All animal experimental procedures carried out here were approved by the Animal Use and Care Committees at the University of Wyoming (Laramie, WY, USA). Adult Akt2 knockout (Akt2−/−) mice were obtained from Prof. Morris Birnbaum at the University of Pennsylvania (Philadelphia, PA, USA) and their wild-type (WT) littermates were used as wild-type controls (11 (link)). All mice were housed in a temperature-controlled room (22.8 ± 2.0°C, 45–50% humidity) under a 12 hr/12 hr light/dark and allowed access to food and water ad libitum. WT and Akt2 knockout mice were divided into two groups: one group with trehalose treatment, mice were first administreated with trehalose (1 mg/g/day) intraperitoneal injection for two days and then given 2% (w/v) trehalose (Sigma-Aldrich Ltd, St. Louis, MO, USA) in drinking water for two months, while the other group were offered regular drinking water (46 (link)).
Trehalose
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, Israel, Macao, France, Sao Tome and Principe, Spain
Trehalose is a disaccharide sugar that is commonly used as a stabilizing agent in various lab equipment and processes. It is a naturally occurring sugar found in various organisms, including yeast, fungi, and plants. Trehalose is known for its ability to protect proteins, enzymes, and other biomolecules from denaturation and damage during storage, transportation, and processing.
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Lab products found in correlation
Sucrose, by Merck Group (55 mentions)
Glucose, by Merck Group (26 mentions)
Mannitol, by Merck Group (17 mentions)
Maltose, by Merck Group (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Sorbitol, by Merck Group (13 mentions)
Nahco3, by Merck Group (12 mentions)
Sodium chloride (nacl), by Merck Group (11 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (11 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (10 mentions)
Hepes, by Merck Group (10 mentions)
Mgcl2, by Merck Group (10 mentions)
Potassium chloride (kcl), by Merck Group (10 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Glycerol, by Merck Group (9 mentions)
Cacl2 2h2o, by Merck Group (9 mentions)
Rapamycin, by Merck Group (8 mentions)
Lactose, by Merck Group (8 mentions)
Galactose, by Merck Group (7 mentions)
Fructose, by Merck Group (7 mentions)
285 protocols using trehalose
1
Trehalose Effect on Akt2 Knockout Mice
2
Ovalbumin Dry Powder Vaccine Preparation
3
Quantification of Trehalose in Fungal Conidia
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The amount of trehalose (α-d-glucopyranosyl α-d-glucopyranoside) in conidia was measured as previously described [28 (link),29 (link)]. Briefly, 108 conidia were suspended in 200 μl of ddH2O and incubated at 95 °C for 20 min. The supernatant was separated by centrifugation. Fifty microliters of supernatant were transferred to a new tube, mixed with an equal volume of 0.2 M sodium citrate (pH 5.5), and incubated with or without (as a negative control) trehalose (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 8 h to generate glucose. After incubation, the amount of glucose generated from the trehalose was assayed with a Glucose Assay Kit (Sigma-Aldrich, St. Louis, MO, USA).
4
Thermal Inactivation of Glucoamylases
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The thermal inactivation of GAM-1 and GAM-2 was studied by incubating the enzyme solution (basic buffer) at the indicated temperatures (60ºC, 70ºC, and 80ºC), and aliquots were removed at specific time points and refrigerated immediately. The residual activity was determined, and the activity of a sample without incubation served as 100%.
The effects of sorbitol and trehalose (Sigma, USA) on glucoamylase were studied by incubating the enzyme solution (basic buffer) in the presence of 1 M sorbitol or 1 M trehalose. As described previously, the thermal inactivation of the samples were determined.
The inactivation rate of the enzyme was calculated by a firstorder expression: ln (residual activity %) = -kt. The k values (inactivation rate constant or first-order rate constant) were calculated from a plot of ln (residual activity %) versus t at a particular temperature. The half-lives (t 1/2 , min) were calculated with the equation t 1/2 =ln2/k [5] . The thermal inactivation energy (∆G) was calculated: ∆G = RTln(k B T/h) -RTlnk [27] . All experiments were conducted in triplicate, and the results are shown as mean values.
The effects of sorbitol and trehalose (Sigma, USA) on glucoamylase were studied by incubating the enzyme solution (basic buffer) in the presence of 1 M sorbitol or 1 M trehalose. As described previously, the thermal inactivation of the samples were determined.
