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Objective To explore the effect of sevoflurane post-conditioning on oxygen-glucose deprivation/reoxygenation(OGD/R) damage of primary hippocampal neurons, oxidative stress and autophagy. Methods Hippocampal neurons were cultured in uterine endometrial rats for(18±0.5) days. After 7 days, the cultured hippocampal neurons were randomly divided into three groups: normal control group(CON group), oxygen-glucose deprivation/reoxygenation(OGD/R) group and sevoflurane post-conditioning group(OGD/R+SEVO group). Primary hippocampal neurons in the CON group were cultured normally; OGD/R group was treated with oxygen-glucose deprivation for 1.5 hours and reoxygenation for 24 hours to establish oxygen and glucose deprivation/reoxygenation injury model; OGD/R+SEVO group was treated with sevoflurane for 1 hour and then reoxygenated for 23 hours. After treatments, the activity of each group of LDH was detected by colorimetric method, TUNEL staining method detected the changes of hippocampal nerve cells in each group, immunofluorescence method detected mitochondrial ROS and the expression of autophagy proteins LC3 B,and Western blot analysis detected the expression of PINK1 and Parkin. Results Compared with CON group, the release of lactate dehydrogenase increased(P<0.01); neuronal apoptosis increased; mitochondrial ROS and the expression of autophagy marker proteins LC3 B increased(P<0.01), PINK1 and Parkin also increased in OGD/R group(P<0.001,P<0.05). Compared with OGD/R group, the release of lactate dehydrogenase was reduced(P<0.05), the apoptosis was inhibited, mitochondrial ROS and the expression of autophagy marker proteins LC3 B were reduced(P<0.05), PINK1 and Parkin protein were reduced(P<0.01,P<0.05) in OGD/R+SEVO group. Conclusion Sevoflurane post-conditioning can protect primary hippocampal neurons by reducing OGD/R-induced excessive autophagy, reducing oxidative stress, and reducing apoptosis, and its mechanism of reducing autophagy may be related to the PINK1/Parkin pathway.

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Objective To investigate the changes of plasma protein expression profile in hyperhomocysteinemia rats and the protective effect of highly expressed angiopoietin 1 in plasma on endothelial cells. Methods The hyperhomocysteinemia animal model was established. The difference in plasma protein content was analyzed by label-free protein spectroscopy. The effects of homocysteine and angiopoietin 1 on endothelial cell migration and proliferation were detected by wound healing and CCK-8 proliferation assay. Results The results of protein profiling showed that 5 proteins were significantly up-regulated and 17 proteins were significantly down-regulated in the plasma of hyperhomocysteinemia rats, among which angiopoietin 1 was significantly up-regulated. In endothelial cells in the superior mesenteric artery of rats, treatment with 30 or 50 μmol/L homocysteine for 24 h significantly inhibited the migration and proliferation. Angiopoietin 1(600 ng/ml) significantly reduced the migration and proliferation of endothelial cells inhibited by 30 μmol/L homocysteine, but had no significant effect on the migration and proliferation of endothelial cells inhibited by 50 μmol/L homocysteine. Conclusion Hyperhomocysteinemia can significantly affect the protein expression profile in plasma. Angiopoietin 1 in plasma can compensate for the damage of vascular endothelial migration and proliferation function caused by homocysteine in a certain concentration range.

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Objective To investigate the effect of miR-143 on the proliferation and apoptosis of prostate cancer cell line PC3. Methods The experiment was divided into non transfected PC3 cells group(PC3 group), transfected with miR-NC group(PC3/miR-NC group) and PC3 cells stably expressing miR-143 group(PC3/miR-143 group), and identified by immunofluorescence and real-time PCR. The effect of miR-143 on the proliferation of PC3 cells by CCK-8 method. The effect of miR-143 on PC3 cells apoptosis was analyzed by flow cytometry. The online analysis software was used to predict the target genes of miR-143, and then the luciferase reporter gene detection plasmid was constructed. The targeted binding sites of miR-143 and target genes were analyzed by luciferase assay. Results The expression level of miR-143 in PC3/miR-143 group was significantly higher than that of PC3 group(P<0.01) and PC3/miR-NC group(P<0.01). CCK-8 test showed that the cell proliferation ability of PC3/miR-143 group decreased significantly compared with PC3 group(P<0.05) and PC3/miR-NC group(P<0.01). The results of flow cytometry showed that the apoptosis level of PC3/miR-143 group was significantly higher than that of PC3 group and PC3/mir-nc group(P<0.01). Online analysis software predicted that miR-143 could target 3 ′-UTR binding to Trefoil Factor 3(TFF3); The results of dual luciferase reporter gene further confirmed that miR-143 could target the 3′-UTR of TFF3. Real time PCR results showed that overexpression of miR-143 could significantly inhibit the expression of TFF3 in PC3 cells. Transfection of TFF3 eukaryotic expression plasmid into PC3/miR-143 group could counteract the effect of miR-143. Conclusion MiR-143 inhibits the expression of TFF3 and the proliferation of PC3 cells by targeting the 3 ′-UTR of TFF3.

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Objective To investigate the effect of salidroside(Sal) on ischemia reperfusion(IR) in isolated rat heart and explore the underlying mechanisms on endoplasmic reticulum stress(ERS) and apoptosis. Methods The isolated rat hearts underwent Langendorff perfusion subjected to IR. After ischemia for 45 min and reperfusion for 2 h, the isolated rat hearts were randomly divided into four groups(n=10): Control group, Sal treatment group(Sal), ischemia reperfusion group(IR) and Sal treatment with ischemia reperfusion group(IR+Sal). Myocardial infarct size was detected by TTC staining. Lactate dehydrogenase(LDH) activity in the coronary flow was determined by ELISA. The cardiac function was evaluated by left ventricular peak systolic pressure(LVSP) and left ventricular end diastolic pressure(LVEDP). The expressions of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax),protein kinase R-like endoplasmic reticulum kinase(PERK), C/EBP homologous protein(CHOP) and activating transcription factor 6(ATF6) were detected by Western blot, and apoptosis was observed by TUNEL staining. Results There was no significant difference in all indexes between Sal group and Control group. Compared with Control group, the infarct size and LDH activity in the coronary flow increased in IR group. The LVSP value decreased, and the LVEDP value increased. The expression of Bax and the TUNEL-positive cells increased, while the expression of Bcl-2 decreased. Meanwhile, the expressions of ERS related proteins including PERK, CHOP and ATF6 also increased. Compared with IR group, the infarct size and LDH activity in the coronary flow increased in IR+Sal group. The LVSP value increased, and the LVEDP value decreased. The expression of Bax and the TUNEL-positive cells decreased, while the expression of Bcl-2 increased. In addition, the expressions of PERK, CHOP and ATF6 decreased. Conclusion Sal can improve myocardial IR injury by inhibiting ERS and apoptosis in isolated rat hearts.

