Janus droplets can function as excellent templates for fabricating physically and chemically anisotropic particles. Here, we report new surfactant-laden Janus droplets with curvature controllability and enhanced stability against coalescence, suitable for fabricating shape-anisotropic polymer microparticles. Using a microfluidic flow-focusing device on a glass chip, nanoliter-sized biphasic droplets, comprising an acrylate monomer segment and a silicone-oil (SO) segment containing a surfactant, were produced in a co-flowing aqueous polyvinyl alcohol (PVA) solution. At equilibrium, the droplets formed a Janus geometry based on the minimization of interfacial energy, and each of the two Janus segments were uniform in size with coefficient-of-variation values below 3%. By varying the concentration of the surfactant in the SO phase, the curvature of the interface between the two lobes could be shifted among concave, planar, and convex shapes. In addition, the Janus droplets exhibited significantly improved stability against coalescence compared with previously reported Janus droplets carrying no surfactant that coalesced rapidly. Finally, via off-chip photopolymerization, concave-convex, planar-convex, and biconvex lens-shaped particles were fabricated. Janus droplets have attracted significant interest recently because of their unique properties originating from their two physically and chemically distinct exposed segments. For example, Janus droplets can be used as templates in the fabrication of various functional microparticles, which are valuable for potential applications as electrically and magnetically driven actuators ,2, interTo date, monodisperse Janus droplets comprised of two miscible  or two iN-isopropylacrylamide) Janus hydrogels  was dissolved in 10-cSt SO  to prepare mixture solutions with concentrations ranging from 0.1 to 5.0 wt.%. For the continuous aqueous phase to produce the surfactant-laden Janus droplets, polyvinyl alcohol  was dissolved in deionized water  to prepare a 2.0 wt.% solution. For the continuous aqueous phase to produce surfactant-free Janus droplets, SDS was dissolved in deionized water to prepare a 0.3 wt.% solution. Oil-soluble dye  was dissolved in the HDDA solution to visually differentiate the two organic phases.Ethanol (>99.5%), acetone (>99%), SDS were purchased from FUJIFILM Wako Pure Chemical Corporation . The acrylate monomer , dynamic viscosity Three syringe pumps  and glass syringes  were used to infuse the fluids into the microfluidic device to generate droplets. The curable segments of the generated Janus droplets were solidified continuously via off-chip photopolymerization using an ultraviolet (UV) light source  with an irradiation distance of approximately 15–20 cm. The products were filtered using a nylon mesh sheet  to remove smaller particles produced from satellite droplets. The particles were lightly washed using acetone and ethanol to remove residual non-curable fluid and dust.Droplet formation in the microfluidic device was monitored using an optical microscope  equipped with a high-speed video camera . The interfacial tensions between the two liquids were measured using the pendant-drop method . We used the software ImageJ  to measure the aperture diameters of Janus droplets. We also used a scanning electron microscope  to observe the shape of the polymer particles. Surface Evolver  was usedQm), SO (Qs), and the PVA solution (Qc) were 0.3, 0.3, and 8.0 mL h−1, respectively, the production rate was ~78 drops s−1  and SO (Ds) segments were measured, and they had narrow size distributions with coefficient-of-variation (CV) values of approximately 2% , Dm increased from 103 to 168 μm with the CV in the range of 1.0% to 2.2%, and Ds decreased from 162 to 75 μm with the CV in the range from 0.8% to 2.4%  [jkγ, jkγ, and jkγ are the three interfacial tensions at the three interfaces . In this study, indices 1, 2, and 3 were for the SO, external aqueous phase, and HDDA, respectively. When indices 1 and 3 for the two phases of droplets satisfy γ12 > γ23, S1 remains negative and the biphasic compound droplets dispersed in the external phase exhibit three morphologies at equilibrium: (1) S1 < 0, S2 < 0, S3 > 0: core-shell droplets (or fully engulfed), where phase 1 is completed engulfed by phase 2; (2) S1 < 0, S2 < 0, S3 < 0: Janus droplets ; and (3) S1 < 0, S2 > 0, S3 < 0: two separate drops. Previously, we reported that biphasic droplets comprised