The problem of drug resistance resulting from the long-term use of antimicrobials is a serious threat to global health,and the emergence of multidrug-resistant bacteria,in particular,has greatly limited therapeutic op-tions.Therefore,the development of new or alternative antimicrobial agents has become an urgent need.Antimi-crobial peptides(AMPs)have been regarded as good alternatives to antibacterial drugs due to their strong antibac-terial activity and unique mechanism of action.At present,some AMPs have completed preclinical studies on drug-resistant bacterial infections and entered the clinical trial stage,while their stability and targeting have been signifi-cantly improved through the optimisation of amino acid modification,nano-delivery system and other technolo-gies,which have gradually become a research hotspot in this field.Therefore,this paper discusses the importance of AMPs in bacterial drug resistance from the biological properties of AMPs,the mechanism of regulating bacteri-al drug resistance and the application of AMPs in the treatment of drug-resistant bacterial infections,with a view to providing a reference for the development of drugs against drug-resistant bacteria and clinical application.

Patients with severe aplastic anemia(SAA)often present with neutropenia,and are particularly susceptible to infections,especially following immunosuppressive therapy.Bacillus cereus is a ubiquitous,opportunistic pathogen capable of colonizing healthy individuals and causing infections through various routes.Neutropenia is a common feature in many hematologic disorders,placing affected patients at higher risk for Bacillus cereus infections.This report describes a rare case of Bacillus cereus bacteremia with multiple abscesses in a patient with SAA during the neutropenic phase following immunosuppressive therapy.Given the rarity of such presentations,relevant literature was reviewed and the diagnostic and treatment process was analyzed in detail,with the aim of providing clinical insights and improving the management of similar cases.

Objective To investigate the mechanism underlying the effect of intra-articular injection of ozonated water on cartilage repair in knee osteoarthritis(KOA)rats via the miR-214-3p/nuclear factor(NF)-κB signaling pathway,to provide a theoretical basis for the clinical application of ozonated water.Methods Sixty male Sprague-Dawley rats were divided randomly into ozonated and experimental groups.Rats in the experimental group were further divided randomly into model,ozonated water,ozonated water+AAV-NC,and adeno-associated virus with ozonated water+AAV-miR-214-3p inhibitor groups.The articular cartilage repair status was evaluated after the respective treatments,using articular cartilage surface gross scoring,hematoxylin/eosin staining,and modified Mankin's score.Expression levels of interleukin(IL)-1β,tumor necrosis factor(TNF)-α,and matrix metalloproteinase(MMP)-13 were detected by enzyme-linked immunosorbent assay(ELISA).Protein expression levels of IκB kinase(IKK)β,p65,and phospho(p)-IκBα were detected in cartilage tissues by Western blot,and gene expression levels of IKKβ,p65,IκBα,and miR-214-3p were detected by real-time polymerase chain reaction(RT-PCR).Results Rats in all groups showed different degrees of cartilage damage compared with the control group,and the scores were significantly lower in the ozonated water and ozonated water+AAV-NC groups compared with the ozonated water+AAV-miR-214-3p inhibitor group(P＜0.05).IL-1β,TNF-α,and MMP-13 levels detected by ELISA were significantly lower in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups(P＜0.05).According to RT-PCR,IKKβ and p65 mRNA levels were significantly down-regulated while IκBα mRNA was significantly up-regulated in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups(P＜0.05).Similarly,IKKβ,p65,and p-IκBα protein expression levels detected by Western blot were significantly down-regulated while IκBα protein expression levels were up-regulated in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups(P＜0.05).Conclusions Ozone hydrotherapy significantly ameliorated cartilage damage in KOA rats,possibly via inhibiting activation of the NF-κB signaling pathway through up-regulation of miR-214-3p expression and promoting cartilage repair in KOA rats.

