Mycobacterium tuberculosis H37Ra was grown at 37°C in Middlebrook 7H9 liquid medium or on 7H11 agar plates supplemented with 10% (v/v) albumin–dextrose–catalase (ADC, Becton Dickinson, Sparks, MD, United States) plus 0.5% (v/v) glycerol, 0.25% (v/v) Tween 80. Plasmids p2NIL and pGOAL19 used in this study were obtained from Addgene (Cambridge, MA, United States). isoniazid (INH), rifampicin (RIF), streptomycin (SM), ethambutol, pyrazinamide (PZA), levofloxacin, amikacin, cycloserine, p-aminosalicylic acid (PAS), clofazimine (CFZ), tetracycline, linezolid, clarithromycin, and piperine were purchased from Sigma-Aldrich (St Louis, MO, United States). The drugs were dissolved in DMSO and further diluted to obtain the desired concentrations in culture media for drug susceptibility testing (see below).
Cycloserine
Manufactured by Merck Group
Sourced in United States
Cycloserine is a laboratory chemical used in various research applications. It functions as an antibiotic and inhibits bacterial cell wall synthesis. Cycloserine is commonly utilized in microbiological studies, biochemical assays, and other scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Isoniazid, by Merck Group (4 mentions)
Ethambutol, by Merck Group (3 mentions)
Clofazimine, by Merck Group (3 mentions)
Rifampicin, by Merck Group (3 mentions)
Clarithromycin, by Merck Group (2 mentions)
Levofloxacin, by Merck Group (2 mentions)
Pyrazinamide, by Merck Group (2 mentions)
Amikacin, by Merck Group (2 mentions)
Linezolid, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Galactose, by Merck Group (2 mentions)
Millex gp pes filter, by Merck Group (2 mentions)
Deuterium oxide, by Merck Group (2 mentions)
Tetracycline, by Merck Group (1 mentions)
Glycerol, by BD (1 mentions)
Piperine, by Merck Group (1 mentions)
Pgoal19, by Addgene (1 mentions)
P aminosalicylic acid, by Merck Group (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
10 protocols using cycloserine
1
Mycobacterium tuberculosis H37Ra Drug Susceptibility
2
Metabolomic Profiling with OGT Inhibitors
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Cells were obtained from ATCC and were maintained according to ATCC guidelines. For metabolomic profiling, cells were plated into either media with a commercial OGT inhibitor (ST045849, TimTec) or a vehicle control (DMSO). O-(2-Acetamido-2-deoxy-D-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), metformin, rotenone, cycloserine and Cl-alanine were obtained from Sigma. OSMI-1 compound was a gift from Professor Suzanne Walker (Harvard Medical School). Cells were allowed to attach for 1 to 3 days, and then treated as indicated in the beginning of the experiment (media was not changed at any point). OGT knockdown was performed with Lipofectamine RNAiMAX reagent according to manufacturer's instructions (Invitrogen) and OGT targeting siRNAs were obtained from Lifetechnologies (s16094 and s16095).
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
Preparation and Enumeration of Candida and Clostridium Strains
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C. albicans strain CKY101, [52 (link)] a virulent strain derived from the sequenced strain SC5314, was used for all studies. Cells were prepared for mouse inoculation by growth at 37 °C in YPD broth (1% yeast extract (BD, Sparks, MD, USA cat. 212750), 2% peptone (Difco, Detroit, MI, USA cat. 0118-17-0), and 2% glucose (Sigma-Aldrich, St. Louis, MO, USA cat. G8270)) [53 (link)] for 21–24 hrs.
C. difficile strain UK1, a NAP1/027/BI human epidemic strain [44 (link)], was used for all studies. Cultures were grown in pre-reduced TY broth (3% tryptose, 2% yeast extract, 0.1% thioglycolate, pH 7.4) [54 (link)]. Spores were isolated as previously described [39 (link)] except that the gradient purification was omitted. For the enumeration of C. difficile vegetative cells and spores in the extracts from mice, the samples were plated on pre-reduced TCCFA plates (Taurocholate (Calbiochem, San Diego, CA, USA cat. 580217), cycloserine (Sigma-Aldrich, St. Louis, MO, USA cat. C6880), cefoxitin (Sigma-Aldrich, St. Louis, MO, USA cat. C4786), fructose (Macron Fine Chemicals, Center Valley, PA, USA cat. 7756-12)) [40 (link)] and incubated at 37 °C for 2 days in an anaerobic chamber. Spores were enumerated by heating the samples at 60 °C for 10 min, followed by plating on pre-reduced TCCFA plates.
