Tigecycline(TGC)is a recently developed broad-spectrum and highly effective glycylcy-cline antibiotic that overcomes traditional tetracycline resistance mechanisms.It is widely used in the treatment of complex skin and intra-abdominal infections.However,the emergence of TGC re-sistance in recent years poses a significant challenge,and its underlying resistance mechanisms re-main incompletely understood,warranting further investigation.This study induced TGC resistance in Klebsiella aerogenes(ATCC-43864)under in vitro conditions and evaluated phenotypic chan-ges,including growth curves,motility,multidrug susceptibility,and ultrastructural alterations,be-fore and after induction.Whole-genome sequencing(WGS)and transcriptome sequencing(RNA-seq)were employed to analyze resistance-related genetic changes,which were further validated.Ef-flux pump inhibition assays and Red homologous recombination were used to investigate the roles of efflux pumps,lon deletion,and rpsM mutation in TGC resistance.A stable resistant strain,CF-T-32,was obtained,with a 32-fold increase in TGC minimum inhibitory concentration(MIC).CF-T-32 exhibited an enlarged periplasmic space between the cell wall and membrane,reduced growth and motility,and no significant changes in susceptibility to 28 antimicrobial agents,including doxy-cycline,gentamicin,and ciprofloxacin.Genomic analysis revealed no significant differences in ge-nome composition or interference from exogenous plasmids between the parental and induced strains.However,two missense mutations were identified in rpsM and lon.Transcriptomic analysis and qPCR validation showed significant upregulation of efflux pump genes(acrA and acrB)in the AcrAB-TolC system and downregulation of ribosomal protein genes(rpsM,rpsJ,and rpsC).Ef-flux pump inhibition and Red homologous recombination experiments demonstrated that the rpsM C287T missense mutation,leading to a Pro96Leu substitution and reduced expression,is likely the primary factor contributing to TGC resistance in the induced strain.

Aim To explore the effect of FTO on adria-mycin resistance in triple-negative breast cancer through the Wnt/β-catenin signaling pathway and to reveal the underlying mechanism.Methods The MDA-MB-231/ADR drug-resistant cell line was constructed using a method of gradually increasing adriamycin concentra-tion with intermittent induction.The half-inhibitory concentration(IC50)of adriamycin for MDA-MB-231 and MDA-MB-231/ADR cells and the expression of FTO were compared.After knocking down FTO in MDA-MB-231/ADR cells,CCK-8,qRT-PCR,colony formation assay,transwell,flow cytometry,and Western blot were used to assess the changes in the IC50 of adri-amycin,cell proliferation,migration,invasion,apopto-sis,and the expression of related proteins.Results FTO was highly expressed in MDA-MB-231/ADR cells.After FTO knockdown,the IC50 value of adriamy-cin in MDA-MB-231/ADR cells decreased,and the a-bilities of proliferation,migration and invasion were weakened.In the FTO knockdown group,the expres-sion levels of Bax,cleaved-caspase3,GSK-3 β proteins and the apoptosis rate significantly increased,while the expression levels of Bcl-2,Wnt5a,β-catenin,c-myc,cyclin D1,and P-gp proteins decreased.Conclusion FTO may inhibit the apoptosis of MDA-MB-231/ADR cells through the Wnt/β-catenin signaling pathway,al-ter P-gp expression,and thereby enhance the resistance of MDA-MB-231/ADR cells to adriamycin.

Tigecycline(TGC)is a recently developed broad-spectrum and highly effective glycylcy-cline antibiotic that overcomes traditional tetracycline resistance mechanisms.It is widely used in the treatment of complex skin and intra-abdominal infections.However,the emergence of TGC re-sistance in recent years poses a significant challenge,and its underlying resistance mechanisms re-main incompletely understood,warranting further investigation.This study induced TGC resistance in Klebsiella aerogenes(ATCC-43864)under in vitro conditions and evaluated phenotypic chan-ges,including growth curves,motility,multidrug susceptibility,and ultrastructural alterations,be-fore and after induction.Whole-genome sequencing(WGS)and transcriptome sequencing(RNA-seq)were employed to analyze resistance-related genetic changes,which were further validated.Ef-flux pump inhibition assays and Red homologous recombination were used to investigate the roles of efflux pumps,lon deletion,and rpsM mutation in TGC resistance.A stable resistant strain,CF-T-32,was obtained,with a 32-fold increase in TGC minimum inhibitory concentration(MIC).CF-T-32 exhibited an enlarged periplasmic space between the cell wall and membrane,reduced growth and motility,and no significant changes in susceptibility to 28 antimicrobial agents,including doxy-cycline,gentamicin,and ciprofloxacin.Genomic analysis revealed no significant differences in ge-nome composition or interference from exogenous plasmids between the parental and induced strains.However,two missense mutations were identified in rpsM and lon.Transcriptomic analysis and qPCR validation showed significant upregulation of efflux pump genes(acrA and acrB)in the AcrAB-TolC system and downregulation of ribosomal protein genes(rpsM,rpsJ,and rpsC).Ef-flux pump inhibition and Red homologous recombination experiments demonstrated that the rpsM C287T missense mutation,leading to a Pro96Leu substitution and reduced expression,is likely the primary factor contributing to TGC resistance in the induced strain.