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Objective To find molecular targets related to the occurrence and development of ovarian cancer induced by whole-exome sequencing and to verify the mechanismin vitrocell experiments. Methods 8 pairs of serous ovarian cancer tissues were sequenced using whole-exome, and another 43 serous ovarian cancer tissues and 156 peripheral blood samples of normal women were performed target gene sequencing. In thein vitroexperiment, the plasmids containing theCREBBPgene L1850 fs and P1373 S mutations were transfected into human serous ovarian cancer cell line A2780 with Lipofectamine 3000, and the plasmids containing wild-typeCREBBPand the empty vector were used as controls. CRP protein expression afterCREBBPL1850 fs and P1373 S mutated was evaluated by Western blot; cell proliferation of L1850 fs and P1373 S mutation was detected by CCK-8 assay; cell migration of L1850 fs and P1373 S mutation was detected by Transwell chamber test; cells invasion of L1850 fs and P1373 S mutation was detected by scratch test. Results Two high-frequency mutation sites ofCREBBPgene on L1850 fs(c.5548 dupC) and P1373 S(c.4117 C>T) in serous ovarian cancer tissues were discovered using whole exome sequencing. L1850 f was a frameshift mutation, and P1373 S was a non-synonymous sequence. In 51 cases of serous ovarian cancer(8 cases of whole exome sequencing, 43 cases of target gene sequencing), the mutation rate ofCREBBPgene was 41.2%, while the mutation rate of normal female peripheral blood was 21.8%, the difference was statistically significant(χ2=7.4,P=0.007). The results of western blotting showed that the L1850 fs mutation could reduce the expression of CBP in ovarian cancer cell, while the P1373 S mutation had no significant effect on CBP protein expression. CCK-8 analysis showed that mutantCREBBPcould promote the proliferation of ovarian cancer cells. Transwell chamber test showed that L1850 fs and P1373 S mutation could promote the migration of ovarian cancer cells. Scratch test analysis showed that L1850 fs and P1373 S mutation could increase the invasion ability of ovarian cancer cells. Conclusion In this experiment, two point mutations ofCREBBPin L1850 fs and P1373 S were found in serous ovarian cancer using whole exome sequencing. Further cell experiments showed that the two mutation could promote the proliferation, invasion and metastasis of ovarian cancer cells by affecting the CBP protein, suggesting thatCREBBPL1850 fs and P1373 S might become new targets for the diagnosis and treatment of ovarian cancer.

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Objective Balb/c mice infected with respiratory syncytial virus(RSV) were used to investigate the metabolic changes in cecal luminal content. Methods A total of 13 female Balb/c mice were randomly divided into Case group(n=7) and control(Ctrl) group(n=6). Animals in Case group were infected with RSV by using intranasal method, while mice in Ctrl group were treated with DMEM medium. Mice were anesthetized with intraperitoneal administration of 10% chloral hydrate and the cecal luminal contents were harvested under sterile conditions. Metabolite concentrations were measured by UPLC-QTOF/MS system. Univariate and multivariate statistical analysis were used to identify differential metabolites between Case and Ctrl groups. Results The overall base peak chromatogram between Case and Ctrl groups had a clear disparity, and PCA and OPLS-DA analysis showed obviously discrepancy based overall metabolomic profile. L-serine, 2-ketobutyric acid, Oleic acid and Chenodeoxycholic acid glycine conjugate were enriched in Case group, whereas L-methionine, L-tyrosine and Nicotinic acid were depleted. Pathway analysis showed lysine degradation, Cysteine and methionine metabolism were enriched. Conclusion Lung injury induced by RSV infection may cause the endogenous metabolism disorder of cecal contents.

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Objective To explore the effects and mechanisms of interfering with transient receptor potential melastatin subfamily member 7(TRPM7) on biological behaviors(proliferation, apoptosis, invasion) of laryngeal carcinoma cells. Methods Human laryngeal carcinoma cells TU212 were culturedin vitro. TRPM7-shRNA plasmid vectors(TRPM7-shRNA1 group, TRPM7-shRNA2 group, TRPM7-shRNA3 group) and negative control shRNA-NC(shRNA-NC group) were constructed. TU212 cells were transfected by liposome transfection method. The cells transfected with empty vector were enrolled as Control group. The level of lactic dehydrogenase(LDH) in TU212 supernatant was detected by ELISA. The level of malondialdehyde(MDA) and activity of superoxide dismutase(SOD) in supernatant were detected by colorimetry. The effects of knocking-down TRPM7 on proliferation and invasion of TU212 cells were detected by CCK-8, clone formation assay and Transwell assay. The expressions of invasion and apoptosis-related proteins were detected by Western blot. Results After transfection, expression levels of TRPM7 mRNA and protein were down-regulated in TRPM7-shRNA1 group, TRPM7-shRNA2 group and TRPM7-shRNA3 group compared with Control group(P<0.05), and the decrease was the most significant in TRPM7-shRNA1 group(P<0.05). In functional experiments, SOD level in TRPM7-shRNA1 group decreased compared with Control group(P<0.05), while MDA and LDH levels increased(P<0.05). The cells proliferation rate and clone formation rate were decreased(P<0.05), the number of invasion cells was reduced(P<0.05), the expressions of N-cadherin and Vimentin proteins were down-regulated(P<0.05), mitochondrial membrane potentials were reduced(P<0.05), Bax/Bcl-2 and cleaved caspase-3/caspase-3 increased(P<0.05). Conclusion Knocking-down TRPM7 can increase oxidative stress level in laryngeal carcinoma cells TU212, inhibit their proliferation and invasion, and induce their apoptosis by mitochondrial pathways.

