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Objective To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.. Methods The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers’ O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested. Results The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL. Conclusion The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.
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Objective:Rapid assessment of the outcome after percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS) is an important clinical issue. In this study, an electrocardiogram (ECG) analysis method based on dynamic learning was proposed.Methods:A total of 203 patients with ACS after successful PCI were enrolled for prospective analysis at the Emergency Department of Qilu Hospital of Shandong University from April 2019 to December 2020. All patients were divided into group without ≥70% postoperative stenosis ( n=72) and group with ≥ 70% postoperative stenosis ( n=131) according to the presence of 70% or more stenosis after PCI. The clinical data of ACS patients were collected and analyzed by χ2 test, t-test, or Mann-Whitney test. ECGs were recorded before and 2 h after PCI, and were dynamically analyzed to generate cardiodynamicsgram (CDG) using dynamic learning. In the group without ≥ 70% postoperative stenosis, the model and CDG index for evaluating myocardial ischemia were obtained by training support vector machine (SVM) using 10 times 10-fold cross-validation. Results:There was no significant difference in clinical data between the two groups. The prediction accuracy and sensitivity of the support vector machine model for myocardial ischemia in group without≥70% postoperative stenosis were 73.61%, and 84.72% respectively. CDG transformed from disorderly to regular after PCI, and CDG index decreased significantly ( P<0.001): 90.28% (65) patients in group without≥70% postoperative stenosis, and 79.39% (104) patients in group with≥70% postoperative stenosis had lower CDG indexes than before PCI. Conclusions:In this study, CDG obtained by dynamic learning can intuitively and effectively evaluate the changes of myocardial ischemia before and after PCI, which is helpful to assist clinicians to formulate the next treatment plan.
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OBJECTIVE： To study the effects of atorvastatin combined with carbon monoxide releasing molecule 3 （CORM-3） on inflammation and oxidative stress indexes in atherosclerotic （AS） vulnerable plaque model rats. METHODS： The rats were randomly divided into control group （normal saline， i.g.）， model group （normal saline， i.g.）， statin group （atorvastatin 2 mg/kg， i.g.）， and statin+CORM-3 group （atorvastatin 2 mg/kg， i.g.+CORM-3 10 mg/kg， i.p.）， with 8 rats in each group. Control group was fed with basal diet， and the right common carotid artery was exposed to surgery without injury and was treated with normal saline instead of drug； other three groups were fed with high-fat diet+right common carotid artery injury+heteroprotein injection to induce AS vulnerable plaque model， for 10 weeks； and then they were given relevant medicine for intervention， once a day， for consecutive 2 weeks. 24 h after last medication， abdominal artery blood was collected； the concentration of LDL-C and HDL-C were determined by fully automatic biochemical analyzer. The levels of hs-CRP， IL-10， MCP-1 and MMP-9 in plasma were detected by ELISA； plasma levels of MDA and oxidized low density lipoprotein （ox-LDL） were determined by chemical colorimetry； the protein expression of heme oxygenase-1 （HO-1） was determined by Western blot. The pathological changes of right common carotid artery were observed under light microscope. RESULTS： Compared with control group， the levels of LDL-C， hs-CRP， MCP-1， MMP-9， MDA and ox-LDL， and protein expression of HO-1 were increased significantly （P＜0.05）， while the levels of HDL-C and IL-10 were decreased significantly in model group （P＜0.05）； the right common carotid artery formed obvious AS plaques. Compared with model group， the levels of LDL-C， hs-CRP， MCP-1， MMP-9， MDA and ox-LDL were decreased significantly in statin group and statin+CORM-3 group in model group （P＜0.05）， while the levels of HDL-C， IL-10 and the protein expression of HO-1 were increased significantly （P＜0.05）. Except for LDL-C and HDL-C， the improvement of other indexes in statin+CORM-3 group was more significant than statin group （P＜0.05）； pathological changes of right common carotid artery in statin group were not obvious， but the pathological changes of rats in statin+CORM-3 group were significantly alleviated and plaque structure also tended to be more stable. CONCLUSIONS： Atorvastatin combined with CORM-3 is better than atorvastatin alone in improving inflammation and oxidative stress indexes of AS vulnerable plaque model rats， and can promote the stability of AS vulnerable plaques.
