Endosomal TLRs (TLR3/7/8/9) are highly analogous innate immunity sensors for various viral or bacterial RNA/DNA molecular patterns. Among them, TLR7, in particular, has been suggested to be a target for various inflammatory disorders and autoimmune diseases including systemic lupus erythematosus (SLE); but few small-molecule inhibitors with elaborated mechanism have been reported in literature. Here, we reported a well-characterized human TLR7-specific small-molecule inhibitor, TH-407b, with promising potency and negligible cytotoxicity through a novel binding mechanism. Notably, TH-407b not only effectively inhibited TLR7-mediated pro-inflammatory signaling in a variety of cultured cell lines but also demonstrated potent inflammation suppressing activities in primary peripheral blood mononuclear cells (PBMCs) derived from SLE patients. Furthermore, TH-407b showed prominent efficacy in vivo, improved survival rate and ameliorated symptoms of SLE model mice. To obtain molecular insights into the TH-407b derivatives' inhibition mechanism, we performed the structural analysis of TLR7/TH-407b complex using cryogenic electron microscopy (cryo-EM) method. As an atomistic resolution cryo-EM structure of the TLR family, it not only of value to facilitate structure-based drug design, but also shed light to methodology development of small proteins using EM. Significantly, TH-407b has unveiled an inhibition strategy for TLR7 via stabilizing its resting/inactivated state. Such a resting state could be generally applicable to all TLRs, rendering a useful method for targeting this group of important immunological receptors.

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 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 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.

Objective To observe the changes of cell cycle,apoptosis and proliferation of endometrial cancer cells after the expression and down-regulation of Ezrin in endometrial cancer cells and to explore whether Ezrin may be a candidate gene for targeted therapy.Methods Endometrial cancer cells were from Shanghai Institute of Cell Research,of Chinese Academy of Medical Sciences in February 2017 and divided into blank control group and siEzrin group according to the intervention methods.Western blot and qRT-PCR was used to detect the expression of Ezrin protein and mRNA in endometrial cell lines.Small interfering RNA (siRNA) was used to transfect HEC-1B cell and down-regulate Ezrin.Cell cycle and apoptosis were detected by flow cytometry.MTT assay was used to detect multiplication.Results Western blot showed that Ezrin protein was expressed in Ishikawa (31.742 ± 5.832)、HEC-1A (16.326 ± 3.135)、HEC-1B(17.636±4.426) and KLE(14.862±5.109) and qRT-PCR showed that mRNA was expressed in Ishikawa (2.513±0.725),HEC-1A (1.655±0.692),HEC-1B (3.237±0.411) and KLE (0.962±0.235) cell lines,and expressed highest in HEC-1B cells (F=6.173,P＜0.05;F=7.042,P＜0.05).Flow cytometry assay showed that compared with blank control group less cells stayed in G1 phase and G2 phase,more stayed in S phase (t=3.118,P＜0.05;t=5.435,P＜0.05;t=3.332,P＜0.05).The apoptotic rate of HEC-1B cells increased from (9.84 ± 2.37) ％ to (17.64 ± 5.96) ％ (t =8.963,P ＜ 0.01) after Ezrin was downregulated.MTT assay showed that the proliferation of HEC-1B cells in 72 h and 96 h siEzrin transfection group was lower than that in blank control group (t =3.209,P＜ 0.05;t =3.726,P＜ 0.05).Conclusion Down-regulating of Ezrin may promote more endometrial cancer cells stay in S phase and promote apoptosis,inhibit proliferation,Ezrin may become target candidate gene in target therapy.

Objective To evaluate the relationship between the degree of brain injury during the perioperative period of liver transplantation and postoperative cognitive dysfunction (POCD).Methods Thirtythree patients,undergoing elective liver transplantation,were enrolled in this study.Before induction of anesthesia (T0),at 5 min before blocking the portal vein (T1),5 min after opening the portal vein (T2),5 min after opening the hepatic artery (T3),and at 24 h after surgery (T4),blood samples were collected from the central vein for determination of the serum concentrations of S1O0β protein and neuron-specific enolase (NSE) by enzyme-linked immunosorbent assay.Patients were divided into POCD group and control group (group C) according to whether POCD happened within 7 days after surgery.Results Compared with the baseline value at T0,the serum concentrations of S100β protein were significantly increased at T2 and T3,and the serum concentrations of NSE was increased at T3 (P＜0.05).There was no significant difference in serum concentrations of S100β and NSE at each time point between group POCD and group C (P＞0.05).Conclusion The degree of brain injury during the perioperative period of liver transplantation is not the dominant factor for the development of POCD in the patients.