Aim To explore the effect of FTO on adria-mycin resistance in triple-negative breast cancer through the Wnt/β-catenin signaling pathway and to reveal the underlying mechanism.Methods The MDA-MB-231/ADR drug-resistant cell line was constructed using a method of gradually increasing adriamycin concentra-tion with intermittent induction.The half-inhibitory concentration(IC50)of adriamycin for MDA-MB-231 and MDA-MB-231/ADR cells and the expression of FTO were compared.After knocking down FTO in MDA-MB-231/ADR cells,CCK-8,qRT-PCR,colony formation assay,transwell,flow cytometry,and Western blot were used to assess the changes in the IC50 of adri-amycin,cell proliferation,migration,invasion,apopto-sis,and the expression of related proteins.Results FTO was highly expressed in MDA-MB-231/ADR cells.After FTO knockdown,the IC50 value of adriamy-cin in MDA-MB-231/ADR cells decreased,and the a-bilities of proliferation,migration and invasion were weakened.In the FTO knockdown group,the expres-sion levels of Bax,cleaved-caspase3,GSK-3 β proteins and the apoptosis rate significantly increased,while the expression levels of Bcl-2,Wnt5a,β-catenin,c-myc,cyclin D1,and P-gp proteins decreased.Conclusion FTO may inhibit the apoptosis of MDA-MB-231/ADR cells through the Wnt/β-catenin signaling pathway,al-ter P-gp expression,and thereby enhance the resistance of MDA-MB-231/ADR cells to adriamycin.

Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

This study aims to analyze the main drug resistance mechanism of Salmonella enteritidis induced by polymyxin in vitro.In this study,the resistance of Salmonella enteritidis CMCC(B)50335(ZK)to polymyxin was induced in vitro,and the growth characteristics,exercise ability,ul-trastructure and sensitivity to 16 antimicrobial agents before and after induction were determined by turbidimetry,semi-solid agar method,transmission electron microscope and disk diffusion method,and the whole genome and single nucleotide polymorphism(SNP)were detected by Illu-mina NovaSeq PE150 method.RT-qPCR was used to detect the differences in the expression levels of six drug-resistant related genes.The recombinant strain △phoPE1-128-1,which induced drug-resistant strain E1-128-1,was constructed by homologous recombination of Red Escherichia coli,and its sensitivity to polymyxin was detected.The results showed that three strains of induced drug-resistant bacteria E1-128-1,E1-128-3 and E2-128-3 were screened out,and the MIC increased by 128,64 and 64 times respectively after drug resistance stability test.Induced drug resistance had no significant effect on the growth ability of the tested bacteria and the sensitivity of 16 antibacte-rial drugs.The exercise ability of E1-128-1was significantly increased,and the cell wall and plasma membrane obviously thicken.There was no significant difference in the genome components of E1-128-1 before and after induction,but eight missense mutations of six drug-resistance related genes,including phoP/phoQ,cpxP,lptD,csrA and acrB,were detected,including four missense mutation sites of phoP,namely Leu185Trp,His189Ser,Thr190Tyr and Ile191His.The corresponding genes were sequenced by PCR,and the results were consistent with those of SNP.RT-qPCR results showed that the expression levels of mutant genes of the three induced strains increased signifi-cantly.Compared with E1-128-1,the MIC of △phoP E1-128-1 decreased to 1 mg/L.It is sug-gested that the mutation and increased expression of phoP gene are important factors for inducing polymyxin resistance of Salmonella CMCC(B)50335 in vitro.

Medication-related osteonecrosis of the jaw (MRONJ) is a refractory disease that can lead to severe destruction of the jaw. As there is no standard protocol for treating MRONJ, various treatments have been studied. Teriparatide has been used as an adjunct therapy for MRONJ. However, its effectiveness has not been sufficiently demonstrated for use as a standard treatment for MRONJ. This study aimed to demonstrate the efficacy of teriparatide in treating MRONJ by presenting two successfully treated cases. Each patient received teriparatide therapy with surgical intervention. The appropriateness of teriparatide use was evaluated based on the patient’s systemic condition, and the administration of teriparatide was supervised by a physician.Complete resolution of the lesion was observed clinically and radiographically in both patients. The first patient underwent implant placement at the lesion site. Due to its anabolic properties and ability to stimulate bone remodeling, teriparatide is an effective adjunctive pharmacological treatment for bone healing before and after surgery with associated beneficial effects on bone and mucosal healing.

