsackievirus A4 (CVA4) strains isolated in Jiangsu from 2015 to 2022.Methods:Throat or anal swab samples were collected from patients with herpangina or hand, foot and mouth disease (HFMD). Real-time PCR was used to detect CVA4. A comprehensive and systematic phylogenetic analysis was conducted based on 72 whole genomes and 99 VP1 sequences of CVA4 strains. Several bioinformatics software including DNAStar, MEGA7.0 and Similarity plots3.5.1 was used for analysis of homology, genetic recombination and amino acid variation sites.Results:Four genotypes (A, B, C and D) and five sub-genotypes (C1-C5) of CVA4 were identified based on the VP1 nucleotide sequences. C2 was the predominant sub-genotype causing HFMD. The Jiangsu strains showed high homology with the CVA4 prototype in the P1 region, and higher identity with other strains of enterovirus group A (EV-A) in the P2 and P3 regions. Genetic recombination analysis revealed that the Jiangsu strains had three genetic recombination patterns with other EV-A epidemic strains in the P2, P3 and 3′-UTR regions. These recombination patterns took place during the sustained and widespread circulation of CVA4 in people and increased the transmissibility of CVA4.Conclusions:This study analyzes the phylogenetic and molecular features of 28 whole genomes of Jiangsu CVA4 strains, which helps to better understand the genomic diversity of CVA4. By analyzing the genetic recombination and amino acid mutations in the VP1 region, this study elucidates the evolution and transmission of CVA4, which is conducive to the control and prevention of CVA4 infection.

Objective:To analyze the whole-genome sequences of coxsackievirus A10 (CVA10) strains isolated in Jiangsu Province from 2015 to 2022 and their molecular epidemiological characteristics.Methods:Forty-five CVA10 isolates circulating in Jiangsu Province during 2015 to 2022 were selected for whole-genome sequencing. Phylogenetic trees were constructed based on the whole genome, VP1, P1, P2 and P3 sequences of CVA10 strains. Bioinformatics software, including DNAStar, MEGA7.0 and Similarity plots3.5.1, was used for analysis of homology, genetic recombination and major amino acid variation sites.Results:The nucleotide and amino acid sequence homology of the whole-genome sequences of 45 CVA10 strains was 90.3%-99.1% and 97.9%-99.8%, respectively. The nucleotide sequence homology of P1 region was the highest (92.1%-100.0%), while the nucleotide sequence homology of P3 region ranged from 84.7% to 100.0%. In contrast to the diversity of nucleotide sequences, the amino acid sequences of each region were conserved. A phylogenetic analysis based on the complete VP1 sequences of CVA10 strains revealed eight genotypes: A-H. The CVA10 isolates in Jiangsu Province and other prevalent strains in China mainly belonged to genogroup C. Results of the phylogenetic analysis based on the whole-genome sequences and complete VP1 sequences were consistent. Phylogenetic analysis bases on different gene segments and Simplot recombination analysis revealed that Jiangsu isolates GD07/Lianyungang/2017 and N180/Suqian/2016 showed high homology with the CVA10 prototype in the P1 region, but had recombination sites with other strains of enterovirus group A in the P2, P3, 5′-UTR and 3′-UTR regions. Compared with the prototype strain AY421767/Kowalik/2004, the Jiangsu isolates showed frequent variations in the VP1 region and many other major amino acid sites, which might result in some imperceptible changes in capsid structure and potential receptor-binding sites.Conclusions:By analyzing the evolution and genetic recombination features of CVA10 strains at the genome level in Jiangsu Province, this study elucidated the influence of genetic recombination and amino acid site mutation on CVA10 infection, providing basic data for the prevention and control of hand-foot-mouth disease in Jiangsu Province.

