Eulalia onto a battery of normal and cancer human cell lines and discovered that the cocktail of proteins is more toxic towards an ovarian cancer cell line (A2780). The secretions’ main proteins were identified by proteomics and transcriptomics: 14-3-3 protein, Hsp70, Rab3, Arylsulfatase B and serine protease, the latter two being known toxins. This mixture of toxins induces cell-cycle arrest at G2/M phase after 3h exposure in A2780 cells and extrinsic programmed cell death. These findings indicate that partial re-activation of the G2/M checkpoint, which is inactivated in many cancer cells, can be partly reversed by the toxic mixture. Protein–protein interaction networks partake in two cytotoxic effects: cell-cycle arrest with a link to RAB3C and RAF1; and lytic activity of arylsulfatases. The discovery of both mechanisms indicates that venomous mixtures may affect proliferating cells in a specific manner, highlighting the cocktails’ potential in the fine-tuning of anti-cancer therapeutics targeting cell cycle and protein homeostasis.As Yondelis joins the ranks of approved anti-cancer drugs, the benefit from exploring the oceans’ biodiversity becomes clear. From marine toxins, relevant bioproducts can be obtained due to their potential to interfere with specific pathways. We explored the cytotoxicity of toxin-bearing secretions of the polychaete  According to the World Health Organization (WHO), cancer is the second leading cause of death globally. The numbers of cases are expected to rise every year despite research on the subject being given top priority . From aAnti-cancer drugs developed from natural products such as toxins and alkaloids nowadays represent 50% of all drugs that are used in chemotherapy . After pEcteinascidia turbinata that competes with enzyme and transcription factors that bind to DNA [Arenicola marina), nicomicin (from Nicomache minor), both peptidic in nature, and proteases from Nereis, all presenting cytotoxicity towards cancer cell lines, seemingly with pro-apoptotic effects , which yielded an IC50 value one order of magnitude lower (0.08 µg. µL−1). Consequently, the ovarian carcinoma cancer cell line was then chosen for further biological characterization towards addressing the mechanisms underlying the loss of A2780 cell viability.The purified extract caused higher antiproliferative effect towards MCF7 and especially A2780 cancer cell lines, compared to normal human primary fibroblasts , Table 250 concentrations, for both 24 h and 48 h (50), revealed further cellular alterations, namely mitochondrial shape changes, nuclear pleomorphisms and an increase in lysosome number.The frequency of apoptotic A2780 cells approximately doubled at ICand 48 h . Howeverand 48 h . Interesand 48 h . Apoptotand 48 h . Moreoveand 48 h . Exposur50 of extracts. The low BAX/BCL-2 expression ratio  and the induction caspase 8 were analyzed in A2780 cells exposed to the ICon ratio A,B, the extracts , and theextracts C clearlyextracts A,B.The effect of the extracts in A2780 cell-cycle progression was evaluated over time of exposure . Cell-cyThe interaction between the human homologs of the main proteins present in extracts is depicted in Eulalia contains a mixture of toxin and non-toxin proteins involved in multiple functions for attacking prey and defense against predators and environmental variables . This cNaja ashei induced cell-cycle arrest and promoted apoptosis with the activation of Caspase 3 and 9 in colorectal carcinoma (HCT116) cells [+ channels expressing cancer cells [The cytolytic properties of animal venoms, poisons and toxungens toward cancer cells has been investigated with promising results, albeit scant comparisons between the differential effects against normal and cancer cells. Bee venom has arguably become one of the most studied and promising cases, with melittin (a 26-amino acid lytic peptide responsible for the painful sting) and phospholipase A being, so far, pinpointed as key substances involved in the promotion of apoptosis . Venoms 6) cells . Additioer cells . NonetheEulalia mucus secretions, arylsulfatase B (ARSB) and serine proteases (SP) are commonly found in venoms [From the five proteins identified from n venoms ,27,28. Sn venoms ,30,31. An venoms . In lympn venoms . In molln venoms . In turnn venoms . In venon venoms ,37,38.Eulalia are not commonly associated with venoms, poisons or toxungens. However, they play paramount roles in cell homeostasis. It is the case of heat shock proteins (HSP). Heat shock protein 70 KDa (HSP70) is usually involved in stress response, helping protein folding (assembly and refolding) in the endoplasmic reticulum [2+-dependent secretion and vesicle docking, thus being essential in exocytic and endocytic membrane trafficking [ZWINT, was discovered to be overexpressed in ovarian cancer, among other genes  related with cell cycle, nuclear division and oocyte meiosis [Other proteins in the toxungenous cocktail of eticulum . In turnfficking ,41. The fficking . The gen meiosis . The 14- meiosis . It mustThe identified proteins, when integrated within protein interaction networks , suggestDrosophila and human cell lines [The RAF proto-oncogene identified in the network is responsible for the regulation of many fundamental cell processes, including proliferation, differentiation, apoptosis, motility and metabolism . The actll lines ,49. RAF1ll lines . The CDCll lines . The disll lines ,52, as sll lines ,43. Addill lines , which iHeat shock proteins 70 controls many housekeeping processes , which iOther homologs from known toxins (PRSS1 and ARSB) appeared, at first sight, to have a smaller contribution to the effects seen in A2780. However, ARSB is known to have its expression reduced in most carcinomas when compared with normal tissues . HoweverFinally, it must be noted that the co-occurrence of apoptosis and cell death by autophagy has already been reported in A2780 cells exposed, e.g., to metal-based compounds ,60,61. BEulalia sp.  were collected from the West Coast of Portugal , in an intertidal rocky beach. Animals were reared in the laboratory in a mesocosm environment recreating their natural habitat, with controlled salinity, temperature and photoperiod . Animals were fed with live mussels, one of the species’ favorite prey.Adult w/v) L-dithiothreitol (DTT) and 1% (v/v) protease inhibitor cocktail . Samples were then filtered through a syringe cellulose acetate filter (0.22 µm). Purification and concentration of extracts was done by dialysis (ultrafiltration) using 3kDa Amicon Ultra centrifugal filters , with Dulbecco’s phosphate-buffered saline  as the vehicle. Total protein content was determined using a Nanodrop 1000 spectrophotometer . Purified extracts were stored at −80 °C until further analyses.Protein was collected from mucous secretions harvested non-invasively by placing the worms in microfuge tubes with filtered sterile seawater, followed by mechanical stimulation of the proboscis, leading to increased secretion. Crude protein extracts were obtained from approximately fifty animals per pool. The crude extracts were immediately diluted ≈ 1:1 in 0.05 M Tris-HCl containing 10% (v/v) and 6% (v/v) acrylamide, respectively. The migration buffer consisted of 25 mM Tris, 192 mM glycine, pH 8.5. Molecular standards (NZYColour Protein Marker II range 11-245 kDa) was purchased from Nzytech. The 2-D separation was obtained by isoelectric focusing (IEF) followed by SDS-PAGE. Briefly, the sample consisted of 100 μL of purified protein extract treated with 2-D Clean-Up Kit , according to manufacturer’s instructions. Precipitated proteins were resuspended in 100 μL of rehydration solution containing 4% (w/v) CHAPS, 7 M urea, 2 M thiourea, 1% (w/v) DTT GE Healthcare, 0.5% (v/v) Immobilized pH Gradient buffer  and 0.002% (w/v) bromophenol blue, and incubated at room temperature for 36 h. After protein quantification using the Pierce 660 nm protein assay reagent , 125 μL of rehydration buffer containing 200 μg of protein were applied onto Immobiline Drystrip IPG strips  with 7 cm and 3 ± 10 non-linear pH. After rehydration, Isoelectric focusing (IEF) was done in an Ettan IPGphor instrument  at a maximum voltage of 5000 V. The IPG strips were afterwards incubated for 15 min in equilibration buffer 1  SDS, 6 M urea, 30% (v/v) glycerol, 1% (w/v) DTT, followed by incubation for another 15 min in equilibration buffer 2  SDS, 6 M urea, 30% (v/v) glycerol, 2.5% (w/v) iodoacetamide). Strips were then placed on top of a 10% SDS-PAGE gel for separation by mass. Both gels were stained with Coomassie Brilliant Blue G-250 to identify most representative spots and bands  for initial separation . The Laend bands  to be piv/v) formic acid plus 5% (v/v) DMSO in water and 0.1% (v/v) formic acid plus 5% (v/v) DMSO in acetonitrile, respectively. The ESI DuoSprayTM ionization source was operated in the positive mode set to an ion spray voltage of 5500 V, 25 psi for nebulizer gas 1 (GS1), 25 psi for the curtain gas (CUR). The rolling collision was used with a collision energy spread of 5. Peptide mass fingerprinting was performed using ProteinPilot 5.0.1 (Sciex) considering the following parameters: cysteine alkylation by acrylamide, digestion by trypsin, and gel-based ID as a special factor. The resulting amino acid sequences were contrasted against UniProtKB and NCBInr databases using Blast [e-values and number of matching peptides per protein.After gel sample destaining and digestion with porcine trypsin, peptides were analyzed on a Eksigent NanoLC Ultra 2D separation system coupled to a TripleTOF 6600 mass spectrometer, all from Sciex . The chromatographic separation by micro-LC was achieved in a ChromXP C18CL column , at 50 °C. The flow rate was set to 5 μL/min and mobile phase A and B were 0.1%  NCBInr using BlastP. The RT-qPCR was then done in a Rotor-Gene 6000 thermal cycler (Corbett Research) using the NZY qPCR Green Master Mix (NZYTech), according to manufacturer instructions. The program was set as: denaturation 94 °C, 45 s (s), annealing 55 °C, 25 s and extension 72 °C, 30 s, with 45 cycles per run. Primer melting analysis was also conducted to verify the specificity of hybridization. Relative expression was determined by the 2−∆∆Ct method [The peptide sequences were contrasted against criptome , depositcriptome . Amplificriptome  by genercriptome , having t method .Homo sapiens homologues were retrieved using BLASTP. Gene network analyses were conducted using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) by setting the confidence cut-off for interaction links between proteins at 0.400 [After the identification of the main proteins by combining proteomics and transcriptomics, at 0.400 .v/v) fetal bovine serum and 1% (v/v) antibiotic/antimycotic solution (Invitrogen), while A2780 (ovarian carcinoma) cells were grown in modified McCoy’s medium: Roswell Park Memorial Institute medium (RPMI) 1630 [http://www.atcc.org) with the exception of A2780 that was purchased from Sigma-Aldrich. All cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% (v/v) CO2 and incubated in the same atmosphere when necessary. Only purified extracts stored for less than 30 days (−80 °C) were used in biological assays and all dilutions (and controls) were prepared in PBS. The same volume of PBS was added to the respective medium of each assay (controls). Total protein content was determined prior to biological assays using a Nanodrop spectrophotometer (Thermo Fisher Scientific). The assays were conducted with cells seeded for 24 h and in the range of 105 cells. mL−1, in the conditions described before, in 6, 24 or 96-well plates, depending on