Plasmid pRPF215, a Himar1 mariner delivery vector30 (link), was conjugated into R20291 from E. coli SD46 and R20291 transconjugants selected on BHI agars supplemented with 250 µg/ml cycloserine (Matrix Scientific, catalog no. 072929), 8 µg/ml of cefoxitin (Chem-Impex International, catalog no. 01490), and 15 µg/ml thiamphenicol (Sigma-Aldrich, catalog no. T0261). To generate Tn mutants, R20291-pRPF215 was grown overnight in BHI broth with 15 µg/ml thiamphenicol, then subcultured into BHI to OD600nm of 0.2. Himar1 was induced with 100 ng/ml anhydrotetracycline (ATc) (Alfa Aesar, catalog no. J66688) for mutagenesis for overnight (15 h). Tn mutants were selected by spreading culture dilutions onto pre-reduced BHI agar containing 20 µg/ml lincomycin. After incubation (24 h), colonies were collected into 96 deep well plates, to amass 7488 independent colonies. The library was screened for metronidazole-susceptible Tn mutants by growing overnight cultures in BHI containing lincomycin and spotting 2 μl inocula onto BHI agars containing 0.5 µg/ml metronidazole and 5 µg/ml of hemin using a 96-Well Bench Top Pipettor. Presumptive metronidazole-susceptible Tn mutants were confirmed by MIC testing and underwent genome and Sanger sequencing to confirm the insertion sites for ermB. Primers and strain constructs used are listed in the Supplementary Data 2 and 3 .
Thiamphenicol
Manufactured by Merck Group
Sourced in United States
Thiamphenicol is a broad-spectrum antibiotic that can be used in the laboratory setting. It functions as an inhibitor of bacterial protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Chloramphenicol, by Merck Group (12 mentions)
Florfenicol, by Merck Group (6 mentions)
Ciprofloxacin, by Merck Group (5 mentions)
Erythromycin, by Merck Group (4 mentions)
Doxycycline, by Merck Group (4 mentions)
Kanamycin, by Merck Group (3 mentions)
Lincomycin, by Merck Group (3 mentions)
Oxytetracycline, by Merck Group (3 mentions)
Chlortetracycline, by Merck Group (3 mentions)
Florfenicol amine, by Merck Group (3 mentions)
Enrofloxacin, by Merck Group (3 mentions)
Tetracycline, by Merck Group (3 mentions)
Danofloxacin, by Merck Group (2 mentions)
Trimethoprim, by Merck Group (2 mentions)
Ofloxacin, by Merck Group (2 mentions)
Norfloxacin, by Merck Group (2 mentions)
Pefloxacin, by Merck Group (2 mentions)
Lomefloxacin, by Merck Group (2 mentions)
Milli q ultrapure system, by Merck Group (2 mentions)
Chloramphenicol d5, by Merck Group (2 mentions)
21 protocols using thiamphenicol
1
Generation of Tn Mutants in C. difficile
2
Multiplex Antimicrobial Residue Detection
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Chloramphenicol (CAP), ciprofloxacin (CIP), florfenicol, (FF), penicillin (PEN), ractopamine (RAC), salbutamol (SAL), sulfadiazine (SUL), and thiamphenicol (TAP) standards were purchased from Sigma Aldrich (99% purity, St. Louis, MO, USA). CAP-conjugated antigen and HRP-labeled CAP antibody were obtained from ZeYang Co. (Beijing, China). SuperSignal chemiluminescence substrate solution was purchased from Thermo Fisher (USA). Milli-Q Synthesis system was obtained from Millipore (Bedford, MA, USA) for water purification. Other reagents (analytical grade) were purchased from Beijing Reagent Corp. (Beijing, China).
3
Molecular Cloning and Mutagenesis Protocols
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Oligonucleotide primers were synthesized by IDT (Coralville, IA) and are listed in Table 3 . PCR reactions were performed using Phusion High-Fidelity Master mix with HF buffer or Phusion Hot Start Flex 2X Master mix (New England Biolabs, Ipswich, MA, USA). Restriction endonucleases, Quick CIP (calf alkaline phosphatase), DNA ligase, and competent E. coli DH10ß cells were purchased from New England Biolabs (Ipswich, MA, USA). Mutations were introduced into the BoNT genes using QuikChange Lightning Multi site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The antibiotics carbenicillin, chloramphenicol, kanamycin, thiamphenicol, erythromycin, and cycloserine were purchased from Sigma-Aldrich (St. Louis, MO). Media reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or DIFCO-BD biosciences (Franklin Lakes, NJ, USA).
