AIM: To investigate the effect and potential mechanism of liraglutide(LIR)combined with neural stem cells(NSCs)in alleviating diabetic retinopathy(DR)in rats.METHODS:A DR rat model was established using a high-fat diet combined with a single intraperitoneal injection of streptozotocin(STZ). Rats in the control group were fed a normal diet and received an intraperitoneal injection of citric acid-sodium citrate buffer. After modeling, the rats were divided into the control group, DR group, DR+LIR group, DR+BMSCs group, DR+NSCs group, DR+BMSCs+LIR group, and DR+NSCs+LIR group. According to the grouping instructions, rats were given intraperitoneal injection of LIR at 200 μg/(kg·d)and intravitreal injection of bone marrow mesenchymal stem cells(BMSCs), or NSCs, or the combined therapy accordingly for 8 wk. Fasting blood glucose(FBG)and serum insulin levels, as well as retinal oxidative stress indexes and serum inflammatory indexes, were detected in each group. Retinopathy was evaluated by hematoxylin-eosin(HE)staining, retinal cell apoptosis was detected by TUNEL staining, SIRT1 expression in retinal tissues was detected by immunohistochemistry, and BDNF expression in retinal tissues was detected by immunofluorescence. The transcriptional levels of cZNF532, miR-29a-3p, SIRT1, TGF-β1, NRF2, Keap1, and BDNF in retinal tissues were detected by qRT-PCR, and the protein expression levels of SIRT1, TGF-β1, SMAD2/3, p-SMAD2/3, NRF2, Keap1, p65, and p-p65 in retinal tissues were detected by Western blot. RESULTS:Compared with the other groups of rats, the retinal morphology of rats in the DR+NSCs+LIR group was significantly improved, the levels of FBG, TNF-α, IL-1β, IL-6 and MDA were decreased(all P<0.05), the serum insulin level and the levels of SOD, CAT, and GSH-Px were increased(all P<0.05), the TUNEL-positive rate of retinal cells was reduced(all P<0.05). The expression levels of cZNF532, SIRT1, NRF2, and BDNF in retinal tissue were up-regulated(all P<0.05), whereas the expression levels of miR-29a-3p, TGF-β1, p-SMAD2/3, Keap1, and p-p65 were down-regulated(all P<0.05). CONCLUSION:LIR combined with NSCs alleviates DR by regulating the cZNF532-miR-29a-3p-SIRT1 network and its downstream TGF-β/SMAD, NF-κB, and Keap1/NRF2 signaling pathways.

The aim of this study is to identify the linear Fc-binding epitope for IgG1 on bovine IgG Fc receptor Ⅱ(boFcγRⅡ)to understand the molecular basis of IgG-Fcγ interaction.The boFcγRⅡ molecules were expressed on cell surface of the boFcγR Ⅱ-transfected COS-7 cells.The extracel-lular domain of boFcγRⅡ was expressed in NS0 cells,and the boFcγRⅡ recombinant protein was purified from ascites by Ni-chelation chromatography.Peptides derived from the membrane-distal extracellular domain(EC2)of boFcγR Ⅱ were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin(BSA).Binding of bovine IgG1 to the different peptides was tested by dot-blot assay,and the IgG-binding peptide was further modified by truncation and mutation to identify the Fc-binding epitope as well as its key amino acids for Fc-binding.The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay,re-spectively.The results showed that boFcγR Ⅱ molecules were stably expressed on surface of the transfected COS-7 cells,which showed about 90％rosetting with IgG1-RBCs.The soluble boFcγRⅡ recombinant protein specifically bound to bovine IgG1.The minimal effective peptide of 122FYQDRKSKIF131 of boFcγRⅡ was able to bind bovine IgG1 specifically,suggesting it repre-sents a linear Fc-binding epitope located in the putative C-C'loop of the EC2 domain on the recep-tor.The Ala-substitution of Phe122,Tyr123,Arg126,Lys127,Ser128,Lys129 or Phe131 within the linear epitope led to a complete loss of its IgG1-binding capability,indicating those residues are critical for IgG1-binding on boFcγRⅡ.The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRⅡ with IC50 of 20.05 μmol/L,and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRⅡ transfected cells with IC50 of 80.15 μmol/L.The re-sults indicate that boFcγRⅡ possesses the linear epitope for Fc-binding,and the Fc-binding pep-tide showed well capability of regulating boFcγR Ⅱ-IgG1 interaction on cell surface,thereby provi-ding a research foundation for understanding the IgG-Fcγ interaction.

