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:Medial sural artery perforator flap (MSAPF) was applied to aesthetically reconstruct wounds in foot, and clinical outcome of this surgical method was explored.Methods:A retrospective analysis was conducted on the data of 10 patients who underwent the surgery of transfer of free MSAPF in reconstruction of foot wounds in the Department of Foot and Ankle Surgery, Xuzhou Renci Hospital from January 2020 to January 2023. The patients were 6 males and 4 females, aged 21 to 52 years, with an average of 42.5 years. The soft tissue defects of the injuries were at 2.0 cm × 4.0 cm to 3.5 cm × 6.0 cm in size, with deep tissues exposure down to the base of wound. MSAPF was used for aesthetic reconstruction of the wounds. The surgical procedures were: (1) The flap was thinned under a microscope and only the subdermal vascular network was kept. (2) Vascular pedicle of the flap was taken as long as possible and had it anastomosed with the proximal dorsal foot artery and vein through a subcutaneous tunnel. (3) Allg?wer-Donati method was applied to suture the skin of flap. (4) Donor site was directly closed in surgery. All patients were entered in the scheduled postoperative follow-up at the outpatient clinic of the surgeon who performed the surgery to evaluate the postoperative effect and observe the survival of flaps. Maryland foot function score and British Medical Research Council (BMRC) sensory function score were used to assess the recovery of flap and limb function.Results:The flaps completely survived, and all the donor and recipient sites had primary healing. All of the 10 patients were included in the postoperative follow-up which lasted 8 to 15 months, with an average of 12 months. The flaps from foot featured a pleasing appearance, with good elasticity and wear-resistant. All patients were satisfied and able to walk normally and bear weight without an occurrence of flap ulceration. At the final follow-up, the therapeutic effect was evaluated according to the Maryland scoring system and achieved the scores at 91 to 98, with 95.6 in average. Nine patients were rated as excellent and 1 as good. The sensory grading by BMRC for the flaps was as follows: the flap sensation of the sutured nerve group recovered to S 3 in 3 cases and S 4 in 2 cases, while the non sutured nerve group only recovered protective sensation, S 2 in 3 cases and S 2+ in 2 cases. Conclusion:By applying MSAPF aesthetics to treat foot wounds, a good appearance has been achieved, with good functional recovery and satisfactory therapeutic effects.

Objective:This study aims to investigate the differences between two groups of patients treated with nucleos（t）ide analogues（NAs）：those with undetectable HBV DNA by ultrasensitive testing and those with HBV DNA below the quantification limit. The findings will provide a basis for the clinical interpretation and rational use of ultrasensitive HBV DNA results.Methods:This cross-sectional study included patients with chronic HBV infection who were treated with NAs between May 2023 and December 2023 at the outpatient clinic and inpatient department of the Hepatology Department，The Second Hospital of Shandong University. All patients had ultrasensitive HBV DNA results that were either undetectable or less than 20 IU/ml. The group with undetectable ultrasensitive HBV DNA was defined as the target not detected（TND）group，while the group with HBV DNA levels below 20 IU/ml was defined as the limit of quantitation（LOQ）group. Data on patients' sex，age，type of NAs used，routine blood tests，liver and kidney function，quantitative hepatitis B serology，ultrasensitive HBV DNA，HBV pregenomic RNA（pgRNA），abdominal ultrasound，CT or MRI imaging were collected. Differences between the two groups in terms of demographic data and HBV markers were analyzed.Results:A total of 827 patients were included in the study，with 536 patients in the TND group and 291 in the LOQ group. Compared with the TND group，the LOQ group had a younger age（ P<0.001），and higher level of ALT，AST，HBeAg positive rate，HBsAg，and HBV pgRNA（all P<0.001）. Subgroup analyses stratified by HBeAg status，cirrhosis status，and sex showed that the LOQ group had significantly higher levels of HBsAg and HBV pgRNA compared to the TND group in all subgroups（all P<0.05）. Conclusion:The LOQ group had significantly higher levels of HBsAg and HBV pgRNA compared to the TND group.