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Objective Eya1 gene lentiviral vectors were constructed, and the effects of overexpression of eye absent 1(Eya1) on 6-OHDA-induced apoptosis of MES23.5 dopaminergic cells were investigated. Methods Lentivirus overexpression of Eya1 gene was constructed and infected MES23.5 cells, then stable Eya1-expression cell lines were screened. MES23.5 dopaminergic cell injury model induced by 6-hydroxydopamine(6-OHDA) was divided into control group, empty vector group and overexpression Eya1 group. Cell counting kit-8(CCK-8), Hoechst staining kit(Hoechst 33258) and Western blot were used to detect the effect of overexpression of Eya1 on apoptosis. Results Enzyme electrophoresis showed that the target gene was recombined into lentivirus vector. Sequencing results showed that Eya1 gene overexpression vector was identical with the Eya1 gene coding sequence. CCK-8 showed that the cell viability of Eya1 overexpressed group was higher than that of control group(P<0.05). Hoechst 33258 showed that the concentration and aggregation level of Eya1 overexpression group was lower than that of control group. The expression of Bcl-2 protein in the overexpression Eya1 group was higher than that in the control group(P<0.05), while that of Bax protein was lower than that of the control. Conclusion The MES23.5 cell line overexpressing of Eya1 was successfully constructed. Eya1 antagonizes the apoptosis of MES23.5 dopaminergic cells induced by 6-OHDA by regulating the expression of Bcl-2 and Bax proteins.

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Objective To explore whether long-term potentiation(LTP) induced by brain-derived neurotrophic factor(BDNF) of hippocampus is involved in the process of maternal separation(MS) leading to impaired cognitive function of offspring in adolescence. Methods The newborn CD-1 mice were randomly divided into maternal separation group(MS group) and control group(CON group). Mice in MS group were separated from the mother mice for 3 h every day from postnatal day 4 to 21 while no intervention was taken in the CON group. The spatial learning and memory ability was assessed using Morris water maze at the age of 3 months. Western blot and real-time quantitative PCR were used to detect the levels of BDNF and BDNF mRNA in the hippocampus. LTP of the hippocampal CA3-CA1 neural pathway was recorded using electrophysiological techniques. Results Compared with CON group, the latency and distance of Morris water maze in maternal separation group were significantly longer(P<0.01). The percentage of time and distance in target quadrant during the memory phase in MS group were obviously lower than those in control group(P<0.05). The results of WB and Real-time quantitative PCR in MS group showed that the levels of BDNF and BDNF mRNA in MS group apparently decreased(P<0.05). Compared with CON group, MS group showed a significantly lower LTP in CA3-CA1 neural pathway(P<0.01). Conclusion The certain intensity of maternal separation can impair learning and memory function in young CD-1 male mice, which may be associated with decreased expression of BDNF and impaired LTP in the hippocampus.

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Objective To observe the effects of nootkatone on depression-like behavior, neurogenesis and protein kinase A(PKA)/cyclic adenosine monophosphate response element-binding protein(CREB)/brain-derived neurotrophic factor(BDNF) signaling pathway in hippocampus of chronic unpredictable stress(CUS) treated mice, and to explore the role and molecular mechanism of nootkatone′s antidepressant effect. Methods Male C57 BL/6 mice were randomly divided into 3 groups: the control group(saline), the CUS group(CUS+saline), the CUS+nootkatone group(CUS+nootkatone). The sucrose preference test and forced swim test were used to evaluate the depression-like behaviors. The mRNA expression of BDNF in hippocampus was measured by RTPCR. The expression levels of BDNF, PKA and phosphorylated cyclic adenosine monophosphate response element-binding protein(p-CREB) in hippocampus were determined by Western blot. The level of neurogenesis was measured by immunofluorescence. Results Compared with the control group, the sucrose preference decreased(P<0.05) and the latency decreased(P<0.05), and immobility time increased in forced swim test(P<0.05) in CUS group, the expression levels of BDNF mRNA(P<0.05) and protein(P<0.05), PKA(P<0.05) and p-CREB(P<0.05) decreased. The sucrose preference of the CUS+nootkatone group increased(P<0.05) and the latency increased(P<0.05), and immobility time decreased in forced swim test(P<0.05), the expression levels of BDNF mRNA(P<0.05) and protein(P<0.05), PKA(P<0.05) and p-CREB(P<0.05) increased in comparison with the CUS group. Compared with control group, the number of hippocampal doublecortin(DCX) labeled neurons decreased(P<0.05) in CUS group. Compared with the CUS group, the number of DCX labeled neurons increased in the CUS+nootkatone group(P<0.05). Conclusion The improvement of depressive symptoms in CUS mice by nootkatone may be related to the neurogenesis in dentate gyrus of hippocampus and the activation of PKA/CREB/BDNF signaling pathway.

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Objective To establish an experimental model of Turner′s tooth caused by trauma in SD rats and observe its surface by scanning electronic microscopes(SEM) and energy dispersive spectrometry(EDS) was performed. Methods 40 SD rats aged one day were randomly divided into four groups(n=10). The control group did nothing, The experimental group was exerted different perpendicular forces(per 5 mm2) on the mandibular anterior alveolar process, which was divided into 5 N force group, 10 N force group and 15 N force group. The SD rats were killed at 30 days old to observe the enamel development of their mandibular central incisors. Meanwhile, SEM and EDS were used to observe normal enamel area and enamel hypoplasia area. Results The control group: all teeth erupted; all enamels developed well. 5 N force group: all teeth erupted; the occurrence rate of enamel hypoplasia was 10%(2 teeth had enamel discoloration). 10 N force group: 1 tooth unerupted; the occurrence rate of enamel hypoplasia was 80%(12 teeth had enamel discoloration and 4 had enamel defects). 15 N force group: 7 teeth unerupted, the occurrence rate of enamel hypoplasia was 60%(3 teeth had enamel discoloration and 9 had enamel defects). There was a statistic difference in the number of unerupted teeth between group 10 N force group and 15 N force group(P<0.05). Under SEM, cracks and rough appeared on the surface of enamel. EDS showed that the Ca and P content in enamel hypoplasia was lower than that in the normal enamel area(P<0.05). Conclusion Tooth trauma can lead to enamel hypoplasia and unerupted teeth. The force of 10 N per 5 mm2is better to establish an experimental model of Turner′s tooth caused by trauma in SD rats. The surface enamel of Turner′s tooth caused by trauma is rough, uneven and the content of calcium and phosphorus decreases.