4
Isolation of Chromosomal Gene Disruption Mutant
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To isolate a chromosomal gene disruption mutant, plasmid pSYCP-cpaAIR was first electrotransformed into C. pasteurianum and transformants were selected using 10 μg ml−1 thiamphenicol. Mosaic colonies containing both wild-type and intron insertion cells were identified with two separate PCRs using primers ltrB.Fw + REN.Fw for one chromosome-intron junction and primers ltrB.Rv + REN.Rv for the adjacent junction. One positive colony was selected for enrichment of the intron disruption by repeated subculturing in selective growth medium. Briefly, a sporulated colony was heat-shocked in 10 ml of 2 × YTG medium, cooled on ice, and supplemented with 10 μg ml−1 thiamphenicol (Sigma-Aldrich; St. Louis, Missouri, United States). Following approximately 24 hours of growth, 0.5 ml was used to inoculate a tube of 10 ml 2 × YTG containing 10 μg ml−1 thiamphenicol. This process was repeated every 12 hours for a total of 10 transfers, at which time serial dilutions were plated onto nonselective 2 × YTG agar. To identify a homogeneous gene disruption colony, colony PCR was performed on enrichment colonies using two gene-specific primers flanking the intron insertion site (REN.Fw + REN.Rv).
5
Comprehensive Veterinary Drug Extraction
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All solvents (HPLC/analytical grade) were purchased from Fluka (Sigma- Aldrich, St. Louis, MO, USA). Trichloroacetic acid (TCA) crystals and the reagents to prepare an EDTA-McIlvaine buffer solution, pH 4 (disodium hydrogen phosphate dihydrate, citric acid monohydrate and EDTA) were purchased from Fluka.
Formic acid (98–100%) was obtained from Riedel-de Haën (Sigma-Aldrich, St. Louis, MO, USA).
The extraction cartridges (Oasis HLB 3 mL, 60 mg) were provided by Waters (Milford, MA, USA).Amoxicillin, ampicillin, benzylpenicillin, cefalexin, cefquinome, ceftiofur, chloramphenicol, chlortetracycline, ciprofloxacin, danofloxacin, dimetridazole, doxycycline, enrofloxacin, florfenicol, florfenicol amine, flumequine, furaltadone, furazolidone, lincomycin, lomefloxacin hydrochloride, marbofloxacin, nalidixic acid, nitrofurazone, oxolinic acid, oxytetracycline, ronidazole, spyramicin, sulphadiazine, sulphadimethoxine, sulphadimidine, sulphamerazine, sulphathiazole, tetracycline hydrochloride, thiamphenicol, tilmicosine, tinidazole, trimethoprim, tylosin, and enrofloxacin d5 as the internal standards (IS) (purity >98%) were used and purchased from Fluka (Sigma-Aldrich, St. Louis, MO, USA).
Formic acid (98–100%) was obtained from Riedel-de Haën (Sigma-Aldrich, St. Louis, MO, USA).
The extraction cartridges (Oasis HLB 3 mL, 60 mg) were provided by Waters (Milford, MA, USA).Amoxicillin, ampicillin, benzylpenicillin, cefalexin, cefquinome, ceftiofur, chloramphenicol, chlortetracycline, ciprofloxacin, danofloxacin, dimetridazole, doxycycline, enrofloxacin, florfenicol, florfenicol amine, flumequine, furaltadone, furazolidone, lincomycin, lomefloxacin hydrochloride, marbofloxacin, nalidixic acid, nitrofurazone, oxolinic acid, oxytetracycline, ronidazole, spyramicin, sulphadiazine, sulphadimethoxine, sulphadimidine, sulphamerazine, sulphathiazole, tetracycline hydrochloride, thiamphenicol, tilmicosine, tinidazole, trimethoprim, tylosin, and enrofloxacin d5 as the internal standards (IS) (purity >98%) were used and purchased from Fluka (Sigma-Aldrich, St. Louis, MO, USA).
6
DNA Oligonucleotide Synthesis and Polyadenylation Assays
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Custom-designed DNA oligonucleotides were purchased from DNA Technology (Aarhus, Denmark). Their sequences and other relevant information are shown in Supplementary Table S1 . Inhibitors used in polyadenylation assays at the final concentrations shown in figures and legends were cordycepin (3′ deoxyadenosine), CCCP, doxycycline, oligomycin (mixture A, B, C), or thiamphenicol (all from Sigma), or puromycin (InvivoGen). Antibodies were from Abcam: anti-mtPAP (ab154555, used at 1:2000); anti-PDE12 (ab87738, 1:500); anti-PNPT1 (PNPase, ab96176, 1:2000) and anti-SUV3L1 (SUV3, ab176854, 1:2000). Anti-GAPDH antibody was from Cell Signaling Technology (14C10, #2118, 1:3000), anti-β-actin from Santa Cruz (C4, sc-47778, 1:200) and HRP-conjugated goat anti-rabbit (111-035-144) and goat anti-mouse (115-035-146) secondary antibodies (1:20 000) were from Jackson ImmunoResearch Inc., the latter being used for detection of the anti-actin signal.