The inactivation rate of the enzyme was calculated by a firstorder expression: ln (residual activity %) = -kt. The k values (inactivation rate constant or first-order rate constant) were calculated from a plot of ln (residual activity %) versus t at a particular temperature. The half-lives (t 1/2 , min) were calculated with the equation t 1/2 =ln2/k [5] . The thermal inactivation energy (∆G) was calculated: ∆G = RTln(k B T/h) -RTlnk [27] . All experiments were conducted in triplicate, and the results are shown as mean values.
5
Trehalose-Stabilized Lyophilization Protocol
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Unless otherwise stated, NP solutions were diluted 3:1 with 20% w/v trehalose (Sigma Aldrich, UK), to give a final concentration of 5% w/v trehalose. Solutions were transferred into a VirTis Wizard 2.0 (SP Scientific, USA) and subjected to freezing for 1 h at -40 o C, before primary drying at -35 o C for 3 h (120 mTorr), -30 o C for 4 h (190 mTorr) and -25 o C for 4 h (190 mTorr). Finally, samples underwent secondary drying at 20 o C for 18 h (190 mTorr for 8 h and 50 mTorr for 10 h). Lyophilised samples were stored at RT until required for experimentation.
6
Electron Microscopy Analysis of Extracellular Vesicles
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EVs were evaluated for morphology and contamination by the Electron Microscopy Center at Indiana University Bloomington. To prepare negative stain grid, 4 μL of sample solution was applied onto a glow-discharged 300-mesh copper grid coated with continuous carbon film (EMS, Hatfield, PA). The sample solution was left for 30 s before blotted with a piece of filter paper. The grid was washed using a 4-μL drop of milli-Q (MilliporeSigma) water and stained with 4 μL of negative stain solution composed of either 1% (w/v) uranyl acetate (EMS) with 0.5% (w/v) trehalose (MilliporeSigma) or 1% (w/v) ammonium molybdate (MilliporeSigma) with 0.5% (w/v) trehalose. Excess stain solution was removed by filter paper and the grid was allowed to air dry. Grids were imaged on a 120-kV JEM-1400Plus (JEOL USA, Peabody, MA) transmission electron microscope equipped with 4 k x 4 k OneView camera (Gatan, Pleasanton, CA).
7
Docetaxel Polymer Conjugate Synthesis
8
Solubilization and Storage of Pharmacological Agents
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Bafilomycin A1 (BafA1), PP242, and Torin1 were obtained from Tocris (R&D Systems, Minneapolis, MN). Chloroquine diphosphate and Actinomycin D (ActD) were obtained from Sigma (St. Louis, MO). Suberanilohydroxamic acid (SAHA or vorinostat) and rapamycin were obtained from LC Laboratories (Woburn, MA). trehalose (dihydrate) was obtained from Sigma or Brooklyn Premium (Brooklyn, NY). BafA1, PP242, Torin1, rapamycin, and SAHA were dissolved in DMSO and stocks were frozen at −20 °C. Chloroquine and trehalose were dissolved in ultrapure Milli-Q water (EMD Millipore) and frozen at −20 °C or filtered (0.22-μm) and stored at 4 °C, respectively. trehalose stocks were made up fresh as needed.
9
Insect Hemolymph Sugar Profiling by RP-HPLC
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The nature of the free sugars in the haemolymph of insects injected with SKs was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Hemolymph (4 µL) from larvae was collected to 600 µL of 70% ethanol and kept at 4 °C. The samples were centrifuged (5 min, 10,000 × g), and the supernatants were used for analysis of hemolymph components by RP-HPLC. Separations were performed using a Dionex Ultimate 3000 chromatographic system, comprising a dual-pump programmable solvent module and a Corona Charged Aerosol Detector (CAD). The supernatants were analyzed on an Asahipak NH2P-50 4E column (250 × 4.6 mm, Shodex, Japan). The samples were eluted with multi-step gradient of ACN concentration and flow rate as following: 0–5 min (86%, 1 mL/min), 5–10 min (83%, 1 mL/min), 10–20 min (81%, 1 mL/min), 20–38 min (81%, 1.4 mL/min) and 38–40 min (86%, 1 mL/min) at 38 °C. Trehalose, glucose, sorbitol, mannitol and inositol (Merck, Germany) were used as standards.
10
Yeast-Based Perfluorocarbon Oxygenation Protocol
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The HSA solution was obtained from Biotest Pharma GmbH (Dreieich, Germany). The yeast (saccharomyces cerevisiae) and perfluorodecalin were obtained from Sigma Aldrich (Steinheim, Germany). Potassium cyanide and trehalose were purchased from Merck KGaA (Darmstadt, Germany). Glucose, disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Aquastar® CombiMethanol and Aqualine™ Complete 5 were purchased from Fisher Scientific (Loughborough, UK).
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