of HDDA and SO without surfactant formed a core-shell geometry at equilibrium in aqueous PVA solution even if their initial morphology was Janus ,22. In cj + γki) , where, γ31 = 2.2 mN m−1, γ12 = 10.8 mN m−1, and γ23 = 1.1 mN m−1, thereby causing S1 < 0, S2 < 0, S3 > 0, forming a core-shell structure [γ31 = 1.63 ± 0.07 mN m−1 and γ12 = 9.61 ± 0.06 mN m−1, while γ23 maintained a similar value of 1.13 ± 0.02 mN m−1 , which is inconsistent with the Janus morphology we confirmed in this study. One possible reason for this discrepancy is that there might be some HDDA molecules at the interface between the SO and the aqueous PVA phase, reducing the interfacial energy at the SO/water interface [γ′12) was estimated using the following equation [α and β are the contact angles, as shown in α = 10.4 ± 1.0° and β = 35.6 ± 0.6° (n = 5) in this case, and we obtained γ′12 ~2.17 mN m−1. This satisfies S1 < 0, S2 < 0, S3 < 0, and the condition for a Janus morphology.To illustrate this point, we measured the three interfacial tensions and calculated the three spreading parameters. In our previous study without a surfactant, we obtained tructure . Meanwhi1 n = 5, . These vequation :(1)γ12′=γ31, γ23, and γ′12  plot in the Janus region, with A, B, and C are in the region for the concave-convex HDDA segment, D nearly on the line of Y = X for the planar HDDA/SO interface, and E in the biconvex HDDA region. These results are in good agreement with the experimental results , the droplets floated at the air-water interface, maintaining their Janus morphology for a short period of time (t = 10 s). However, at t = 40 s, we found that many Janus droplets were already lost, suggesting their rupturing due to coalescence. In contrast, for the surfactant-laden Janus droplets in the aqueous PVA solution, no coalescence was observed at t = 40 s, suggesting better stability . Such a reduction in interfacial tension might also be responsible for the improved stability against coalescence. A similar phenomenon was reported in the literature, where the presence of phosphatidylcholine as a surfactant in the oil phase at a concentration of 3.0 wt.% reduced the interfacial tension at the oil/water interface to 0.5 mN m−1, resulting in more stable O/W Janus droplets with greater resistance to coalescence [This significantly improved stability of the surfactant-laden Janus droplets might be due to the molecular size of PVA, which is much larger than the SDS molecules, thereby causing steric hindrance to stabilize the droplets . This co3 mN m−1 ,13. Meanlescence . Qm/Qs = 1/2 and 2/1 were solidified, although the transition of the inner curvature between concave and convex surfaces was at 1.0 wt.% for Qm/Qs = 1/2 and 5.0 wt.% for Qm/Qs = 2/1. All the shapes of the particles agreed well with the shape of the HDDA segments in the precursor droplets as well as the results predicted by modeling (Polymer particles of various lens shapes were prepared from the surfactant-laden Janus droplets shown in modeling .Polymer particles of various microlens shapes can be prepared from similar surfactant-free ,13 or suThe presented approach could be further enhanced for the fabrication of more shape-anisotropic particles through the variation of the surfactant types, their concentrations, and the number of inputs for the curable or non-curable phases . MoreoveIn this study, we synthesized surfactant-laden Janus droplets comprised of two immiscible curable and non-curable segments with high stability against coalescence and tunable morphologies, suitable for the fabrication of lens-shaped microparticles. Monodisperse biphasic droplets of an acrylate monomer and SO containing surfactant were produced in an aqueous PVA solution via microfluidic flow focusing. Unlike similar biphasic droplets without surfactants that form a core-shell geometry, we found that the produced droplets had a uniform Janus geometry at equilibrium. Furthermore, the curvature radii of the interface between the two Janus lobes could be varied by varying the concentration of the loaded surfactant. In addition, the produced surfactant-laden Janus droplets had considerably higher stability against coalescence than the surfactant-free Janus droplets in the aqueous SDS solution. We believe that the presented approach provides a new route for fabricating novel particulate materials with precisely designed curvatures, which hold considerable potential as elements in micro optical applications.


