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.

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.

[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 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.

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 study the masseter motor evoked potential(MEP)in patients with sleep bruxism(SB)and in healthy con-trols.Methods:30 subjects with SB and 30 healthy controls were included.MEPs were obtained by transcranial magnetic stimulation (TMS).Tests were done during daytime when the subjects were awake.The data were statistically analysed.Results:In the patients AMT was 55(52,55)%,latency of c-MEP (6.7 ±1.3)ms,the amplitude of c-MEP 0.19(0.15,0.29)mV,latency of r-MEP (2.3 ±0.4)ms,the central conduction time(CCT)4.4(3.3,5.2)ms.In the control subjects AMT was 52(52,55)%,latency of c-MEP (6.4 ±0.7)ms,the amplitude of c-MEP 0.23(0.17,0.28)mV,latency of r-MEP (2.4 ±0.4)ms,CCT 4.0 (3.4,4.4) ms.No significant difference was found between the 2 groups in the measurements evoked by TMS.Conclusion:The MEP after TMS in patients with SB is similar to that of healthy subjects,indicating that the excitability of the cortical motor system is not changed in bruxism subjects,at least when evaluated by TMS.

Objective To screen the effective silencing targets of P21 gene at the cellular level in rhesus monkey . Methods To detect the expression of P21 gene in COS-7 cells ( derived from the kidney of African green monkey , Cerco-pithecus aethiops).Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and in-serted into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into COS-7 cells respective-ly.The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by West-ern blot assay .Results Four gene-silencing sequences were screened that lied in 541-561 bp, 542-562 bp, 215-239 bp, and 624-648 bp of the rhesus monkey P21 mRNA.Their silencing rate was (91.82 ±3.21)%, (82.47 ±2.48)%, (81.31 ±2.69 )% and ( 87.35 ±4.59 )%, and the protein expression was ( 11.97 ±0.70 )%, ( 20.22 ±0.65 )%, ( 23.21 ± 0.63)%and (14.42 ±0.86)%, respectively.Conclusions Four effective silencing target sequences are screened at cel-lular level , which can be used in gene silencing research of rhesus monkeys .

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 .

Objective:To establish a method for the determination of content and related substances of troxipite tablets by HPLC. Methods:A Waters Symmetry-C18 (150 mm × 4. 6 mm,5 μm) column was used. The mobile phase was methanol -0. 4% phosphoric acid solution (50∶50). The flow rate was 1. 0 ml·min-1. The detection wavelength was 260nm. The column temperature was 35℃and the injection volume was 20 μl. Results:The linear range of troxipite was 3-75 μg·ml-1(r=0.999 7). The average recovery was 99. 7%,RSD=0. 95%(n=9). Conclusion:The method is simple, accurate and specific, and can be used in the quality control of troxipite tablets.