Pure ethanol

For this study, a specific soda beverage was selected; the brand remained confidential to avoid potential bias or misinterpretation. Beverage samples were collected from retailers in four major US cities: Atlanta (A), Chicago (C), Los Angeles (L), and Washington, DC (W). The samples contained three types of packaging materials: aluminum (A), glass (G), and plastic (P). At least three samples from each city and packaging material combination were examined and labeled numerically as 1, 2, and 3. The labeling convention used was a combination of letters and numbers, following the “city-material-sample number” format, such as WP for a Washington DC-plastic combination or CA2 for the second sample from Chicago in an aluminum container.
Prior to filtering, the soda samples were left unsealed for 60 min to allow the release of carbon dioxide. The filtration setup consisted of a detachable syringe filter (Ks-Tek, 25 mm) in combination with a polytetrafluoroethylene (PTFE) membrane filter paper (Deschem, 5.0 μm pore size, 25 mm diameter), and a 50-mL glass syringe (Tomopal) equipped with a metal Luer lock. This assembly creates an effective, straightforward, and efficient vacuum filtration system.
Each soda container underwent three water rinses after filtration using 20 mL of MP-free water per rinse, and the wash was filtered to capture any remaining MPs. This procedure is also essential for removing water-soluble contaminants from filter paper. Following this, the filter paper was subjected to three ethanol rinses using 10 mL of pure ethanol (99.5 %, Sigma Aldrich) each time, to cleanse the solids and eliminate water traces.
The filter paper with MPs was then transferred to a 50 mL glass bottle containing 20 mL of pure ethanol (99.5 %, Sigma Aldrich) and sealed with an aluminum cap. Each sample was agitated at 3000 rpm for 5 min using a vortex mixer (Four E's Scientific Laboratory) to ensure even distribution of the MPs in ethanol. The filter paper was removed from the bottle and the samples were left to evaporate until the volume was reduced to approximately 1 mL.
This concentrated solution was carefully added dropwise onto a MirrIR low-E glass microscope slide (Kevley Technologies), targeting a 12-mm diameter area on the slide designated for testing. To hasten the evaporation of the remaining ethanol, the slides were heated to a temperature of approximately 100 °C. Once dry, the IR-glass slides bearing MPs were prepared for in-depth analysis using an LDIR analyzer.

Lipofectamine 3000 (Cat# L3000001), RPMI medium 1640 (Cat# 11875119), DMEM medium (Cat# 11965092), 0.05% trypsin-EDTA (Cat# 25300054), phosphate-buffered saline (PBS; Cat# 10010023), fetal bovine serum (Cat# 26140079), opti-MEM (Cat# 31985070), RIPA buffer (Cat# 89900), and bicinchoninic acid (BCA) Protein Assay Kit (Cat# 23227) were purchased from Thermo Fisher Scientific. Saturated phenol (pH 4.5; Cat# 97064-716) was purchased from VWR. Pure ethanol (Cat# E7023) and protease inhibitor cocktail (Cat# P8340) were purchased from Sigma-Aldrich. CellTiter-Glo 2.0 Cell Viability Assay Kit (Cat# G9241) was purchased from Promega. miRCURY LNA hsa-miR-7-5p mimic (Cat# 339174 YM00472714-AGA) and negative control (Cat# 339174 YM00479902-AGA) were purchased from Qiagen. Direct-zol RNA miniPrep Kit (Cat# R2061) was purchased from Zymo Research. All primers (Supplemental Table S3) were synthesized and purchased from Integrated DNA Technologies. iTaq Universal SYBR Green Supermix (Cat# 1725121), TGX Stain-Free FastCast Acrylamide Kit (7.5%; Cat# 1610181, and 10%; Cat# 1610183), Clarity Western Enhanced Chemiluminescence Substrates (Cat# 1705061), blotting-grade blocker (Cat# 1706404), and polyvinylidene difluoride (PVDF) membranes (Cat# 1620177) were purchased from Bio-Rad. All other chemicals and organic solvents of analytical grade were purchased from Thermo Fisher Scientific, VWR, or Sigma-Aldrich. All laboratory supplies and reagents used in our protocols are DNase and RNase free.

All procedures employed had prior approval and were in strict conformity with the guidelines of the National Center for Laboratory Animal Sciences.
Citral was delivered to the rats using pure ethanol (Sigma-Aldrich, Bangalore, India) as the vehicle. A total of 50 male Sprague-Dawley rats, 4 weeks of age were matched for body weight and randomly allocated to the experimental group (n=30, 78–82 g), which received citral and the modified diet, the drug control group (n=10, 75–84 g), which received the vehicle and modified diet, and the diet control group (n=10, 72–84 g), which received none. The experimental group was further divided into 3 subgroups A, B, and C, which received 10, 15, and 20 mg/kg body weight citral, respectively. The rats were housed in small plastic cages (approx. 40 cm × 15 cm) under conditions of controlled lighting (12:12 h light:dark cycle) and temperature, 24°C-25°C and humidity 45%–65%.
In the beginning, the experimental and drug control groups were subjected to dietary manipulation. To develop obesity they were given free access to an energy-intense, palatable, “fattening” diet, and water ad libitum similar to the one designed by Rolls et al.[15 (link)] The diet consisted of three commonly consumed energy-intense foods (flavored potato chips (Pepsico Inc.), chocolate chip cookies (Parle Ltd.), and butter flavored cookies (Britannia Ltd.). The diet and its approximate composition and energy content are given in Table 1.
The food intake was measured by weighing the leftovers on a daily basis for each cage. To ensure absence of vitamin deficiency, a vitamin supplement from Glaxo Smith Kline was added to the bottle of water.
The diet control group was fed the standard laboratory diet made in-house from milk, grams, nuts, and pulses (homogenized and made into small pellets), which were placed on the cage. The approximate calorific value of this composition was established at 8.0 kJ/g. After a period of 6 weeks, the rats on modified diet were found to be significantly heavier compared with the diet control group.
From the seventh week onward, the experimental group was divided into 3 subgroups (A, B, and C) who were administered citral, once daily in the morning, as a solution of citral (wt/mL = 0.8928) and absolute alcohol (Sigma–Aldrich) in the ratio of 1:10 by oral gavage. The doses of the 3 groups were 10, 15, and 20 mg/kg body weight for groups A, B, and C, respectively. After 4 weeks of citral administration, rats were subjected to metabolic experiments.