7
Evaluation of Antibacterial Activity
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Direct antibacterial activity was determined by the microdilution method as described [36 ]. In practice, 20 mg of each plant extract were dissolved in 100 μL of DMSO and then completed to 5 mL with broth to obtain a stock concentration of 4 mg/mL (2% DMSO). Then, this solution was diluted to obtain an entry concentration of 512 mg/L with a concentration of 0.2% in DMSO. This solution was transferred to the wells of the first column of the 96-well plates (200 μL/well) for serial dilution with Muller-Hinton II (MHII). One hundred (100 μL) of the bacterial suspension (5 × 105 CFU/mL) was added to each well to obtain a final volume of 200 μL, with a final DMSO concentration of 0.1%. The plates were incubated for 18 hours at 37 °C without agitation with closed lid. The minimal inhibitory concentrations (MICs) of samples were observed after the addition 40 µL of 0.2 mg/mL of iodonitrotetrazolium chloride. MIC values were recorded as the lowest concentration of the sample that totally inhibited bacterial growth. The reference antibiotics used in the present work included chloramphenicol, florfenicol, thiamphenicol, ciprofloxacin, cefepime, and ceftazidime (Sigma-Aldrich). Objectives were to detect molecules that were able to modulate the bacterial permeability of the bacterium in terms of penetration and efflux.
8
Analytical Standards for Veterinary Pharmaceuticals and Heavy Metals
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Chemical standards for ciprofloxacin, enrofloxacin, lomefloxacin, norfloxacin, norfloxacin-d5, ofloxacin, pefloxacin, chlortetracycline, doxycycline, oxytetracycline, tetracycline, tetracycline-d6, chloramphenicol, chloramphenicol-d5, thiamphenicol, florfenicol, florfenicol amine, metronidazole, metronidazole-d4, malachite green, malachite green-d5, Leucomalachite green, and leucomalachite green-d5 were all purchased from Sigma-Aldrich with purity grade more than 95% (St. Louis, MO, USA). The standard solutions of arsenic (As) and cadmium (Cd) were brought from Agilent Technologies (Santa Clara, CA, USA). HPLC-grade acetonitrile (ACN), methanol (MeOH), formic acid (FA), and ethyl acetate were brought from Merck Chemicals Ltd. (Darmstadt, Germany). Ultrapure HNO3 (68.0–70.0%, Certified ACS) was obtained from Fisher Scientific (Ottawa, ON, Canada). Ammonia solution, hydrochloric acid, sodium chloride, and ethylenediaminetetraacetic acid disodium salt (Na2EDTA) with analytical grade were all purchased from Sinopharm (Beijing, China). Ultrapure water was produced through the Milli-Q ultrapure system (Millipore, Bedford, MA, USA).
9
Antimicrobial Susceptibility of Bacterial Strains
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Six pathogenic bacterial strains were purchased from China Veterinary Culture Collection (CVCC, Beijing, China). The strains were E. coli K88, E. coli CVCC 245, S. aureus CVCC 26003, S. aureus ATCC 25923, Micrococcus luteus (M. luteus) CVCC 28001, and Listeria monocytogenes (L. mono.) CVCC 1599. Neomycin sulfate, chloramphenicol, thiamphenicol, kanamycin, and penicillin G were all purchased from Sigma (San Francisco, CA, USA).
10
Preparation of Antibacterial Compounds
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Chloramphenicol (≥98%; C0378-5G), thiamphenicol (>99.9%; T0261-15), and kanamycin sulfate (KAN) (K1377) were purchased from Sigma-Aldrich and prepared as 50 mg/mL stock solutions in ethanol (amphenicols) or water, filter sterilized, and stored at −20°C. Bratton-Marshall reagent (N-[1-naphthyl]ethylenediamine HCl) (98+%; AC423990250), sodium nitrite (97% minimum; AA1424422), ammonium sulfamate (98+%; AC423390050), riboflavin 5′-monophosphate sodium salt (FMN) (93.0+%; R00235G), and florfenicol (NC1617017) were purchased from Fisher Scientific and prepared as described below. β-Nicotinamide reduced tetrasodium salt (NADPH) was purchased from Dot Scientific (>93%; DSN20140-0.1), stored dry at −20°C, and prepared fresh for all experiments. Amino-Chloramphenicol was purchased from Toronto Research Chemicals (A622670) as an HCl salt and prepared by dissolution in water. Amino-Chloramphenicol solutions were stored at −20°C for less than 1 month. Sch-24893 (Fig. 1b ) was synthesized by the Northwestern University ChemCore, validated by proton and carbon NMR in dimethyl sulfoxide (DMSO) (Fig. S1A and B), and prepared as a 50 mg/mL stock solution in ethanol.
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