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Objective To explore the role and mechanism of long noncoding RNA(lncRNA) AC005062.1 in colorectal cancer(CRC) cell proliferation and apoptosis. Methods The analysis of GSE84983 and GSE 104364 was used to indicate the expression level lncRNA AC005062.1 in CRC. The tumor tissues(tumor group) and adjacent tissues(control group) of patients undergoing CRC surgery in the hospital tumor surgery department were collected. Eight pairs of tissues were randomly selected, and qPCR was used to detect the expression of lncRNA AC005062.1 in CRC tissues and paired adjacent tissues in the two groups. After using siRNA to down-regulate the expression of lncRNA AC005062.1 in CRC cells, CCK-8 assay was used to detect cell proliferation in two groups, flow cytometry was used to detect cell cycle and apoptosis in two groups, wound scratch test was used to detect cell migration in two groups, and the lncRNA was determined. The database was used to compare the localization of lncRNA AC005062.1 and MACC1 genes on staining. The expression levels of MACC1 transcription and protein levels in the control group and tumor group were detected by qPCR and Western blot, respectively. The lncRNA AC005062.1 in CRC cells was down-regulated, and the expression levels of MACC1 transcription and protein levels in the two groups of cells were detected by qPCR and Western blot, respectively. Results The database analysis of GSE84983 and GSE104364 and the detection results of the two groups indicated that the lncRNA AC005062.1 was highly expressed in CRC. After down-regulation of lncRNA AC005062.1, the results of CCK-8 experiments showed that the proliferation rate of HT29 cells in the down-regulation group decreased. Flow cytometry showed that the number of apoptosis of HT29 cells in the down-regulated group increased, the proportion of G1 phase increased, and the proportion of S phase and G2 phase decreased. Western blot experiments showed that the expressions of cleaved-caspase3 and Bax in the down-regulated HT29 cells increased, while the expression of Bcl-2 decreased. Wound scratch experiments showed that the migration rate of HT29 cells in the down-regulated group was lower than that in the control group. The analysis of UCSC to compare the location of lncRNA ac005062.1 and MACC1 on chromosomes showed that they were located very close together. MACC1 was highly expressed in CRC and down-regulated lncRNA AC005062.1 expression in HT29 cells could reduce the expression of MACC1. Conclusion lncRNA AC005062.1 can inhibit the proliferation, cycle and migration of HT29 cells, and promote its apoptosis by regulating the expression of MACC1 in CRC.

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Objective To screen the differential genes and pathways of female and male C57 BL/6 J mice by transcriptome sequencing, and to lay a foundation for further exploring the molecular mechanism of behavioral differences between male and female mice. Methods 8-week-old female and male mice of the C57 BL/6 J strain were completely isolated from the mouse hippocampus, and total RNA was extracted and reverse transcribed to construct a cDNA library, and 50 single-ended mice were generated on the BGIseq500 platform(BGl-Shenzhen, China) The base reads were transcriptome sequenced. After sequencing, based on GO and KEGG databases, combined with the phyper function in R language to screen, correct and enrich the data, calculatePvalue, and then perform FDR correction onPvalue to obtainQvalue and analyze different annotated genes based on the hypergeometric test method. The expression status was analyzed, and the differential genes and pathways in the hippocampus of mice of different genders were screened out. Results Through the comparison of male and female differences, 325 differential genes were screened, including 233 up-regulated genes and 92 down-regulated genes. The functions of these differential genes were mainly enriched in long-term potentiation(LTP), calcium signal pathway, nicotine addiction and other processes. There were 362 junctions and 1 703 interaction edges in the female-male differential gene interaction network, and 10 core genes selected: lysine demethylase 5 D(Kdm5 d), cyclin-dependent kinase 5(Cdkl5), Cell output mediator(Cle1), dopamine receptor 6 a3(Slc6 a3), cassette transcription factor(Fox), precursor mRNA processing factor 4 B(Prpf4 b), glycine receptor 4(Glur4), calcium ion receptor 2(Camk2), DNA and RNA binding protein family(Son), serotonin receptor 5 b(Htr5 b). Conclusion The selected differential genes and signal pathways may lay the foundation for explaining the molecular mechanism of the differences in behavior between male and female mice.

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Objective To investigate the correlation of carriage and expression of quorum sensing(QS) system regulatory genes and virulence genes with drug resistance inPseudomonas aeruginosastrains isolated from clinical sources. Methods A total of 97 strains ofPseudomonas aeruginosaisolated from various clinical specimens in the laboratory were collected. Firstly, Drug-resistance were detected by the vitek2-compact instrument method and the standard disc diffusion method, 4 QS system-regulated genes and 7 virulence genes were detected by polymerase chain reaction(PCR). After that, the expression of regulatory genes LasI, Rh1 R and virulence genes exoS and PCN were detected by real-time fluorescence quantitative PCR(qRT-PCR), and moreover linearly analysis was used to analyze the correlation of the two types of genes. Results The resistance rates of 97 strains to 11 antibacterial drugs. The resistance rate of tobramycin, gentamicin, and amikacin was less than 10%, and the minimum amikacin was 2.06%. The resistance rate to the remaining drugs was all above 11%, and the highest was imipenem 25.77%, and 43 strains were multi-drug resistant bacteria, and the respiratory-derived strains were more resistant to antimicrobials. The results of PCR gene detection showed: the detection rates of the four QS system-regulated genes LasI, LasR, Rh1 I and Rh1 R were all 100%; the highest detection rate of 7 virulence genes was exoT 98.97%(96/97),and the lowest was exoU 13.40%(13/97),PCN 43.30%(42/97),and the rest were above 86%. The strains without virulence genes were resistant to 6 antibiotics including ceftazidime, and the strains with 7 virulence genes were only resistant to ceftazidime; among the PCN, exoS, and exoU positive strains, the proportion of non-multidrug resistantPseudomonas aeruginosa(NMDR-PA)was higher; PCN+ strains were more resistant to piperacillin/tazobactam and the difference was statistically significant; compared with exoS+/exoU-strains, the types of antibiotics resistant to exoS-/exoU+ strains decreased significantly. The relative expression levels of regulatory genes rh1 R and LasI in NMDR-PAwere higher and the difference of LasI was statistically significant; the expression of the regulatory gene rh1 R and the virulence gene exoS and the regulatory gene LasI and the virulence gene PCN showed a positive linear relationship. Conclusion The drug-resistance ofPseudomonas aeruginosais still serious and multi-drug resistance. QS system regulatory genes exist in the strain stably, except for exoU and PCN, the other virulence genes exoS, exoT, exoY, ToxA and LasB have higher carrying rates, the regulatory genes rh1 R and LasI positively regulate the expression of virulence genes, both QS system regulatory genes and virulence genes affect drug resistance inPseudomonas aeruginosa.

