Coxsackievirus A16(CVA16);VP1 sequence;Isolation;Identification;Evolutionary analysis;Genetic characteristics Objective To analyze the genetic characteristics of the entire VP1 gene of Coxsackievirus A16(CVA16) strains isolated from the feces of patients with hand,foot and mouth disease(HFMD) in Yunnan Province in 2019.Methods The virus was isolated from human embryonic lung diploid fibroblast(KMB-17) cells and African green monkey kidney(Vero)cells,and the primers for the complete VP1 gene sequence of CVA16 were designed.The target fragment was amplified by RT-PCR and sequenced;the complete VP1 sequence was analyzed by softwares such as MEGA 7.0 and Geneious 9.0.2.Results A total of 26 CVA16 strains were isolated,including eight KMB-17 isolates and 18 Vero isolates.Twenty CVA16isolates were randomly selected for analysis,and three isolates were found to have Bla and 17 B1b genotypes;the nucleotide and amino acid homology of 17 CVA16 B1b isolates were 93.8%—100% and 98.3%—100%,and the nucleotide and amino acid homology with other domestic isolates was 91.1 %—99.2% and 97.3%—99.0%,respectively;the nucleotide and amino acid homology of the three Bla isolates was 98.0%—98.1% and 99.3%,and those with other domestic Bla isolates was 88.7%—98.1% and 98.3%—99.7%,respectively;17 B1b isolates and other three Bla isolates showed the nucleotide and amino acid homology of 87.4%—88.4% and 97.3%—98.7%.Conclusion The CVA16 prevalent in Kunming in 2019 belonged to Bla and B1b genotypes,with B1b as the main strain,and all of them were prevalent strains in the mainland of China.

ObjectiveTo compare the susceptibility of human rhabdomyosarcoma RD cells,human embryonic lung diploid KMB-17 cells and Vero cells to enterovirus(EV)isolation and culture,in order to provide experimental basis for subsequent isolation of EV genotypes.MethodsSixty stool samples of patients with hand-foot-mouth disease(HFMD)diagnosed in a hospital in Kunming in 2019 were collected,and EV strains were isolated and cultured with RD,KMB-17 and Vero cells respectively. The positive samples were detected by RT-PCR,sequenced and blasted by using NCBI BLAST software to identify EV genotypes.ResultsA total of 112 EV strains were isolated,belonging to 11 genotypes respectively. Among them,26strains of Echo-11,6 strains of Echo-6 and Echo-30,2 strains of CV-A4 and CV-A10,1 strain of CV-A6,CV-A16 and Echo-18 were isolated from RD cells;42 strains of Echo-11,3 strains of Echo-6 and 2 strains of Echo-30 were isolated from KMB-17 cells;6 strains of CV-B1,5 strains of CV-B3,4 strains of Echo-11,3 strains of CV-A16 and 1 strain of CV-B2were isolated from Vero cells.ConclusionRD cells were susceptible to most EV genotypes,KMB-17 and Vero cells were the most susceptible to Echo virus and CV-B virus,respectively.

Objective To analyze the genetic characteristics of the entire VP1 gene of Coxsackievirus A16(CVA16) strains isolated from the feces of patients with hand,foot and mouth disease(HFMD) in Yunnan Province in 2019.Methods The virus was isolated from human embryonic lung diploid fibroblast(KMB-17) cells and African green monkey kidney(Vero)cells,and the primers for the complete VP1 gene sequence of CVA16 were designed.The target fragment was amplified by RT-PCR and sequenced;the complete VP1 sequence was analyzed by softwares such as MEGA 7.0 and Geneious 9.0.2.Results A total of 26 CVA16 strains were isolated,including eight KMB-17 isolates and 18 Vero isolates.Twenty CVA16isolates were randomly selected for analysis,and three isolates were found to have Bla and 17 B1b genotypes;the nucleotide and amino acid homology of 17 CVA16 B1b isolates were 93.8%—100% and 98.3%—100%,and the nucleotide and amino acid homology with other domestic isolates was 91.1 %—99.2% and 97.3%—99.0%,respectively;the nucleotide and amino acid homology of the three Bla isolates was 98.0%—98.1% and 99.3%,and those with other domestic Bla isolates was 88.7%—98.1% and 98.3%—99.7%,respectively;17 B1b isolates and other three Bla isolates showed the nucleotide and amino acid homology of 87.4%—88.4% and 97.3%—98.7%.Conclusion The CVA16 prevalent in Kunming in 2019 belonged to Bla and B1b genotypes,with B1b as the main strain,and all of them were prevalent strains in the mainland of China.