After centrifugation, almost all motility parameters were significantly altered, as observed by computer-assisted sperm analysis. A polarographic assay of oxygen consumption showed a progressive decrease in mitochondria respiration from the gentlest to the strongest protocol. By laser scanning confocal microscopy, significant reduction of mitochondrial membrane potential, at any tested protocol, and time-dependent effects, at the same centrifugal force, were found. Increased DNA fragmentation index at any tested protocol and time-dependent effects at the same centrifugal force were found, whereas increased protein carbonylation was observed only at the strongest centrifugal force. These results provide more comprehensive understandings on centrifugation-induced effects on cryopreserved stallion sperm and suggest that, even at a weak force for a short time, centrifugation impairs different aspects of equine sperm metabolism and functionality.Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300×  Assisted reproductive technologies (ARTs) consist of all procedures for handling female and male gametes and leading to embryo production aimed to overcome infertility in humans. Conventional sperm selection techniques used in human ARTs are density gradient centrifugation (DGC), which separates spermatozoa according to their density, and swim up (SU), which separates spermatozoa according to their motility ,2. Such The horse is a suitable animal model for studying the efficacy of sperm selection methods and, due to the use of a wide range of ARTs ,13,14, iThe role of mitochondria in sperm motility and quality has been emerging in recent years ,32,33,34In this context, considering that information reported to date on the effects of centrifugation on sperm quality are controversial and mostly focused just on motility assessment, the aim of this study was to investigate the effects of different centrifugation protocols on cryopreserved stallion sperm motility and oxidative status in terms of oxygen consumption, MMP and oxidation-related injuries.All chemicals were purchased from Sigma-Aldrich unless otherwise indicated.7 sperm cells/mL, came from the Istituto Nazionale Fecondazione Artificiale, Cadriano, Granarolo Emilia, Bologna . Straws were rapidly thawed in water bath at 37 °C for 30 s and diluted in different solutions according to the experimental design. Subsequently, the total volume was split in 5 different tubes which were subjected to the following experimental conditions: i) no centrifugation (control); ii) centrifugation at 300× g for 5 min; iii) centrifugation at 300× g for 10 min; iv) centrifugation at 1500× g for 5 min; v) centrifugation at 1500× g for 10 min. Each experiment was performed in three independent replicates.Equine sperm samples (0.4 mL/straw) from a single stallion of proven fertility and frozen at the concentration of 8 × 106 sperm cells/mL) in prewarmed (37 °C) sperm-Chatot, Ziomet and Bavister medium ; the percentage of progressively motile spermatozoa ; the average path velocity , calculated as the curvilinear trajectory of the sperm head; the straight-line rectilinear velocity , calculated as the velocity of the sperm head along the trajectory between the first and the last spotted position; the curvilinear velocity , calculated as the velocity of the sperm head along the total covered distance; the beat-cross frequency , the average rate at which the curvilinear path crosses the average path; two velocity ratios, straightness (STR) related to the linearity of the average path and linearity (LIN) related to the linearity of a curvilinear path, calculated as the ratio between VSL and VAP and between VSL and VCL, respectively; the amplitude of the lateral head displacement . Furthermore, sperm velocity distribution was analyzed by identifying four sperm cell movement subcategories based on equine-specific VAP cut-off, as reported in the HTM-IVOS CASA system, version 12.3 software: percentage of rapid cells ; percentage of medium speed cells ; percentage of slow cells  and percentage of static cells . Sperm motility parameters were defined as previously reported in the IVOS software manual and in [Sperm motility was analyzed by the HTM-IVOS Computer-Assisted Sperm Analyzer (CASA), . Sperm samples were resuspended . Then, l and in ,44.g for 10 min and resuspended in isotonic salt medium . Oxygen uptake by hypotonically treated