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Objective To study the antibacterial properties of nano-silver-containing polyelectrolyte multilayer films assembled with heparan sulfate(HEP) and chitosan(CHI) by layer-by-layer self-assembly technology(LBL) on porous titanium surface. Methods Negatively charged HEP and positively charged CHI were alternately adsorbed by LBL to form polyelectrolyte multilayer films, which were deposited on the surface of porous titanium, and then silver ions were loaded on the multilayer films under alkaline conditions. The surface was evaluated by scanning electron microscopy, X-ray photoelectron spectrometer, a measuring instrument for contact angle and inductively coupled plasma emission spectrometer. The antibacterial effect was evaluated by inhibition zone, turbidity method, live/dead bacterial staining and coated plate method. Results Material characterization methods showed polyelectrolyte multilayer films were successfully deposited on the surface of titanium, and nano-silver particles were loaded. Antibacterial experiments showed that the silver-loaded modification significantly improved the antibacterial ability of the coating. Conclusion Silver-loaded HEP-CHI polyelectrolyte multilayer films are deposited on the surface of modified titanium by LBL, which can inhibit the adhesion of bacteria.

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Objective To investigate the effects of long-term hyperglycemia induced ultrastructure of left ventricular myocardium in streptozotocin-induced type 1 diabetes mellitus(T1 DM) cynomolgus monkeys. Methods The T1 DM cynomolgus monkeys were induced by intravenous administration of streptozotocin as the model group. The normal cynomolgus monkeys were distributed to the control group. Ultrastructural morphometric features of left ventricular myocardium in the model group and the control group were obtained using transmission electron microscope and quantitatively analyzed. Results Ultrastructural observations indicated that the numbers of mitochondrion(P<0.01), the relative electron density of mitochondrion(P<0.000 1), the volume density(P<0.001) and the surface area density(P<0.05) of mitochondrion were observed to decrease in the cytoplasm of left ventricular cardiomyocytes of the model group compared to the control group. The ration surface and the average area of mitochondrion showed no difference between two groups. Massive lipid droplets were identified in the cytoplasm of left ventricular cardiomyocytes of the model group. Thickening of basement membrane(P<0.000 1) and decreasing of pinocytotic vesicles(P<0.000 1) were observed in the capillary endothelial cells in left ventricular myocardium of the model group compared to the control group. Conclusion The results indicate that long-term hyperglycemia induces left ventricular cardiomyocytes and capillary endothelial cells ultrastructural damage in TIDM cynomolgus monkeys.

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Objective To explore the effect of melatonin(MLT) on human umbilical vein endothelial cell(HUVEC) injury induced by high glucose and to explore its probable molecular mechanism. Methods HUVEC were cultured and divided into three groups: control group, model group and treatment group. The control group(Ctrl group) was treated with DMEM medium at a normal low glucose level(5.5 mmol/L glucose), and the model group(HG group) was treated with DMEM medium at a high glucose level(30 mmol/L glucose). Treatment group(HG+MLT group) was treated with DMEM medium at a high glucose level containing 100 μmol/L melatonin. The method of MTT was used to detect the effects of high glucose and melatonin on HUVEC proliferation. The release of lactate dehydrogenase(LDH) in cell culture supernatant was determined. The production of reactive oxygen species(ROS) was detected by flow cytometry. Hoechst 33342/PI double-staining was used to detect cell apoptosis and necrosis. Western blot was used to detect the effects of high glucose and melatonin on endoplasmic reticulum stress and cell pyroptosis related protein expression in HUVEC. Results Compared with the control group, cell proliferation level of the model group decreased, LDH releasing in cell culture supernatant increased, ROS production increased, the blue and red fluorescence increased in the double-staining, the expressions of GRP78, ATF4, CHOP, IRE1α, PERK, ATF6α, NLRP3, cleaved caspase-1, IL-1β increased. Compared with model group, cell proliferation level of the treatment group increased, LDH releasing in cell culture supernatant decreased, ROS production decreased, the blue and red fluorescence decreased in the double-staining, the expressions of GRP78, ATF4, CHOP, IRE1α, PERK, ATF6α, NLRP3, cleaved caspase-1, IL-1β decreased. Conclusion MLT can alleviate HUVEC damage induced by high glucose, and the probable mechanism may be related to the regulation of endoplasmic reticulum stress and cell pyroptosis.

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Objective To investigate the expression of lysophosphatidic acid(LPA) in mouse silicosis model and its effect on epithelial-mesenchymal transition(EMT) of mouse lung epithelial(MLE-12) cells. Methods 20 C57 BL/6 male mice were randomly divided into the control group and the model group. The control group was given normal saline, and the model group was given nasal drip of 50 μl silicon dioxide(SiO2) suspension with 100 mg/L every day for 7 consecutive days. They were killed on the 28 th day. Partial lung tissues were taken. Immunohistochemistry was used to observe the expression of lysophosphatidic acid receptor 1(LPAR1), and Western blot was used to detect the protein expression of α-smooth muscle actin(α-SMA),Type Ⅰ collagen( COLⅠ) and LPAR1; the proliferation of MLE-12 was detected by solution cell proliferation assay; scratch test was used to detect the migration ability of SiO2on MLB-12 cells. MLE-12 cells were divided into control group, SiO2stimulation group and inhibitor group, and the expression levels of LPARI and EMT related proteins were detected by Western blot. Results Western blot detection showed that the expression of α-SMA and COLⅠin the lung tissue of mice from the model group increased, and the model was established successfully; immunohistochemistry showed that the expression of LPAR1 was positive in the epithelial cells around the trachea and bronchus of the model group mice, showing bright brown; Western blot detection found that the expression of LPAR1 protein in the lung tissue of mice from the model group was higher than that from the control group(P<0.05); cell proliferation assay and scratch test showed that SiO2could significantly promote the proliferation and migration of MLE-12 cells; Western blot showed that the expression of LPAR1 and interstitial marker Vimentin protein increased in SiO2stimulation group(P<0.05), while the expression of epithelial marker E-cadherin protein decreased(P<0.05), and the difference was statistically significant compared with the control group and the inhibitor group(P<0.05). Conclusion The expression of LPA increased in mouse silicosis model, which can promote the proliferation and migration of MLE-12 cells by regulating EMT process and exacerbates the process of silicosis in mice.

