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Objective:To understand the plague epidemic characteristics in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province, and to provide scientific basis for innovative prevention and control of the plague in combination with local conditions. Methods:A retrospective study was used to collect the monitoring data of the natural foci of plague in Gansu Province from 2011 to 2018 (from the epidemic surveillance files of the Center for Disease Control and Prevention of Gansu Province and direct network reporting information). Descriptive epidemiological method was used to analyze the plague epidemic characteristics of natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province from 2011 to 2018, including the distribution of host animals, pathogenic and serological testing of the plague bacteria, and the epidemic characteristics of human plague. Results:From 2011 to 2018, the total average marmot density in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province was 0.21/hm 2, of which Tianzhu County had the highest average marmot density of 0.58/hm 2, and Jiayuguan City had the lowest average marmot density of 0.01/hm 2. A total of 381 strains of Yersinia pestis were isolated in the foci, of which 4 were isolated from human corpses, 298 were host animals, and 79 were infectious vectors. Among them, the top 3 counties (cities) of isolated strains were Aksai County (38.85%, 148 strains), Subei County (31.50%, 120 strains) and Yumen City (16.27%, 62 strains). A total of 6 860 marmot serum, 1 769 dog serum were tested, the F1 antibody positive rates were 2.70% (185/6 860), 8.42% (149/1 769); and the F1 antigen positive rate of 814 animal materials was 4.30% (35/814), respectively. There were 4 times of human plague, 4 cases occurred and 4 cases died; 3 times occurred in Subei County and 1 time in Yumen City. The onset months were July, September, November and December. Active contact with infected animals such as shepherd dogs was the main route of infection, and migrant herders were the key occupation population. Conclusions:The animal epidemic situation in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province is active, and the plague presents different epidemic states in different regions. The prevention and control measures should be taken according to local conditions and guided by classification to strictly prevent the occurrence and transmission of the plague.

Objective To apply the DNA barcoding technology for identification of rodent animals and to establish a rodent animal DNA barcode database in Gansu Province.Methods A total of 54 rodent animals were detected.DNA barcoding technology was used to analyze the DNA mitochondrial cytochrome C oxidase subunit Ⅰ (CO Ⅰ) gene sequence in Gansu Province.Results The intra-specific genetic distance was 0-2％ while the interspecific distance ranged from 18％ to 30％.Eight major clusters were apparently showed on a Neighbor joining tree.Conclusion DNA barcoding technology could overcome the shortcomings of the morphological identification,so it could be used to identify the rodent animals and has important implications for disease control and prevention in the natural focus of Gansu Province.

Objective:To study the genotyping and regional distribution characteristics of Yersinia pestis by single nucleotide polymorphism (SNP) in Gansu Province. Methods:A total of 52 strains of Yersinia pestis isolated from Himalaya Marmot plague foci and Spermophilus alaschanicus plague foci in Gansu Province from 1962 to 2017 were selected for culture and extraction of DNA. The genomic DNA of Yersinia pestis was sequenced by the second generation of Illumina PE150 to identify the SNP sites. The species characteristics of Yersinia pestis in Gansu Province was determined by the Kimura-2-parameter model of neighbor joining of Mega 10.0 software based on the SNP sites. The molecular evolutionary tree of the groups was determined by Hasegawa-Kishino-Yano model of maximum likelihood method according to the SNP sites. Results:A total of 103 SNP sites were identified in 52 strains of Yersinia pestis in Gansu Province, including 28 intergenic loci, 43 non-synonymous mutations, 31 synonymous mutations and 1 nonsense mutation. The 52 strains of Yersinia pestis were divided into 2 biotypes and 3 groups, which were ancient type (1.IN2, 3.ANT) and medieval type (2.MED). Among them, 35 strains belonged to 1.IN2 group, 13 strains belonged to 3.ANT group, and 4 strains belonged to 2.MED group. The 1.IN2 group was further divided into 5 subgroups: the groups of Yuerhong Town and Dangchengwan Town in Subei County, the groups of Mati Town and Dahe Town in Sunan County, and the group of Xiahe County. The 3.ANT group was further divided into 2 subgroups: the groups of Hongliuwan Town in Aksay County and Machang in Dangchengwan Town of Subei County. Conclusion:The SNP method can be used to genotype Yersinia pestis from different plague foci in Gansu Province, which has certain regional characteristics.

