Mycoplasma pneumoniae(MP) is a common pathogen found in respiratory tract infections of children and is susceptible to macrolides, tetracycline, aminogly-cosides, and quinoloines . Of all these antibiotics, macrolides is the frist choice for children. However,in recent years, strains which are resistant to common drugs have been selected in vitro and isolated from patients Point mutation at antibitics target site of these 8trains is one of the molecular mechanisms.The study of MP resistance can provide theoretical guidance for rational choice of antibiotics and application.

Objective To explore the role of ripply1 in zebrafish dorsal-ventral development .Methods Using ze-brafish whole-mount in situ hybridization to examine the ripply1 expression pattern in early embryo development .To analyse the expression pattern changes of dorsal-ventral marker genes at shield stage and the morphological changes at 24 hpf (hours post-fertilization) after overexpression of ripply1 by injecting synthetic mRNA at 1-cell stage.Using Tol2 transposon technology to obtain a ripply1 promoter driven GFP transgenic fish and to identify promoter region that recapitulates endoge -nous ripply1 expression pattern .Results The in situ hybridization results revealed that ripply1 specifically expresses in the future dorsal region at shield stage .Overexpression of ripply1 caused an enhanced expression of dorsal marker genes and a reduction of ventral marker genes .Embryos overexpressing ripply1 also showed severely dorsalized phenotype , with enlarged head, reduced ventral yolk extension , and shortened posterior trunk and tail regions , and the formation of a secondary trunk axis.Transgenic fish revealed the maternal expression of ripply1 and suggested that a 1.2 kb promoter-driven GFP is able to recapitulate the endogenous gene expression pattern .Conclusion ripply1 may participate in the early development of dor-sal-ventral axis in zebrafish embryo .

Based on the characteristics of salt.alkalinized soil in northeastem China,twenty-five kinds of salt alkaline conditions with different salinities and pH were simulated by mixing NaCl,NaHCO3,Na2SO4,and Na2CO3,in various proportions and applied to sunflower (Helianthus annuus L.) seedlings to investigate the coordinative effects of salt and alkali stresses on its antioxidant enzyme system.The soil was conditioned with a salt concentration range between 50 to 250 mmol/L and pH values from 7.12 to 10.46.Several physiological indexes of stressed seedlings were measured,including the activities of superoxide dismutase (SOD),catalase (CAT),and pemxidase (POD),as well as the content of malondialdehyde (MDA).The results showed that the responses of the antioxidant enzyme system in sunflowers were influenced by salinity and alkalinity,which all three antioxidant enzymes exhibited a rise-drop pattern as salinity increased,whereas their responses to alkalinity appeared to be diverse:decreased for SOD and CAT,and increase for POD along when increasing alkalinity.A two-way analysis of variance (ANOVA) showed that the effects of salinity and alkalinity on the activities of the three enzymes were significant (P＜0.001).The effect of salinity on POD and SOD was greater than that of alkalinity,whereas the effect of alkalinity on CAT was greater than that of salinity.The interrelation of salinity and alkalinity on each antioxidant enzyme was significant (P＜0.001) except for SOD.The correlation and stepwise regression analyses between the activity of the antioxidant enzyme system and the MDA content were significant to difierent extend,SOD was a dominant factor,and POD was neglectable.

Objective To developed A laboratory diagnosis of Moraxella catarrhalis by an laboratories diagnostic method real-time fluorescence quantitative PCR assay. Methods The specific primers and probes were designed based on the sequence of outer membrane protein CopB(copB)gene in Moraxella catarrhalis,and the Taqman probe RT-PCR method was developed to detect the Moraxella catarrhalis.The standard plasmids ex-tracted from the Moraxella catarrhalis standard strains were used to constitute the standard samples,and compared with these standard samples,the sensitivity of the fluorescence quantitative PCR assay was tested by the estab-lished standard curves.The specificity of the fluorescence quantitative PCR assay was tested by the DNA samples of other bacterias in the laboratory.Meanwhile,321 throat swab samples from inpatient and outpatient child pa-tients,with asthma infection were collected as clinical samples to validate the fluorescence quantitative PCR as-say.Results The standard curve was drawn in the real-time PCR by the Taqman fluorescence reporter.During the sensitivity tests,the newly-developed real-time fluorescence PCR could detect at least 10 copies of Moraxella catarrhalis,and could successfully distinguish several DNAs of the pathogens.On the basis of the validation result of the 321 throat swab samples,there are 25 Moraxella catarrhalis with 7.79 % positive rate.Conclusion The fluorescence quantitative PCR assay is of great sensitivity and specificity,and it can be widely used for the detec-tion of Moraxella catarrhalis.