spermatozoa (10 × 106 sperm cells/sample) was measured at 37 °C by using a Clark-type oxygen probe  in the presence of mitochondrial respiratory substrates  and 0.76 µM of adenosine diphosphate (ADP). The rate of oxygen uptake by sperm mitochondria (V) was expressed as nmol O2 mL−1 × minute−1. The respiratory control ratio (RCR), an index of mitochondrial respiration efficiency, was calculated by dividing V3 (rate of oxygen uptake measured in the presence of respiratory substrates + ADP) by V4 .Sperm samples were subjected to hypotonic treatment for 1.5 h on ice as previously described . Later o6 sperm cells/sample) were resuspended in prewarmed (37 °C) PBS containing 200 nM of the probe and incubated for 15 min at 37 °C. The cell-permeant MitoTracker probe contains a mildly thiol-reactive chloromethyl moiety for labelling mitochondria and it is distributed in sperm inner mitochondrial membrane proportionally to mitochondria activity [Staining procedure with MitoTracker Green FM  and Laser Scanning Confocal Microscopy (LSCM) assessment of mitochondrial activity were performed as previously described by Moscatelli et al. . Sperm sactivity ,46. SampSperm sample were subjected to hypotonic sperm treatment (as described above) and three freezing/thawing cycles. The total amount of proteins was then quantified by the Bradford protein assay (Bio-Rad). The volume corresponding to 20 µg of proteins from sperm lysates was resuspended in sample buffer and used for oxidative protein damage evaluation by quantifying carbonyl groups on protein side chains using the OxyBlot TM Protein Oxidation Detection Kit (Merck Millipore), according to the manufacturer’s instructions and as previously reported by De Riccardis et al. . Briefly6 sperm cells/sample) were fixed in 4% PBS-buffered paraformaldehyde for 15 min at room temperature. Fixed spermatozoa were centrifuged (300× g for 5 min) to remove the fixative and then permeabilized with 0.25% Triton X-100 for 20 min. Samples were centrifuged again and washed twice with deionized water. Subsequently, 25 µL of each sample were spotted on a poly-l-lysine-coated glass slide and air dried. Sperm samples were incubated at 37 °C in a humidified chamber with TUNEL reagents consisting of the terminal deoxynucleotidyl transferase (TdT) enzyme, EdUTP nucleotides and the Alexa Fluor 488 picolyl azide dye. After incubation, sperm samples were washed with 3% Bovine Serum Albumin (BSA) in PBS. Total sperm nuclei were stained with 5 μg/mL Hoechst 33258 in 3:1 (v/v) glycerol/PBS, mounted on microscope slides, covered with cover-up micro slides, sailed with nail polish and kept at 4 °C in the dark until observation. Positive and negative controls were included in each replicate. Spermatozoa were observed by using a 63 × oil-immersion objective under a confocal laser microscope (LEICA TCS SP8) equipped with a 405 nm continuous wave diode laser for sperm nuclei observation (blue fluorescence) and a 488 nm continuous wave Argon laser for TUNEL positive cells observation (green fluorescence). Fluorescent emission was detected in the spectral window between 420 nm and 480 nm and between 500 nm and 550 nm, for Hoechst 33258 and Alexa Fluor 488 respectively. For each sample, 5 randomly fields were analyzed. DNA fragmentation index (DFI) was determined as the percentage of labelled cells (TUNEL positive) to the total cell number (Hoechst 33258) as previously reported by Hoogewijs et al. [Sperm DNA fragmentation was assessed by using the Click-iT Plus TUNEL Assay , following the manufacturer’s instructions. Briefly, sperm sample  to compare MitoTracker Green FM fluorescence intensity signals, motility parameters, RCRs and densitometric values between each treatment group and control. Percentages of spermatozoa showing DNA fragmentation were compared among groups by Chi-square test. Differences were considered significant when p < 0.05, for 300× g for 5 min vs. control; p < 0.0001, for 1500× g for 5 and 10 min respectively vs. control; p < 0.0001 vs. control; p < 0.0001 vs. control; Total motility was impaired at almost any tested centrifugation protocol (g for 5 min (p < 0.01), at 300× g for 10 min and 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control; VSL was significantly reduced after centrifugation at 300× g for 5 min (p < 0.001), at 300× g for 10 min and 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control; VCL was