the endpoint.HCT116 , A549 (lung adenocarcinoma), human primary fibroblasts, MCF7 (breast carcinoma) and K562 (myelogenic leukemic carcinoma) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% (MI) 1630 ,74. All -3 to 1.6 µg. µL−1) for the estimation of the half-maximal inhibitory concentration (IC50). Cells were incubated for 48 h with each diluted extract. Viability was determined with the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay , using 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt (MTS). Quantification of the formazan dye formed in viable cells was done spectrophotometrically by measuring absorbance at 490 nm with a Bio-Rad microplate reader Model 680  [Cell viability was determined by subjecting all cell lineages to successive dilutions of the purified protein extract  ,74,75. T50), 24 h (IC50) and 48 h . After incubation, cells were washed with PBS and fixed with fresh 4% (m/v) formaldehyde , for 10 min in the dark. Cells were then incubated with the dye (provided by Sigma-Aldrich), prepared as 5 μg. mL−1 in PBS. After 10 min, slides were washed with PBS and mounted. Fluorescent nuclei (100 per sample) were sorted in a DM 2500 LED model microscope, adapted for epifluorescence with an external EL6000 light source . The assay was performed in triplicate for each timeline. The results are expressed as the mean percentage of apoptotic nuclei.Apoptosis was determined based on the Hoechst 33258 labelling assay. Cells were seeded on sterilized coverslips and incubated for 24 h to obtain an adherent monolayer. Cells were then incubated with the purified extract for 12 h  was used as a positive control ,74. By t2 with 6 × 105 cells. mL−1 for 24 h and exposed to the purified extract (IC50 dose), plus PBS (control) and incubated for 48 h. Cells were then scraped, washed in PBS, resuspended in cell lysis buffer and incubated on ice for 30 min. Total protein in lysates was quantified as previous and adjusted to 200 µg/50 µL buffer. Each microplate well was loaded with 200 µg protein/50 µL 50 µL of 2× Reaction Buffer with DTT  and 5 µL of IETD-pNA substrate (4 M). After incubation (2 h), absorption at 400 nm was quantified on a 680 Microplate Reader .The activity of Caspase 8 was determined to assess the extrinsic apoptotic pathway using the Caspase 8 assay kit from Abcam, following manufacturer instructions. Briefly, cells were plated on T-flasks with an area of 25 cm50, 1/5 IC50 and IC50 (0.08 µg. µL−1), plus controls. Hydrogen peroxide (50 µM) was used as a positive control. Cells were harvested after 48 h, washed twice with PBS before resuspension in pre-warmed (37 °C) PBS containing 100 µM 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) and incubation at 37 °C for 20 min, in the dark. Fluorescence intensity was measured on an Eclipse Ti inverted microscope equipped with a DS-QiMc camera and adapted for epifluorescence , with fixed exposure time for all samples. The green fluorescent signal was normalized and analyzed using ImageJ [Intracellular reactive oxygen species (ROS) were determined using the KITNAME (Sigma-Aldrich), as previously described . Brieflyg ImageJ .50, 1/5 IC50 and IC50 diluted in fresh medium and exposed for 48 h. Cells were then stained with the JC-1 staining solution for 20 min at 37 °C in the dark. Image acquisition and analysis was done as above, based the ration between green and red fluorescence.The mitochondrial transmembrane potential was analyzed using the bistrimethinecyanine iodide (JC-1) dye . After seeding and incubation cells were treated with the protein extract at the concentrations of 1/10 IC50, diluted in fresh medium (RPMI), and incubated for 1 h and 3 h. Cells were then trypsinized and washed with PBS and RPMI medium (without phenol red) before incubation with BCECF-AM (2 µM in the same medium) for 20 min, at 37 °C. After washing in the culture medium, data was collected using an Attune acoustic focusing flow cytometer (ThermoFisher Scientific) and analyzed using FCS Express 6.0 Flow Cytometry . The results were normalized for cell counts and the respective control. The assays were performed in duplicate and technical duplicates were included to minimize technical error. Doxorubicin (0.4 mM) was used as a positive control in all assays.The plasmatic transmembrane potential was analyzed using 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester (BCECF-AM) dye (Invitrogen). After seeding and incubation, cells were treated with the purified protein extract at the concentration of IC50 diluted in fresh RPMI medium for 1 and 3 h. After washing with RPMI (without phenol red), cells were incubated with BCECF-AM (µM) for 20 min, washed again and incubated with propidium iodide (2.5 µg. mL−1) for 10 min. Cells were then washed and visualized in RPMI (without phenol red) under an Eclipse Ti fluorescence microscope, equipped with a DS-QiMc camera .After seeding and incubation, cells were treated with the protein extract at the concentration of IC2 with 4 × 105 cells. mL−1. After incubation for 24 h to allow adhesion, cells were treated with fresh medium supplemented with purified extract at the concentration of IC50 or PBS (control) and incubated for 48 h. The cell monolayer was washed three times with PBS and the cells were detached by scraping. The cells were pelletized by centrifugation at 500× g for 5 min (4 °C). The following procedure was adapted from Vinhas et al. [Cells were plated on T-flasks with an area of 75 cms et al. , using as et al.  followin5 cells per well and synchronized in early S-phase using a thymidine double block [g for 5 min at 4 °C, pelleted cells were washed with 1 mL cold PBS and centrifuged at 3000× g for 5 min at 4 °C. The cells were then suspended in 100 µL cold PBS and completely disaggregated by soft pipetting before gently adding 1 mL ice-cold ethanol (80% (v/v)). After at least 16 h at 4 °C, the cells were pelletized by centrifuging at 5000× g for 10 min at 4 °C and the supernatant discarded. The cells were treated for 30 min with RNase A 50 µg. mL−1 at 37 °C and propidium iodide was added to a final concentration of 25 µg. mL−1. Data was collected using an Attune acoustic focusing cytometer (ThermoFisher Scientific) and analyzed using FCS Express 6 Flow Cytometry (De Novo Software).Cells were seeded in a 6-well plate at 1 × 10le block . After t5 cells. mL−1 for 3 h, 6 h and 12 h and at 0.375 × 105 cells. mL−1 for 48 h. Cells were then fixed in 4% (v/v) formaldehyde, permeabilized with 0.1% (v/v) Triton X-100 and blocked with 1% (w/v) bovine serum albumin (BSA). The cells were probed with Alexa Fluor 488 Phalloidin (2.4% (v/v) in 1% (w/v) BSA, 20 min) to stain actin filaments, followed by Hoechst 33258  at 5 μg. mL−1 in PBS (15 min) as the nuclear counterstain. Alpha tubulin was tagged with the DM1A anti-α-tubulin monoclonal antibody (Sigma-Aldrich), diluted to 1:200 in 1% (w/v) BSA. After incubation o/n at 4 °C, the primary antibody was tagged with anti-mouse TRITC-conjugated polyclonal secondary antibody (Sigma), diluted to 1:64 in 1% (w/v) BSA (1 h). Cells were then washed with Tris-buffered saline (pH 7.6) with Tween 20 (TBST) and mounted with PBS. The cells were then examined with an Eclipse Ti fluorescence microscope (Nikon Instruments), as described previously.Cells were seeded for 24 h in 24-well plates at 1 × 106 cells. mL−1, were exposed to different concentrations of the purified extract , plus controls (PBS), for 48 h and additionally to IC50 for 1 h and 3 h. After exposure, cells were scraped and processed for transmission electron microscopy (TEM) as described in Vogt et al. [v/v) glutaraldehyde in 0.1 M phosphate buffer and post-fixed in 2% (w/v) osmium tetroxide in 0.1M phosphate buffer, overnight. Pellets were washed in ultrapure water and dehydrated in a progressive series of acetone. Pelletized cells were embedded in Epon resin (Sigma-Aldrich) using propylene oxide as intermediate. Thin sections (50–60 nm) were obtained with an Ultracut E ultramicrotome Leica. Sections were collected onto copper and nickel mesh grids and contrasted with 2% (w/v) aqueous Uranyl Acetate and Reynold’s Lead Citrate [Cells, seeded in 24-well plates with a density of 10t et al. , with mo Citrate . Grids w50) was estimated using least squares regression and the results are expressed as IC50 ± standard error of estimates (SEE). Normality and homoscedasticity were evaluated using Shapiro-Wilk’s and Levene’s tests, respectively. When assumptions were met for parametric analyses, we employed ANOVA based on F and Dunn’s tests for multiple comparisons, otherwise the non-parametric Kruskal–Wallis H statistic was used, followed by Student’s T-test and Mann-Whitney’s U  for paired-sample tests. Statistics were computed using R 3.5 [The half maximum inhibitory concentration (ICng R 3.5 . The sigIn large part due to the number and variety of compounds therein, toxin-bearing secretions of marine invertebrates may have cytotoxic properties beyond the functions acquired by means of natural selection. The present study showed that proteins present in the mucus secreted by a toxungenous marine worm, five of which were positively identified, can specifically cause cell-cycle arrest by a mechanism that is specific to A2780 ovarian adenocarcinoma cells among a panel of six human cell lines, normal included. Even though the precise mechanisms still need further exploration, the effects on A2780 cells seem to derive from the collision of two interaction networks: one centered in cell-cycle arrest as a consequence of dysregulation of protein expression and folding, with a link to RAB3C oncoprotein function; another centered in the lytic activity of arylsulfatases, common enzymes in animal venoms and poisons. The mechanisms, however, are complex and the exact quantities of each individual component in the extract may vary. Nevertheless, the variation in this case seems to be neglectable, as the usage of biological replicates gave similar results. Thus, the consistency of the findings supports the potential of mixed proteins in venoms against aggressively-proliferating cells, as seen, e.g., for bee venoms, steering toward a novel approach to anti-cancer treatments while highlighting the immense potential of the oceans as a source of novel therapeutics.


