Whatman

Well-developed cultures of the SGI16 isolate (10 days old) were fragmented into small pieces and completely covered with ethyl acetate. Maceration was carried out twice in order to recover the maximum of produced bioactive molecules. The crude extracts were filtered through Whatman N° 01 paper (11 µm) and then concentrated to dryness using a rotary evaporator (Heidolph, Germany) [38 ].

Filters for targeted-NanoSIMS (i.e., CARDFISH + NanoSIMS) analyses were conducted on sub-samples of the ¹⁵N₂ incubations described above. Volumes of 500 ml, 50 ml, and 10 ml for the SUSP, SS, and FS fractions, respectively, were filtered onto 0.2 µm polycarbonate filters (Nuclepore, Whatman, Maidstone, UK), fixed with 16% microscopy grade paraformaldehyde (1.6% final concentration) and stored at −80 °C. These filters were used to identify Gammaproteobacteria cells using a CARD-FISH assay (see below). CARD-FISH positively stained cells were then mapped for single-cell isotope ratio measurements using nanoSIMS (see Section “Single-cell N2 fixation rates”). Gammaproteobacteria were chosen as targets due to its prevalence in these waters^{38 ,39 (link)}.
Filter scissions were embedded in 0.1% ultrapure agarose (Life Technologies, Carlsbad, CA, USA). This was followed by two-step permeabilization using a 10 mg ml⁻¹ lysozyme and 60 U ml⁻¹ achromopeptidase solution incubated at 37 °C for 1 h and 30 min, respectively. Hybridization was carried out with horseradish peroxidase-labeled oligonucleotide probes (Biomers.net Inc., Ulm/Donau, Germany) targeting Gammaproteobacteria at 46 °C. The probes used to target Gammaproteobacteria were GAM42A, as named in probebase^{40 (link)}. Following the hybridization at 46 °C the filters were washed with washing buffer (i.e., 47.775 ml Milli-Q + 700 µl 5 M NaCl + 1 ml 1 M TRIS HCl + 0.5 ml 0.5 M EDTA + 25 µl 20% SDS) at 48 °C to remove unincorporated probes. The tyramide signal amplification (TSA) step consisted of Alexa 488 fluorophore (Biomers.net, Ulm, Germany) diluted in amplification buffer (final concentration: 1X PBS, 1 mg ml⁻¹ blocking agent, 2 M NaCl, 100 mg ml⁻¹ dextran sulfate) and hydrogen peroxide (0.0015% final concentration). After the TSA step, filters were washed with 1X PBS, 0.01 M HCl and rinsed with autoclaved Milli-Q water. Filters were then dried and counter-stained with 4’,6-diamidino-2-phenylindole (DAPI) with ProlongTM Diamond Antifade Mountant (Molecular Probes, Eugene, OR, USA). Filter slices were visualized on a Zeiss Axioplan epifluorescence microscope (Oberkochen, Germany) to check for positive hybridized cells on particles. Filters were then gently washed with Milli-Q water and placed upside down on a silicon wafer (1.2 × 1.2 cm, with a 1 × 1 mm raster, Pelotec SFG12 Finder Grid substrate, Ted Pella, Redding, CA, USA), then frozen at −80 °C for ~5 min. Filters were gently removed from the wafers while still frozen, facilitating the transfer of cells and particles to the wafer. Wafers were then stored at −20 °C until further analyses. Before nanoSIMS analysis, the wafers were allowed to dry before mapping target cells using an epifluorescence microscope with 10, 40, and 60X dry objectives, by targeting DAPI (Ex: 350 nm/Em: 465 nm) and Alexa488 (Ex: 488 nm/Em: 591 nm) on a Zeiss Axioplan epifluorescence microscope (Oberkocken, Germany) at UCSC. Finally, the particles containing positively stained cells by the CARD-FISH assay were counted and their size measured using the Zeiss ZEN microscopy software.

One gram of the sample was added to 10 mL MeOH/H₂O (50:50, v/v; acidified to pH 2 with 0.1 M HCl) and shaken in the orbital shaker–incubator ‘ES-20’ (Biogen Científica, S.L.) (25 °C, 250 rpm, 20 min), with a ceramic homogenizer. After this, centrifugation was performed in a 5810R centrifuge (Eppendorf) (20 °C, 4000 rpm, 10 min) and the supernatant liquid was removed and retained. A second and third extraction were carried out, following the same procedure, and the newly collected fraction was pooled with the previous one. Then, 10 mL of acetone/H₂O (70:30, v/v) was added to the pellet and shaken in an orbital shaker (25 °C, 250 rpm, 20 min), with a ceramic homogenizer, and the steps described above were repeated. A final extraction with acetone/H₂O (70:30, v/v) occurred. All the fractions from each extraction step were pooled and filtered using Whatman grade 1 cellulose paper. Subsequently, the samples were concentrated in a Multivapor™ P-12 system (Buchi) to a volume of 20 mL.