OBJECTIVE To explore the protective effect of 2,3,5,4’-tetrahydroxy stilbene-2-Ο-β-D-glucosid(TSG) on hearing injury in acetaminophen(APAP) induced guinea pigs. METHODS The ear injury of guinea pigs was induced by APAP, after treatment with TSG, the auditory brainstem response(ABR) and distortion product otoacoustic emission(DPOAE) of guinea pigs were tested to judge the hearing loss, the injury of cochlear hair cells was observed by immunofluorescence, and the liver and kidney tissues of guinea pigs were observed by pathology to judge the toxicity of TSG to liver and kidney. The damage of HEI-OC1 auditory nerve cells induced by APAP was used as the study of ear injury cells. The cell survival and apoptosis were detected by MTT method, cell morphology observation, and flow cytometry, the expression of Birc5 gene was detected by fluorescence quantitative PCR. RESULTS Compared with the model group, on the 14th day, the ABR hearing threshold of guinea pigs decreased significantly, the difference was more significant when the DPOAE test frequency was 2 kHz(P<0.01). The acoustic startle reflex test showed that the amplitude of startle reflex decreased significantly at 100 dB(P<0.05). The gap-prepulse inhibition experiment showed that the amplitude of startle reflex decreased at 10, 40, 50 ms. The guinea pig cochlea was peeled off and the integrity of hair cells was observed after immunofluorescence staining. Compared with the model group, the outer hair cells in TSG group were arranged orderly without loss, and the inner hair cells were basically arranged orderly. Pathological observation showed that TSG had certain hepatotoxicity, but the nephrotoxicity was not obvious. The cell experiment found that the half inhibitory concentration of APAP on HEI-OC1 auditory nerve cells was 800 µmol·L-1. When 10 µmol·L-1 TSG intervened in APAP induced cell injury, the apoptosis rate decreased to 16.0%, which was significantly lower than 25.5% in model(APAP) group. In APAP group, the expression level of Birc5 in HEI-OC1 cells decreased significantly, and TSG treatment could significantly increase the expression level of Birc5. CONCLUSION TSG 15 mg·kg-1 can protect guinea pigs from hearing loss induce by 100 mg·kg-1APAP for 14 d. The protective effect is related to TSG inhibiting APAP induces auditory nerve cell apoptosis and promoting the expression of apoptosis inhibitor Birc5.

Gallic Acid/pharmacology* ; Humans ; Senotherapeutics ; Stem Cells

Objective The prognostic role of PD-L1 expression in surgically resected lung adenocarcinoma(ADC)remains controversial. The aim of present study was to investigate the prognostic value of PD-L1 combined with CD8 +TILs expression in pa-tients with resectable lung adenocarcinoma. Methods A total of 104 patients with lung adenocarcinoma who underwent radical resec-tion were enrolled at the Affiliated Tumor Hospital of Harbin Medical University from January 2009 to December 2012. Immunohisto-chemistry was used to detect the expression of PD-L1 and CD8 +TILs in primary tumor specimens. Its clinical pathological factors and its relationship with prognosis were statistical analysis. Results The positive expression rate of PD -L1 in tumor cells was 38. 5% (40/104),and the positive rate of CD8 +TILs was 44. 2% (46/104). There was a significant correlation between the expres-sion of CD8 +TILs and TNM stages of patients with resectable ADC. The Kaplan-Meier survival analysis showed that low expression of PD-L1 and high expression of CD8 +TILs were significantly associated with disease -free survival ( DFS) and overall survival ( OS),and could be used as an independent predictors for predicting prognosis of patients with resectable ADC. Conclusion The ex-pression of PD-L1 and CD8 +TILs in the postoperative specimens can help to judge the prognosis of patients. This study has certain significance for immunotherapy of PD-1/PD-L1 pathway in patients with surgically resected ADC.

Objective: To analyze the molecular epidemiology, genetic variations and evolution of enterovirus 71 (EV71) strains isolated in Jiangsu Province from 2009 to 2018. Methods: Statistical methods were used to analyze the data about epidemiological characteristics and results of pathogen detection in cases with EV71 infection in Jiangsu Province from 2009 to 2018. The complete VP1 sequences of 80 EV71 strains were amplified and sequenced for analysis of diversity and phylogenesis. Results: A total of 41 858 enterovirus-positive hand, foot and mouth disease cases were reported in Jiangsu Province from 2009 to 2018. EV71 was the predominant pathogen, accounting for 36.52%, and responsible for most of the severe cases. However, the percentage of EV71 among all pathogens gradually decreased over time. EV71 infection reached the peak in April to June and mainly occurred in children aged six months to five years old with higher incidence in males than in females. In terms of regional distribution, EV71 infections were characterized by area clustering in Jiangsu Province, mainly detected in Nanjing, Suzhou, Wuxi and Lianyungang. The 80 EV71 isolates belonged to C4a genotype. Nucleotide differences between them and three vaccine strains (H07, FY23 and FY7VP5)were 0.6%-5.5%, 0.8%-5.7% and 1.9%-6.9% and amino acid difference were 0-1.4%, 0.3%-2.0% and 0.3%-2.0%, respectively. Amino acid mutations in the epitopes of the 80 EV71 strains did not marked by years or regions. Conclusions EV71 strains showed obvious epidemiological characteristics in time, population and regional distribution in Jiangsu Province from 2009 to 2018.All of the 80 EV71 isolates belonged to C4a subgenotype. The nucleotide sequences between them and the vaccine strains varied greatly, but the homology of amino acids was relatively high, indicating the existence of some synonymous mutations and no risk of antigenic drift. This study would provide reference for EV71 vaccination in Jiangsu Province.