A new supramolecular electrochemical sensor for highly sensitive detection of dopamine (DA) was fabricated based on supramolecular assemblies of mixed two surfactants, tetra-butylammonium bromide (TBABr) and sodium dodecyl sulphate (SDS), on the electrodeposition of gold nanoparticles on graphene oxide modified on glassy carbon electrode (AuNPs/GO/GCE). Self-assembled mixed surfactants (TBABr/SDS) were added into the solution to increase the sensitivity for the detection of DA. All electrodes were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The supramolecular electrochemical sensor (TBABr/SDS⋅⋅⋅AuNPs/GO/GCE) showed excellent electrocatalytic activity toward the oxidation of DA. Under the optimum conditions, the concentration of DA was obtained in the range from 0.02 µM to 1.00 µM, with a detection limit of 0.01 µM (3s/b). The results displayed that TBABr/SDS⋅⋅⋅AuNPs/GO/GCE exhibited excellent performance, good sensitivity, and reproducibility. In addition, the proposed supramolecular electrochemical sensor was successfully applied to determine DA in human serum samples with satisfactory recoveries (97.26% to 104.21%). Dopamine  is a neurotransmitter that plays an important role in communication in the central nervous system. Low levels of DA may cause neurological disorders including Alzheimer’s and Parkinson’s ,2. Thus,2-hybridization carbon materials, including graphene oxide (GO) and reduced graphene oxide (rGO). They have gained enormous attention for use as a modifier on the surface of electrode owing to their high electronic conductivity and electrocatalytic activity [Many types of materials have been employed to modify electrodes such as metal nanoparticles ,16,17,18activity ,28, as wactivity ,30. GO hactivity ,32,33,34activity . Besidesactivity ,36,37,38activity ,40,41,42activity ,44.Supramolecular assembly or supramolecule can be simply produced by self-assembly processes from amphiphiles containing both hydrophobic and hydrophilic parts. Different types of aggregation can be formed such as nanostructures, micelles, or vesicles depending on the composition and shape of amphiphiles in aqueous medium . SurfactIn the present work, a novel supramolecular electrochemical sensor based on mixed surfactants on gold nanoparticles/graphene oxide modified glassy carbon electrode, TBABr/SDS⋅⋅⋅AuNPs/GO/GCE, was fabricated to detect DA. Morphology and electrochemical properties of the modified electrodes were investigated using scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The electrochemical properties of modified electrodes were performed using the differential pulse voltammetry (DPV) technique. The TBABr/SDS⋅⋅⋅AuNPs/GO/GCE electrode showed a high peak and potential for the determination of DA. Moreover, the supramolecular electrochemical sensor showed a high level of sensitivity and excellent performance in stability and reproducibility. The proposed supramolecular electrochemical sensor was applied to human serum for the determination of DA.The surface morphology of the AuNPs/GO/GCE was characterized by SEM. 2) greater than 0.990, indicating a surface confined process [In addition, the accumulation of AuNPs on surface of GO/GCE was confirmed using CV. The modified electrode, AuNPs/GO/GCE, was run in the buffer solution (pH 7.0) for 10 cycles.  process .The electrochemical behavior of 0.80 µM DA was investigated using DPV with a bare GCE (a), a GO/GCE (b), an AuNPs/GO/GCE (c), and TBABr/SDS⋅⋅⋅AuNPs/GO/GCE (d), as shown in dl) with the parallel combination of the charge transfer resistance (Rct) and the diffusion impedance (W) [ct) at the GCE was estimated to be 254 Ω, which dropped to 45 Ω at the GO/GCE and to 15 Ω at the AuNPs/GO/GCE. Meanwhile, Rct of TBABr/SDS⋅⋅⋅AuNPs/GO/GCE was 10 Ω, providing much lower electron transfer resistance on the surface of TBABr/SDS⋅⋅⋅AuNPs/GO/GCE.The interfacial properties of the modified electrode surface were studied by electrochemical impedance spectroscopy (EIS). The impedance data was obtained from the modified Randles circuit consisting of the interfacial capacitance (Cance (W) . Figure To enhance the performance of the electrochemical sensor, the surfactants are an important parameter that can accelerate the diffusion of DA to the surface of the electrode. Several types of mixed surfactants to form the supramolecular assembly were studied including cationic surfactants such as TBABr, DTAB, TTAB, and CTAB, and anionic surfactants such as SDS. It was found that the oxidation peak current of DA was decreased in the presence of supramolecular assemblies of DTAB/SDS, TTAB/SDS, and CTAB/SDS. Meanwhile, the oxidation peak current of DA increased and gave the highest response with the supramolecular assemblies of TBABr/SDS see .To form the supramolecular assemblies of mixed surfactants between TBABr and SDS, the concentrations of surfactants were studied. The concentrations of two surfactants were studied in the range of 0.04–0.12 mM (fixed molar ratio of TBABr/SDS of 1:1), which is lower than its critical micellar concentration (CMC) (CMC of SDS is 8.3 mM and CMC of TBABr is 588 mM) ,50,51. TΓ) can be calculated by the Laviron equation [n represents the number of electrons involved in the reaction (n = 2), A is the surface area of the GCE electrode (0.07 cm2), Ipa is the peak current of DA (0.8 µM), Γ is the surface coverage concentration (mol/cm2), F is Faraday constant , and ν is the scan rate (50 mV/s). The surface coverage of the electroactive species (Γ) can be estimated to be approximately 2.00 × 10−8 mol/cm2 for the supramolecular system (TBABr/SDS⋅⋅⋅ AuNPs/GO/GCE), which was greater than the Γ value with the absence of the supramolecular system (8.36 × 10−9 mol/cm2). The possible mechanism for the detection of DA using the supramolecular electrochemical sensor (TBABr/SDS⋅⋅⋅ AuNPs/GO/GCE) is described in The comparison of sensitivity of electrochemical sensor (AuNPs/GO/GCE) was tested with and without the supramolecular assemblies of mixed surfactants of the TBABr/SDS system . It was equation  with the2 = 0.9939)  B. The ob studied . The W1/s is the standard deviation of blank and b is the slope of the calibration curve. The LOD and LOQ were found to be 0.01 µM and 0.06 µM, respectively. In addition, the proposed supramolecular electrochemical sensor (TBABr/SDS⋅⋅⋅AuNPs/GO/GCE) is more sensitive and provides a lower LOD than the other published works  at a potential range of 0.0 V to 0.60 V in PBS buffer by DPV. The peak current of DA increased linearly with the increasing DA concentrations in the range of 0.02 µM to 1.00 µM (Y = 156.43 × −3.1470 and Y = 481.86 × −130.12), with a correlation coefficient of 0.9960 and 0.9910, respectively . The deted works .The proposed supramolecular electrochemical sensor was applied to detect the DA in standard human serum samples. The standard DA solution was added into diluted human serum (at the ratio of human serum: 0.1 M PBS pH 7.0 of 1:4) with three different concentrations  before the detection. The results are summarized in To evaluate the selectivity of the supramolecular electrochemical sensor (TBABr/SDS⋅⋅⋅AuNPs/GO/GCE) for the detection of DA, the effect of some commonly existing interferents was studied, including ascorbic acid (AA), uric acid (UA), citric acid, cysteine, lysine, and glucose, which were expected to be found in human serum samples . The intn = 7) in real samples using the supramolecular electrochemical sensor as TBABr/SDS⋅⋅⋅AuNPs/GO/GCE. The precision, which is expressed in terms of relative standard deviation (RSD) for the peak current of DA, was found to be less than 3.0% for intra-day and 8.0% for inter-day, indicating the TBABr/SDS⋅⋅⋅AuNPs/GO/GCE electrode had high reproducibility. When the modified electrode was used for 15 days, the peak current of DA at the same concentration was slightly decreased by about 10%, demonstrating that the supramolecular electrochemical sensor showed good stability.The peak current of 0.80 µM DA was evaluated for seven measurements , ascorbic acid , uric acid , sodium hydroxide , sodium phosphate monobasic dihydrate , sodium phosphate dibasic  and phosphoric acid , potassium hexacyanoferrate (III) (K3Fe(CN)6, Sigma–Aldrich), citric acid , potassium chloride , N,N-dimethylformamide , l-cysteine , lysine (Fluka Chemie AG), glucose (Sigma-Aldrich), N,N-dimethylformamide , sodium dodecyl sulfate , cetyltrimethylammonium bromide , trimethyltetradecyl ammonium bromide , dodecyltrimethy–lammonium bromide , N,N-dimethylformamide , and tetrabutylammonium bromide . Deionized water (18.2 MΩ) was used in all experiments.All chemicals used were of analytical-reagent grade and obtained from different sources: dopamine , gold (III) chloride trihydrate  was prepared daily by dissolving in water. Phosphate buffers with various pH were prepared by mixing the 0.1 M NaHThe surface morphology of the modified electrodes was conducted by scanning electron microscopy . All electrochemical experiments, including cyclic voltammetry (CV), differential pulse voltammetry, and electrochemical impedance spectroscopy (EIS), were performed on a AutoLab PGSTAT302N  at room temperature. A glassy carbon electrode (GCE) modified with GO and deposited AuNPs (AuNPs/GO/GCE) was used as working electrodes, Ag/AgCl (3 M NaCl) as reference electrode , and a platinum wire as counter electrode  to complete a three-electrode system.Graphene oxide was prepared from graphite powder by Hummers’ method with some modifications ,62. Five2SO4 solution containing 1.0 mM HAuCl4. The electrochemical deposition of the AuNPs was conducted for 40 s at −1.0 V  based on supramolecular assemblies of mixed surfactants, tetra-butylammonium bromide (TBABr), and sodium dodecyl sulphate (SDS), on gold nanoparticles/graphene oxide modified glassy carbon electrode (TBABr/SDS⋅⋅⋅AuNPs/GO/GCE). The supramolecular assemblies of mixed cationic and anionic surfactants, TBABr and SDS, can increase the electrochemical performance of the determination of DA based on the hydrophobic interaction and electrostatic attraction. The low LOD of DA was displayed as 0.01 µM (3s/b). The proposed supramolecular electrochemical sensor has good sensitivity and stability. In addition, TBABr/SDS⋅⋅⋅AuNPs/GO/GCE was applied to detect DA in human serum samples with satisfactory recovery (97.26–104.21%).


Water stress was applied by withholding irrigation in the phenological phase of male pre-anthesis. Additionally, two contrasting inbred lines, P7 (superior line) and L75 (low performer) were compared for grain yield (GY) and expanded popcorn volume (EPV), selected from previous studies, were tested under greenhouse conditions. In the field, no genotype x water condition x crop season (G×WC×CS) interaction was observed, whereas GY (−51%), EPV (−55%) and leaf greenness (SPAD index) measured 17 days after anthesis (DAA) (> −10%) were highly affected by water limitation. In general, root traits  presented G×WC×CS interaction, which did not support their use as selection parameters. In relation to leaf senescence, for both WS and WW conditions, the superior inbred lines maintained a stay-green condition (higher SPAD index) until physiological maturity, but maximum SPAD index values were observed later in WW (48.7 by 14 DAA) than in WS (43.9 by 7 DAA). Under both water conditions, negative associations were observed between SPAD index values 15 and 8 days before anthesis DBA), and GY and EPV (r ≥ −0.69), as well as between SPAD index 7, 17, and 22 DAA, and angles of brace root (AB), number of crown roots (NC) and crown root density (CD), in WS (r ≥ −0.69), and AB and CD, in WW (r ≥ −0.70). Lower NC and CD values may allow further root deepening in WS conditions. Under WS P7 maintained higher net photosynthesis values, stomatal conductance, and transpiration, than L75. Additionally, L75 exhibited a lower  carbon isotope composition value than P7 under WS, confirming a lower stomatal aperture in L75. In summary, besides leaf greenness, traits related to leaf photosynthetic status, and stomatal conductance were shown to be good indicators of the agronomic performance of popcorn under water constraint.Climate change is expected to intensify water restriction to crops, impacting on the yield potential of crops such as popcorn. This work aimed to evaluate the performance of 10 field cultivated popcorn inbred lines during two growing seasons, under well-watered (WW) and water stressed (WS) (ψ Since the pre-industrial period, anthropogenic activities mainly associated with increased greenhouse gas emissions, have caused an increase of about 1°C in the average global temperature. Depending on future emissions, this increase may reach up to 1.5°C between 2030 and 2052, which tends to aggravate the frequency of extreme droughts and rainfall events worldwide . This wiPopcorn sales are worth approximately one hundred million dollars annually in the USA, and there is also a high demand in Brazil for this product . The seeThe initial step is the evaluation of germplasm with identification of predictor characteristics for drought adaptation. Traditionally, secondary characteristics associated with higher grain yield are considered, such as a shorter interval between male and female anthesis , late le2 availability to the RuBisCO carboxylation sites, and thus C-assimilation  and chlorophyll concentrations (SPAD index) . The anaMaize plants under water stress have less crown roots, increasing water acquisition through other roots located in deeper soil layers , since a7), whose genealogy is derived from germplasm adapted to tropical  and temperate/tropical conditions   .The experiments were conducted at the Experimental Station of the Antônio Sarlo Agricultural State College in Campos dos Goytacazes, RJ, Brazil , in the crop seasons (CS) of 2016 and 2018, during the dry seasons (April and August).The experimental design was completely randomized blocks with three repetitions for both well-watered (WW) and water-stressed (WS) conditions. The plots comprised two 4.40-m lines, spaced 0.20 m between plants and 0.80 m between lines (44 plants per plot).