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1/4 MIC Contezolid, 1/4 MIC Linezolid and DMSO control group of Staphylococcus aureus biofilm quantitative proteomics comparative study to observe the global expression of protein Contezolid, Linezolid treatment groups and DMSO control group

ABSTRACT: S. aureus isolate in stable phase were inoculated into 15 ml TSBG plate culture, which contained 1/4 MIC of contezolid and linezolid treatment (DMSO as control) with 3 technical replicates in each group,. Incubate at 37 ° for 16 h and centrifuge at 5,000 rpm for 5 min to collect bacteria, including the formed biofilm bacteria. Wash with 1 ml PBS in water twice, transferred to a 1.5 ml EP tube, centrifuge and discard the supernatant. Lyse the cells by bead-beating at 4 °C in RIPA lysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS), The protein reduction, alkylation and tryptic digestion were followed as filter-aided sample preparation (FASP). The digestion products were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive plus mass spectrometer.

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Zewen Wen

PROVIDER: PXD040923 | iProX | Thu Mar 16 00:00:00 GMT 2023

REPOSITORIES: iProX

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Project description:Cortical rat neuronal culture, lysis and proteolytic digestion Primary rat cortical neurons were generated from E18 Sprague-Dawley rat embryos with minor modifications (Swanger, Mattheyses et al. 2015, Henderson, Greathouse et al. 2019). Neurons were cultured at a density of 4x105 cells per well in 12-well culture plates (Fisher Scientific, catalog no. 353043). Neurons were cultured in Neurobasal medium (Fisher Scientific, catalog no. 21103-049) supplemented with B27 (Fisher Scientific, catalog no.17504-044). Culture maintenance included a half media change every 2-3 days. At DIV 14, neurons were either treated with 150 ng/mL recombinant NRN1 protein (Abcam, ab69755) or vehicle treated with diH2O for 6 hours. NRN1 concentration was chosen based on published data that identified a plateau in exogenous NRN1 induced effects on transient potassium currents at 150 ng/mL (Yao et al., 2012). After 6 hours neurons were washed 2x with 1 mL 1X phosphate-buffered saline (PBS). To harvest cells, 1 mL 1X PBS + protease inhibitor (Fisher Scientific, catalog no. 78426) was added and cells were centrifuged for 2300rpm for 5 minutes at 4°C. Cell pellets were lysed in 200uL 8M urea buffer and HALT protease and phosphatase inhibitor cocktail (1x final concentration). Lysates were sonicated with a probe sonicator 3 times for 10 s with 10 s intervals at 30% amplitude and cleared of cellular debris by centrifugation in a tabletop centrifuge at 18,000 rcf for 3 minutes at 4° C. Protein concentration was determined by BCA assay and one-dimensional SDS-PAGE gels were run followed by Coomassie blue staining as quality control for protein integrity and equal loading before proceeding to protein digestion. Protein homogenates (50 µg) were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and then treated with 1 mM dithiothreitol (DTT) at 25°C for 30 minutes, followed by 5 mM iodoacetimide (IAA) at 25°C for 30 minutes in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25°C for 2 hours and further digested overnight with 1:50 (w/w) trypsin (Pierce) at 25°C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.

Project description:Purpose: The goals of this study are to compare NGS-derived gingiva transcriptome profiling (RNA-seq) of rats with or without experimental periodontitis and to explore the effect of curcumin/ZNPs hydrogel on the transcription of gingiva in rats with periodontitis. Methods: Six-week-old male Sprague-Dawley rats weighing 250–300 g (Dashuo company, China) were randomly divided into five groups (n = 5 per group): i) healthy control (Sham); ii) experimental periodontitis (EP); iii) experimental periodontitis treated by curcumin hydrogel (EP+Cur); iv) experimental periodontitis treated by ZNPs hydrogel (EP+ZNPs); v) experimental periodontitis treated by curcumin/ZNPs hydrogel (EP+Cur/ZNPs). Animals were anesthetized with isoflurane (5% induction, 2% maintenance, sealed until euthanized) and intramuscularly administered 0.1 mL penicillin (40,000 U/mL) for 7 days. EP was established by ligature with 4–0 silk thread around the bilateral maxillary first molars. Three days after ligature, P. gingivalis ATCC 33277 (107 CFU/mL) was smeared onto silk twice. Rats in the EP+Cur group were treated by curcumin hydrogel (Dalian Meilun Biotech Co., Ltd), EP+ZNPs group were treated by ZNPs hydrogel (Shanghai aladdin Biochemical Technology Co., Ltd), and EP+Cur/ZNPs group were treated by curcumin/ZNPs hydrogel. Total RNA was extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc.). The TruSeq Stranded total RNA with RiboZero Plus kit (IIlumina, San Diego, CA) was used to obtain a sequencing library from 1μg of total RNA. Eukaryotic mRNA sequencing was performed on Illumina Novaseq 6000 by Shanghai Majorbio Bio-pharm Technology Co., Ltd. Raw gene counts with a minimum of two counts per million in at least one sample were used for downstream analyses. Differentially expressed genes were determined by the DESeq2 Bioconductor/R package. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Transcriptome analysis of 25 samples was completed in this project, and a total of 184.45 Gb Clean Data was obtained. The Clean Data of each sample reached 5.92 Gb and above, and the Q30 base percentage was above 92.75%. The Clean Reads of each sample were compared with the designated reference genome, and the comparison rate ranged from 93.38% to 95.1%. A total of 26188 expressed genes were detected in this analysis, including 22736 known genes and 3452 new genes; 57,961 expressed transcripts, including 29332 known transcripts and 28629 new transcripts. Conclusions: Our study represents the first detailed analysis of gingival transcriptomes of rats treated by Cur/ZnO gel, with biologic replicates, generated by RNA-seq technology.

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1/4 MIC Contezolid, 1/4 MIC Linezolid and DMSO control group of Staphylococcus aureus biofilm quantitative proteomics comparative study to observe the global expression of protein Contezolid, Linezolid treatment groups and DMSO control group

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