Medication-related osteonecrosis of the jaw (MRONJ) is a refractory disease that can lead to severe destruction of the jaw. As there is no standard protocol for treating MRONJ, various treatments have been studied. Teriparatide has been used as an adjunct therapy for MRONJ. However, its effectiveness has not been sufficiently demonstrated for use as a standard treatment for MRONJ. This study aimed to demonstrate the efficacy of teriparatide in treating MRONJ by presenting two successfully treated cases. Each patient received teriparatide therapy with surgical intervention. The appropriateness of teriparatide use was evaluated based on the patient’s systemic condition, and the administration of teriparatide was supervised by a physician.Complete resolution of the lesion was observed clinically and radiographically in both patients. The first patient underwent implant placement at the lesion site. Due to its anabolic properties and ability to stimulate bone remodeling, teriparatide is an effective adjunctive pharmacological treatment for bone healing before and after surgery with associated beneficial effects on bone and mucosal healing.

Medication-related osteonecrosis of the jaw (MRONJ) is a refractory disease that can lead to severe destruction of the jaw. As there is no standard protocol for treating MRONJ, various treatments have been studied. Teriparatide has been used as an adjunct therapy for MRONJ. However, its effectiveness has not been sufficiently demonstrated for use as a standard treatment for MRONJ. This study aimed to demonstrate the efficacy of teriparatide in treating MRONJ by presenting two successfully treated cases. Each patient received teriparatide therapy with surgical intervention. The appropriateness of teriparatide use was evaluated based on the patient’s systemic condition, and the administration of teriparatide was supervised by a physician.Complete resolution of the lesion was observed clinically and radiographically in both patients. The first patient underwent implant placement at the lesion site. Due to its anabolic properties and ability to stimulate bone remodeling, teriparatide is an effective adjunctive pharmacological treatment for bone healing before and after surgery with associated beneficial effects on bone and mucosal healing.

Objective:To investigate the role of group 3 innate lymphoid cells (ILC3) in skin wound healing, and to explore the underlying mechanisms.Methods:Twenty-four 5-week-old male C57BL/6 mice were randomly and equally allocated into 3 groups: the skin wound + ILC3 inhibitor group (referred to as ILC3 inhibitor group), the skin wound group, and the control group, with 8 mice in each group. Four days before the establishment of the wound model, mice in the ILC3 inhibitor group were intraperitoneally injected with 1 &mu;g of ILC3 inhibitor every 2 days for a total of 2 doses, mice in the skin wound group were injected with an equal volume of physiological saline solution, and mice in the control group were fed normally. To establish a mouse skin wound model, a full-thickness circular incision with a diameter of 0.6 cm was made around the midpoint of the dorsal midline using a biopsy punch after the intraperitoneal injection of anesthetics, which was histologically confirmed to be a full-thickness injury. The size of the wounds was observed and recorded, photographs of the wounds were taken on days 0, 1, 3, 5, 7, and 9 after wounding, and corresponding wound healing rates were calculated. On day 9 after wounding, tissue samples were collected from the wound edges, and subjected to flow cytometry analysis to quantify ILC3 infiltrating around the skin wound, and hematoxylin and eosin (HE) staining was performed to assess the healing status of the skin wounds. Real-time quantitative polymerase chain reaction (qRT-PCR) was conducted to determine the mRNA expression of vitamin D receptor (VDR), Notch1, tumor necrosis factor-alpha (TNF-α), interleukin (IL) -17A, IL-17F, and IL-22 in the wound-edge tissues, and Western blot analysis to determine their protein expression. Statistical analysis was carried out by using one-way analysis of variance and t test. Results:On day 9 after wounding, the skin wound group showed an increased number of ILC3 in the wound-edge tissues (5.31% ± 1.47% vs. 3.10% ± 0.54%, P < 0.01), increased mRNA and protein expression of TNF-α, IL-22, IL-17A, and IL-17F (all P < 0.05), but decreased mRNA and protein expression of VDR (both P < 0.05) compared with the control group; the protein expression of Notch1 was significantly higher in the skin wound group than in the control group ( P < 0.05), but there was no significant difference in its mRNA expression between the two groups ( P > 0.05). On days 1, 3 and 5, the wound healing rates were significantly higher in the ILC3 inhibitor group (45.17% ± 9.90%, 61.58% ± 11.61%, 75.61% ± 9.12%, respectively) than in the skin wound group (25.87% ± 10.96%, 47.78% ± 13.81%, 64.55% ± 10.29%, respectively, all P < 0.05). On day 9, the ILC3 inhibitor group showed a decreased number of ILC3 around the wound (2.69% ± 0.95%, P < 0.01), decreased mRNA and protein expression of TNF-α, IL-22, IL-17A, and IL-17F in the wound-edge tissues (all P < 0.05), but increased mRNA and protein expression of Notch1 and VDR in the wound-edge tissues (all P < 0.05) compared with the skin wound group. On day 9 after wounding, histopathological examination with HE staining revealed continuous and intact epithelial structure, as well as dense and neatly arranged collagen fibers in the ILC3 inhibitor group, and the structures of hair follicles, blood vessels, and sebaceous glands were similar to those in the control group. Conclusions:Skin ILC3 infiltrated local wounds and were involved in the skin wound healing process through inflammatory factors such as TNF-α, IL-17A, IL-17F, and IL-22. Downregulating the number of ILC3 may promote skin wound healing by activating VDR and Notch1, as well as inhibiting the TNF-α signaling pathway and the expression of downstream inflammatory factors.