C. difficile strain UK1, a NAP1/027/BI human epidemic strain [44 (link)], was used for all studies. Cultures were grown in pre-reduced TY broth (3% tryptose, 2% yeast extract, 0.1% thioglycolate, pH 7.4) [54 (link)]. Spores were isolated as previously described [39 (link)] except that the gradient purification was omitted. For the enumeration of C. difficile vegetative cells and spores in the extracts from mice, the samples were plated on pre-reduced TCCFA plates (Taurocholate (Calbiochem, San Diego, CA, USA cat. 580217), cycloserine (Sigma-Aldrich, St. Louis, MO, USA cat. C6880), cefoxitin (Sigma-Aldrich, St. Louis, MO, USA cat. C4786), fructose (Macron Fine Chemicals, Center Valley, PA, USA cat. 7756-12)) [40 (link)] and incubated at 37 °C for 2 days in an anaerobic chamber. Spores were enumerated by heating the samples at 60 °C for 10 min, followed by plating on pre-reduced TCCFA plates.
5
D-Depleted Medium for Mutant Selection
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Bacterial cultures were grown in Mineral Salts (MS) medium [35 (link)] complemented with either 10.1 mM glucose or 217.3 mM glycerol. To make a D-depleted medium, the components of MS (17.3 mM K2HPO4, 51.5 mM KH2PO4, 7.5 mM (NH4)2SO4, 1.44 mM Na-citrate, 0.41 mM MgSO4, 2 μM FeSO4) were added in the form of crystals to D-depleted water (≤1 ppm deuterium oxide; Sigma-Aldrich, St. Louis, MO, USA), followed by the carbon source (glucose crystals or 99% glycerol) and filtered sterile using a 0.22 μm pore-size Millex®GP PES filter (Merck-Millipore, Carrigtwohill, Ireland). For the cycA tests, sterile vitamin B1 injection (Zentiva, Prague, Czech Republic) was directly added to obtain a 2.5 μg/mL thiamine concentration. Control medium was made from D-depleted medium by re-complementing with D2O (99.9 atom%; Sigma-Aldrich, St. Louis, MO, USA) to obtain 150 ppm deuterium concentration. MS components were provided by Molar Chemicals Ltd. (Budapest, Hungary). Mutant selection was carried out on 1.5% agar-MS plates, prepared using water of 150 ppm D. The plates contained glucose as the carbon source (glycerol for ackA and galK tests), and one of the following selective agents: cycloserine (0.04 mM), chloroacetate (10 mM) or galactose (10 mM) (all obtained from Sigma-Aldrich, St. Louis, MO, USA).
6
Evaluating Synergistic Drug Combinations for Tuberculosis
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For drug combination studies, MABA was used utilizing similar methodology as described above. For the two drug combination studies, isoniazid (Sigma-Aldrich, St. Luis, MO, USA) at 0.2 µg/mL, rifampicin (Sigma-Aldrich) at 0.026 ng/mL, cycloserine (Sigma-Aldrich) at 5.5 µg/mL, and clarithromycin (Sigma-Aldrich) at 0.09 µg/mL concentrations were used. For the three-drug combination studies, isoniazid at 0.05 µg/mL and rifampicin at 0.012 ng/mL concentrations, respectively, were used. The combination effect of compounds was determined by calculating the combination index (CI). CI was calculated as the sum of the individual drug effect divided by the combination effect of the drugs. If CI values were <1.0, this indicated a synergistic effect (i.e., the combined effect was greater than the sum of their individual effects). If CI = 1.0, this indicated an additive effect (i.e., the combined effect was equal to the sum of their individual effects) and if CI >1.0, this indicated an antagonistic effect (i.e., the combined effect was less than the sum of their individual effects) [64 (link),65 (link),66 (link)].