Objective:To investigate the optimal serum antibody titer in acute stage for the diagnosis of Mycoplasma pneumoniae (MP) infection by passive agglutination, and to evaluate the clinical diagnostic value of different antibody titers.Methods:A cross-sectional study.Eighty-eight pairs of clinical serum samples were collected from children with MP infection treated at the Department of Pediatrics in Shengjing Hospital of China Medical University from December 2016 to February 2017 and Children′s Hospital of Baotou in November 2019.The four-fold change of the double serum specific antibody titer was used as the gold standard, and the receiver operating characteristic (ROC) curve was plotted.When detecting the single serum in acute stage, different antibody titers were used as positive criteria to evaluate their clinical application value in the diagnosis of MP infection and find the most appropriate serum antibody titer as the diagnostic cut-off value.Results:(1)When the serum specific antibody titer ≥1∶40 was used as the positive criterion, the sensitivity was 72.9%, the area under the ROC curve was 0.817, and the specificity was 87.5%, which might cause overdiagnosis.When the serum specific antibody titer ≥1∶160 was used as the positive criterion, the specificity was 97.5%, the area under the ROC curve was 0.775, and the sensitivity was 52.1%, which might cause missed diagnosis.When the serum specific antibody titer ≥1∶80 was used as the positive criterion, the sensitivity was 60.4%, the specificity was 97.5%, and the area under the ROC curve was 0.823, overall performing better compared with the said two criteria.(2)After the disease lasted at least 5 days, blood samples were collected.About 72.5% of the children had antibodies, and 60.0% of the children had antibody titers ≥1∶80.Conclusions:(1)When the passive agglutination method is used to detect MP infection, antibody titer ≥1∶80 is recommended as the diagnostic standard.However, in clinical practice, the diagnosis of MP infection depends on clinical and other laboratory test results.(2) It is appropriate to collect blood samples on 5-7 days of illness.If MP infection is clinically suspected, and an antibody titer of 1∶40 is also suggestive, it can perform cooperative diagnosis based on molecular biology lab results or retest at a shorter interval.

Objective To investigate the antibacterial effect of Fusidic acid on Mycoplasma pneumoniae and antibiotic resistant Mycoplasma pneumoniae in vitro.Methods Twenty-eight clinical strains of Mycoplasma pneumoniae isolated from patients with respiratory tract infection at Beijing Friendship Hospital Affiliated to the Capital University of Medical Sciences from January to December 2016 and 2 Mycoplasma pneumoniae reference strains were enrolled.The minimum inhibitory concentration (MIC) of Fusidic acid and Azithromycin were determined by using micro-dilution ration method.The chessboard method was used to check the antibacterial effect of combination between Fusidic acid and Azithromycin.The antibacterial activity of the Fusidic acid was evaluated by measuring the antibacterial rate of different concentrations.Results One isolate showed no mutation in 23SrRNA,26 isolates had one point mutation in loci 2063 and 1 isolate had one point mutation in loci 2064 among the 28 clinical isolates.The findings by micro-dilution method results showed that the MIC values of all the clinical isolates with mutations associated with macrolide resistance to Azithromycin were ＞ 1.000 0 mg/L,and the MIC values of all the clinical isolates with no mutations to azithromycin were ＜ 0.500 0 mg/L.The findings by micro-dilution method results showed that the MIC value of Fusidic acid for Mycoplasma pneumonia and drug resistance Mycoplasma pneumoniae was 1.000 0 mg/L.The Fractional Inhibitory Concentration index of Fusidic acid and Azithromycin combination was ≤0.500 0 mg/L.When the concentration of the Fusidic acid was lower than or equal to 32 MIC,the antibacterial effect of Fusidic acid against Mycoplasma pneumoniae increased with its higher concentration.When the concentration of the Fusidic acid was lower than or equal to 8 MIC,the longer the strain was exposed to the drug,the stronger antibacterial effect was against Mycoplasma pneumoniae.Conclusion If the treatment of Mycoplasma pneumoniae infection is not effective or the infection of patient is combined with bacteria,the application or combination of Fusidic acid may inhibit pathogenic bacteria effectively.Of course,how to use Fusidic acid in clinical treatment needs further study and discussion.