−1. Soil water was monitored using three Decagon MPS-6 sensors  installed at a depth of 0.20 m in the planting line between two plants. The WW condition received irrigation at soil field capacity (−0.01 MPa), whereas in WS conditions, irrigation was withheld 15 days before male anthesis (June 22nd to June 26th during both years). Under WS, the soil reached permanent wilting point (−1.5 MPa) 77 days after sowing (DAS) (grain filling stage) in the 2016 experiment, and at 68 DAS  and 90 DAS (grain filling stage) in the 2018 experiment   Figure 1−2 s−1 in 2016 and 2018, respectively , 60 kg ha−1 P2O5 (triple superphosphate), and 60 kg ha−1 K2O (potassium chloride). Cover fertilization (30 DAS) included 100 kg ha−1 N (urea).Planting fertilization included 30 kg ha−1. Grain yield (GY) was obtained after threshing the ears of each plot, which were corrected to 13% humidity (kg ha−1). The EPV was obtained by multiplying GY and PE (m3 ha−1). The row number per ear (RNE) and grain number per row (GNR) were determined by counting, ear diameter (ED) was estimated with a Vernier caliper (mm), and ear length (EL) was measured with a ruler (cm). The characteristics 100GW, PE, GY, and EPV were measured in all plants in the plot. GNR, RNE, ED, and EL were measured using a random sample of six plants per plot.The 100-grain weight (100GW) was determined by averaging the weight (g) of two samples of 100 grains per plot. Popping expansion (PE) was measured by the mass of 30 g of grains placed in a microwave oven in a kraft bag for 2 min, with the volume of popcorn quantified in a 2000-ml beaker and the ratio of the popped volume being divided by 30 g and expressed in ml gRoot architecture was evaluated based on the methods used by Leaf greenness (SPAD index) was estimated using three readings from the middle third of the leaf counted from the apex and below the flag leaf using a portable SPAD-502 chlorophyll meter . The SPAD index was measured on eight different dates according to male anthesis: 15 (S1) and 8 (S2) days before anthesis (DBA), during male anthesis (S3), and 7 (S4), 17 (S5), 22 (S6), 35 (S7), and 42 (S8) days after anthesis (DAA). SPAD index was evaluated in six plants per plot, with three readings per plant.ij = µ + gi + bj + eij, where mean (µ) and genotype effect (g) were considered fixed and block (b) and error (e) were considered random.Analysis of variance was performed for each water treatment and year considering the linear model: yijk = µ + (b/l)/wcjkm + gi + csj + wck + gaij + gwcik + cswcjk + gcswcijk + eijk, where the mean (µ), the effect of genotype (G), crop (CS), water condition (WC), G × CS interaction (gcs), G × WC interaction (gwc), CS × WC (cswc) interaction, and G × CS × WC interaction (gcswc) were considered fixed and the block within WC (b/l) and error (e) were considered random.The experiments were then jointly analyzed considering different water treatments and years using the following mathematical model: yThe variables that showed significant differences between genotypes with at least 5% probability from the F-test were analyzed with the Tukey’s test at the same level of significance.Regression analysis was performed for the SPAD index variable after analysis of variance using the quadratic model and considering significance at 5% probability from the F-test.rg) estimates were calculated according to t-test.Subsequently, genotypic correlation coefficient , in a greenhouse at the Faculty of Biology, University of Barcelona, Spain, using two inbred lines (P7 and L75) identified as drought tolerant and sensitive, respectively, in the 2016 and 2018 field experiments in Brazil. Three seeds from each lineage were germinated in substrate in PVC tubes of 14 cm diameter and 150 cm length, which were kept at field capacity (FC). The substrate consisted of 80% perlite and 20% peat with the addition of a slow-release fertilizer, which in absolute values corresponded to 144.7 kg N haThe tubes were longitudinally divided into two halves held together with adhesive tape. The lower part of the tube was tied with wire and closed with a vase of the same diameter, with eight holes to avoid loss of substrate. In addition, the tubes were covered with plastic to reduce evapotranspiration.Thinning was performed 15 days after germination, retaining one plant per tube. The experiment was arranged in completely randomized blocks under the two water conditions, with three repetitions each. The plants were cultivated under a rain shelter that allowed the control of water availability. The temperature and humidity inside the greenhouse followed a seasonal pattern as shown in For field capacity evaluation the tubes were fully irrigated before plant sowing, and left for 72 h to drain the excess irrigation water. After this time, the cylinders were weighed. Field capacity was calculated from the weight difference between the dry substrate and after draining excess water.In the WW condition, the tubes were kept at FC until the time of the evaluations, which were conducted during the pre-anthesis period. For this, every 2 to 3 days before irrigation, the tubes were weighed and received the corresponding amount of water to return to FC. In the WS condition, limited irrigation was imposed 15 days after the emergence of seedlings. The amount of water available in the tubes gradually reduced according to plant consumption, until reaching 35% of FC (40 DAS); thereafter, they were maintained for 15 days until harvest. The decrease in moisture in the tubes was homogeneous as the plants that consumed the most water received the precise amount of irrigation to return to the substrate water conditions of the plants that consumed the least. In both WCs, the weight of the plants was disregarded for the calculation of the water capacity of the tubes.2 g−1) ratio. For this, of 1.65 cm diameter leaf discs were collected from the last developed leaf of each plant and dry mass determined as above.After harvest, plants were collected and dried  to determine shoot biomass . The specific leaf area (SLA) was calculated using the leaf area (cm²) to dry leaf mass (cm https://support.pix4d.com/hc/en-us/articles/202559809-Ground-sampling-distance-GSD). We have used the following equation: The leaf area (m²) was calculated from photographs of each individual plant. The images were acquired with a Sony α6000 DSLR , 24.5-megapixel resolution camera with a 23.5 x 15.6 mm sensor size, native resolution of 6,000 × 4,000 pixels and equipped with a 35-mm focal length lens. The pixel size was calculated using the Ground Sample Distance (GSD) calculator tool developed by Pix4D , were coated with a colorless nail base coat. After drying for 10 min, the dried coat layer was removed with adhesive tape and transferred to a glass slide. The number of stomata (s) and epidermal cells (e) were counted at ×40 magnification. Each foliar replica  was counted in three microscope fields, according to the methodology described by −2), ECD is epidermal cell density (mm−2), and 0.152 mm−2 is the surface area of the microscope (0.22 mm radius).The stomatal and epidermal cell densities were calculated using the following equations: The stomatal index  of each leaf face was calculated using the following equation: ® meter .Foliar chlorophyll, flavonoid, and anthocyanin epidermal levels and the nitrogen balance index were evaluated from the middle third of the last leaf developed at the time of harvest, using a portable Dualexam and 2:00 pm, gas exchanges were evaluated using an infrared gas analyzer, model LI-6400 , equipped with a light source . During the evaluations, the PAR was set to 1500 μmol m−2 s−1, CO2 concentration to 400 µmol mol−1, relative humidity to between 55% and 60%, and temperature to 25°C. Net photosynthetic rate (A), stomatal conductance (gs), and transpiration rate (E) were evaluated from the last developed leaf of each plant. The instantaneous (WUEinstant = A/E) and intrinsic (WUEintrinsic = A/gs) water use efficiencies were also calculated.One day before harvest, between 11:00 cum is the total amount of transpired water of each plant . Irrigation was controlled during the whole growth cycle, which allowed estimation of the Tcum values.The agronomic water use efficiency was calculated using the equation 13C:12C) isotope ratio was measured in leaf dry matter using an elemental analyzer  coupled to an isotope ratio mass spectrometer  operating in a continuous flow mode. Samples of 0.7–0.8 mg of leaf dry matter from each plant, together with reference materials were weighed and sealed into tin capsules. Measurements were performed at the Scientific Facilities of the University of Barcelona. Isotopic values were expressed in composition notation (δ) as follows: δ13C = [(13C/12C) sample/(13C/12C) standard]–1, where “sample” refers to plant material and “standard” to international secondary standards of known 13C:12C ratios  calibrated against Vienna Pee Dee Belemnite calcium carbonate with analytical precision (standard deviation) of 0.15‰. The last fully developed leaf of each plant was used.The stable carbon  were collected from the same leaves as those used for gas exchange evaluation to obtain the relative water content according to the methodology proposed by After harvesting the shoots, the roots were also collected, being abundantly washed with water to remove perlite and peat, rinsed with distilled water and superficially dried with paper towels. Finally, the root material was cut into five equal sections, each 0.30 m long, obtained from the upper surface of the tubes to the lower end, in the following layers: 0–30 cm (a), 30–60 cm (b), 60–90 cm (c), 90–120 cm (d), and 120–150 cm (e).The root sections were stored separately in a paper envelope and dried . Afterward, the ratio between the dry biomass of the aerial parts and the roots was calculated.sec, g m−3) was estimated by: sec is the dry biomass of the roots in the soil section (g); R is the tube radius (0.07 m), and L is the length of the section (0.30 m long) (The root weight density in each section of soil (RWD m long) .ijk = observation of the i-th genotype in the j-th environment in the k-th block; μ = general constant; Gi = fixed effect of i-th genotype; B / WCjk = random effect of the k-th block within WCj; WCj = fixed effect of the j-th environment; GWCij = fixed effect of the interaction between the i-th genotype and the j-th WC; and ϵijk = random error. Then, the effects of the inbred lines were estimated for each trait along with the estimates of the genotypic correlation coefficient.Analysis