The aim of this study is to identify the linear Fc-binding epitope for IgG1 on bovine IgG Fc receptor Ⅱ(boFcγRⅡ)to understand the molecular basis of IgG-Fcγ interaction.The boFcγRⅡ molecules were expressed on cell surface of the boFcγR Ⅱ-transfected COS-7 cells.The extracel-lular domain of boFcγRⅡ was expressed in NS0 cells,and the boFcγRⅡ recombinant protein was purified from ascites by Ni-chelation chromatography.Peptides derived from the membrane-distal extracellular domain(EC2)of boFcγR Ⅱ were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin(BSA).Binding of bovine IgG1 to the different peptides was tested by dot-blot assay,and the IgG-binding peptide was further modified by truncation and mutation to identify the Fc-binding epitope as well as its key amino acids for Fc-binding.The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay,re-spectively.The results showed that boFcγR Ⅱ molecules were stably expressed on surface of the transfected COS-7 cells,which showed about 90％rosetting with IgG1-RBCs.The soluble boFcγRⅡ recombinant protein specifically bound to bovine IgG1.The minimal effective peptide of 122FYQDRKSKIF131 of boFcγRⅡ was able to bind bovine IgG1 specifically,suggesting it repre-sents a linear Fc-binding epitope located in the putative C-C'loop of the EC2 domain on the recep-tor.The Ala-substitution of Phe122,Tyr123,Arg126,Lys127,Ser128,Lys129 or Phe131 within the linear epitope led to a complete loss of its IgG1-binding capability,indicating those residues are critical for IgG1-binding on boFcγRⅡ.The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRⅡ with IC50 of 20.05 μmol/L,and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRⅡ transfected cells with IC50 of 80.15 μmol/L.The re-sults indicate that boFcγRⅡ possesses the linear epitope for Fc-binding,and the Fc-binding pep-tide showed well capability of regulating boFcγR Ⅱ-IgG1 interaction on cell surface,thereby provi-ding a research foundation for understanding the IgG-Fcγ interaction.

Objective:To establish a method of dual nueleic acid test strips for rapid detection of Bacillus cereus cytK and nhe toxin genes based on polymerase chain reaction(PCR)and colloidal gold technique,and to evaluate its specificity,sensitivity,repeatability and stability.Methods:Bacillus cereus DNA was extracted by boiling method.Specific primers were designed with Bacillus cereus cytK and nhe as the target genes.Clonal transformation was used to identify the PCR products.The optimal labeling amounts of colloidal gold-labeled streptavidin,quality control line(C line),cytK detection line(T1)and nhe detection line(T2)were determined.The nucleic acid test strips were assembled and its specificity,sensitivity,reproducibility and stability were evaluated.Results:The DNA concentration of Bacillus cereus was 248 mg·L-1,and the purities were 1.8-2.0.After cloning and plasmid sequencing,the similarities between the PCR products and the sequences of cytK and nhe registered in the GenBank database were 100％.Under the condition of pH 7.0,the optimal amount of streptavidin labeling per 200 μL of colloidal gold solution was 6.0 μL;the optimal marking amount was 2.00 g·L-1 for the quality control line(C line),0.550 g·L-1 for cytK gene detection line(T1)and 0.2 g·L-1 for nhe gene detection line(T2).In the specificity test,positive result on the test strips was seen only for Bacillus cereus,and no cross-reactivity was observed for Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Bacillus subtilis,which were consistent with the electrophoresis results.Sensitivity assay showed that even when DNA concentration was reduced to 10-2 mg·L-1,three bands(C line,T1 line and T2 line)could be observed,and the detection limit of the test strip was one-tenth of agarose gel electrophoresis(10-1 mg·L-1).The nucleic acid test strips were verified by different operators in different laboratories,and the results were consistent.The stability of the test strips was verified at the 6th,9th and 12th months,and the results showed good stability.Conclusion:The dual nucleic acid test strip method established in this study can simutaneously detect the cytK and nhe toxin genes of Bacillus cereus with high sensitivity and specificity,achieving short-term visual detection.