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Objective To investigate the clinicopathogenical significance of phosphorylation type of c-jun N-terminal kinase(p-JNK)and muhidmg resistance protein P-glycopretein(P-gP),multidrug resistance proteinl(MRP1)and lung resistance protein(LRP)in gastric cancer. Methods The expression of p-JNK,P-gP,MRP1 and LRP was detected in a tissue microarray containing 168 spots of gastric cancer tissue and 27 spots of normal gastric tissue by immunohistochemistry.Results The positive expression rate of p-JNK,P-gp,MRP1 and LRP in gastric cancer wag 45.8%,51.8%,45.8%and 55.4%respectively,which Was significantly higher than that in normal gastric tissue(P＜0.05).The p-JNK expression correlated with depth of invasion(P＜0.01),histology grade(P＜0.05),vessel invasion (P＜0.01),lymph node metastasis and distant metastasis(P＜0.01).The expression of p-JNK,P-gp and MRP1 in a positive relationship(P＜0.05).Kaplan-Meier analysis revealed a significant impact on survival by p-JNK,P-gp and MRPl(P＜0.05). Conclusion The p-JNK expression in gastric cancer is correlated with malignant biological behavior and may be involved in the chemotherapeutic resistance by upregulating the expression of P-g[ and MRP1.
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Streptomyces hygroscopicus NND-52 producing Azalomycin B was treated with UV, UV -- LiC1, acridine orange ,the best treatment dose was determined. The yields of several strains which were obtained were more than three times thaat of CK, and the yield of strain A13 was 1100mg/L,they were stable. Through comparing the treating methods, it was shown that the later two methods were more effective
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AIM: To explore the effects of heme oxygenase-1(HO-1) protein expression induced by ginkgo biloba extract(EGB761) in rat vascular smooth muscle cells(RVSMC) and the correlative cell signaling pathway.METHODS: The RVSMC lines were revived.Serial passage to 6 generation was carried out and divided into different groups.The cells were treated respectively with vehicle,purely EGB761,EGB761 plus zinc protoporphyrin IX or other specific inhibitors of cell signaling pathway.Western blotting method was used to detect the expression of HO-1 in RVSMC.RESULTS: EGB761 induced HO-1 protein expression in a dose dependent manner.ZnPPⅨ and genitein significantly inhibited HO-1 protein expression induced by EGB761(0.10?0.01,0.07?0.01 vs 0.61?0.07,P0.05,respectively).CONCLUSION:(1) EGB761 significantly induces HO-1 protein expression in RVSMC,and the effect can be inhibited by a specific HO inhibitor ZnPPⅨ.(2) The HO-1 protein expression induced by EGB761 in RVSMC is mediated by tyrosine protein kinase pathway.
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Streptomyces hygroscopicus NND 52 producing Azalomycin B was treated with UV,UV+LiCl,acridine orange,the best treatment dose was determined. The yields of several strains which were obtained were more than three times thaat ofCK, and the yield of strain A13 was 1100mg/L,they were stable.Through comparing the treating methods, it was shown that the later two methods were more effective
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Objective To observe the characteristics of biology and pathogenicity of the third stage Anisakis simplex larvae ( L3). Methods The survival time of the L3 in various conditions was observed and the pathological change after experimental infection in rats was examined. Results The results showed that the L3 frozen at -20℃ for 10 - 12 h can be killed. In the temperature range of 4 - 10 ℃ , the L3 can survive for over 8 months. The L3 was very active at 37℃ , and was killed at the high temperature over 40℃ in a very short time. The ingredients for sashimi cannot kill the L3. The experiment of rats infected by the L, showed that about 15% -25% of the L3 penetrated into the gastrointestinal wall or migrated into the peritoneal cavity in 2 days. After 3d the L4 was not infectious, and died automatically in 7 - 10 days and could not develop into adults. The animals can be easily infected when the stomach was empty. The pathological study showed that the primary infection was a kind of reaction to foreign body, while that of the re - infection was allergic. Conclusion The L3 has a strong resistance to low temperature and to ingredients , it can be killed by freezing at -20℃ in 24 hours. The L3 can not mature in the body of terrestrial mammals but causes pathological change in the stomach and allergy.[