The problem of drug resistance resulting from the long-term use of antimicrobials is a serious threat to global health,and the emergence of multidrug-resistant bacteria,in particular,has greatly limited therapeutic op-tions.Therefore,the development of new or alternative antimicrobial agents has become an urgent need.Antimi-crobial peptides(AMPs)have been regarded as good alternatives to antibacterial drugs due to their strong antibac-terial activity and unique mechanism of action.At present,some AMPs have completed preclinical studies on drug-resistant bacterial infections and entered the clinical trial stage,while their stability and targeting have been signifi-cantly improved through the optimisation of amino acid modification,nano-delivery system and other technolo-gies,which have gradually become a research hotspot in this field.Therefore,this paper discusses the importance of AMPs in bacterial drug resistance from the biological properties of AMPs,the mechanism of regulating bacteri-al drug resistance and the application of AMPs in the treatment of drug-resistant bacterial infections,with a view to providing a reference for the development of drugs against drug-resistant bacteria and clinical application.

Objective: To construct microRNA(miR)-22 gene knockout(miR-22-/-) mice using CRISPR/Cas 9 technology, to breed miR-22-/- mice and to identify their genotypes. Methods : In this experiment, CRISPR/Cas 9 technology was used to construct miR-22-/- genetically engineered mice. After gene identification, the F0 generation miR-22-/- mice were mated with wild-type mice in the same litter to obtain F1 generation miR-22-/- mice. The miR-22 knockout efficiency was analyzed at the RNA level by real-time fluorescence quantitative polymerase chain reaction(qPCR). Western blot was used to detect the interaction between miR-22 and target genes. Results : miR-22-/- mice were successfully constructed using CRISPR/Cas 9 technology, gene identification was performed on the bred mice, and three stable genotypes of miR-22+/+,miR-22+/-, and miR-22-/- were identified. The real-time fluorescence quantitative PCR detection results confirmed that miR-22-/- mice showed almost no expression of miR-22 in the heart, liver, lung, kidney, spleen, and thymus tissues compared to wild-type mice in the same litter. Western blot analysis showed that the relative expression level of NLRP3 protein in miR-22-/- mouse tissues was lower than that in wild-type mice. Conclusion A miR-22-/- mouse model is successfully constructed, and stable genetic homozygous miR-22-/- mice is obtained. This indicates that miR-22 has an inhibitory effect on the downstream target gene NLRP3.

Objective: To explore the breeding and genotyping of CCR2 knockout mice, and to verify the applicability of the polymerase chain reaction(PCR) method for genotype detection of CCR2 knockout mice. Methods: The introduced CCR2 pure male mice and wild-type female mice were mated and bred to produce the offspring generation, the obtained F1 generation heterozygous mice were continued to be mated. DNA was extracted by clipping the tail tissues of the mice at the age of 2 weeks, the target gene fragment was amplified by PCR, and the genotypic results were determined by agarose gel electrophoresis. The proportion of purebred progeny carrying the CCR2 knockout gene was increased by genetic crosses, the effect of CCR2 knockout in the progeny mice was verified by using Western blot against major immune cells and key organs, and flow cytometry was used to detect whether the knockout of the CCR2 gene had any effect on the function of the immune system by targeting the major immune cells. Results: CCR2 knockout mice were successfully bred and characterized, and three genotypes of F2 generation mice were obtained: CCR2+/+, CCR2+/-, and CCR2-/-. The offspring genotypes were identified by PCR, and Western blot showed extremely low CCR2 protein expression in CCR2 knockout mice. Flow analysis showed that CCR2 knockdown reduced the expression of CD4+T and Th1 cells in mouse spleen-derived T cells, but did not affect macrophage function. Conclusion Correct breeding and identification are important ways to get the pure CCR2 knockout mice, and PCR method for identifying mouse genotypes is simple, fast and reliable.

Objective: To constructCSF1R+/-mice and to analyze their genotypes, so as to provide animal model basis for disease pathological mechanism and drug target. Methods : A linearized targeting vector was designed according to Cre/Loxp system. A Loxp site was inserted upstream of the 5th exon of theCSF1Rgene, and a neomycin resistance box with Loxp sites on both sides was inserted downstream of the 5th exon. The linearized targeting vector was electroporated into embryonic stem cells. The correctly targeted embryonic stem cells were injected into the blastocysts of C57BL/6J mice to obtain chimeric mice, which were bred with Zp3-Cre mice. The newborn mice were numbered 9-14 days after birth and their tails were cut. The DNA of the mice was extracted, and the genotype of the mice was identified by polymerase chain reaction and agarose gel electrophoresis. The expression of CSF1R in mouse macrophages was detected by flow cytometry. The expression of CSF1R in mouse tissues was detected by Western blot. Results: The results of agarose gel electrophoresis showed that 453 bp bands were amplified in wild type mice, and 453 bp and 650 bp bands were amplified in heterozygous mice. The results of flow cytometry showed that the expression of CSF1R in peritoneal macrophages and bone marrow-derived macrophages of CSF1R heterozygous mice was lower than that of WT group(P<0.05). The results of Western blot showed that the expression of CSF1R in spleen, kidney and brain tissue of CSF1R heterozygous group was lower than that of WT group(P<0.05). Conclusion CSF1R+/-mice are successfully constructed, reproduced and identified, which provides an animal model basis for further revealing the potential mechanism of CSF1R in immune regulation.