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 explore the role of the SerpinB5 and β-catenin in occurrence and development of the primary hepatocellular carcinoma (HCC). Methods The expressions of SerpinB5 and β-catenin protein and mRNA in carcinoma tissues and paracancerous tissues of 60 patients with primary HCC were detected by immumohistochemistry and real-time quantitative reverse transcriptional polymerase chain reaction (RT-PCR) methods. Results The positive expression rate of SerpinB5 protein and SerpinB5 mRNA in carcinoma tissues were significantly lower than those in paracancerous tissues:25.0%(15/60) vs. 63.3%(38/60) and 1.12 ± 0.43 vs. 5.19 ± 0.39, and there were statistical differences (P<0.01). The positive expression rate of β-catenin protein and β-catenin mRNA in carcinoma tissues were significantly higher than those that in paracancerous tissues: 65.0%(39/60) vs. 31.7%(19/60) and 4.23 ± 0.25 vs. 1.19 ± 0.17, and there was statistical difference (P<0.01). Decreased SerpinB5 expression was associated with higher serumα-fetoprotein level, larger tumor size, poor differentiation, advanced TNM stage, capsule invasion and tumor thrombosis (P < 0.01 or 0.05). Increased β-catenin expression was associated with poor differentiation, advanced TNM stage, capsule invasion and tumor thrombosis (P < 0.01 or < 0.05). The correlation analysis result showed that SerpinB5 had negative correlation withβ-catenin (carcinoma issues:r=-0.346, P=0.001;paracancerous tissues:r=-0.258, P = 0.024). Conclusions The abnormal expression of SerpinB5 and β-catenin may contribute to the progression and biologically malignant behavior of primary HCC, and SerpinB5 and β-catenin exists synergistic effect in the occurrence and development of primary HCC.

OBJECTIVETo observe autophagy induced by starvation in non-small cell lung cancer A459 and 95D cells.

METHODSA549 and 95D cells in logarithmic growth in 1640 medium were cultured in Earle's balanced salt solution (EBSS) for 0, 1, 2, 3, 4 or 5 h. Autophagosome formation in the cell culture was observed by MDC fluorescent staining, and the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in the cells were detected using Western blotting.

RESULTSCompared with the control cells, the cells with prolonged starvation showed increased MDC-positive cells and autophagosome formation. The expression of Beclin-1 and the LC3-II/LC3-I ratio also increased as the starvation prolonged, reaching the peak levels at 3 h and 4 h, respectively.

CONCLUSIONAutophagy can be induced by starvation in A549 and 95D cells in correlation with the expression of autophagy-related proteins LC3 and Beclin-1. These cell models of nutritional deficiency-induced autophagy may allow for a better understanding of the role of autophagy in the development of non-small cell lung cancer.

Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Humans ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism

Objective To investigate the clinical features and therapeutic strategies of one patient with meningioangiomatosis ( MA ) complicated with intracranial aneurysm formation. Methods The clinical data of one patient with aneurysm-formed MA admitted to the Department of Neurosurgery,the First Affiliated Hospital of Sun Yat-sen University was analyzed retrospectively. The patient received emergency craniotomy and middle cerebral artery aneurysm clipping and evacuation of intracranial hematoma. The meningeal tissue around the aneurysm was taken for pathological examination. Results Microscopy revealed meningeal thickening in the patient with MA accompanied with intracranial aneurysm. Spindle cells around the meninges and perivascular tissue of cortical hyperplasia formed an annular and fence-like structure. The results of immunohistochemistry showed the hyperplastic vessels and perivascular fibroblast like spindle cells diffuse vimentin-positive;the hyperplastic vascular endothelial cells CD99,CD34 were positive;and the residual glial cells in the lesions showed glial fibrillary acidic protein positive. Conclusion MA complicated with intracranial aneurysm is rare. The vascular condition is poor. The shape of aneurysm is irregular,and the risk of bleeding is high. Excise the involved meningeal tissue and clip the aneurysm with microsurgical operation is the resonable treatment.