Objective: To construct evaluation index system of training effectiveness for new recruiting nurses based on the theory of Kirkpatrick Model. Methods: The draft of evaluation index system of training effectiveness for new recruiting nurses was drawn up through literature survey and interview method first, and then it was constructed by using Delphi method and statistical analysis. Results: Through two rounds of expert consultations, the experts′ suggestions were revised and summarized, and then the evaluation index system was constructed, including 4 grade I indexes, 14 grade Ⅱindexes, and 56 grade Ⅲ indexes. The positive coefficients of the two rounds of expert consultation were 96.15% and 100.00%, respectively, with authoritative coefficient of 0.877. The Kendall Harmony Coefficients of importance and operability of grade I, Ⅱ, and Ⅲ indexes were 0.640, 0.378, 0.296, and 0.707, 0.515, 0.416, respectively (P< 0.01). Conclusions The enthusiasm, authority level and coordination degree of the experts were all at a high level. The evaluation index system of training effectiveness for new recruiting nurses was of high importance and operability.

Objective To determine the volume reduction of the primary olfactory cortex (POC) in patients with Alzheimer disease (AD) and investigate the potential relationship of functional olfactory activation and anatomical atrophy changes. Methods Twelve patients with AD, eight patients with mild cognitive impairment (MCI) and twenty normal controls (NC) underwent standardized UPSIT (University of Pennsylvania smell identification test) behavior smell test and neuropsychological tests. Then all of the subjects underwent the high resolution MRI and an olfactory fMRI scan on a 3T system. Volumetric measurement of the POC was conducted and the areas were also saved as a ROI which would be used during the processing of fMRI data to get the activation voxels in local region. The Kruskal-Wallis rank test was used to examine the significance of POC volume and activation in three groups, If P-value was less than 0.05,Bonferroni method was used for multiple comparisons. The correlation between the anatomical volume and functional activation was analyzed with partial correlation adjusted for age. Results The POC volume of NC, MCI and AD groups were 3024--4734, 1409--4553 and 1561--3759 mm~3, and the medians were 3749, 2752 and 2156 mm~3. The activation voxels of each group were 0--2360, 0--2160 and 0--100 mm~3, while the medians were 430, 40 and 0 mm~3. There were significant differences of both POC volume atrophy and activation between the three groups, with a trend of reduction from NC to MCI to AD group (H is 14.942 and 16.587, both P<0.05). The volume of olfactory activation at POC was highly correlated with the volume of POC (r=0.364, P=0.023). Conclusions In this study, we explored the morphological and functional changes in the POC. It is revealed that POC suffers prominent local atrophy and dysfunction as well as hippocampus in AD. These results can provide neuropathological and neurofunctional bases for olfactory deficit in Alzheimer Disease.

Objective Corneal endothelial decompensation is caused by many corneal diseases. It often results in severe clinical complications. Endothelial keratoplasty (EK) is a new therapy for corneal endothelial decompensation. This study aimed to investigate a new approach to establishing corneal endothelial decompensation animal model with Descemetorhexis technique in order to better understand the tissue response to EK. Methods Thirty New Zealand white rabbits were randomly divided into three groups according to different surgical procedures; corneal endothelial cells (CEC), Descemet's membrane and corneal endothelial cells (DM + CEC) as well as Descemet' s stripping with endothelial keratoplasty(DSEK) group and 10 eyes for each. The right eyes of rabbits were as surgery eyes. Other 10 rabbits were as DSEK donors. Corneal transparency, anterior chamber response and graft location were examined once per day for two weeks under the slit lamp. Comeal thickness was measured by ultrasound biomicroscope. Corneal endothelial cells were analyzed using vital staining with alizarin red and trypan blue in 2, 4 and 8 weeks after operation. Results The cornea in DM + CEC group remained opaque throughout the observation period. In CEC and DSEK group, corneal clarity was gradually restored and corneal thickness was significantly less than that in the DM + CEC group during the postoperative 8 weeks. There were significant differences in corneal thickness between the DM + CEC group and CEC group or DSEK group during the postoperative 8 weeks (P ＜0. 05). The vital staining showed that most Descemetorhexis area was not covered by endothelial cells even 2 months after surgery. Conclusion A new corneal endothelial decompensation model is successfully established for the study of corneal endothelial keratoplasty, which is helpful for understanding the wound-healing of rabbit corneal endothelium after Descemel' s membrane damage.

Acupuncture and Tuina plus functional training were used to treat forty cases of shoulder impingement syndrome. Twenty-seven cases were cured clinically, accounting for 67.5%; 9 cases had marked effectiveness, accounting for 22.5%; 3 cases had effectiveness, accounting for 7.5%; 1 case obtained no effect, accounting for 2.5%; the total effective rate was 97.5%.