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Objective To investigate the effect of demethylase obesity-associated protein(FTO) on the expression of tumor necrosis factor-α-stimulating gene-6(TSG-6) mRNA demethylation in keloid tissue and its role in scar formation. Methods Fresh keloid tissue(experimental group) and normal skin tissue(control group) were randomlyselected, and the expression level of TSG-6 in the experimental group and control group was detected by immunofluorescence method. A probe expressing TSG-6 mRNA sense and antisense chain were constructed for RNA pulldown, followed by Western blot detection of samples in each group with N6-methyladenine(m6A) antibody. Real-time fluorescence quantitative polynucleotide chain reaction(qPCR) and Western blot were used to detect the expression of TSG-6 mRNA demethylase and methylase in the experimental group and control group. The expression levels of FTO in experimental group and control group were detected by immunofluorescence assay. Results Immunofluorescence results showed that the expression level of TSG-6 in the experimental group was lower than that in the control group(P<0.01). Pulldown test results of biotin-labeled TSG-6 RNA showed that the m6A expression level of TSG-6 mRNA in the experimental group was lower than that in the control group(P<0.01). The results of qPCR showed that the expression level of FTO mRNA in the experimental group was higher than that in the control group(P<0.01). Western blot analysis showed that FTO protein expression in the experimental group was higher than that in the control group(P<0.001). Immunofluorescence results showed that the expression level of FTO in experimental group was higher than that in control group(P<0.001). Conclusion The demethylase FTO catalyzes the demethylation of TSG-6 mRNA and promotes the formation of keloid, which may provide a new idea and direction for clinical treatment of keloid.

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Objective To establish and evaluate the pyroptosis model of rat chondrocytes induced by tumor necrosis factor-α(TNF-α). Methods A two-step enzymatic digestion method was used to obtain rat articular chondrocytes, inverted phase contrast microscope was used to observe the morphological structure of chondrocytes. Toluidine blue staining and type Ⅱ collagen(Col Ⅱ) staining were used to identify chondrocytes. Different mass concentrations of TNF-α(5, 10, 20, 40 ng/ml) were used to establish the pyroptosis model with TNF-α(0 ng/ml) as the control group. Cell viability was detected by CCK-8 method and proteins of pyroptosis signal were detected by Western blot. The levels of IL-1β and IL-18 in cultured supernatants were examined by ELISA kits. The expression of gasdermin D(GSDMD) in chondrocytes was detected by immunofluorescence. Scanning electron microscope was used to observe the morphological changes of chondrocyte. Results Compared with the control group, the cell viability of rat chondrocytes gradually decreased with the increase of TNF-α concentration and the expression of nucleotide-binding and oligomerization domain-like receptor containing protein 3(NLRP3), caspase-1, GSDMD and Phospho-NF-κB p65(P-p65) proteins increased. Furthermore, TNF-α(20 ng/ml) could up-regulate the expression of matrix metalloproteinase-13(MMP-13), the fluorescence expression of GSDMD and the levels of IL-1β and IL-18 while the expression of Col Ⅱ was distinctly reduced. What′s more, the articular chondrocytes were swollen,and the microstructure was destroyed.Conclusion TNF-α(20 ng/ml) can cause the swelling and death of rat chondrocytes, degradation of cartilage matrix and activation of pyroptosis signaling pathway. The pyroptosis model of rat chondrocytes was successfully established.

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Objective To analyze the effect of miR-138 regulating TrkA-TRPV1 signaling pathway on cartilage damage in knee osteoarthritis(KOA) model rats. Methods Forty healthy SD rats were selected and divided into normal group, model group, silent group and overexpression group with 10 rats in each. Behavioral and cartilage histological scores of rats in each group were compared. The expression levels of inflammatory factors[TUMOR necrosis factor-α(TNF-α), interleukin(IL)-1, IL-6]in articular fluid and bone metabolism markers[matrix metalloproteinase(MMP-13), type Ⅱ collagen(Col Ⅱ)], TrkA, TRPV1 and Mir-138 in cartilage tissue were detected. The effect of Mir-138 on cartilage injury in KOA rats was analyzed. Results Compared with normal group, behavioral and cartilage histological scores of model group, silent group and overexpression group increased(P<0.05). Compared with overexpression group, behavioral and cartilage histological scores of model group and silent group increased(P<0.05). Compared with normal group, the levels of TNF-α, IL-1 and IL-6 and the expressions of MMP-13, TrkA and TRPV1 increased in model group, silent group and overexpression group, while the expressions of Col II and Mir-138 decreased(P<0.05). Compared with overexpression group, the levels of TNF-α, IL-1 and IL-6 and the expressions of MMP-13, TrkA and TRPV1 increased in model group and silent group, while the expressions of Col II and Mir-138 decreased(P<0.05). Conclusion miR-138 can alleviate cartilage injury in KOA model rats, and its mechanism may be related to the inhibition of TrkA-TRPV1 signaling pathway.

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Objective To investigate the difference in invasiveness between vulvovaginal candidiasis(VVC) and recurrent vulvovaginal candidiasis(RVVC) strains, and to explore the effect of strains with different secreted aspartate protease(Sap) on vaginal local immunity. Methods Vaginal secretions of VVC and RVVC patients were collected for fungus identification.Candida albicans(C.albicans) were taken for drug sensitivity analysis, 25 S rDNA genotyping, and detection of secreted aspartate protease(Sap) and phospholipase(Plb) activity.C.albicanswith different Sap activity were cultured with vaginal epithelial cells, and the levels of Interleukin(IL)-4, IL-8, IL-17 in cell supernatant were measured by ELISA. Results Sap activity of VVCC.albicanswas stronger than that of RVVC. After infection withC.albicans, the secretion of IL-4, IL-8 and IL-17 by vaginal epithelial cells was higher, and IL-8 and IL-17 appeared earlier and lasted longer.C.albicanswith strong Sap enzyme activity could stimulate vaginal epithelial cells to secrete more cytokines. Conclusion Except for the Sap activity, there was no significant difference in the invasiveness between VVC and RVVC strains.C.albicanscould activate the vaginal host immune response in a short time. The lower Sap activity ofC.albicansin RVVC patients may be related to the protective factors secreted by the vaginal epithelium.

