The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.

Based on the theory of circulation of qi,a theoretical model of the circulation of phase fire in the human body was con-structed.It is proposed that cancer toxicity is the key factor that triggers the disorder of phase fire and hinders the circulation of phase fire.The disorder of phase fire and poor circulation are important pathogenesis of the occurrence and development of cancer toxicity.With the principle of strengthening healthy qi and eliminating evil qi,the Wenzi Jiedu method is used to treat tumors,which plays an important role in warming and nourishing,regulating yin and yang,and eliminating cancer toxicity.The combination of war-ming,nourishing and detoxification can promote the return of the phase fire and make the human body's generation and transformation active,providing a new treatment idea for TCM diagnosis and treatment of tumors with mainly cold poison or mixed cold and heat.

Based on the theory of circulation of qi,a theoretical model of the circulation of phase fire in the human body was con-structed.It is proposed that cancer toxicity is the key factor that triggers the disorder of phase fire and hinders the circulation of phase fire.The disorder of phase fire and poor circulation are important pathogenesis of the occurrence and development of cancer toxicity.With the principle of strengthening healthy qi and eliminating evil qi,the Wenzi Jiedu method is used to treat tumors,which plays an important role in warming and nourishing,regulating yin and yang,and eliminating cancer toxicity.The combination of war-ming,nourishing and detoxification can promote the return of the phase fire and make the human body's generation and transformation active,providing a new treatment idea for TCM diagnosis and treatment of tumors with mainly cold poison or mixed cold and heat.

AIM To study the chemical constituents from Pyrolae Herba and their activity against pathogenic bacteria associated with sports injuries.METHODS Silica gel and Sephadex LH-20 were used for isolation and purification,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The antibacterial activity was detected by filter paper method.RESULTS Twenty five compounds were isolated and identified as manuleoside H(1),5-hydroxy-3,7-dimethoxy-4,-methyl flavone(2),5-hydroxy-3,7,4'-methoxy-flavone(3),5-hydroxy-7,3',4'-trimethoxy-flavone(4),5,7-dihydroxy-3-methoxy-flavone(5),7.5,4'-trihydroxyflavone(6),ethyl vanillate(7),clovandiol(8),hydroxydihydrobovolide(9),zanthopyranone(10),eugenin(11),berchemol(12),3,7-dimethoxy-5,3',4'-trihydroxyflavone(13),2,5-dimethyl-7-hydroxychromone(14),rutalinium(15),formononetin(16),8-hydroxy-4',7-dimethoxy-isoflavones(17),isoline(18),dearabinosyl pneumonanthoside(19),3,5-dihydroxycinnamic acid(20),2,4,6-trihydroxyacetophenone(21),p-isopropyl benzoic acid(22),geraldone(23),ethylsyringin(24),4-hydroxyphenylethyl-2'-hydroxypropionate(25).Compounds 8,10,12,16 and 24 showed bacteriostatic zone on pathogenic bacteria susceptible to sports injuries.CONCLUSION All the compounds are isolated from Pyrolae Herba for the first time.Compounds 8,10,12,16 and 24 have inhibitory activity against pathogenic bacteria susceptible to sports injuries.

The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.