Objective:To discuss the effect of quorum sensing-related gene expression on biofilm formation and drug resistance in clinically multidrug-resistant Pseudomonas aeruginosa,and to clarify the mechanism of enhacing drug resistance.Methods:A total of 77 strains of Pseudomonas aeruginosa were collected.Based on drug resistance,the strains were divided into multidrug-resistant group and sensitive group.The optimal biofilm formation conditions were determined using the microtiter plate method;biofilm formations of the stains in both groups was observed under an optical microscope;crystal violet staining was used to semiquantitatively detect biofilm formation ability of P.aeruginosa in both groups;microbroth dilution method was used to determine the minimal inhibitory concentration(MIC)values of the quorum sensing inhibitor(C-30)against Pseudomonas aeruginosa in both groups;RNA was extracted from two groups using a commercial kit,while RNA from planktonic state and biofilm state of multidrug-resistant strains was extracted using modified TRIzol method;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of quorum sensing-related genes(lasR/I,RhlR/I,PqsR/A)of the stains in multidrug-resistant group and sensitive group,as well as before and after adding the quorum sensing inhibitor C-30.Results:Among 77 strains of Pseudomonas aeruginosa,56 were multidrug-resistant(multidrug-resistant group)and 21 were fully sensitive(sensitive group).Optimal biofilm formation occurred at a bacterial concentration of 1.5×108 CFU·mL-1 with 48 h incubation.The biofilm positivity rate was 91%,with strongly positive,moderately positive,weakly positive,and negative biofilms accounting for 16%,34%,41%,and 9%,respectively.The biofilm positivity rate in multidrug-resistant strains was 96%,and biofilm formation ability in multidrug-resistant group was higher than that in sensitive group(P<0.05).When the concentration of C-30 was 8 mg·L-1 the biofilm formation in most Pseudomonas aeruginosa was inhibited,with enhanced suppression at higher concentrations.The absorbtion(A)value of both planktonic-state and biofilm-state RNA ranged from 1.8 to 2.0.The RT-qPCR results showed that compared with planktonic state,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA of the stains in biofilm state were significantly increased(P<0.01).Compared with non-inhibitor group,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA in biofilm state of inhibitor-treated group were significantly decreased(P<0.01).Conclusion:High expression of quorum sensing-related genes in multidrug-resistant P.aeruginosa promotes biofilm formation,thereby enhancing drug resistance.

Objective:To establish a rapid detection method for pathogenic microorganisms by combining loop-mediated isothermal amplification(LAMP)and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 12a(Cas12a)(CRISPR-Cas12a)system,and to evaluate its efficacy for detecting the thermolabile hemolysin(tlh)gene of Vibrio parahaemolyticus(Vp).Methods:Using the tlh gene of Vp as the target gene,LAMP primers and CRISPR RNA(crRNA)were designed to construct and optimize the optimal concentration ratio of each component in the LAMP-CRISPR detection system.Bacillus cereus,Staphylococcus aureus,and Escherichia coli were used as control groups,and the specificity,sensitivity,reproducibility and positive conformity rate were verified to establish a rapid LAMP-CRISPR/Cas12a method for detecting the tlh gene of Vp.Results:The method specifically detected Vp,while Bacillus cereus,Staphylococcus aureus,and Escherichia coli yielded negative results.The DNA extraction concentration of Vp was 190.67 mg·L-1 with an A(260)/(A280)ratio of 1.84.Under the reaction conditions of 37℃ with 80 cycles for 40 min using quantitative PCR(qPCR)method,when the concentrations of Cas12a protein and crRNA in the LAMP-CRISPR/Cas12a system were 50 nmol·L-1,the visual brightness and relative fluorescence intensity peaks were high.The sensitivity of LAMP CRISPR/Cas12a for detecting Vp DNA concentration could reach 10-6 mg·L-1.The reproducibility test results showed that different experimenters had consistent results in different experimental environments and times.Conclusion:The established LAMP-CRISPR/Cas12a method can rapidly detect the tlh gene of Vp with high sensitivity and specificity,and can achieve short-term visual detection in the field.