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Objective To investigate the effect of tripartite motif containing 59(TRIM59) on the biological function of human colorectal cancer HCT116 cells and its possible mechanism. Methods The recombinant TRIM59 interference plasmid(si-TRIM59) was constructed and transfected into colorectal cancer cell line HCT116 by liposome transfection. The transfection efficiency was verified by RT-qPCR and Western blot. The effects of TRIM59 gene silencing on the proliferation, colony-formation ability and cell cycle of colorectal cancer cells were detected by CCK-8 method, colony formation assays and flow cytometry respectively. Western blot was used to detect the expression level of CDK4 and cyclinD1.Results The expression levels ofTRIM59mRNA and protein in colorectal cancer cells were significantly higher than those in normal colorectal mucosa cells(P<0. 05). After knockdown of TRIM59expression, the proliferation activity and colony forming ability of HCT116 cells were significantly inhibited(P<0. 05), and the cell cycle was arrested in G0/G1 phase. At the same time, the expression levels of CDK4and CyclinD1 significantly decreased(P<0. 05).Conclusion TRIM59was highly expressed in colorectal cancer cells. Down regulation ofTRIM59expression can inhibit the proliferation and clone formation of colorectal cancer cells, suggesting thatTRIM59may become a new target for gene therapy of colorectal cancer.

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Objective To rapidly identify triazole(fluconazole, voliconazole, iriconazole) drug resistance and sensitiveCandida tropicalusing matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry(MALDI-TOF MS) platform data analysis and machine learning algorithms. Methods A total of 191Candida tropicalwere collected from various clinical specimens, 71 of which were triazole-resistantCandida tropicaland 120 were triazole-sensitiveCandida tropicalstrains. Data acquisition was performed using the MALDI-TOF MS platform, and the mass and charge ratio features of resistant and susceptible strains were classified and selected based on the Mann-Whitney Rank-sum Test(Mann-WhitneyU-test) and the importance score obtained by the Random Forest(RF) algorithm. The classification model was constructed using the RF algorithm and a nonlinear support vector machine with a radial basis function kernel(RBF-SVM), calculating the accuracy, sensitivity, specificity, F1 value and the area under the subject worker curve(AUC) of the RBF-SVM model under the same experimental data to evaluate the model discrimination performance. Results All strains obtained 76 unique mass spectrum peaks after analysis on the MALDI-TOF MS platform. Among them, six peaks 3 481,7 549,6 500,3 048,6 892,2 596 m/z were selected as the model feature peaks established by the feature dimensionality reduction treatment. The accuracy of both the RBF-SVM and RF models was 0.84, and the AUC scores were 0.930 5 and 0.927 3, respectively. Conclusion Machine learning algorithms combined with the MALDI-TOF MS platform for data analysis can serve as a possible method to rapidly distinguish triazole-resistantCandida tropicaland triazole-sensitive strains.

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Objective To investigate the effect of modified hyaluronic acid(HA) hydrogel-controlled release brain-derived neurotrophic factor(BDNF) on the growth, differentiation, and apoptosis of rat embryonic neural stem cells(rFNSCs). Methods The experiment took rat embryonic stem cells as the research object. The experiments were divided into three groups, blank control group [rFNSCs co-cultured with poly(lactic-co-glycolic acid)(PLGA)and HA],conditional control group(rFNSCs co-cultured with BDNF-PLGA and HA), and experimental group(rFNSCs co-cultured with BDNF-PLGA and modified hyaluronic acid hydrogel that is Dex-HA). ELISA was used for detecting the release curve of BDNF in the controlled-release scaffold. The Live/Dead assay detected the apoptosis of rFNSCs in each group, CCK-8 experiment was used to test the cell proliferation activity of rFNSCs after coculture with hydrogel. The differentiation ratios of rFNSCs in different groups were detected by immunocytochemical staining.Results ELISA result showed that the controlled-release scaffold could release BDNF for 14 days.Live/Dead result showed the number of rFNSCs apoptotic in experimental group was less than blank control group and conditional control group. CCK-8 experiment result showed that the cell proliferation activity of rFNSCs in the experimental group was showed stronger survivability than blank control group and conditional control group. The difference was statistically significant(P<0. 05). Moreover, immunocytochemical staining showed that the experimental group hydrogel could promote the neuronal differentiation of rFNSCs and reduce their differentiation into astrocytes in vitro compared with the blank control group and the conditional control group. The difference was statistically significant(P<0. 05).Conclusion modified hyaluronic acid hydrogel controlled release brain-derived neurotrophic factor can promote the growth and differentiation of rFNSCs and reduce their apoptosis.

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Objective To investigate the optimal setting value of the minimum segment area(MSA) based on the Monaco planning system in the static intensity modulated radiotherapy(IMRT) plan for cervical cancer to improve the accuracy of radiotherapy planning for cervical cancer. Methods A retrospective collection of 10 patients with cervical cancer was performed using the Monaco treatment planning system to design fixed five-field static intensity modulation plans with MSA of 1, 2, 4, 10, 20, 50, 80, and 100 cm2. Each patient received eight radiotherapy plans. The radiotherapy plan with an MSA of 2 cm2was used as the control group to compare the radiotherapy plans with other MSA settings. Under the premise that other optimization objective functions and constraints were the same, only the set value of MSA was changed, and the statistical methods for analyzing of variance and post-hoc comparison were used to study the impact of MSA on radiotherapy plans. Results When MSA was in the range of 10～20 cm2, compared with the control group, the dose of target area and organ-at-risk did not change significantly, but the number of monitor units and subfields began to decrease. When MSA starts from 50 cm2, compared with the control group, the maximum dose(D2%) and the average dose(Dmean) in the target area both increased, and the uniformity index(homogeneity index, HI) and conformity index(conformity index, CI) began to deteriorate. Except for the small intestine average dose(Dmean) that changed slightly with MSA, the exposure to other organs at risk increased with the increase of MSA(P<0.05); the number of monitor units and subfields generally decreased with the increase of MSA. Conclusion In the design of a static intensity modulation plan for cervical cancer based on a Monaco treatment planning system, the optimal setting range for the MSA setting value is 10～20 cm2.