OBJECTIVE To carry out regular monitoring of carbapenem-resistant Klebsiella pneumoniae(CRKP)contamination status in the environment of intensive care unit(ICU)and take targeted prevention and control measures so as to reduce the incidence of hospital-associated infections with multidrug-resistant organisms(MDROs).METHODS The surfaces of surroundings of the patients who were colonized and infected with CRKP in the ICU of grade A tertiary hospital of Shanghai and the hands of relevant staff were sampled by stages from Jan 1,2021 to Jun 30,2024.The distribution of the CRKP strains in the surroundings were analyzed according to the locations positive for CRKP,and the disinfection measures were accordingly and continuously modified.The trend of isolation rate of CRKP strains from the ICU patients was analyzed during the time period when the measures were implemented.RESULTS Totally 266 environmental samples were collected during the baseline period(from Jan.1 2021 to Dec.31 2021),265 during intervention period(from Jan.1 2022 to Dec.31 2023),274 during con-solidation period(from Jan.1 to Jun.30 2024);the isolation rates of the CRKP strains were 4.51％,4.91％and 3.65％,respectively.The isolation rate of the strains was highest from the bed unit(10.40％),followed by the article for public use(6.74％),articles used by health care workers(2.98％)and diagnosis and treatment arti-cles(1.91％).The isolation rate of CRKP of the patients was 24.75％during the baseline period,15.48％during the intervention period,5.69％during the consolidation period,showing a continuously downward trend(x2=30.330,P＜0.001).CONCLUSION It is necessary to regularly carry out the environmental monitoring of CRKP strains,seek for the weak links of environmental disinfection and implement the intensified prevention and control measures so as to reduce the incidence of CRKP infection,which may provide theoretical bases for effective control of the CRKP strains.

OBJECTIVE To carry out regular monitoring of carbapenem-resistant Klebsiella pneumoniae(CRKP)contamination status in the environment of intensive care unit(ICU)and take targeted prevention and control measures so as to reduce the incidence of hospital-associated infections with multidrug-resistant organisms(MDROs).METHODS The surfaces of surroundings of the patients who were colonized and infected with CRKP in the ICU of grade A tertiary hospital of Shanghai and the hands of relevant staff were sampled by stages from Jan 1,2021 to Jun 30,2024.The distribution of the CRKP strains in the surroundings were analyzed according to the locations positive for CRKP,and the disinfection measures were accordingly and continuously modified.The trend of isolation rate of CRKP strains from the ICU patients was analyzed during the time period when the measures were implemented.RESULTS Totally 266 environmental samples were collected during the baseline period(from Jan.1 2021 to Dec.31 2021),265 during intervention period(from Jan.1 2022 to Dec.31 2023),274 during con-solidation period(from Jan.1 to Jun.30 2024);the isolation rates of the CRKP strains were 4.51％,4.91％and 3.65％,respectively.The isolation rate of the strains was highest from the bed unit(10.40％),followed by the article for public use(6.74％),articles used by health care workers(2.98％)and diagnosis and treatment arti-cles(1.91％).The isolation rate of CRKP of the patients was 24.75％during the baseline period,15.48％during the intervention period,5.69％during the consolidation period,showing a continuously downward trend(x2=30.330,P＜0.001).CONCLUSION It is necessary to regularly carry out the environmental monitoring of CRKP strains,seek for the weak links of environmental disinfection and implement the intensified prevention and control measures so as to reduce the incidence of CRKP infection,which may provide theoretical bases for effective control of the CRKP strains.

AIM To study the chemical constituents from Pyrolae Herba and their activity against pathogenic bacteria associated with sports injuries.METHODS Silica gel and Sephadex LH-20 were used for isolation and purification,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The antibacterial activity was detected by filter paper method.RESULTS Twenty five compounds were isolated and identified as manuleoside H(1),5-hydroxy-3,7-dimethoxy-4,-methyl flavone(2),5-hydroxy-3,7,4'-methoxy-flavone(3),5-hydroxy-7,3',4'-trimethoxy-flavone(4),5,7-dihydroxy-3-methoxy-flavone(5),7.5,4'-trihydroxyflavone(6),ethyl vanillate(7),clovandiol(8),hydroxydihydrobovolide(9),zanthopyranone(10),eugenin(11),berchemol(12),3,7-dimethoxy-5,3',4'-trihydroxyflavone(13),2,5-dimethyl-7-hydroxychromone(14),rutalinium(15),formononetin(16),8-hydroxy-4',7-dimethoxy-isoflavones(17),isoline(18),dearabinosyl pneumonanthoside(19),3,5-dihydroxycinnamic acid(20),2,4,6-trihydroxyacetophenone(21),p-isopropyl benzoic acid(22),geraldone(23),ethylsyringin(24),4-hydroxyphenylethyl-2'-hydroxypropionate(25).Compounds 8,10,12,16 and 24 showed bacteriostatic zone on pathogenic bacteria susceptible to sports injuries.CONCLUSION All the compounds are isolated from Pyrolae Herba for the first time.Compounds 8,10,12,16 and 24 have inhibitory activity against pathogenic bacteria susceptible to sports injuries.