Objective:To study the clustered regularly interspaced short palindromic repeats (CRISPR) loci polymorphism of Yersinia pestis and its area distribution in Gansu province. Methods:A total of 203 strains of Yersinia pestis isolated from 1962 to 2014 were selected for the culture and extraction of DNA. Three pairs of CRISPR primers were used to amplify the strain DNA by PCR, and the PCR products were sequenced. The groups and genotypes of strains were determined according to the spacer and spacer arrangement of CRISPR loci in the strain. Cluster analysis was done by using the software BioNumerics 5.10. Results:A total of 16 spacers, including 9 species of YPa loci, 4 species of YPb loci and 3 species of YPc loci, were found in the 203 strains of Yersinia pestis. A new spacer of a1′ was found. The 203 strains were divided into 5 CRISPR genotypes and classified into 5 CRISPR clusters (Cb2, Ca7, Ca7′, CaΔ5′ and Ca35′). Each cluster showed significant area-specific characteristics, Cb2 was mainly distributed in Huining country and Pingchuan district, Ca7 was mainly found in Aksai Kazak autonomous country, Ca7′ was mainly found in Xiahe country, Ca35′ was mainly found in Subei Mongolia autonomous county and Yumen city and CaΔ5′ was mainly distributed in Sunan Yugur autonomous county. Conclusions:The strains from different plague foci in Gansu were distinguished by CRISPR, all kinds of clusters showed the obvious area specific characteristics. It is important to study the evolution of Yersinia pestis in Gansu and trace the molecular biology origin of human plague.

ObjectiveTo investigate the antitumor effect and mechanism of Guiqi Yiyuan ointment on Lewis lung cancer mice based on the extracellular regulatory protein kinase （ERK） signaling pathway. MethodsA Lewis lung cancer mouse model was established. Except for the blank group， the model mice were randomly divided into the model group， Guiqi Yiyuan ointment low， medium， and high dose groups， and the extracellular ERK1/2 inhibitor group， with 10 mice per group. The Guiqi Yiyuan ointment was administered by gavage at doses of 1.75， 3.5， 7.0 g·kg^-1·d^-1 for the low， medium， and high dose groups， respectively. The ERK1/2 inhibitor group was given the ERK1/2 inhibitor LY3214996 （100 mg·kg^-1·d^-1） by gavage. The treatment was administered for 14 consecutive days， after which samples were collected. Tumor histopathological changes were observed using hematoxylin-eosin （HE） staining. Transmission electron microscopy was used to observe ultrastructural changes in tumor cells. Immunofluorescence was performed to measure the phosphorylation of ERK1/2 （p-ERK1/2） and the expression of programmed cell death ligand-1 （PD-L1） in tumor tissues. Western blot and real-time quantitative polymerase chain reaction （Real-time PCR） were used to detect the expression of p-ERK1/2， PD-L1， the autophagy marker Beclin-1， the autophagic protein p62， and the microtubule-associated protein light chains LC3Ⅰ and LC3Ⅱ at both the protein and gene levels. ResultsCompared with the model group， the average tumor weight was significantly reduced in the low and medium dose groups of Guiqi Yiyuan ointment （P<0.05）， and markedly reduced in the high dose and inhibitor groups （P<0.01）. Tumor cells in all treatment groups became progressively irregular， with ruptured nuclei and expanded areas of cell disintegration and necrosis. The number of organellar ablations in tumor tissues increased， and the number of autophagic vesicles also increased in all groups. The mean fluorescence intensity of p-ERK1/2 and PD-L1 was reduced in the low and medium dose groups of Guiqi Yiyuan ointment （P<0.05）， and significantly reduced in the high dose and inhibitor groups （P<0.01）. The mRNA expression of ERK1/2， PD-L1， Beclin-1， and p62 was reduced in the medium dose group （P<0.05）， while LC3Ⅰ/Ⅱ mRNA expression was elevated （P<0.05）. In the high dose and inhibitor groups， mRNA expression of ERK1/2， PD-L1， Beclin-1， and p62 was significantly reduced （P<0.01）， while LC3Ⅰ/Ⅱ mRNA expression was significantly increased （P<0.01）. Protein expression of p-ERK1/2， PD-L1， Beclin-1， and p62 was reduced in the medium dose group （P<0.05）， and LC3Ⅰ/Ⅱ protein expression was elevated （P<0.05）. In the high dose and inhibitor groups， protein expression of p-ERK1/2， PD-L1， Beclin-1， and p62 was significantly reduced （P<0.01）， while LC3Ⅰ/Ⅱ protein expression was significantly elevated （P<0.01）. ConclusionGuiqi Yiyuan ointment may inhibit the activation of the ERK signaling pathway， downregulate the expression of p-ERK1/2， promote autophagic flux in tumor cells， and regulate the expression of PD-L1， thereby exerting an inhibitory effect on tumor growth in Lewis lung cancer mice.