Objective To sequence the whole genome and analyze the genetic characteristics of an echovirus 11(E-11)strain isolated from stool samples of patients with hand, foot and mouth disease(HFMD) in Kunming, Yunnan Province in2019, so as to provide a reference for the prevention and control of E-11.Methods The virus was isolated using Vero cells,the RNA from cytopathic products was extracted for RT-PCR and sequenced, and the whole genome sequence was analyzed by software such as MEGA 7.0, Geneious 10.0 and Simplot 3.5.1.Results The 19V30322/YN/CHN/2019 isolate was sequenced and determined to be E-11, which belongs to the D5 subtype of the same genotype D as the E-11 epidemic strain in Guangdong and Hubei, China in recent years, with a full length of 7 434 nt and encoding a polyprotein of 2 195 amino acids. 19V30322 showed 81. 8%-98. 3% nucleotide identity and 94. 5%-99. 3% amino acid similarity with the domestic isolate E-11, and recombined with Coxsackievirus A10, E-6, Coxsackievirus B4 and Coxsackievirus B5 during the evolutionary process.Conclusion The 19V3/YN/CHN/2019 isolate is the D5 subtype of E-11 and is a recombinant strain.

Objective To study the clinical value of ultrasonic calcifications in differential diagnosis of thyroid papillary carcinoma in Hashimoto''s thyroiditis background,to provide basis for clinical diagnosis and treatment.Methods The clinical data of 800 patientss with thyroid nodules who were examined by pathological examination were retrospectively analyzed.All patients underwent ultrasound examination,180 cases with Hashimoto''s thyroiditis were included in the study group,620 cases without Hashimoto''s thyroiditis were included in the control group.The gold standard was the result of pathology.The value of ultrasonography and calcifications for the identification of Hashimoto''s thyroiditis and thyroid papillary carcinoma was compared.Results The average diameter of the study group was (10.78±2.16)mm,which of the control group was (11.98±3.25)mm,there was no significant difference(t=2.153,P=0.083).The accurate rate of ultrasound in the differential diagnosis of benign thyroid nodules and papillary thyroid carcinoma in the study group was 76.67％(138/180),which in the control group was 80.81％(501/620),the difference was not statistically significant(χ2=0.898,P=0.098).There was no significant difference in coarse calcification rate and micro calcification rate of malignant nodules between the two groups(χ2=0.558,P=0.175).In the study group,the rate of micro calcification in malignant nodules was 12.07％,which was significantly higher than 53.13％ in benign nodules(χ2=32.142,P=0.001).In the control group,the micro calcification rate and coarse calcification rate of malignant nodules were 49.28％ and 12.08％,respectively,which were higher than 19.13％ and 6.05％ of benign nodules (χ2=67.368,7.056,P=0.001,0.009).The coarse calcification rate of benign nodules and malignant nodules in the study group were 6.25％ and 11.21％,respectively,there was no significant difference(χ2=1.098,P=0.062).Conclusion Whether associated with Hashimoto''s thyroiditis has no influence in the ultrasound differential diagnosis of thyroid papillary carcinoma,more micro calcification occurs in malignant nodules,but without the background of Hashimoto''s thyroiditis,coarse calcification can also be seen in malignant nodules.

[Summary] Pruritus is one kind of skin disease that has itching symptom with no primary skin lesions , may develop excoriation , crust and pigmentation secondary . Pruritus is a common skin complication affecting on patients with diabetes ,which is also one of the clues of early diagnosis .This article focus on the etiology ,pathogenesis ,clinical manifestation ,prevention and treatment of diabetes patients complicated with skin pruritus .

Objective To study the effect of high frequency stimulation (HFS) in pedunculopontine nucleus (PPN) on the neuronal activities of globus pallidus internus (Gpi) in Parkinson’s disease (PD) model rats, and the mechanisms there-of. Methods Seventy male Sprague-Dawley rats were divided into two groups, control group (n=30) and PD model group (n=40). PD rat model was established by the injection of 6-OHDA into substantia nigra pars compacta (SNc) on the right side of the brain with stereotactic technique. Electrophysiological recordings were made in anaesthetized rats to investigate the ef-fects of HFS-PPN on the firing rate of the GPi neurons. Brain microdialysis combined with high-performance liquid chroma-tography was applied to detect glutamate (Glu) andγ-aminobutyric acid (GABA) levels in GPi. Results HFS-PPN caused an excitatory reaction of the majority of neurons recorded in the GPi in PD model group and control group. The mean firing rate of GPi excited neurons was significantly increased (P﹤0.01). The levels of Glu were reduced under HFS-PPN and the levels of GABA were not affected (P>0.05).Conclusion HFS-PPN heightened the electrical activity of GPi neurons and re-duced the level of Glu. These excitatory effects were probably realized by PPN-GPi direct path or other indirect path.