Objective:To evaluate the efficacy of adjusted Narcotrend Index (NI) value guidancecombined with small-dose esketamine for program-controlled closed-loop target-controlled infusion (TCI) system.Methods:Forty-eight American Society of Anesthesiologists Physical Status classificationⅠ or Ⅱpatients, regardless of gender, aged 18-55 yr, with body mass index of 18-25 kg/m 2, scheduled for elective laparoscopic surgery under general anesthesia, were assigned to control group (group C, NI baseline value median 36) and esketamine group(group E, NI baseline value median 46) using a random number table method, with 24 cases in each group. Anesthesia induction and maintenance were carried out using effect-site concentration TCI(Schnider model for propofol infusion and Minto model for remifentanil infusion). After the NI value was maintained at 26-46 during anesthesia maintenance, a small dose of esketamine was given (as an intravenous bolus 0.2 mg/kg, followed by an infusion of 5 μg·kg -1·min -1for 30 min) in group E, and the equal volume of normal saline was given instead in group C. Program-controlled closed-loop TCI was then started, and the target effect-site concentrations of propofol and remifentanil were adjusted every 5 min according to the corresponding preset NI baseline value. The main outcome measures were the percentage of time of NI value maintained in the target range within 1 h after administration of esketamine. Secondary outcome measures were the consumption of propofol and remifentanil, postoperative recovery time, incidence of nausea and vomiting, pain and shivering within 1 h after surgery. Patients were followed for intraoperative awareness on 2nd day after operation. Results:The performance of the program-controlled closed-loop TCI systems was within the safe clinical threshold, with no intraoperative awareness occurred in both groups. The consumption of propofol and remifentanil was significantly reduced in group E as compared to group C( P<0.05). There were no statistically significant differences in the percentage of time of NI value maintained in the target range, postoperative recovery time and incidence of adverse reactions between the two groups ( P>0.05). Conclusions:Adjusted NI value guidance combined with small-dose esketamine provides better efficacy when used for program-controlled closed-loop TCI system.

Objective To discover the host factor interacting with severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)papain-like protease(PLpro)and explore the potential mechanism.Methods The second-generation proximity-dependent biotin identification(BioID2)approach combined with mass spectrometry analysis was used to search for the potential host factors.Immunofluorescence and co-immunoprecipitation(Co-IP)assay were used to verify the interactions between DEAD-box helicase 3(DDX3)and PLpro.The influence of PLpro on DDX3-inhibitor of kappa B kinase ε(IKKε)-TANK-binding kinase 1(TBK1)and DDX3-mitochondrial antiviral signaling protein(MAVS)complexes was also investigated by Co-IP.The effect of PLpro on interferon-β(IFN-β)immune pathway and the protease activity on substrates were studied via luciferase activity assay.Results DDX3 could co-locate and interact with PLpro intracellularly.PLpro might possibly inhibit both the formation of DDX3-MAVS complex and the interactions between DDX3-IKK-ε-TBK1.PLpro could negatively regulate type Ⅰ interferon pathway.Overexpression of DDX3 could lead to a significant increase in the cleavage activity of PLpro/PLP-TM that might be significantly decreased in case of inventions with DDX3 expressions.Conclusion DDX3 may be one of the host factors that interact with SARS-CoV-2 PLpro.PLpro negatively regulates IFN-β immune pathway induced by DDX3,which may provide a favorable immune environment for virus replication.