Nalgene rapid flow filter

After the incubation period, cultures were filtrated with Nalgene™ Rapid-Flow™ Filters of 0.45 μm pore size (Thermofischer Scientific, Waltham, MA, USA) to remove microorganisms. Filtrates were then extracted with 70 mL of ethyl acetate and shaken on a Universal Shaker SM 30 B Control Edmund Bühler^® (Thermofischer Scientific, Waltham, MA, USA) set at 150 rpm overnight. The organic phase was recovered and evaporated until dry under a rotavapor set at 60 °C. Samples were resuspended with 2 mL of acetonitrile/water (30/70, v/v) mixture and filtered through 0.45 µm PTFE syringe filters (Sigma Aldrich, St. Quentin Fallavier, France). Samples were conserved at 4 °C until further analysis. T-2 toxin was analyzed by Gemini C18 columns, 150 mm × 4.6 mm, 3 μm and a pre-column with the same characteristics (Phenomenex). As for PLA, T-2 toxin was detected and quantified using HPLC-DAD (Dionex, Sunnyvale, CA, USA) according to the methodology described by Medina et al. [59 (link)]. T-2 toxin quantification was calculated according to a standard calibration curve with concentrations ranging between 0.2 and 50 μg/mL.

Individual sharks were captured by using a handline or drumline and secured alongside the boat. Handling time was limited to 30 min, at which point the sharks were released safely back to the water. Microbiome samples were taken from three distinct anatomical locations (skin, gills, and cloaca) by gently rubbing a sterile swab (COPAN Diagnostics, United States) against the shark’s organs. In addition, an environmental sample (1.5-L surrounding seawater) was collected near each shark captured. The swabs and the seawater samples were kept sterile in a cool box until arrived at the laboratory. At the laboratory, environmental samples were filtered using a single use “Nalgene RapidFlow Filters” 0.2 μm (Thermo Scientific, cat no. 566–0020, Israel). All swabs and filtered environmental samples were stored at −20°C for further work. In total, 27 sharks (15 female dusky and 12 male sandbar sharks) were sampled, including 77 shark microbiome samples and 12 environmental samples (Supplementary Table S1). Mean ambient seawater temperature was measured in 10 m intervals by four acoustic receivers (Thelma Biotel, Norway) positioned around the power and desalination plant’s warm water plume.

Thiopental sodium was obtained from IE Ulagay-Turkey. We purchased DEN (Cas Number # 55-18-5) and phosphate buffer sodium (PBS) from Sigma-Aldrich (Oakville, ON, Canada; St. Louis, MO, USA, Cas Number # 7558-80-7). A Thermo Nalgene™ Rapid-Flow™ filter was obtained from Thermo Fisher Waltham (Canada; Waltham, MA, USA, Cas Number # 64-19-7). A Sephadex LH20 column was purchased from Merck (Darmstadt, Germany, Cas Number # 9041-37-6). Hematoxylin and eosin (H&E) were obtained from Merck (Darmstadt, Germany, Cas Number # 17372-87-1). Malondialdehyde (MDA) (Cat. No. E-BC-K025-M), Klotho (Cat. No. E-EL-M3051), PPAR-gamma (PPARγ) (Cat. No. E-EL-M0893), Bax (Cat. No. E-EL-M0178), Bcl-2 (Cat. No. E-EL-M0175), and caspase-3 (Cat. No. E-EL-M0238) were obtained from (Elabscience, Houston, TX, USA).

3BNC117, 10-1074, and SF12 heavy and light chains were previously cloned into human antibody expression plasmids [22 (link),23 (link),24 (link)]. Antibodies were produced by transfection of 293-6E cells (National Research Council Canada) using branched polyethylenimine (PEI) 25kDa (Sigma-Aldrich, Munich, Germany). Cells were maintained at 37 °C and 6% CO₂ FreeStyle 293 Expression Medium (Thermo Fisher, Darmstadt, Germany) and 0.2% Penicillin/Streptomycin 7 days post transfection, the cell culture supernatant was harvested, filtered with a 0.45 μM Nalgene Rapid Flow filter (Thermo Fisher, Darmstadt, Germany), and incubated overnight at 4 °C with Protein G Sepharose 4 Fast Flow (GE Healthcare, Solingen, Germany) overnight. Antibody bound Sepharose beads were washed on chromatography columns (BioRad, Duesseldorf, Germany) and antibodies were eluted using 0.1M Glycine pH = 3 and immediately buffered in 1M Tris pH = 8. Thereafter, buffer exchange to PBS was performed using 50 kDa Amicon Ultra-15 spin columns (Millipore, Darmstadt, Germany) and the antibodies were sterile filtered using 0.22 μm Ultrafree-CL columns (Millipore, Darmstadt, Germany) and stored at 4 °C.

SHEDs were a generous gift from BioEden (BioEden, TX, USA). Cell culture medium was an EV-depleted general medium, composed of Mesenchymal Stem Cell Basal Media (Lonza, UK) supplemented with 15% EV-depleted Fetal Bovine Serum (FBS) (Invitrogen, UK), 1% L-ascorbate 2-phosphate (Sigma-Aldrich, Poole, UK), 1% L-glutamine (Invitrogen, UK), and 1% Antibiotic-Antimycotic (Invitrogen, UK). FBS was inactivated at 59.5°C for 30 min, followed by ultracentrifugation at 100,000 g for 18 h at 4°C (Sorvall Discovery 100SE, UK) and filtration of the resulting supernatant through a 0.2 μm Nalgene™ Rapid-Flow™ filter (Thermo Fisher Scientific, UK) to obtain EV-depleted FBS. Upon thawing, SHEDs were seeded into T175 culture flasks (Sarstedt, Germany) at 10,000 cells/cm² and incubated at 37°C, in 5% CO₂ and 95% humidity, with medium changes performed every 2-3 days.