A culture of biocontrol T. harzianum culture was made using PDA media, and after that, 2 mL of T. harzianum suspension at (3 × 10⁸ cfu/g) concentration was prepared. After a 7‐day incubation period under aseptic circumstances at a temperature of 28°C, two bits of mycelium of 8 mm discs were introduced to 150 mL of potato dextrose broth [13 (link)]. For 16 days, the culture was shaken at 250 rpm at 25°C with an orbital shaker. The Whatman filter paper was used for the filtration of the biomass. The filtered material of T. harzianum, which contains various metabolites, enzymes, and reproductive structures to synthesize NPFe NPs, was used. The dried substance was discarded, and the aqueous filtrate was filtered to a final concentration of 1 × 10⁻³ mol L⁻¹, forming the biocontrol NPFe using T. harzianum.

The affinity of metal-free DT14D and metal-tagged [^natGa]Ga/[^natIn]In/[^natLu]Lu-DT14D (Supplementary Materials) for the human NTS₁R was evaluated via competitive binding assays. These assays were performed using the [¹²⁵I]I-Tyr³-NT radioligand in membrane homogenates obtained from WiDr cells, which were stored at −80 °C in 100 µL aliquots of Tris/EDTA buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4) [56 (link)]. On the day of the experiment, the membrane homogenates were thawed, combined, and diluted in ice-cold binding buffer (BB: 50 mM HEPES, 5.5 mM MgCl₂, 0.1 mg/mL bacitracin, 1% w/v BSA, pH 7.4). Each of the above ligands (including NT as a control) was incubated in triplicate at increasing concentrations (ranging from 10⁻¹³ M to 10⁻⁶ M in BB, 30 µL) with [¹²⁵I]I-Tyr³-NT (214 pM in 70 µL BB) and membrane homogenate (300 µL in BB) for 1 h at 22 °C in an Incubator-Orbital Shaker unit (MPM Instr. SrI, Bernareggio, Milan, Italy). Following incubation, the samples were filtered rapidly through glass fiber filters (Whatman GF/B, Brandel Inc., Gaithersburg, MD, USA), presoaked in BB for at least 1 h, using a Brandel Cell Harvester (Adi Hassel Ingenieur Büro, Munich, Germany). The filters were washed with ice-cold washing buffer (WB, 10 mM HEPES pH 7.4, 150 mM NaCl), collected individually, placed in separate RIA tubes, and measured for their radioactivity content in a gamma counter (automated multi-sample well-type instrument with a NaI(Tl) 3″ crystal, Canberra Packard Cobra Quantum U5003/1, Auto-Gamma^® counting system; Canberra Packard Central Europe GmbH, Schwadorf, Austria). The 50% inhibitory concentration (IC₅₀) was determined with nonlinear regression analysis using a one-site model in PRISM GraphPad Software (10.2 version, San Diego, CA, USA). The results are presented as the mean IC₅₀ values ± standard deviation (sd) from three independent experiments, each performed in triplicate.

The synthesis of copper nanoparticles (CuNPs) using fennel extract involved several critical parameters, including the concentration of copper sulfate, the pH of the solution, the temperature, and the duration of the reaction. These parameters were optimized to achieve the desired particle size, stability, and yield of CuNPs. Copper sulfate pentahydrate (CuSO4·5H2O) and other chemicals were purchased from Sigma-Aldrich (analytical grade) and used without further purification. Fennel seeds were collected, air-dried to remove moisture, and ground into a fine powder using a mechanical grinder. The bioactive compounds were extracted from the powdered fennel seeds by maceration in ethanol (70% v/v) for 48 hours at room temperature, with intermittent shaking to ensure maximum yield. The extract was then filtered using Whatman No.1 filter paper and the solvent was evaporated under reduced pressure using a rotary evaporator at 40°C to obtain a concentrated extract. For the synthesis of copper nanoparticles, the concentrated fennel extract was mixed with an aqueous solution of copper sulfate under controlled conditions. The reaction mixture was maintained at a temperature of 60°C for 3 hours and stirred continuously at 400 rpm. To facilitate the reduction process, the pH of the solution was adjusted to 10 using sodium hydroxide (NaOH). The change in color of the solution from blue to dark brown indicated the formation of copper nanoparticles. The reaction was carried out for 3 hours to complete reduction of copper ions.
Then, the copper nanoparticles were isolated from the solution by centrifugation at 10,000 rpm for 15 minutes. These precipitates were then washed three times with deionized water and ethanol to remove contaminants. To stabilise the nanoparticles, the pellet was suspended in deionized water and sonicated for 20 min. Copper nanoparticles were gathered and oven dried at 50°C for 12h to obtain fine nanoparticle powder ready for further characterization and testing [10 (link)].