Objective To investigate the role of Staphylococcus aureus enterotoxin B (SEB) in non-IgE mediated activation of mast cells (MCs) by in vitro co-culture of laboratory of allergic disease 2 (LAD2) cells and SEB.Methods The LAD2 cells were incubated with SEB at different concentrations of 0.01,0.1,1,10 and 100 μg/ml,A23187 positive control and negative control separately for 30 minutes.Then,effects of SEB on the morphology of MCs were observed by using a light microscope,and culture supernatants of the above incubation systems were collected.The concentration of tryptase released from MCs was analyzed by enzymatic activity assay,and the level of histamine was detected by enzyme-linked immunosorbent assay (ELISA).Results After 30-minute co-culture of LAD2 cells and SEB,MCs showed larger size,obscure boundaries,increased number of protuberances on the cell surface and decreased refractivity,with a radial burr fin-like appearance.After 30-minute co-culture of LAD2 cells and SEB at different concentrations of 0.01,0.1,1,10 and 100 μg/ml,the concentrations of tryptase in the culture supematants were 4.116 ± 0.651,5.344 ± 0.874,3.806 ± 0.459,1.309 ± 0.247,0.310 ± 0.199 ng/ml respectively.Additionally,the tryptase levels were significantly higher in the 0.01-,0.1-,1-μg/ml SEB groups than in the negative control group(1.538 ± 0.490,all P ＜ 0.05),and gradually decreased along with the increase of SEB concentrations.The histamine levels in the 0.01-,0.1-,1-,10-and 100-μg/ml SEB groups were 242.409 ± 63.915,522.491 ± 73.466,550.926 ± 84.466,334.397 ± 33.640,226.527 ± 5.678 ng/ml respectively.In the 0.01-,0.1-,1-μg/ml SEB groups,the levels of histamine released from MCs were gradually increased along with the increase of SEB concentrations,and were significantly higher than those in the negative control group (146.436 ± 3.100,all P ＜ 0.05).However,with the continued increase of SEB concentrations,the histamine levels gradually decreased.Conclusion SEB can directly activate MCs by a non-IgE mediated mechanism,followed by morphologic changes of MCs and release of tryptase and histamine.

Objective To explore the effect of let-7c-1 on the learning and memory of PTZ-induced epileptic rats and its relevant mechanism.Methods A model of temporal lobe epilepsy (TLE) was induced via PTZ kindling in SD male rats.The epileptic rats were divided into epilepsy group,agomir-control group,let-7c-1 agomir group (12 rats for each).Twelve rats were served as a negative control group.The behavior and the expression levesl of let-7c-1,Bcl-2 protein and Caspase3 were evaluated at 28 days following PTZ.Results Compared to the negative group,the escape latency of epilepsy group was prolonged and the crossing times as well as the quadrant total distance in the target were reduced (P＜0.05).However,those parameters were not significantly different between the epilepsy group and the agmoir-control group (P＞0.05).Compared to the agmoir-control group,the escape latency of let-7c-1 agomir group was prolonged and the crossing times as well as the quadrant total distance in the target were reduced (P＜ 0.05).The expression levels of let-7c-1 and let-7c-1 were 1.35±0.32 in agmoir-control group and 62.53±21.01 in agomir group (F=50.97,P＜0.05).The expression levels of let-7c-1 were higher in let-7c-1 agomir group than in other groups (P＜0.05).Compared to the negative group,the expressions of Bcl-2 protein in other groups were decreased (P＜0.05) and the Caspase3 protein were increased (P＜0.05).Compared to the agomir-control group,the expression of Bcl-2 protein was significantly decreased and the expression of Caspase3 protein was significantly increased in let-7c-1 agomir group (P＜0.05).Conclusions The present study shows that let-7c-1 may impair the learning and memory of PTZ-induced epileptic rats through decreasing the Bcl-2 protein and increasing Caspase3 protein in the hippocampus.

Objective:To study the effect of interference TIPE3 on the colon cancer cell growth by transfecting SW480 colon cancer cells with the TIPE3 interference plasmid were detected.Methods:Transfecting the constructed TIPE3-shRNA-pSIREN-RetroQ plasmid to SW480 cells.To determine the highest interference efficiency plasmid ,the mRNA and protein levels of recombined plasmid were detected by RT-PCR and Western blot separately and tested the cell proliferation with CCK 8.Meanwhile,apoptosis rate of SW480 cells was determined by flow cytometry assay with AnnexinV-FITC/PI.To further determined the effects of recombined plasmid on cell development ,the level of protein involved in proliferation and apoptosis were detected by Western blot .Results:The most effecient in-terference plasmids were successfully constructed.We found that the cell survival rate decreased when interference TIPE 3 gene express-ing in colorectal cancer cells .Flow cytometry indicated that interefering the expression of TIPE 3 would increase the sensitivity of SW 480 cell to apoptosis induced by aDR5ScFv.The results of Western blot showed that low expression of TIPE 3 would activate caspase3 and downregulate the expression of p-AKT,p-PDK1 and PCNA.Conclusion:Interference TIPE3 could promote apoptosis and inhibit prolif-eration in SW480 colon cancer cells .