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 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.

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.
Amino Acid Sequence ; Animals ; Mice ; Phylogeny ; Pore Forming Cytotoxic Proteins ; Protein Structure, Secondary ; Ranidae

Ubiquitin specific peptidase 28 (USP28) is closely associated to the occurrence and development of various malignancies, and thus has been validated as a promising therapeutic target for cancer therapy. To date, only few USP28 inhibitors with moderate inhibitory activity have been reported, highly potent and selective USP28 inhibitors with new chemotypes remain to be discovered for pathologically investigating the roles of deubiquitinase. In this current study, we reported the synthesis and biological evaluation of new [1,2,3]triazolo[4,5-]pyrimidine derivatives as potent USP28 inhibitors. Especially, compound potently inhibited USP28 (IC = 1.10 ± 0.02 μmol/L,  = 40 nmol/L), showing selectivity over USP7 and LSD1 (IC > 100 μmol/L). Compound was cellularly engaged to USP28 in gastric cancer cells. Compound reversibly bound to USP28 and directly affected its protein levels, thus inhibiting the proliferation, cell cycle at S phase, and epithelial-mesenchymal transition (EMT) progression in gastric cancer cell lines. Docking studies were performed to rationalize the potency of compound . Collectively, compound could serve as a new tool compound for the development of new USP28 inhibitors for exploring the roles of deubiquitinase in cancers.

ABSTRACT:Objective To assess the effect of prolonged laparoscopic surgery on peritoneal mesothelial cells and fibrinolysis in humans.Methods We examined prospectively 1 6 consecutive patients who underwent laparoscopic surgery (LAP)and 2 1 patients who underwent conventional open surgery (OP)for high-medium rectal cancer with curative intent.During the procedure,biopsy of the parietal peritoneum was made before operation and at 45 min,90 min,and 120 min after operation.The tissue-type plasminogen activator (tPA)and plasminogen activator inhibitor-1 (PAI-1 )were determined by enzyme-linked immunosorbent assay in peritoneal tissues.The cellular injury was detected by LDH assay.The proliferation was quantified by MTT assay.Results PAI-1 activity in the peritoneal tissue was significantly lower in LAP group than in the OP group.tPA activity decreased after 45min of open surgery,but there was no significant change in the LAP group.With time extension,the LDH activity increased and the proliferation of the mesothelial cells decreased.Conclusion Preservation of a prolonged hypofibrinolytic state by inhibition of PAI-1 up-regulation during LAP may predispose patients to less postoperative peritoneal adhesion. The cellular injury becomes apparent and the proliferation is inhibited during prolonged laparoscopic surgery.

Calculation of cardiac hemodynamic parameters is based on accurate detection of feature points in impedance cardiogram. According to these parameters, doctors can determine heart conditions, so it is very important to accurately detect the feature point of impedance differential signals. This article presents a process in which we used wavelet threshold method to de-noise signals, and then detected the feature points after six layers wavelet decomposition by using bior3. 7. The experimental data were collected from healthy persons in our laboratory and twenty two clinical patients in Chongqing Daping Hospital by using KF_ICG instrument. The results indicated that this method could precisely detect feature points whether it was from healthy people or clinical patients. This helps to achieve the application of noninvasive detection cardiac hemodynamic parameters in clinical treatments by using impedance method.
Cardiography, Impedance ; Electric Impedance ; Heart ; physiology ; Hemodynamics ; Humans ; Wavelet Analysis

Objective To analyze the clinical and CT manifestations of lung involvement of microscopic polyangiitis (MPA). Methods The clinical manifestations,laboratory ANCA examinations and CT features of 16 patients with lung involvement of MPA were retrospectively reviewed. Results (1) Clinical manifestations: 11 cases had hemoptysis or bloody sputum. Eight cases, who first presented with lung symptoms, were misdiagnosed with other lung diseases. All cases had mulfiorgans injuries involved kidney, cardiovascular and endocrine system, etc. (2) Laboratory examinations: all cases were pANCA positive and 14 cases were MPO-ANCA positive. (3) CT examinations: all cases had interstitial changes, 15 cases were interstitial predominately and 1 case was parenchymal predominately. Eight cases had pulmonary interstitial fibrosis and 11 cases had consolidation and 6 of them had both interstitial and consolidation. Two case had accompanied multiple nodulesand one of them had multiple cavitates. Six cases had mediastinal lymphoadenopathy. Conclusions Most of MPA patients have clinical manifestations of hemoptysis and bloody sputum, the CT examination show interstitial lung disease. Middle or advanced age population presented with above-mentioned manifestations should be alert to MPA, whether or not they have kidney and other organs injury.