Objective:To investigate drug resistance gene in Mycoplasma pneumoniae(MP) and the distribution of 13 respiratory pathogens in bronchoalveolar lavage fluid(BALF) of children with Mycoplasma pneumoniae pneumonia(MPP).Methods:A total of 100 BALF of children with MPP in Peking University Third Hospital and Peking University First Hospital from January 2018 to January 2019 were collected.Fluorogenic quantitative PCR was used to detect nucleic acid and it′s drug resistance gene of MP and multiple PCR method was adopted to detect influenza A virus, influenza A virus-H 1N 1, influenza A virus-H 3N 2, influenza B, human parainfluenza virus, adenovirus, human bocavirus, human rhinovirus, Chlamydia pneumoniae, human metapneumovirus, MP, human coronavirus, and respi-ratory syncytial virus gene, and the results were compared by using Chi square test. Results:In 100 BALF samples, MP and drug resistance gene were detected by fluorogenic quantitative PCR.Totally, 83 cases (83.00%) were MP positive and 78 cases (93.98%) were drug resistant.All of them had the point mutations A2063G in V region of 23S rRNA domain.A total of 13 kinds of respiratory pathogens were detected by multiplex PCR method, and 89 cases (89.00%) were positive.Totally, 79 cases (79.00%) were MP positive, of which 74 cases (74.00%) detected only MP, and 5 cases (5.00%) detected MP combined with other pathogens.Other pathogens were detected in 10 cases (10.00%). The virus detection rate of 0-4 years old group was higher than that of >4-6 years old group ( P=0.042) and >6 years old group ( P=0.002), and the differences were statistically significant. Conclusions:MP can be detected in most BALF samples of MPP children, the drug resistance phenomenon is serious, and the main point mutation is A2063G.There were other respiratory pathogens and 2 or 3 pathogens were detected in a small number of BALF samples.

Objective To clone the HnGUA gene from Hirudo nipponia and conduct bioinformatics analysis,protein prokaryotic expression analysis and gene differential expression analysis.Methods Based on the transcriptome data of H.nipponia in the previous study,the full-length cDNA of HnGUA was cloned by rapid amplification of cDNA ends(RACE),and bioinformatics analysis was performed.The prokaryotic expression vector was constructed,transformed into Escherichia coli BL21(DE3)competent cells and the expression of recombinant protein was induced by IPTG.The qPCR was used to further analyze the tissue-specific expression of HnGUA.Results The size of HnGUA gene was 504 bp,containing an open reading frame(ORF)of 231 bp and encoding 76 amino acids.Its protein molecular weight and isoelectric point are 8.17 kDa and 4.44,respectively.Multiple sequence alignment analysis showed that HnGUA was highly homologous to genes in other leech species that encode inhibitory proteins.The results of the prokaryotic expression analysis showed that the constructed pET32a-HnGUA vector could be successfully expressed in E.coli BL21(DE3),and the SDS-PAGE results showed that the induced recombinantly expressed HnGUA protein was around 6 kDa,which was basically consistent with the predicted protein size.The results of the Real-time PCR revealed spatial and temporal differences in the expression profiles of HnGUA,with high levels of expression detected in the skin and crop tissues.Conclusion This study represents the first successful cloning of the HnGUA gene from H.nipponia and the expression of the corresponding recombinant protein in E.coli.It provides a foundation for future exploration of the biosynthetic pathways and molecular regulatory mechanisms of active small anticoagulant molecules in leeches.

Objective To discuss the perioperative care and wound protection of xenotransplantation from genetically edited pigs to monkeys, with the goal of improving the success rate of such experimental procedures. Methods From October 2022 to October 2023, perioperative care and wound protection were performed on 7 recipient rhesus monkeys undergoing xenotransplantation of genetically edited pig tissues and organs. Customized wound protective garments were designed based on monkeys' size and surgical area to protect the wounds, alongside meticulous perioperative care. This included preoperative preparation and medication, intraoperative monitoring of physiological indicators and anesthesia management, and postoperative care comprising wound protection, observation and monitoring, and nutritional support. Results All seven monkeys successfully underwent xenotransplantation. With the aid of protective garments and detailed care, all surgical wounds healed by first intention, and postoperative recovery was satisfactory. Conclusion Proper care and wound protection during xenotransplantation from genetically edited pigs to monkeys not only promote wound healing, but also alleviate pain and harm to animals. This has significant implications for advancing experimental research in pig-monkey xenotransplantation and enhancing animal welfare.