of variance was performed for each water condition (WS and WW) and for the interaction genotype by WC. The analysis followed the statistical model: The inbred lines showed significantly different agronomic characteristics in both WCs and CS, except for 100GW in WW-2018. Comparing WCs, expressive reductions (> 20%) occurred in 100GW (23.39), PE (29.31), GY (56.70), and EPV (68.27) in CS 2016 and in GY (44.58), EPV (50.61), and GNR (26.15) in CS 2018. There were significant differences between CS (except for PE), WC, and G. The CS × WC interactions for the variables GNR and ED, as well as the CS × G interactions for the variables GY, ED, and EL and the WC× G interactions for the variables 100GW, RNE, GNR, ED, and EL were not significant. The CS × WC × G interaction was significant only for PE Table 1.p > 0.05). The CS × WC × G interaction was significant only for S7 (p < 0.01)  occurred for S5 (11.26), S6 (18.33), S7 (28.23), and S8 (50.85) in 2016; and S2 (14.85), S3 (17.41), S4 (13.72), S7 (13.26), and S8 (21.59) in 2018. There were significant differences for CS, WC, and G when considering the SPAD variable for all evaluation dates. The CS × WC interactions for SPAD in the evaluations at S1 and S5, as well as the CS × G interactions at S4 and S7 and WC × G interactions at S1, S2, S3, S4, S5, S7, and S8 were not significant ( < 0.01) Table 1.In CS 2016, the inbred lines presented significant differences in all the root characteristics and in both WCs. In 2018, the variables AC, NA, DA, DB, and DC under WS conditions, and AB and AC under WW conditions, expressed significant differences between genotypes. Comparing the WS and WW conditions, significant increases were observed in the variables AB (31.13), AC (21.48), DB (19.83), and DC (47.90) in 2016, and decreases in DA (15.61) and DB (11.82) in 2018.p > 0.05). All root variables had significant WC × G interactions. The CS × WC × G interaction was significant for AC, NA, NB, DA, and DB (p < 0.01)  Table 1.Under WS and WW conditions for CS evaluated in relation to 100GW, L65 presented the highest mean estimates. The inbred lines L71, L76, P6, and P7 had the highest means for PE in CS 2016 and L61, L71, P6, and P7 in CS 2018. The highest GY and EPV means, at the same time for different WCs and CS, were obtained for P2, P3, P6, and P7 Figure 4The highest mean values for RNE, occurring simultaneously in different WCs and CS, and were expressed by L61, L71, L76, and P2; for GNR by P2 and P7; for ED by L76, P2, and P3; and for EL by P2 and P7 Figure 5Unlike L61, L65, and L75, the P2, P6, and P7 inbred lines expressed better agronomic performance for GY and EPV in both WCs and CS. In greenhouse studies and with a greater number of physiological traits, the genotype P7 was notable as being superior and L75 as inferior.Considering the different WCs and CS together, L61, L65, and L76 showed the highest estimates for AB, whereas L61, L75, and L76 were notable with the highest means for AC Figure 6p < 0.01)  Table 1.0), date of occurrence of the male anthesis, was 41.36 in WS and 44.17 in WW. In WS, the maximum value of the SPAD index was 41.39, verified at both DAA. Whereas in WW, the maximum value observed was 45.32, verified at 11 DAA , the value corresponding to the starting point .The variables PE, S7, AC, NA, NB, DA, and DB, which showed significant CS × WC × G interaction Table 1,rg were in the pairs S1 × S2, S1 × S3, S3 × S4, S3 × S5, S3 × S6, S4 × S5, S4 × S6, and S5 × S6, all also positive. Although with positive estimates, no significant rg was observed for root variables ; GY × S1, EPV × S1, RNE × S1, GNR × S1, and ED × S1 (negative); 100GW × S2 (positive); GY × S2, EPV × S2, RNE × S2, GNR × S2, ED × S2, and EL × S2 (negative); 100GW × S3 (positive); RNE × S3 (negative); GY × S5, EPV × S5, EL × S5, and RNE × S8 (positive). No significant positive rg were observed between agronomic and root variables, although there was a tendency for negative correlations between them. Among the different SPAD reading dates, the following negative rg were observed for the root variables: S4 × AB, S4 × NC, S4 × DC, S5 × AB, S5 × NC, S5 × DC, S6 × AB, S6 × NC, and S6 × DC  in relation to L75. There was a mean SLA increase of approximately 21.8% when comparing WS with WW. The leaf area (LA), independently of WC, was higher in P7, with values of 0.51 m² and 0.63 m² in WS and WW, respectively. In both WCs there was a difference in the comparison between means. L75 had a greater decrease in LA, corresponding to 32.5% Table 2.Among the variables related to stomatal characteristics, significant differences were observed between adaxial stomatal density and abaxial stomatal density, in both WCs; and for abaxial stomatal density only in WW. The comparison between WCs, in general, showed increases for P7 and decreases for L75 when considering these variables. The joint analysis showed neither statistical differences between G and WS nor significant G × WC interactions Table 2.In foliar pigments, statistical differences were observed only in chlorophyll and NBI in the WW condition. Chlorophyll and NBI decreased under WW compared with WS conditions, being more pronounced for P7. However, there was also a slight increase of anthocyanin in the same lineage. The joint analysis for both WC showed statistical differences only for chlorophyll and NBI Table 2.s, and E  of biomass was −13.16‰ for P7, and −13.75‰ for L75 (significant), which were slight lower values than the −12.18‰ and −12.37‰ observed in their respective WW plants (not significant)  Table 2.Regardless of water condition, P7 plants had higher RWD values in the different soil strata. Significant difference between inbred lines for RWDa in both WCs and for RWDb and RWDc in WS, as well as for RWDe in WW was observed. Considering the comparison between WCs, a mean RWD decrease was observed in the different strata, being 53.4% for P7 and 57.20% for L75.The shoot/root ratio of dry biomass increased from 3.80 to 4.93 when comparing the WW to WS condition, without significant differences between these inbred lines.In general, root characteristics in the joint analysis showed significant differences within G and WC. However, there was no significant G × WC interaction.Soil water limitation most affected GY and EPV yield components. Water limitation implemented during pre-anthesis and grain filling reduced the yield covariates to a lesser extent. Under dry conditions, the reduction in the number of grains per ground area has been indicated as being responsible for GY reduction in common maize . Pollen The variables of economic interest, i.e., PE, GY, and EPV, had a significant WC × G interaction, which pointed to different responses among the evaluated genotypes in the different implemented WCs. Interactions of this nature interfere with selection gains and cultivar recommendation for specific environments . In thesThe SPAD index can be used for the indirect selection of the most productive plants in the WS condition, as there was no significant WC × G or CS × WC × G interaction, whereas a positive association with GY and EPV was found Table 1.Root systems had phenotypic plasticity in different WCs, which implies significant WC × G and CS × WC × G interactions. Therefore, in addition to the difficulty in measuring root traits, the occurrence of interactions is an obstacle to improving the use of root characteristics in plant breeding , which iTo understand the physiological traits inherent to the most productive lines under water limiting conditions, the genotypes were grouped using on the best results for GY and EPV. Soil water depletion reduced the SPAD index regardless of genotypic groups. Foliar senescence induction is a plant response to water deficit conditions , being cRegardless of the phenotypic class, the maximum SPAD index values were attained later (11 DAA) in WW than in WS (2 DAA) condition. Furthermore, the genotypes with greater productivity were more stay-green , as shown by the higher SPAD values recorded during grain filling. In contrast, genotypes with lower productivity reached higher SPAD index values earlier, irrespective of water conditions, thus reflecting an association between higher SPAD index values and higher yield values. These finding were in line with reports that higher SPAD index values, together with a senescence delay, are likely to contribute to the maintenance of C-assimilation and the mobilization of photoassimilates to the reproductive organs, therefore, increasing agricultural productivity .The GNR, ED, and EL components are important selection characteristics for yield and EPV increments, especially in popcorn plants under adequate water availability  and duriThe choice of an appropriate phenological stage for SPAD index measurements is crucial, since phenotyping methodologies should be used in critical stages associated with grain production . In thisA root system adapted to specific abiotic soil stress conditions represents an agronomic advantage with positive impact on yield . NotablyThe highest SPAD values at 7, 17, and 22 DAA were associated with the lowest root values for the variables AB and DC, regardless of water availability. As for the NC variable in WS, there was a negative association with the same SPAD measurement dates . In fact, the lowest NC value, as well as the decreased branching density of the lateral root, may have improved the water absorption capacity in the soil, which was indirectly reflected in a better leaf status and therefore higher SPAD values. Notably, the most productive genotypes under lower water availability are expected to have steeper angles to the soil horizon , which wWhen comparing the two water availability levels, the superior genotype (P7) showed higher SB and LA values, and a smaller proportional LA decrease under WS when compared to WW. Higher biomass accumulation before flowering and increased leaf size may result in a larger photosynthesizing area, which would increase C-assimilation . SLA sho2  or by osmotic adjustment . The higintrinsic and WUEinstant, together with WUEagron and RWC, did not help to differentiate the evaluated inbred lines. In fact, WUEinstant and WUEintrinsic are leaf scale characteristics mainly associated with transpiration. Therefore, an increase in these variables can be associated with reduced gs, which in turn can reduce photosynthesis, growth, and productivity (agron has been suggested to be a variable more associated with the effective use of water (WUEuctivity . Therefoof water . However13C (carbon isotope composition) than P7, indicating that L75 stomata were more frequently closed throughout the crop development  under water stress than P7.This study was conducted in two parts. Firstly, field experiments (in 2016 and 2018) were designed to understand which secondary characteristics were most important to explain the higher yield and EPV values (main characteristics). As a result, we identified GNR, ED, and EL, as adequate traits for plant selection. With the same objective, we sought to identify important root characteristics. Among these, at the 25 cm depth evaluated, it was not possible to diagnose root characteristics that could be indicative of satisfactory agronomic performance. The high root plasticity response to different WCs may have been responsible for this finding. There was an association between the highest SPAD index values and the highest yield values, favoring the use of this characteristic for indirect selection. Second, complementing the field experiments, two contrasting inbred lines were studied under greenhouse conditions. Under WS at the end of the