Objective To analyze the clinical and CT manifestations of the chest and abdomen lymphangioleiomyomatosis (LAM). Methods The clinical and CT manifestations of 13 patients with LAM proved histopathologically were reviewed retrospectively.Results Twelve patients onset with intrapulmonary manifestations all had progressive dyspnea, other symptoms included pneumothorax of recurrent attacks, chest distress, hemoptysis, cough, chylothorax and so on. During the course of disease, 12 patients had no extrapulmonary symptoms, abdominal great goiter was found unintentionally in the rest one without any intrapulmonary symptoms. Pectoral CT manifestations included sporadic or asystematic cysts in pulmones with size of 2-20 mm, and most had thin and clear capsule wall. The lung parenchyma among cysts was mostly normal. Four patients had pneumatocele, 2 had pleural effusion. Abdominal CT was performed in 10 patients and 7 had abnormal findings: renal angiolipoleiomyoma (ALL) in 3 including one had retroperitoneal multiple lymphangiomyomas and effussion and seroperitoneum, another 2 had multiple liver ALL and spleen accretion. The rest 4 patients included retroperitoneal lymphadenectasis in 2, seroperitoneum in one, as well as retroperitoneal lump and spleen accretion in one patient.Conclusion Pectoral and abdominal symptoms in LAM are not specific, but the CT manifestations somehow specific, which are helpful to the identification and early diagnosis of LAM.

BACKGROUND: The activation of hypothalamus-pituitary-adrenal cortex axis may play key role in the increasing expression of hypothalamic corticotropin-re-leasing hormone (CRH) during stress reaction. However by what way to induce the CRH expression in hypothalamic neuron, and whether CRH can activate hypothalamic neurons are still not very clear.OBJECTIVE: To observe the changes of intracellular cyclic adenosine monophosphate (cAMP) and cytoplasmic Ca2+ concentration in the hypothalamic neurons cultured in vitro due to exogenous CRH stimulation.DESIGN: Comparative observation experiment.SETTING: Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA; Department of Neurosurgery , Tiantan Hospital, Capital University of Medical Sciences MATERIALS: This experiment was carried out in the Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA between December 1999 and March 2002. Hypothalamus was obtained from fetus rat at pregnancy of 17 days for the in vitro culture of hypothalamic neurons.METHODS: Hypothalamic neurons were co-cultured with exogenous CRH,with or without pretreatment with specific CRH 1 receptor antagonist -CP-154526. hypothalamic neurons were randomized into: ① CRH (10-12,10-10, 10-8, 10-6 mol/L) stimulation group. ② CP-154526(500 μmol/L)pretreatment aud CRH ( 10-12, 10-10, 10-8,10-6 mol/L) stimulation group. ③Hypothalamic neurons in corresponding normal control group were exposed to the isotonic saline stimulation. PTI fluorescence image system was used to determine and analyze the change of cytoplasmic free Ca2+ concentration in hypothalamic neurons due to exogenous CRH stimulation and RIA was used to detect the neuronal cAMP content.MAIN OUTCOME MEASURES: ①Cytoplasmic free Ca2+ concentration in hypothalamic neurons. ②cAMP content in hypothalamic neurons.RESULTS: The cytoplasmic free Ca2+ concentration and cAMP content were relatively lower in the hypothalamic neurons in normal control group,which obviously increased due to CRH stimulation [(240±22),(153±11)nmol/L; (3.26±0.19),(0.44±0.02) pmol/dish,P ＜ 0.01];CP-154526 could remarkably suppress the CRH (10-6 mol/L)induced increase in cytoplasmic free Ca2+ concentration and cAMP content in hypothalamic neurons [Ca2+ concentration: (240±22),(171±16)nmol/L; cAMP content:(3.26±0.19), (2.33±0.21) pmol/dish, P ＜ 0.01].CONCLUSION: CRH can directly act on hypothalamic neurons via type 1-receptor,thereby increase the cytoplasmic free Ca2+ concentration and cAMP content in hypothalamic neurons,playing the key role in the modulation of the synthesis and secretion of CRH during the activation of hypothalamic neurons.