Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug resistant strains of P. aeruginosa and E. coli.
BackgroundType 2 innate lymphoid cells (ILC2s), characterized by secreting type 2 cytokines, regulate multiple immune responses. ILC2s are found in different tumor tissues and ILC2-derived interleukin (IL)-4, IL-5, and IL-13 act on the cells in tumor microenvironment to participate in tumor progression. ILC2s are abundant in colorectal cancer (CRC) tissue, but the role of ILC2s in CRC remains unclear. MethodsILC2s were sorted from the spleen using microbeads combined with flow cytometry and tumor infiltrating CD8 + T cells were isolated from tumor tissue by microbeads. Flow cytometry and immunofluorescence were used to detect the percentage of ILC2s and CD8 + T cells in the spleen and CRC tissue. Effects of IL-9 and IL-9-stimulated CD8 + T cells on CT26 cells were measured by proliferation, apoptosis, and migration assays in vitro. GEPIA was used to detect the ILC2s chemokines in CRC tissue and adjacent normal tissue. ResultsWe found that ILC2s were increased in CRC tissue compared with the adjacent normal tissue. In vitro experiments showed that IL-9 could activate CD8 + T cells to promote the death of CT26 cells. ILC2s were the main IL-9-secreting cells in CRC tissue as shown by flow cytometry analysis. In vivo experiments showed that neutralizing ILC2s promoted the tumor growth, while tumor inhibition occurred by intravenous injection of IL-9. ConclusionsOur results demonstrated that ILC2-derived IL-9 activated CD8 + T cells to promote anti-tumor effects in CRC.Th9 cells inhibit the development of melanoma and breast cancer by secreting 33]. In addition, Th9-derived IL-9 promotes the expansion of CD8 + T cells through IL-9 receptor (IL-9R) in CRC [34]. However, the main IL-9-producing cell subset in CRC remains unclear.Here we investigated the role of ILC2s and IL-9 in the CRC progression. The IL-9-producing cell subset in CRC tissue was also detected. We hypothesized that ILC2-derived IL-9 could exert an antitumor role in CRC. Materials And Methods Cell line and cell cultureThe murine CRC cell line CT26 was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in a humidified incubator with 5% CO2 at 37 °C. AnimalsFemale BALB/c mice (6-8 weeks, 18-22 g) were purchased from the Animal Center of Yangzhou University (Yangzhou, China) and the Animal Center of Jiangsu University (Zhenjiang, China). Mice were housed in cages and bred in pathogen-free rooms with a temperature of 23 °C ± 2 °C and relative humidity of 55%±10%. All animal experiments complied with the protocol of Jiangsu University Animal Ethics and Experimentation Committee.
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