experiment, P7 maintained higher values of leaf net photosynthetic rates, stomatal conductance, and transpiration than L75. Thus, single-leaf gas exchange measurements shortly before the harvest proved to be adequate indicators for the evaluation the agronomic performance of popcorn under soil water limitation conditions. However, gas exchange measurements are not amenable for high throughput phenotyping when a large number of genotypes are evaluated. In that case, a trait which can be used to evaluate stomatal conductance throught the crop cycle, such as the stable carbon isotope composition on dry leaf matter, was shown to indicate that L75 maintained the stomata closed for longer periods  – Finance Code 001. Funding was also provided by FAPERJ, with the project E26/201.813/2017 to SK and E26/202.761/2017 to AA. Funding to JR by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through the research units UID/UIDB/00239/2020 (CEF), and UIDP/04035/2020 (GeoBioTec) is also greatly acknowledged. JA acknowledges the support of Catalan Institution for Research and Advanced Studies , through the ICREA Academia Program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chlorococcum amblystomatis lipidome using hydrophilic interaction liquid chromatography-high resolution electrospray ionization- tandem mass spectrometry (HILIC–HR–ESI–MS/MS). The Chlorococcum amblystomatis strain had a lipid content of 20.77% and the fatty acid profile, determined by gas chromatography-mass spectrometry, has shown that this microalga contains high amounts of omega-3 polyunsaturated fatty acids (PUFAs). The lipidome identified included 245 molecular ions and 350 lipid species comprising 15 different classes of glycolipids (6), phospholipids (7) and betaine lipids (2). Of these, 157 lipid species and the main lipid species of each class were esterified with omega-3 PUFAs. The lipid extract has shown antioxidant activity and anti-inflammatory potential. Lipid extracts also had low values of atherogenic (0.54) and thrombogenic index (0.27). In conclusion, the lipid extracts of Chlorococcum amblystomatis have been found to be a source of lipids rich in omega-3 PUFAs for of great value for the food, feed, cosmetic, nutraceutical and pharmaceutical industries.There is a growing trend to explore microalgae as an alternative resource for the food, feed, pharmaceutical, cosmetic and fuel industry. Moreover, the polar lipidome of microalgae is interesting because of the reports of bioactive polar lipids which could foster new applications for microalgae. In this work, we identified for the first time the  Microalgae represent a sustainable4 and versatile6 resource with exploitation potential for different industries 7. These unicellular organisms are found not only in aquatic systems but also in terrestrial systems, with a wide range of catalogued species9. Microalgae are valued for their photosynthetic efficiency, low water requirement, fast growth and high biomass production10, which allows efficient biomass production with lower resource consumption that meet industry’s needs. Also, microalgae grow with greater photosynthetic efficiency than higher plants and do not require arable land to be cultivated11. Microalgae have been used for different applications, namely, as direct food, as food nutrients, for animal feed, as sources of bioactive products for the pharmaceutical industry, and as a source of lipids for the production of biodiesel5. For example, microalgae can consume nutrients that are in excess in the agri-food industry waste streams, such as nitrogen and phosphorous12.. In terms of nutrients, they are a rich source of essential amino acids, vitamins, minerals, pigments and lipids, namely polyunsaturated fatty acids (PUFAs)13. They are considered a promising source of bioactive components due to their antioxidant, anti-inflammatory, anti-obesity, anti-tumour, antiviral and anti-bacterial properties14. Among the bioactive compounds described, phospholipids (PL) seem to be interesting ingredients as the main vector of omega-3 fatty acids (FA)15 and glycolipids (GL) as promising bioactive phytochemicals16. There is a large body of evidence suggesting that omega-3 PUFAs esterified in polar lipids have important physiological properties. For example, PL and GL containing omega-3 FA, were associated with antioxidant and anti-inflammatory properties, and with cardiovascular protection17 and also that PLs esterified in omega-3 PUFAs are excellent delivery vehicles19. In addition, GLs with PUFAs have shown anti-inflammatory potential with nitric oxide release inhibitory activity21 while DGDGs and SQDGs from algae showed chemotherapeutic potential22 and SQDG esterified in EPA also displays an anti-proliferative effect23. Very recent studies have focused on the identification of polar lipids from the most commercially used microalgae, such as Chlorella sp. and Chlorella vulgaris25, Nannochloropsis oceanica and Nannochloropsis oculata27, and Phaeodactylum tricornutum28. However, polar lipids are scarcely studied compared to other ingredients derived from microalgae, an even larger number of microalgae lipidome remains unknown, unexplored or poorly studied, hampering the full biotechnological exploitation of this resource.Reducing the impact of climate change is one of the main challenges of this decade, which has increased the demand to move to more environmental and sustainable resourcesChlorococcum sp. is a green microalga which belongs to the phylum Chlorophyta, class Chlorophyceae, order Chlorococcaceae and family Chlorococcaceae, and it can be cultivated under photoautotrophic and mixotrophic conditions29. The biotechnological potential of this microalga remains poorly explored29. Chlorococcum sp. has been described as a useful tool in carbon sequestration, lipid flocculation in wastewater30 and carotenoid production31, a good source for the production of biodiesel32 and useful for the food industry34. The production of lutein from Chlorococcum citriforme has already been patented35. Regarding its biochemical composition, a total biomass dry weight content of proteins of 13.08–40%36, carbohydrates of 3.42–40%36, chlorophylls of 0.4–1.5%32, carotenoids of 3.2–12.6 mg/g37 and lipids of 8.71–32.3%40 has been described. In addition to its high lipid content, the FA composition of the Chlorococcum strains studied to date, included good amounts of omega-3 fatty acids33 suggesting the possibility of added-value lipids that needs to be explored.27. As such, the complete lipidome has been reported for certain green microalgae belonging to the phylum Chlorophyta, such as Haematococcus sp.27, Chlamydomonas sp.41, Scenedesmus sp.24, Chlorella sp.42 and Chlorella kessleri43. Microalgal lipids have received much attention due to their high abundance of omega-3 and omega-6 PUFAs, and, more recently, due to their polar lipids content , in which the PUFAs are esterified. These glycolipids are plastid lipids, therefore abundant in green microalgae. Also, these lipids were reported to have desired bioactive properties, such as antibacterial, antioxidant and anti-inflammatory activities16. PUFAs were already described in Chlorococcum sp.39, however, the polar lipidome of this microalga remains to be described, preventing its full exploitation. Thus, to fill this gap, in the present work, we have analysed and characterized in detail the polar lipidome of Chlorococcum amblystomatis, cultivated in autotrophic conditions, using a lipidomic approach based on the hydrophilic interaction liquid chromatography-electrospray ionization-high resolution mass spectrometry (HILIC–HR–ESI–MS). We also explored the bioactive potential of lipid extracts of Chlorococcum amblystomatis to inhibit COX-2 activity and its antioxidant properties, using commercial kits, to promote valorisation of this microalga as a source of bioactive lipids for the food, feed, cosmetics or pharmaceutical industries.The interest of knowing the lipidome of green microalgae, either to optimize the production of biodiesel or to produce lipids with added value, has led to the complete profiling of several microalgaeChlorococcum amblystomatis corresponded to an average yield of 20.77 ± 0.57% of microalgal biomass. The fatty acid composition of Chlorococcum amblystomatis identified by GC–MS showed that C16:0 was the most abundant FA (23.0 ± 0.8%), followed by C18:3 n-3 (19.4 ± 0.6%), C18:0 (13.9 ± 2.3%), C16:4 n-3 (11.1 ± 0.6%), C20:5 n-3 (8.9 ± 0.6%) and C16:1 n-9 (7.5 ± 0.3%) , thrombogenic (TI), hypocholesterolemic/hypercholesterolemic (h/H) and the PUFA n-6/ PUFA n-3 indices were calculated. The AI and TI of the lipid extracts were 0.5 and 0.3, respectively, while the h/H ratio was 1.4 and the n-6/n-3 ratio was 0.1.To assess the potential health benefits of m/z of the fragment ions used to identify each molecular ion and each molecular species can be found in Supplementary Table The polar lipid profile was revealed by hydrophilic interaction liquid chromatography-high resolution electrospray mass spectrometry (HILIC–HR–ESI–MS and HILIC–HR–ESI–MS/MS)  (Supplementary Table The PL classes identified included phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE), identified in the HILIC–HR–ESI–MS analysis as the molecular ion [M+H]+ Fig. ; and pho. − Fig. . The onlm/z 184 (for PC and LPC) and fragment ions arising from the neutral loss of 141 Da (for PE and LPE)48. On the other hand, the complementary analysis of the molecular ions on the HILIC–HR–ESI–MS/MS spectra acquired in negative ion mode, observed as [M+CH3COO]− ions for PC and LPC and [M−H]− ions for PE and LPE, allowed to assign the fatty acyl composition. This was done by observing the presence of the RCOO−carboxylate fragment anions of the fatty acids esterified in the glycerol backbone48.As previously indicated, the identification of the lipid species was based on the mass accuracy, retention time and interpretation of MS/MS data. The interpretation of the MS/MS data of the molecular ions of the PC, LPC, PE and LPE species allowed to confirm their identity (Supplementary Table m/z 171 (for PG) and m/z 241 (for PI and PI-Cer), and identify the fatty acyl chains by the presence of RCOO−carboxylate anions (for PG and PI)48 (Supplementary Table m/z 241. The most abundant species in each phospholipid class were: PC(34:2) identified as PC(16:1–18:1) and PC(16:0–18:2); LPC(16:1); PE(34:4) identified as PE(16:0–18:4); LPE(16:0); PG(34:3) identified as PG(16:0–18:3); and PI(32:1) identified as PI(16:0–16:1).The HILIC–HR–ESI–MS/MS spectra of the negative molecular ions belonging to the PG, PI and PI-Cer classes allowed to confirm the presence of the corresponding polar heads by observing for each species the product ions at − ions and of RCOO−carboxylate hydroxyl anions was indicative of the formation of hydroxy derivatives50. No product ions with oxidized polar head groups were observed 45.Among the PG molecular ions identified, a few oxidized PG species were found Table , the mos+ ions  and ketene (–R=C=O) derivatives47 (Supplementary Table The betaine lipids identified in the HILIC–HR–ESI–MS data included the classes of monoacylglycerol-trimethylhomoserine (MGTS) and diacylglycerol-trimethylhomoserine (DGTS), and were identified in positive ion mode as [M+H]ns Table , Fig. 3.2O and the product ions formed due to the loss of oxidized fatty acyl chains (acid and keto derivatives). Also, the product ion characteristic of betaine lipids at m/z 236 was present in all MS/MS spectra, therefore no product ions with oxidized polar head groups were observed.The most abundant ions in each class of betaine lipids were MGTS 