Objective:This study aims to investigate the differences between two groups of patients treated with nucleos（t）ide analogues（NAs）：those with undetectable HBV DNA by ultrasensitive testing and those with HBV DNA below the quantification limit. The findings will provide a basis for the clinical interpretation and rational use of ultrasensitive HBV DNA results.Methods:This cross-sectional study included patients with chronic HBV infection who were treated with NAs between May 2023 and December 2023 at the outpatient clinic and inpatient department of the Hepatology Department，The Second Hospital of Shandong University. All patients had ultrasensitive HBV DNA results that were either undetectable or less than 20 IU/ml. The group with undetectable ultrasensitive HBV DNA was defined as the target not detected（TND）group，while the group with HBV DNA levels below 20 IU/ml was defined as the limit of quantitation（LOQ）group. Data on patients' sex，age，type of NAs used，routine blood tests，liver and kidney function，quantitative hepatitis B serology，ultrasensitive HBV DNA，HBV pregenomic RNA（pgRNA），abdominal ultrasound，CT or MRI imaging were collected. Differences between the two groups in terms of demographic data and HBV markers were analyzed.Results:A total of 827 patients were included in the study，with 536 patients in the TND group and 291 in the LOQ group. Compared with the TND group，the LOQ group had a younger age（ P<0.001），and higher level of ALT，AST，HBeAg positive rate，HBsAg，and HBV pgRNA（all P<0.001）. Subgroup analyses stratified by HBeAg status，cirrhosis status，and sex showed that the LOQ group had significantly higher levels of HBsAg and HBV pgRNA compared to the TND group in all subgroups（all P<0.05）. Conclusion:The LOQ group had significantly higher levels of HBsAg and HBV pgRNA compared to the TND group.

Objective:Medial sural artery perforator flap (MSAPF) was applied to aesthetically reconstruct wounds in foot, and clinical outcome of this surgical method was explored.Methods:A retrospective analysis was conducted on the data of 10 patients who underwent the surgery of transfer of free MSAPF in reconstruction of foot wounds in the Department of Foot and Ankle Surgery, Xuzhou Renci Hospital from January 2020 to January 2023. The patients were 6 males and 4 females, aged 21 to 52 years, with an average of 42.5 years. The soft tissue defects of the injuries were at 2.0 cm × 4.0 cm to 3.5 cm × 6.0 cm in size, with deep tissues exposure down to the base of wound. MSAPF was used for aesthetic reconstruction of the wounds. The surgical procedures were: (1) The flap was thinned under a microscope and only the subdermal vascular network was kept. (2) Vascular pedicle of the flap was taken as long as possible and had it anastomosed with the proximal dorsal foot artery and vein through a subcutaneous tunnel. (3) Allg?wer-Donati method was applied to suture the skin of flap. (4) Donor site was directly closed in surgery. All patients were entered in the scheduled postoperative follow-up at the outpatient clinic of the surgeon who performed the surgery to evaluate the postoperative effect and observe the survival of flaps. Maryland foot function score and British Medical Research Council (BMRC) sensory function score were used to assess the recovery of flap and limb function.Results:The flaps completely survived, and all the donor and recipient sites had primary healing. All of the 10 patients were included in the postoperative follow-up which lasted 8 to 15 months, with an average of 12 months. The flaps from foot featured a pleasing appearance, with good elasticity and wear-resistant. All patients were satisfied and able to walk normally and bear weight without an occurrence of flap ulceration. At the final follow-up, the therapeutic effect was evaluated according to the Maryland scoring system and achieved the scores at 91 to 98, with 95.6 in average. Nine patients were rated as excellent and 1 as good. The sensory grading by BMRC for the flaps was as follows: the flap sensation of the sutured nerve group recovered to S 3 in 3 cases and S 4 in 2 cases, while the non sutured nerve group only recovered protective sensation, S 2 in 3 cases and S 2+ in 2 cases. Conclusion:By applying MSAPF aesthetics to treat foot wounds, a good appearance has been achieved, with good functional recovery and satisfactory therapeutic effects.

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.