7
Cycloser ine Stability Protocol
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Eight concentrations of cycloserine (Sigma-Aldrich Co., St Louis, MO, USA) from 2 μg/mL to 512 μg/mL in MH medium were prepared by two-fold serial dilution and were sub-packed into 29 8-strip PCR tubes (cap attached) with 100 μL/tube. At the same time, 256 μg/mL cycloserine solution was also placed into 29 1.5mL Eppendorf (EP) tubes at 1 mL/tube. All tubes were stored at ˗20 °C. Every day, one strip and one EP tube were taken out and placed in the 37°C incubator. After 29 days, the solutions in the PCR tubes were used for DST and the solutions in the EP tubes were used for ultra-performance liquid chromatography (UPLC) and liquid chromatography tandem mass spectrometry (LC-MS).
8
Anti-TB Drug Combination Screening
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Anti-TB drugs amoxicillin/clavulanate (AC 4:1 wt:wt ratio), clofazimine (CFZ), cycloserine (CYS), ethambutol (EMB), isoniazid (INH), linezolid (LZD), moxifloxacin (MXF), para-aminosalicyclic acid (PAS), prothionamide (PRO), pyrazinamide (PZA), and rifampicin (RIF), rifapentine (RPT), and streptomycin (STR) were purchased from Sigma-Aldrich. Bedaquiline (BDQ) was purchased from Asclepia (Belgium). PA-824 (PA824) was provided by the Global Alliance for TB Drug Development; SQ-109 (SQ109) was provided by Sequella, Inc; and delamanid (DLM) was provided by Otsuka Pharmaceutical Co. As described in detail previously [4 (link)], the various PRS experimental regimens and the Standard Regimen (INH, RIF, EMB, PZA) were mixed in sterile polypropylene 96-well deep well plates (Nunc) using Microlab STAR Line liquid handling workstation (Hamilton) operated by Venus One software.
9
Synthesis of Antitubercular Compounds
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1599 and 1810 were synthesized as described (Figure S1).3 (link) Isoniazid, rifampicin, pyrazinamide, ethambutol, levofloxacin, kanamycin, amikacin, ethionamide, ofloxacin, cycloserine, rifapentine and clofazimine were purchased from Sigma-Aldrich. Linezolid was purchased from 21CEC. Moxifloxacin was obtained from Bayer. PA-824 (Pretomanid) was obtained from the Global Alliance for TB Drug Development. Bedaquiline (Sirturo) was provided by Johnson and Johnson. SQ109 was synthesized as described.9 (link)
10
Deuterium-Depleted Media for Mutant Selection
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Bacterial cultures were grown in Mineral Salts (MS) medium [34] complemented with either 10.1 mM glucose or 217.3 mM glycerol. To make a D-depleted medium, the components of MS (17.3 mM K 2 HPO 4 , 51.5 mM KH 2 PO 4 , 7.5 mM (NH 4 ) 2 SO 4 , 1.44 mM Na-citrate, 0.41 mM MgSO 4 , 2 M FeSO 4 ) were added in the form of crystals to D-depleted water (≤1 ppm deuterium oxide; Sigma-Aldrich, St. Louis, MO, USA), followed by the carbon source (glucose crystals or 99 % glycerol) and filtered sterile using a 0.22 m pore-size Millex ® GP PES filter (Merck-Millipore, Carrigtwohill, Ireland). For the cycA tests, sterile vitamin B1 injection (Zentiva, Prague, Czech Republic) was directly added to obtain a 2.5 g/mL thiamine concentration. Control medium was made from D-depleted medium by recomplementing with D 2 O (99.9 atom %; Sigma-Aldrich, St. Louis, MO, USA) to obtain 150 ppm deuterium concentration. MS components were provided by Molar Chemicals Ltd.
(Budapest, Hungary). Mutant selection was carried out on 1.5 % agar-MS plates, prepared using water of 150 ppm D. The plates contained glucose as the carbon source (glycerol for ackA and galK tests), and one of the following selective agents: cycloserine (0.04 mM), chloroacetate (10 mM) or galactose (10 mM) (all obtained from Sigma-Aldrich, St. Louis, MO, USA).
(Budapest, Hungary). Mutant selection was carried out on 1.5 % agar-MS plates, prepared using water of 150 ppm D. The plates contained glucose as the carbon source (glycerol for ackA and galK tests), and one of the following selective agents: cycloserine (0.04 mM), chloroacetate (10 mM) or galactose (10 mM) (all obtained from Sigma-Aldrich, St. Louis, MO, USA).
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