18:4) and DGTS (34:4) assigned as DGTS (18:4–16:0) and DGTS (18:3–16:1) , monogalactosylmonoacylglycerol (MGMG), digalactosyldiacylglycerol (DGDG) and digalactosylmonoacylglycerol (DGMG), identified as [M+NH4]+ ions  or 359 Da (for DGDG), which corresponds to the loss of the carbohydrate moiety combined with the loss of NH347. The assignment of the fatty acyl composition was corroborated by the presence of product ions corresponding to each fatty acyl group as acylium plus 74 [RCO+74]+ ion47 (Supplementary Table − ions of the acidic glycolipids (SQDG and SQMG), when available, showed the product ion of the sulfoquinovosyl polar head group at m/z 225 and the fatty acyl composition was confirmed by the neutral loss of fatty acyl chains as carboxylic acid (-RCOOH) and by the presence of carboxylate RCOO− anions , SQDG (34:3-OH) identified as SQDG (18:3-OH-16:0) and SQDG (34:4-OH) identified as SQDG (18:4-OH-16:0). HILIC–HR–ESI–MS/MS analysis of all oxidized GL species suggested that a hydroxyl group was present in the fatty acyl group (Supplementary Table The most abundant species in each class of glycolipids were: MGDG (34:7), identified as MGDG (16:4–18:3) and MGDG (18:4–16:3); MGMG (16:4); DGDG (34:3), identified as DGDG (16:0–18:3) and DGDG (18:1–16:2); DGMG (18:3); and SQDG (32:1), identified as SQDG (16:0–16:1). The only identified glycolipid species belonging to the SQMG class was identified by accurate mass measurements and by the presence on the MS/MS spectrum of the product ion corresponding to the polar head at Chlorococcum amblystomatis, with a relative amount greater than or equal to 1.5%. Glycolipids were the group of lipids with the largest number of species (101). The most abundant species was MGDG (34:7), assigned as MGDG (16:3–18:4) and MGDG (16:4–18:3), and the second most abundant species was SQDG (16:0–16:1).The relative quantification of the identified species was carried out as described in the methods section. Figure 54. The use of one internal standard is acceptable for semi-quantification because the ionization efficiency of polar lipids depends mainly on the polar head, while the length of the chain and the degree of unsaturation contribute little56. However, we would like to point out the limitations of our inter-class quantitation approach, because despite the similar retention time, the response factors for each class, even between phospholipids, tend to differ in ESI–MS, which prevents a robust and accurate quantification53. Normalized data were then used to semi-quantify the abundances of the identified molecular ions. For a clearer overview of the composition of the polar lipidome of Chlorococcum amblystomatis, we have summed up the relative percentage of all the detected lipid species belonging to glycolipids, betaines and phospholipids , we have observed an inhibition of 87.5 ± 0.1 of COX-2 activity  was 76.25 ± 1.02 Trolox µmol g−1 of lipid extract. In contrast, the lipid extract which resulted in a 50% inhibition (IC50) of ABTS•+ was 37.69 ± 4.44 µg mL−1 and the expressed TE activity of 428.81 ± 54.32 Trolox µmol g−1 of lipid extract. The antioxidant potential of lipid extracts from ays Fig. , as descChlorococcum amblystomatis is a green microalga with great potential to be a sustainable resource of commercially important essential lipids29, with promising applications in several industrial sectors33. In our work, the lipid extracts represented 20.77 ± 0.57%, of the dry weight of the biomass, which is consistent with the lipid content reported for Chlorococcum (8.71–32.3%)20. This wide range of lipid content determined by different studies is consistent with the variation in lipid content due to growing conditions40 and the use of different extraction methods54.n-3), C18:0, C16:4 (n-3), C20:5 (n-3) and C16:1–9, representing respectively 23%, 19%, 14%, 11%, 9% and 7% of the total FA pool. It is important to note that this is the first time that FA C16:4 (n-3) and C20:5 (n-3) have been reported in Chlorococcum amblystomatis. The values of relative abundance collected in the present work are different from those reported in the literature to Chlorococcum sp39. Such differences could result from differences in growth conditions 55, different strains, different species, different derivatization methods or even GC detectors (FID or MS) 56. Chlorococcum amblystomatis extracts were rich in omega-3 PUFAs, representing 43.2%, with a high contribution of the most abundant FA, C18:3 (n-3), C16:4 (n-3) and C20:5 (n-3). Omega-3 PUFAs are known to have a variety of health benefits, such as preventing chronic diseases, as they are associated with anti-inflammatory and antioxidant protection, benefits for the cardiovascular system, prevention of breast cancer, improvement of neurological capacities and visual development59. To understand the beneficial impact on health and assess the nutritional quality of Chlorococcum amblystomatis, we calculated the values of AI (0.5), TI (0.3), h/H (1.4), and n-6/n-3 (0.1). The AI and TI indexes are commonly used to assess the potential of the matrix to stimulate platelet aggregation, and the lower the indexes the more beneficial it is in reducing the prevalence of heart disease 60, and have already been used to assess these benefits in microalgae, seaweeds and fish65. Our results show that these indexes of the lipid extract of Chlorococcum amblystomatis were similar to those observed in fish oils67. Compared to Spirulina platensis, Nannochloropsis gaditana, Nannochloropsis oculata and Porphyridium tricornutum, Chlorococcum amblystomatis had lower AI values. Their AI values ranged between 0.6 and 1.7 61. As for the TI index, Chlorococcum amblystomatis had lower values than Spirulina platensis, Nannochloropsis gaditana and Porphyridium Cruentum61, which were 1.5, 3.8 and 0.6, respectively. The higher values for h/H and the lower n-6/n-3 ratio  reinforce the nutritional potential of the lipid extracts of Chlorococcum amblystomatis.The FA profile identified by GC–MS included as the most abundant FA: C16:0, C18:3 (Chlorococcum amblystomatis. The polar lipids identified included glycolipids (GL), phospholipids (PL) and betaine lipids (BL). Data from the literature indicate that algae are a valuable and promising source of polar lipids with bioactive properties, namely, polar lipids esterified to commercially important PUFAs, e.g. eicosapentaenoic acid (EPA). Our profiling made it possible to identify 245 molecular ions, corresponding to a minimum of 350 molecular species: 89 molecular ions of PL, 101 molecular ions of GL , 54 molecular ions of BL and 1 inositol phosphoceramide. These ions were distributed by 15 classes of lipids: PL classes were LPC, PC, LPE, PE, PG and PI; sphingolipid classes were PI-Cer, BL classes were MGTS and DGTS; and GL classes were MGMG, MGDG, DGMG, DGDG, SQDG, and SQDG.The profiling of the total lipid extract using high-resolution HILIC–ESI–MS and MS–MS approaches, made it possible, for the first time, to describe the polar lipid composition, in Chlorococcum amblystomatis, and both carried omega-3 fatty acids, including EPA. In a previous study in which anti-inflammatory activity was addressed, PG (16:0–20:5) was suggested to promote down-regulated iNOS activity in LPS-stimulated macrophages68.About 45% of the lipid species detected were esterified to omega-3 fatty acids. Some of these species caught our attention because of their richness in omega-3 PUFAs. The PG (34:3) and the PG (36:5), respectively assigned to PG (16:0–18:3) and PG (16:0–20:5) are two species of PL among the most abundant of Chlorococcum amblystomatis there are high amounts of PLs esterified to omega-3 PUFAs and it has been suggested that the PLs are an excellent delivery vehicle for PUFAs19, this will favour its valorization for food and nutraceutical formulations, where they can be used as value-added ingredients, and also in the cosmetic industry, as they can be used to create moisturizing emulsions 69. Interestingly, oxidized phosphatidylglycerols (PG) with bonded oxylipins have been identified in this study. Some studies have suggested the anti-inflammatory potential of oxylipins 70, as reported for Nannochloropsis gaditana and Chlamydomonas debaryana71. Indeed, we observed a COX-2 inhibitory activity from the lipid extracts of Chlorcococcum amblystomatis, to which these oxylipins could have contributed. Consequently, extracts rich in polar lipids from Chlorococcum amblystomatis could constitute an interesting opportunity for the nutraceutical or pharmaceutical industries.Since in Chlorococcum amblystomatis, include as the most abundant species, DGTS (34:4), DGTS (32:4), DGTS (36:7), DGTS (34:7), DGTS (34:3), DGTS (40:10) and DGTS (36:6). Moreover, DGTS (40:10) was identified as DGTS (20:5–20:5), a molecular species associated with anti-inflammatory activity, by reducing the production of nitric oxide (NO) 72. However, BL remains poorly studied to date, and little is known about its bioactive potential.The BL identified in the polar lipidome of Chlorococcum amblystomatis was particularly rich in glycolipids  which represented approximately half of the polar lipids content , MGDG (20:5–16:1), MGDG (20:5–18:2), MGDG (20:5–20:5), DGDG (20:5–16:0), DGDG (20:5–18:2), DGDG (18:4–16:0) and MGDG (16:4–18:3), were also identified in the Chlorococcum amblystomatis lipidome. These GL species were associated with anti-inflammatory activity by down-regulating the iNOS78.The polar lipidome of ent Fig. . There iChlorococcum amblystomatis, and were linked to anti-inflammatory 79 and antiviral activities 80, respectively. Also, SQDG (16:0–20:4) and SQDG (16:0–18:4), identified in extracts of Chlorococcum amblystomatis, have been reported for their antiproliferative properties82. Finally, SQMG (16:0), although detected in low abundance, has been reported to have antimicrobial and antitumor activities 83.Regarding sulfur-containing lipids, SQDG (34:3), assigned as SQDG (16:0–18:3), and SQDG (32:0), were among the most abundant SQDG identified in Chlorococcum amblystomatis may contribute to the antioxidant activity observed in the DPPH and ABTS radical scavenging assays, as some of the reported polar lipids were previously associated with antioxidant activity 73. Natural antioxidants are highly sought after for their biological effects 84 and their wide applications in the food and pharmaceutical sectors 85. As such, microalgal biomass extracts have been widely explored regarding their potential antioxidant activity87. Ethanolic and aqueous extracts are mainly composed of phenolic compounds, while methanolic extracts are rich in polar lipids 88. In a recent study, the DPPH assay was employed to evaluate antioxidant activity on 9 microalgae. The methanolic extracts of the different microalgae strains showed antioxidant activity, with percentage inhibition of DPPH ranging from 15 to 45%  86. In this work, the dichloromethane:methanol extracts of Chlorococcum amblystomatis resulted in a percentage of inhibition of 36% at 200 µg mL−1, displaying a value better compared to 7 strains of microalgae from the reported work.The polar lipids of 289. In the present work, the lipid extracts of Chlorococcum amblystomatis exhibit a COX-2 inhibiting activity. It is the first time that such a response has been described in Chlorococcum sp. However, recent work, also measuring COX-2 inhibition, compared the anti-inflammatory potential of aqueous and ethanolic extracts of two Tetraselmis sp. strains and a Skeletonema sp. strain 81. The highest value of anti-inflammatory activity (82 ± 2%) was measured for the ethanolic extract of Skeletonema sp. at a concentration of 1 mg mL-1. Our results showed that the COX inhibition of dichloromethane: methanol extracts of Chlorococcum amblystomatis was 87.5 ± 0.1%, at a concentration of 50 μg mL−1. These results are consistent with the hypothesis that Chlorococcum amblystomatis has a higher COX-2 inhibitory power than Tetraselmis sp. and Skeletonema sp. The high COX-2 inhibitory activity of Chlorococcum amblystomatis extracts results in promising anti-inflammatory potential. Future studies should explore the use of lipid extracts of Chlorococcum amblystomatis in inflammatory cells to explore this anti-inflammatory potential.COX-2 is an important component of inflammation, associated with pro-inflammatory activity, responsible for the production of prostaglandin EChlorococcum amblystomatis was characterized for the first time in this study. The Chlorococcum amblystomatis strain used revealed a high content of omega-3 PUFAs. PUFAs are associated with several health benefits, such as the prevention of cardiovascular disease. The HILIC–MS/MS lipidomic approach identified 245 molecular ions of polar lipids, in Chlorococcum amblystomatis, revealing to be a microalga rich in glyco- and betaine lipids. Some of the identified polar lipids have already been reported with biological activity, for example, DGTS (20:5–20:5), SQDG (16:0–18:3), MGDG (20:5–18:2) and DGDG (20:5–18:2) which were associated with anti-inflammatory activity. In addition, extracts rich in polar lipids had COX-2 inhibiting activity and antioxidant activity. In conclusion, due to its chemical, biochemical, bioactive, and health-promoting properties, the lipid extracts of Chlorococcum amblystomatis have been found to be of high value for application in food, feed, cosmetic, nutraceutical, and pharmaceutical applications.The polar lipidome of 2Cl2), were purchased from Fisher Scientific Ltd. . All other reagents were purchased from major commercial sources. Milli-Q water  was used. Phospholipid internal standards 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (dMPE), 1,2-dimyristoyl-sn-glycero-3-phospho-(10-rac-glycerol) (dMPG), 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (dMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol (dPPI), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) were purchased from Avanti Polar Lipids, Inc. , according to the methodology previously described35. DPPH• was purchased from Aldrich . 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS•+) was obtained from Fluka . The 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich . The cyclooxygenase (COX-2) inhibitory screening assay was performed using a commercial kit, Cayman test kit-701080 from Cayman Chemical Company . All the other reagents and chemicals used were of the highest grade of purity commercially available.HPLC grade methanol (MeOH), ethanol absolute, and dichloromethane  reactor, which was later sequentially scaled as follows: 0.25 m3 L to a 0.5 m3 L to a 1 m3 Flat Panels. Four of the later reactors were used as inoculum of a 10 m3 photobioreactor (PBR). The reactor was operated for 21 days, exposed to the environmental light and temperature conditions, at an average temperature of 15.5 °C and light irradiance of 20.10 MJ m−2 day−1. pH was maintained constant, 7.0–8.0, by pulse injections of CO2 and the temperature was kept under 28 °C by a sprinkler-like irrigation system. After growing period, the biomass was industrially collected by centrifugation and further spray-drying.Portugal Chlorococcum amblystomatis biomass using the solvent mixture. The suspension was centrifuged  at 2000 rpm for 10 min and the supernatant was collected in a new pre-weighed glass tube. This process was repeated four times until the extraction solvent lost the green colour. The combined supernatants were dried under a stream of nitrogen.Lipid extraction was carried out using a mixture of dichloromethane: methanol solvents (DM) . The lipids were extracted from 25 mg of lyophilized 92. The extracts were redissolved in 2 mL of dichloromethane, and 1 mL of methanol and 0.75 mL of Milli-Q water were added. The mixture was vortexed for 2 min followed by phase separation by centrifugation at 2000 rpm for 10 min. The organic phase was collected in a new pre-weighed tube, and the aqueous phase was reextracted with 2 mL of dichloromethane, two more times. The combined organic phases were dried under a stream of nitrogen and weighted.The Folch extraction method was used to the obtained dried supernatantsEach series of extracts was repeated five times and the total lipid content was determined by gravimetry. The yield of lipids extracted from dry biomass extracts (DW) was calculated as follows Eq. :1\docume93. This screening assay directly measures the amount of prostaglandin F2α generated from arachidonic acid  in the cyclooxygenase reaction. This assay was carried out according to the instructions described by the supplier of the assay kit, using an aliquot of test extract or DMSO. For this assay, lipid extracts of Chlorococcum amblystomatis  were dissolved in DMSO and the final volume of reaction was 1000 μl. Positive and negative controls were provided by the assay kit protocol. The positive control used inactivated COX-2 enzyme, and negative control used the enzyme with 100% initial activity without any inhibitor. The assay was performed in three replicates. Interferences were considered by subtracting COX-2 inhibition from the blank assays. The results were expressed as a percentage of inhibited COX-2.A commercial cyclooxygenase (COX-2) inhibitory screening assay kit—Cayman test kit-701080 —was used to assess their anti-inflammatory potential •) was evaluated as described previously95 with some modifications. Briefly, 150 µL of an ethanolic dilution of the extracts  or 150 µL of the Trolox standard solution  were mixed in triplicate with 150 µL of a DPPH• working solution in ethanol . The mixture was incubated for 120 min and the absorbance was measured at 517 nm every 5 min . A control was prepared by replacing the DPPH• solution with ethanol. The antioxidant activity, expressed as a percentage of inhibition of the DPPH radical, was calculated using the following equation  was evaluated using a method previously described96 with some modifications. Briefly, 150 µL of an ethanolic dilution of the extracts  or 150 µL of the Trolox standard solution  were mixed in triplicate with 150 µL of an ABTS•+ working solution in ethanol . The mixture was incubated for 120 min and the absorbance was measured at 734 nm every 5 min . A control was prepared by replacing the ABTS•+ solution with ethanol. The antioxidant activity, expressed as a percentage of inhibition of the ABTS radical (IC40 substituted by IC50), was calculated using Eq. (DPPH• substituted by AbsABTS•+) and expressed in Trolox Equivalents  according to the methodology previously described 90. A volume of 2.0 μL of a hexane solution containing FAMEs and 1.03 μg mL−1 of methyl nonadecanoate  as internal standard was injected in a chromatography-mass spectrometry (GC–MS)  equipped with a DB-FFAP column with the following specifications: 30 m long, 0.32 mm internal diameter, and 0.25 μm film thickness . The GC equipment was connected to an Agilent 5977B Mass Selective Detector operating with electron impact ionization at 70 eV and a scanning range of m/z 50–550 (1 s cycle in a full scan mode). The following conditions were used: helium as carrier gas (constant flow 1.4 mL min−1), inlet temperature 220 °C, detector temperature 230 °C, injection volume 2 μL (splitless). The oven temperature was programmed as follows: 58 °C for 2 min, 25 °C min−1 to 160 °C, 2 °C min−1 to 210 °C, 30 °C min−1 to 225 °C (held for 20 min). The data acquisition software used was GCMS5977B/Enhanced MassHunter. Data were analysed using Agilent MassHunter Qualitative Analysis 10.0 software. The identification of FA was carried out by comparison of the MS spectrum with the NIST chemical database library and confirmed with the literature reports. Five independent replicates were injected.The fatty acid methyl esters (FAMEs) were prepared from total lipid extracts of 97The atherogenic, thrombogenic and hypocholesterolemic/hypercholesterolemic indices (h/H) were calculated as the content ratio of saturated fatty acids (SFA)/unsaturated FA, as monounsaturated fatty acids (MUFAs) and omega-3 and omega-6 PUFAs, using the following formula , as proposed by Ulbricht and Southgate 35. The dried samples were dissolved in CH2Cl2 to a final concentration of 1 ug uL-1. From each sample, a volume of 10 µL (10 µg of lipid extract) was taken and transferred to an appropriate vial, followed by the addition of 86 µL of a solvent system composed of two mobile phases in a proportion of 90% v/v eluent B and 10% v/v eluent A and 4 µL of a mixture of internal standards —0.02 μg, LPC—0.02 μg, dPPI—0.08 μg, dMPG—0.012 μg, dMPS—0.04 μg). The composition of eluent B was 60% v/v of acetronitrile, 40% v/v of methanol, and 5 mM of ammonium acetate and the composition of eluent A was 50% v/v of acetonitrile, 25% v/v of methanol, 25% v/v of water, and 5 mM ammonium acetate.The polar lipidome was determined according to the methodology previously described−1 and a temperature of 35 °C. The following gradient was applied: 10% A (0–2 min), 10–90% A (2–15 min), 90% A (15–17 min). The mass spectrometer with Orbitrap technology was operated simultaneously in positive (electrospray voltage 3.0 kV) and negative (electrospray voltage − 2.7 kV) modes, with the following configuration: high resolution with 70,000, AGC target of 1 × 106, capillary temperature 250 °C, and sheath gas flow of 15 U. The tandem mass spectrometry experiments were performed according to the following configuration: resolution of 17,500, AGC target of 1 × 105, with one full scan mass spectrum and 10 data-dependent MS/MS scans. The cycles were repeated continuously throughout the experiments with the dynamic exclusion of 60 s and an intensity threshold of 1 × 104. Normalized collision energy (CE) ranged between 25, 30, and 35 eV. Data acquisition was performed using the Xcalibur data system . Five independent biological replicas were carried out.The lipids were separated by hydrophilic interaction liquid chromatography (HILIC) using a microbore Ascentis Si column  and a high performance-liquid chromatography (HPLC) system  with an autosampler coupled to the Q-Exactive hybrid quadrupole Orbitrap mass spectrometer . A 5 µL aliquot of each sample mixture was injected into the HPLC column, at a flow rate of 50 µL min98: this approach consists of the localization of the species according to the retention time of internal standards . The identification was carried out according to a standard approach in our laboratory Relative quantification was performed by exporting the values of the peak areas to a computer spreadsheet . To normalize the data, the peak areas of the extracted ion chromatograms (EIC) of each lipid molecular species were divided by the EIC peak areas of the selected internal standards. Relative abundances were calculated using dMPC, dMPE, SM17:0/d18:1), LPC, dPPI, dMPG and dMPS as internal standards. To normalize DGTS and MGTS we used PE as internal standard, for SQDG we used PG as internal standard, and for MGDG, DGDG, DGMG and MGMG, we used Ceramide as internal standard. For clear visualization, normalized data was transformed in percentage. The normalized data was calculated as follows  was calculated as follows Eq. :9\documeRaw abundances, normalized data and relative quantification can be found on the additional spreadsheet (Supplementary file Supplementary Information