Patients with severe aplastic anemia(SAA)often present with neutropenia,and are particularly susceptible to infections,especially following immunosuppressive therapy.Bacillus cereus is a ubiquitous,opportunistic pathogen capable of colonizing healthy individuals and causing infections through various routes.Neutropenia is a common feature in many hematologic disorders,placing affected patients at higher risk for Bacillus cereus infections.This report describes a rare case of Bacillus cereus bacteremia with multiple abscesses in a patient with SAA during the neutropenic phase following immunosuppressive therapy.Given the rarity of such presentations,relevant literature was reviewed and the diagnostic and treatment process was analyzed in detail,with the aim of providing clinical insights and improving the management of similar cases.

Objective To investigate the mechanism underlying the effect of intra-articular injection of ozonated water on cartilage repair in knee osteoarthritis(KOA)rats via the miR-214-3p/nuclear factor(NF)-κB signaling pathway,to provide a theoretical basis for the clinical application of ozonated water.Methods Sixty male Sprague-Dawley rats were divided randomly into ozonated and experimental groups.Rats in the experimental group were further divided randomly into model,ozonated water,ozonated water+AAV-NC,and adeno-associated virus with ozonated water+AAV-miR-214-3p inhibitor groups.The articular cartilage repair status was evaluated after the respective treatments,using articular cartilage surface gross scoring,hematoxylin/eosin staining,and modified Mankin's score.Expression levels of interleukin(IL)-1β,tumor necrosis factor(TNF)-α,and matrix metalloproteinase(MMP)-13 were detected by enzyme-linked immunosorbent assay(ELISA).Protein expression levels of IκB kinase(IKK)β,p65,and phospho(p)-IκBα were detected in cartilage tissues by Western blot,and gene expression levels of IKKβ,p65,IκBα,and miR-214-3p were detected by real-time polymerase chain reaction(RT-PCR).Results Rats in all groups showed different degrees of cartilage damage compared with the control group,and the scores were significantly lower in the ozonated water and ozonated water+AAV-NC groups compared with the ozonated water+AAV-miR-214-3p inhibitor group(P＜0.05).IL-1β,TNF-α,and MMP-13 levels detected by ELISA were significantly lower in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups(P＜0.05).According to RT-PCR,IKKβ and p65 mRNA levels were significantly down-regulated while IκBα mRNA was significantly up-regulated in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups(P＜0.05).Similarly,IKKβ,p65,and p-IκBα protein expression levels detected by Western blot were significantly down-regulated while IκBα protein expression levels were up-regulated in cartilage tissues from rats in the ozonated water and ozonated water+AAV-NC groups compared with the model and ozonated water+AAV-miR-214-3p inhibitor groups(P＜0.05).Conclusions Ozone hydrotherapy significantly ameliorated cartilage damage in KOA rats,possibly via inhibiting activation of the NF-κB signaling pathway through up-regulation of miR-214-3p expression and promoting cartilage repair in KOA rats.

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods Ac-cording to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were de-signed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19～20 days.Positive F0 mice were mated with C57BL/6J mice to ob-tain stable F1 Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtain Spi1flox/flox mice.Spi1flox/flox mated with Lyz2-Cre+mice to obtain Spi1flox/+/Lyz2-Cre+mice,and then mated with Spi1flox/flox,the Spi1flox/flox/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electro-phoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus provi-ral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1flox/flox homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre+.Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macropha-ges(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and West-ern blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-spe-cific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.