L. donovani (MHOM/ET/2009/GR356 clone IV) promastigote growth and 91S ribosome purification has been performed as previously described12 (link). Ribosome complexes with mRNA, three tRNA molecules and paromomycin were assembled by sequential addition of an mRNA fragment (
Paromomycin sulfate
Manufactured by Merck Group
Sourced in United Kingdom
Paromomycin sulfate is a white or off-white, crystalline powder. It is a broad-spectrum antibiotic used in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Neomycin sulfate, by Merck Group (3 mentions)
Hygromycin b, by Duchefa Biochemie (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Loperamide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Gene pulser 2, by Bio-Rad (1 mentions)
Lambda carrageenan, by Merck Group (1 mentions)
Hi fcs, by Merck Group (1 mentions)
Schneider s insect medium, by Merck Group (1 mentions)
Rpmi medium, by Merck Group (1 mentions)
Evans blue, by Merck Group (1 mentions)
Chloroquine diphosphate, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
2 deoxystreptamine dihydrobromide, by Merck Group (1 mentions)
Disodium tetraborate, by Merck Group (1 mentions)
Nitromethane ch3no2, by Merck Group (1 mentions)
Nalidixic acid, by Merck Group (1 mentions)
Mrs sorbitol agar, by Merck Group (1 mentions)
Mrs agar, by Merck Group (1 mentions)
13 protocols using paromomycin sulfate
1
Leishmania Ribosome Purification and Assembly
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L. donovani (MHOM/ET/2009/GR356 clone IV) promastigote growth and 91S ribosome purification has been performed as previously described12 (link). Ribosome complexes with mRNA, three tRNA molecules and paromomycin were assembled by sequential addition of an mRNA fragment (CACC AUG UUCAAA, GE Dharmacon) containing the leishmanial Kozak sequence41 (link) (highlighted in bold) a P-site start codon (AUG, underlined) and an A-site Phe codon (UUC), P-site tRNAfmet (E.coli, Sigma), A-site tRNAphe (E.coli, Sigma) and Paromomycin sulfate (Sigma) at 1:100:5:5:100 stoichiometric ratio. Complex assembly has been performed at 26 °C in ribosome conservation buffer (20 mM HEPES–KOH pH 7.6, 100 mM KOAc; 10 mM Mg(OAc)2, 10 mM NH4OAc, 2 mM β-mercaptoethanol and 1:40 dilution of RNAsin U (Promega)) with relaxation times of 30 min after the addition of each complex component. Ribosome final concentration was 125 nM.
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L. donovani (MHOM/ET/2009/GR356 clone IV) promastigote growth and 91S ribosome purification has been performed as previously described12 (link). Ribosome complexes with mRNA, three tRNA molecules and paromomycin were assembled by sequential addition of an mRNA fragment (
2
Microfluidic Cultivation and Analysis of Algae
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Two separate units of the “Tulip” microfluidic device were loaded with CC-4533 and Crvtc2-1 cultures of 1 µg Chl(a + b)/mL in TAP or HSM medium, at a flow rate of 80 µL/h, provided by a syringe pump (Model No. 4000, New Era Pump Systems Inc.,Farmingdale, NY, USA). The flow rate was kept constant during the entire experiment.
For filling up the “Pot” microfluidics device containing seven different trap types, we loaded the CC-124 algae culture (1 µg Chl(a + b)/mL) at a flow rate of 80 µL/h for 60 min using a syringe pump. Following this step, TAP medium was provided continuously, at a flow rate of 180 µL/h until the end of the experiment. For filling up the “Pot” microfluidics device with Traps II and VI, we set an initial flow rate of 80 µL/h for approx. 40 min and then the inlet tube was flushed with TAP medium and then the flow rate was kept at 180 µL/h until the end of the experiment.
Illumination was provided by white LED spot microscope lamps at an intensity of approx. 180 µmole photons m−2s−1 on the surface of the microfluidic device with 18 h light/6 h dark cycles. In order to prevent bacterial contamination, Hygromycin B (Duchefa Biochemie, Haarlem, The Netherlands) and paromomycin sulfate (Sigma Aldrich, Burlington, MA, USA) were added to the TAP medium at final concentrations of 1 µg/mL each before loading the cells into the microfluidic device.
For filling up the “Pot” microfluidics device containing seven different trap types, we loaded the CC-124 algae culture (1 µg Chl(a + b)/mL) at a flow rate of 80 µL/h for 60 min using a syringe pump. Following this step, TAP medium was provided continuously, at a flow rate of 180 µL/h until the end of the experiment. For filling up the “Pot” microfluidics device with Traps II and VI, we set an initial flow rate of 80 µL/h for approx. 40 min and then the inlet tube was flushed with TAP medium and then the flow rate was kept at 180 µL/h until the end of the experiment.
Illumination was provided by white LED spot microscope lamps at an intensity of approx. 180 µmole photons m−2s−1 on the surface of the microfluidic device with 18 h light/6 h dark cycles. In order to prevent bacterial contamination, Hygromycin B (Duchefa Biochemie, Haarlem, The Netherlands) and paromomycin sulfate (Sigma Aldrich, Burlington, MA, USA) were added to the TAP medium at final concentrations of 1 µg/mL each before loading the cells into the microfluidic device.
3
In Vitro Permeability Assay Using Caco-2 Cells and Mouse Liver Microsomes
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Paromomycin sulfate (Cat # P9297) was purchased from Sigma, Germany. Loperamide (Cat # L4762) was purchased from Sigma, Germany. Caco-2 cell line was procured from National Centre for Cell Science, Pune, India. Pooled mouse liver microsomes (MLMs) (Cat # 452220) were purchased from BD Gentest, MA, USA (B6C3F1– pool of ~ 100 mice, 9–10 weeks of age; Cat # 452220). Dulbecco's Modified Eagles medium (Cat # D5671), trypsin-EDTA solution (Cat # T4049) and Hank's buffered salt solution (HBSS) Buffer (Cat # H6648) were purchased from Sigma, Germany. Fetal Bovine Serum (Cat # 14-502F) was purchased from Lonza, Walkersville, MD, USA. Glasswares such as T-75 flasks and pipettes were procured from Grenier-Bio-one, Germany. Mill cell-24 well PET membrane 1 μm plates (Cat # PSRP010 R5) were from Millipore Corporation, Billerica MA.
4
Establishing Stable Cell Lines
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Conjugating cells were subjected to transfection by electroporation using a Gene Pulser II (Bio-Rad, Hercules, CA) as described previously (Iwamoto et al., 2014 (link), 2015 (link)). The resulting cell suspension was cultivated for 18 h and then treated with paromomycin sulfate (Sigma-Aldrich, St Louis, MO) at 120 µg/ml when using pIGF1, pIGF1C, pEGFP-neo4 and p2xmNeon_6xmyc_Neo4 vectors, or puromycin dihydrochloride (Fermentek, Jerusalem, Israel) at 200 µg/ml when using a pmCherry-pur4 vector. Cadmium chloride was also added at 0.5 µg/ml to induce the expression of drug-resistant genes for pEGFP-neo4, p2xmNeon_6xmyc_Neo4, and pmCherry-pur4 vectors. Resistant cells usually appeared within a few days after the drug was added. We checked that at least five independent clones (i.e. grown in five different wells) exhibited the same intracellular localization of each GFP–Nup.
5
Evaluation of Antileishmanial Drugs In Vivo
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L. major MHOM/SA85/JISH118 and L. mexicana MNYC/BZ/62/M379 parasites were cultured in Schneider's insect medium (Sigma, UK) supplemented with 10% heat-inactivated fetal calf serum (HiFCS; Sigma UK). These were passaged each week at a 1:10 ratio of existing culture to fresh media in 25-ml culture flasks without a filter and incubated at 26°C. For infection of mice, stationary-phase parasites were centrifuged for 10 min at 2,100 rpm and 4°C. The supernatant was removed and the pellet resuspended in RPMI medium (Sigma, UK). Cell number was estimated by microscopic counting with a Neubauer hemocytometer. AmBisome (LAmB; Gilead, UK) was reconstituted with 12 ml sterile water (as per the manufacturer's instructions) to yield a stock solution of 4 mg/ml and diluted in 5% aqueous dextrose to achieve a drug dose of 25 mg/kg. Paromomycin sulfate (Sigma) was prepared in phosphate-buffered saline (PBS) to yield 50-mg/kg doses. Lambda carrageenan (Sigma) and Evans blue (Sigma) 0.5% (wt/vol) solutions were made up in phosphate-buffered saline (PBS; Sigma). The drug preparations were stored at 4°C during the experiments.
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6
In vitro Antileishmanial Drug Assays
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For the in vitro drug assays, stocks of paromomycin sulfate (20 mM [aq]; Sigma, UK), MF (20 mM [aq]; Paladin Inc., UK), amphotericin B deoxycholate (5.2 mM [aq]) (Fungizone; Gibco, UK), and chloroquine diphosphate (10 mM [aq]; Sigma, UK) were prepared, aliquoted, and kept at −20°C until use. From the same original drug batches, solutions in PBS (0.9% NaOH [pH 7.4]; Sigma, UK) were made for the rodent experiments (mean weight per animal of 20 g) at concentrations of 50 mg/kg PM (5.797 mg/ml), 25 mg/kg CQ (4.032 mg/ml), and 50 mg/kg PM plus 25 mg/g kg CQ (coadministration of the same doses).
7
Cultivation of Wild-type and Transgenic P. morum
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The wild-type P. morum strain SAG 32.96 was obtained from the Culture Collection of Algae at Goettingen University (SAG), Germany. Cultures were maintained in Jaworski medium (JM) [47 ] at 29°C in an 8 h dark/16 h light (~10,000 lux) cycle [11 (link),20 (link)]. Cultures were grown in 10 ml glass tubes, 50 or 300 ml Erlenmeyer flasks, or 1,000 ml Fernbach flasks. The glass tubes had caps that allow for gas exchange, and Erlenmeyer and Fernbach flasks were aerated via Pasteur pipettes with approximately 50 cm3 sterile air/min [11 (link),20 (link)]. Transgenic strains that express the aphVIII gene were grown in JM in the presence of 1 μg paromomycin/ml (paromomycin sulfate, Sigma-Aldrich, St. Louis, MO).
8
Generation of Diploid Chlamydomonas Strains
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The strains used in this study are listed in Table 1 . To generate diploid strains, single mutant haploid strains with the AC17 allele were crossed with strains CC-5908 or CC-5909 with the genotype ac17; aphviii-TG (S1 Table ) that are mating-type plus or mating-type minus, respectively. Mutant strains with reciprocal selectable genotypes were mated and then plated onto acetate-free plates supplemented with 10 μg/ml paromomycin sulfate (Sigma Aldrich, P-8692, lot #101K1943). Plates were incubated in constant light at 25°C for 3–5 days until colonies appeared. Each diploid colony was screened by PCR for heterozygosity at the mating-type locus before further analysis. Available PCR markers were used to genotype strains when appropriate. Primers are listed in S1 Table . Strain CC-5485 with aphviii insertions was used to obtain strains CC-5908 and CC-5909. The aphviii insertion in CC-5485 does not cause a motility phenotype [85 (link)]. The oda11 strain CC-2672 from the Chlamydomonas Resource Center has a second hit that results in short cilia. This strain was backcrossed to wild-type to remove it.
9
NMR Spectroscopy of Aminoglycoside Antibiotics
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Deuterium oxide (99.97%
D2O), DCl (20% concentration solution in D2O),
and NaOD (30% concentration solution in D2O) were purchased
from Goss Scientific (U.K.). Neamine free-base was purchased from
Carbosynth (U.K.). 2-Deoxystreptamine dihydrobromide, neomycin sulfate,
paromomycin sulfate, streptomycin potassium hydrogen phthalate, disodium
tetraborate, sodium trimethylsilylpropanoate (TMSP), and nitromethane
(CH3NO2) were purchased from Sigma–Aldrich
(U.K.).
D2O), DCl (20% concentration solution in D2O),
and NaOD (30% concentration solution in D2O) were purchased
from Goss Scientific (U.K.). Neamine free-base was purchased from
Carbosynth (U.K.). 2-Deoxystreptamine dihydrobromide, neomycin sulfate,
paromomycin sulfate, streptomycin potassium hydrogen phthalate, disodium
tetraborate, sodium trimethylsilylpropanoate (TMSP), and nitromethane
(CH3NO2) were purchased from Sigma–Aldrich
(U.K.).
10
Aminoglycoside Compounds Synthesis Protocol
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