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Objective To investigate the relationship between the expression of p-Stat3 and Survivin in gastric cancer and the clinical characteristics and prognosis of patients, and to establish the prediction model of postoperative survival of patients combined with alanine aminotransferase(ALT) and aspartate aminotransferase(AST). Methods Data of 133 patients undergoing gastric tumor resection were collected and followed up. The expression of p-STAT3 and Survivin in gastric cancer and precancerous tissues were detected by immunohistochemical method. Kaplan-Meier method was used to draw the survival curve. Logistic regression model combined with receiver operating characteristic curve(ROC) curve was used to describe the predictive value of multi-index combined detection on postoperative survival status of patients. The prediction model of the histogram was established using Survival and RMS packages in R Studio software. Results The expressions of p-Stat3 and Survivin in gastric cancer tissues were higher than those in precancerous tissues. The expression of p-Stat3 in gastric cancer tissues was positively correlated with the depth of invasion and TNM stage, while the expression of Survivin in gastric cancer tissues was positively correlated with the degree of differentiation and the depth of invasion. The higher the expression levels of p-Stat3 and Survivin, the worse the prognosis. The lower the ALT level, the worse the prognosis. Survivin, ALT and AST were the optimal combination for predicting postoperative survival in patients with gastric cancer. Conclusion The expression of p-Stat3 and Survivin plays an auxiliary role in the diagnosis of gastric cancer. P-Stat3, Survivin and ALT are correlated with the prognosis of patients with gastric cancer and have predictive value. Survivin, ALT and AST combined model has a big value to predict the postoperative survival prognosis of gastric cancer patients, which can provide a reference for clinical practice.

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Objective To analyze the clinical features, the efficacy of131I therapy and its effect on granulopoiesis in Graves′ hyperthyroidism(hyperthyroidism) patients with neutropenia. Methods 144 hyperthyroidism patients were retrospectively studied after131I therapy, among which 42 cases(HT group) accompanied neutropenia due to hyperthyroidism itself, 52 cases(ATD group) appeared neutropenia after anti-thyroid drug(ATD)treatment, and the remaining 50 cases(control group) were hyperthyroidism patients with normal neutrophil count. The clinical features, the efficacy of131I treatment for hyperthyroidism and the change of neutrophil count after131I treatment were analyzed and compared in three groups.Results Mild neutropenia was commoner in both the HT group and the ATD group. The curative rate of hyperthyroidism in the HT group, the ATD group and the control group were80. 95%, 84. 62% and 84. 00%, respectively. There was no significant difference in the efficacy of131I therapy among the three groups. Compared with the baseline value before treatment, neutrophil count increased in all three groups 2-4 weeks after131I treatment(allP<0. 05). The neutrophil recovery rates of the HT group and the ATD group were 61. 90% and 84. 62%, respectively. The ATD group had a higher recovery rate(P<0. 05).Conclusion Mild neutropenia is commoner in hyperthyroidism patients with neutropenia due to either hyperthyroidism itself or ATD treatment. Normative131I treatment for hyperthyroidism patients with neutropenia is safe and effective.

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Objective To analyze the CD4+T cell count level of newly diagnosed HIV infected patients in Jiangsu province, and to understand their immune status and disease progression so as to provide scientific basis for HIV prevention and control strategy in the whole province. Methods Flow cytometry was used to detect the absolute count of CD4+T lymphocytes in newly diagnosed patients who had not initiated ART in 2020. Multivariate Logistic regression was used to analyze the associated factors of CD4+T cell count. Results In 2020, there were 2 175 new diagnosed cases with HIV infection in Jiangsu Province. Patients under 30 years old, infected by homosexual transmission, unmarried, with college degree or above, students, diagnosed in counseling and testing and floating population had a higher absolute count level of CD4+T cell count(P<0.05). Multivariate Logistic regression analysis showed that people aged ≥30 and diagnosed in medical institutions were more likely to be presented lately(P<0.05). Conclusion In recent years, the intervention strategy for men who had sex with men(MSM) in Jiangsu province had achieved remarkable results.In the future, much more attention should be paid for the population over or equal to 30 years old, married or divorced, infected by heterosexual transmission, with high school education or below, farmers or migrant workers, retirees and diagnosed in medical institutions so that the PITC service should be promoted, and the publicity and intervention should be strengthened to present cases as soon as possible.

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Objective To construct nomogram to predict the risk of bone metastasis in patients with non-small cell lung cancer(NSCLC). Methods The clinical data of NSCLC patients diagnosed in the hospital were retrospectively analyzed, including the occurrence of bone metastasis, age, gender, pathological type, smoking status, PS score, TN stage, metastasis of other sites before bone metastasis, carcinoembryonic antigen(CEA) level, alpha fetoprotein(AFP) level, serum calcium(Ca2+), serum phosphorus(P), alkaline phosphatase(ALP) level, which were determined by univariate and multivariate logistic regression analysis. Receiver operating characteristic curve(ROC) and decision curve analysis were used, DCA was used to verify the accuracy and clinical benefit of the model, and nomogram was used to visualize the model. Results Area under the ROC curve(AUC) showed that in the modeling group(n=138) and the validation group(n=92), the AUC value predicted by combined indicators(age, gender, pathological type, CEA, ALP)(modeling group=0.792, validation group=0.629) was higher than that predicted by single indicator. Conclusion The prediction model constructed in this study has good effect and can provide reference for clinical screening of high-risk patients with bone metastasis of NSCLC.

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To explore the clinical value of arthrography in children with Jacob Ⅱ type humeral humeral lateral condylar fracture. A retrospective collection of eligible children with a total of 85 patients was recorded in this study. According to the arthrography results, the children were divided into JA-JD four groups. The variance analysis and t text analyzed the correlation between gender, side, age, time from injury to operation, fracture displacement degree and treatment. There was no significantly difference between gender, side, age, time from injury to operation and treatment(P>0.05). The degree of fracture displacement in group JA was significantly lower than group JB [(2.58±0.41)vs(3.32±0.50),P<0.05]. The intraoperative arthrography adds valuable information for the surgical treatment of children with Jacob II humeral lateral condylar fracture. When the fracture displacement below 3.2 mm in X-ray, it is feasible to perform closed reduction and percutaneous pinning.