OBJECTIVES: To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. METHODS: L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. RESULTS: The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. CONCLUSIONS The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.
Listeria monocytogenes/isolation & purification* ; Gold Colloid ; Bacterial Toxins/genetics* ; Polymerase Chain Reaction/methods* ; Hemolysin Proteins/genetics* ; Bacterial Proteins/genetics* ; DNA, Bacterial/genetics* ; Food Microbiology ; Heat-Shock Proteins

Objective:To establish a method for rapid detection of OXA and par C resistance genes of Acinetobacter baumannii(Ab)by double nucleic acid colloidal gold strip and to develop kit.Methods:DNA of Ab was extracted by heating and boiling method.OXA and par C genes sequences of Ab were selected as target gene fragments based on NCBI.Primers were designed and labeled with 6-FAM,digoxin and biotin,respectively.Drug resistance gene detection reagents were developed,and nucleic acid gold test strips were used for rapid and visual detection.Molecular cloning and sequencing techniques were used to clone positive control samples and evaluate specificity,sensitivity and stability of kit.Results:DNA concentration and purity of Ab extracted by boiling method were good.Homology between cloned and sequenced plasmid DNA and gene sequence in GenBank database was 100%,respectively.Speci-ficity of kit was good,with only Ab showing positive results and other bacterial genera showing negative results;DNA concentration of Ab in double nucleic acid colloidal gold test strip decreased to 10-3 ng/μl,a red line still appeared,whose sensitivity was 10 times higher consistent with minimum detection limit of electrophoresis 10-2 ng/μl;test kits were tested at 3rd,6th and 9th months,and showed good stability.Conclusion:Double resistance detection kit established in this study can simultaneously detect OXA and par C resis-tance of Ab,who has advantages of high sensitivity,strong specificity,rapid and simple,and provides a new method for detection of carbapenem and quinolone antibiotic resistance of Ab.

A 11-year old female patient with severe thalassemia, receipt a lentivirus-based cell and gene therapy (CGT) therapy in Shenzhen Children′s Hosptial on July 27th, 2021. At the two follow-up visits after discharge, patient were continuously tested positive for HIV screening through HIV Ag/Ab Combo assay (chemiluminescence Immunoassay), and the viral load results of HIV-1 nucleic acid testing (NAT) were both>5 000 copies/ml. The patient can be diagnosed with HIV infection according to the National Guideline for Detection of HIV/AIDS(2020 Revised Edition). The thorough investigation findings and supplementary experiment results indicated that the false-positive HIV-1 NAT results was caused by cross-reactivity between the target sites detected by conventional HIV-1 NAT reagents and the lentiviral vectors fragments integrated into the genome of patient′s hematopoietic stem/progenitor cells. In conclusion, it is important for laboratories to select appropriate HIV-1 NAT testing platforms which won′t cause cross-reactivity for the testing of samples from patients who have been treated with HIV-derived vectors. It is also recommended to design and develop NAT testing platforms with multiple target regions labeled by different fluorescents for HIV NAT supplementation experiment to reduce the risk of false-positive diagnoses of HIV infection.

We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.
African Swine Fever Virus/genetics* ; Animals ; DNA ; Immunity, Innate ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine