E. coli cell extracts were prepared from the BL21 Star (DE3) strain with a high‐throughput sonication method, as described previously.
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8 protocols using sterile syringe filter
1
High-Throughput E. coli Cell Extract Preparation
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E. coli cell extracts were prepared from the BL21 Star (DE3) strain with a high‐throughput sonication method, as described previously.6 Briefly, overnight cultures grown in Luria‐Bertani (LB) medium at 37°C 220 rpm were diluted 1000 times with 1 L of 2× YTPG medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 7 g/L K2HPO4, 3 g/L KH2PO4, and 18 g/L glucose; adjusted to pH 7.2 with KOH) and incubated at 37°C and 220 rpm until an optical density at 600 nm (OD600 nm) of 0.5 was reached. T7 RNA polymerase was then induced by the addition of 1 mM of isopropyl β‐D‐1‐thiogalactopyranoside during incubation. Cell cultures were harvested after an OD600 nm of 3.0 was reached. Cell pellets were suspended in 1 ml of S30 buffer per gram of cells and lysed on ice with a Q125 sonicator (Qsonica). Cell debris and insoluble components were removed by two rounds of centrifugation for 10 min at 14,000 × g at 4°C, and supernatants were filtered through a 0.2‐μm sterile syringe filter (Corning). The crude extracts were stored at −80°C until use.
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E. coli cell extracts were prepared from the BL21 Star (DE3) strain with a high‐throughput sonication method, as described previously.
2
Purification and Radiolabeling of RNA Substrates
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L-21 ScaI ribozyme (E) was
transcribed and gel-purified
according to reported procedures.49 (link) Care
was taken to avoid RNA damage from ultraviolet shadowing, as previously
described.50 (link) Guanosine (G) was purchased
from Sigma-Aldrich (St. Louis, MO) with a purity of ≥98%, and
3′-aminoguanosine [G(2′N)] and 3′-amino-2′-deoxyguanosine
[G(2′H,3′N)] were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA) and were of the highest purity commercially available
(≥98%). 2′-Amino-3′-deoxyguanosine [G(2′N,3′H)]
was a gift from J. W. Szostak (Harvard University, Cambridge, MA).
The oligonucleotide substrates, CCCUCUA (rSA) and CCCUCdUA (−1d,rSA),
were purchased from Integrated DNA Technologies (Redwood City, CA),
5′-32P-radiolebeled using [γ-32P]ATP (MP Biomedicals, Santa Ana, CA) and T4 polynucleotide kinase
(New England Biolabs, Ipswich, MA) according to the manufacturer’s
protocol, and gel-purified as previously described.51 (link) Buffers and salts were purchased from Sigma-Aldrich. All
nonradioactive reagents were passed through a 0.2 μm sterile
syringe filter (Corning, Corning, NY) prior to use.
transcribed and gel-purified
according to reported procedures.49 (link) Care
was taken to avoid RNA damage from ultraviolet shadowing, as previously
described.50 (link) Guanosine (G) was purchased
from Sigma-Aldrich (St. Louis, MO) with a purity of ≥98%, and
3′-aminoguanosine [G(2′N)] and 3′-amino-2′-deoxyguanosine
[G(2′H,3′N)] were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA) and were of the highest purity commercially available
(≥98%). 2′-Amino-3′-deoxyguanosine [G(2′N,3′H)]
was a gift from J. W. Szostak (Harvard University, Cambridge, MA).
The oligonucleotide substrates, CCCUCUA (rSA) and CCCUCdUA (−1d,rSA),
were purchased from Integrated DNA Technologies (Redwood City, CA),
5′-32P-radiolebeled using [γ-32P]ATP (MP Biomedicals, Santa Ana, CA) and T4 polynucleotide kinase
(New England Biolabs, Ipswich, MA) according to the manufacturer’s
protocol, and gel-purified as previously described.51 (link) Buffers and salts were purchased from Sigma-Aldrich. All
nonradioactive reagents were passed through a 0.2 μm sterile
syringe filter (Corning, Corning, NY) prior to use.
3
Bacterial Growth Conditions Optimization
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Each bacteria was cultured to log phase at 37ºC in 3 mL Luria-Bertani (LB, pH 7.4) broth with no supplementation. Bacterial suspensions were diluted 1:100 and plated 100 μl into a 96-well plates containing LB broth with 1% glucose (Sigma-Aldrich). The plates were incubated at 37ºC with agitated shaking (AS) at 250 rpm) or no agitation (static environment (SE)) for 6-72 hours or incubated in LB broth with different pH environments (pH 5 to 8) for 24-48 hours. Bacterial growth was determined by optical density at 600 nm (OD6oo) using a spectrophotometer. Furthermore, serial dilutions of culture were performed and plated for colony forming units (CFU).
For adjusting different pH in LB broth, either hydrochloric acid or sodium hydroxide was used to either decrease or increase the pH, respectively, to 5, 6, 7 and 8. Media was then filtered through a 0.2 μm sterile syringe filter (Corning, Inc., Corning, NY). Each bacterium was added into aliquots of sterile pH-adjusted media and pH was measured using pH indicator strips (EM Science, Cherry Hill, NJ) before and after incubation.
For adjusting different pH in LB broth, either hydrochloric acid or sodium hydroxide was used to either decrease or increase the pH, respectively, to 5, 6, 7 and 8. Media was then filtered through a 0.2 μm sterile syringe filter (Corning, Inc., Corning, NY). Each bacterium was added into aliquots of sterile pH-adjusted media and pH was measured using pH indicator strips (EM Science, Cherry Hill, NJ) before and after incubation.
4
Plasma Microvesicle Quantification by NTA
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Analysis of the concentrations and sizes of MVs in plasma was performed by nanoparticle tracking analysis (NanoSight 300; NTA, NanoSight Ltd., Amesbury, UK). For the analysis, 100 μL of plasma was centrifuged at 5,000 g for 20 min to remove platelets and apoptotic bodies. The plasma was then diluted to 200 μL with phosphate-buffered saline (PBS), which was filtered through a 0.2-μm sterile syringe filter (Corning, NY). Samples were then analyzed by NanoSight 300 in accordance with the manufacturer's instructions.
5
Polymeric Micelle Formulation of Fenbendazole and Rapamycin
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FEN/RAPA-loaded polymeric micelles were prepared from three polymers by the freeze-drying method (Figure 1 ).57 ,58 Various FEN/RAPA ratios were dissolved along with the polymers in 1 mL tert-butanol and stirred in water at 60°C for 3 min. Then 1 mL DW was added and the FEN/RAPA-polymer mixture was vortexed, quickly frozen at −70°C for 1 h, put in a freeze-dryer (Advantage Pro; SP Scientific, Warminster, PA, USA), and lyophilized for 24 h. Then 1 mL DW at 60°C was added to rehydrate the mixture. The FEN/RAPA-loaded micelle solution was centrifuged at 16,600 x g for 5 min at 4°C (Hanil Science Inc., Gimpo, Korea) and the supernatant was collected and sterilized and purified using a non-pyrogenic sterile syringe filter with 0.2 μm pore size (Corning, NY, USA).![]()
Fenbendazole (FEN)/rapamycin (RAPA)-loaded micelle preparation using freeze-dryer.
6
Quantification of Fecal Short-Chain Fatty Acids
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SCFA levels (i.e., acetate, propionate, and butyrate) in the feces were measured using gas chromatography-mass spectrometry (GC-MS), as previously described by Guard et al. [16 (link)] with some modifications. Briefly, fecal samples were weighed, lyophilized (cryodos-50; Telstar, Barcelona, Spain), and diluted 1:5 in extraction solution, ethyl acetate. After homogenization for 30 minutes at room temperature, the fecal suspensions were centrifuged (5810 R; Eppendorf, Hamburg, Germany) for 20 minutes at 2,100×g and 4°C. Supernatants were collected using sterile syringe filters (Corning Inc., Corning, NY, USA). A gas chromatograph (Clarus 600; Perkin-Elmer) coupled to a mass spectrometer (Clarus 600 C; Perkin-Elmer) was used for chromatographic separation and detection of SCFAs in the samples. The GC temperature program was as follows: 40°C for 0.1 minute, increased to 70°C at 5°C/min, 70°C for 3.5 minute, increased to 160°C at 20°C/min, and finally increased to 280°C for 3 minute at 35°C/min. The total run time was 20.53 minute, with a solvent delay of 5 minute.
7
Residual Inhibitory Activity Assay
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Residual inhibitory activity in fish and film in treatments C and D was determined by centrifuging homogenate (7,500 × g for 15 min) at 4°C and filtering the supernatant using sterile syringe filters (0.45 μm, Corning, Germany). The agar diffusion method and a quantitative critical dilution micro-method (Tahiri et al., 2004 (link)) were used with L. innocua HPB13.
8
Delivery of dsRNA to Asian Citrus Psyllid
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We used the bioassay called ‘in planta system’ (iPS)17 to deliver dsRNA to ACP. Briefly, citrus flushes were collected from potted plants, washed in 0.2% bleach for 10 min and rinsed for 3 min by submersion in autoclaved or filtered water. The base of stems were cut with a razor blade, ~45° angle, while submerged in water. The flush was then transferred to a 1.5 mL tube containing 0.5 mL water. A dsRNA solution (i.e. 100 ng of dsRNA in 300 μL of water) was added to the tube, the tube top was wrapped with Parafilm™ (American National Can™, Neenah, WI 54956) and placed under artificial lighting to stimulate absorption of the dsRNA solution. After the plant flush absorbed the entire dsRNA solution, the tube was filled with filtered water (0.45 μm, Sterile Syringe Filters, Corning®). The treated flushes in the tubes were then transferred to a cage and 15 adult ACP, of mixed genders, were added to each cage (Fig. 5 ). Each treatment (ACP-dsRNA, dsGFP and water control) consisted of four cages, and experiments were repeated three times. Mortality was scored daily for 15 days. Data for total mortality was analyzed with analysis of variance (ANOVA) and t-Test, with P < 0.05 of probability.
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