significantly reduced after centrifugation at 300× g for 10 min (p < 0.01) and at 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control. Additionally, velocity ratios were significantly reduced at any tested centrifugation protocols (g for 5 min (p < 0.01), at 300× g for 10 min (p < 0.001) and at 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control; LIN was significantly reduced at any tested centrifugation protocol (p < 0.0001 vs. control). However, BCF was significantly reduced at the strongest tested centrifugation protocols  were significantly reduced at almost any tested centrifugation protocol A. In detrotocols B. In parg for 5 min vs. 300× g for 10 min and 1500× g for 5 min and 1500× g for 10 min, p > 0.05).For any motility parameter analyzed, no significant differences were found between samples centrifuged at the same centrifugal force and different times (300× g for 5 min (p < 0.05 vs. control), 1.4 ± 0.1min for 300× g for 10 min (p < 0.01 vs. control) 1.2 ± 0.1 and 1.2 ± 0.0 for 1500× g for 5 and 10 min respectively (p < 0.001 vs. control). No statistical difference was found between samples centrifuged at the same centrifugal force and different times .Subsequently, we investigated the effects of different centrifugation protocols on RCR values, which are an index of mitochondrial respiration efficiency. RCR values were significantly reduced showing a linearity by increasing force and time of the centrifugation protocol . In detan = 172, n = 291, n = 232, n = 196, n = 174 for control, 300× g for 5 min and 10 min, 1500× g for 5 min and 10 min, respectively. Similarly to sperm mitochondrial respiration, sperm MMP was significantly reduced at any tested centrifugation protocols in terms of fluorescence intensity  and after centrifugation at 1500× g for 5 min vs. 10 min .We performed OxyBlot analysis in order to determine post-translational modification of sperm cellular proteins due to oxidative stress A. Quantig for 5 min (p < 0.05), at 300× g for 10 min and 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control. Interestingly, by comparing groups centrifuged at the same centrifugal force but different times, DFI significantly increased after centrifugation at 300× g for 5 min vs. 10 min (p < 0.0001) and after centrifugation at 1500× g for 5 min vs. 10 min , widely used to process sperm samples for ICSI ,49, and g or 900× g, while these parameters were altered after centrifugation at 4500× g [g for 10 min and in progressive motility after centrifugation at 400× g and 600× g for 10 min [As a first analysis, we evaluated sperm motility by CASA, and we found that almost any motility parameter was altered after centrifugation, including progressive motility and percentage of rapid cells useful for sperm fertilizing ability and VCL, VAP, LIN and STR that give information about sperm linearity and hyperactivation related to capacitation. Different results were obtained in previous studies in the stallion. Hoogewijs et al. found that, compared to uncentrifuged samples, centrifugation of semen resulted in a better sperm quality . Len et  4500× g . These c 4500× g . Anotherr 10 min . These rr 10 min , as wellr 10 min  and the r 10 min .g and 600× g for 10 min in rats sperm cells and after JC-1 staining [Sperm mitochondria have been recently indicated as biomarkers of sperm health and fertility , though staining . As descstaining ,37,38. Tg but we observed an increase in DFI.Aberrant mitochondria function may lead to enhanced ROS formation which in turn may damage cell molecules such as proteins and DNA . For thiTaken together, our data suggest that centrifugation, even weak and brief, impairs different aspects of stallion sperm functionality with negative impact on its fertilizing ability. Moreover, damage due to centrifugation is particularly severe in sperm samples with initial low quality. Even if a possible solution could be represented by the use of lower g forces, previous studies in other species demonstrated that centrifugation forces lower than those used in the present study, even not affecting sperm motility and MMP , induce


Campylobacter jejuni (CJ) is the most prevalent zoonotic pathogen of chicken meat and related products, which may lead to gastroenteritis and autoimmune diseases in humans. Although controlling this bacterium is important, CJ strains resistance against traditional antibiotic therapy has been increased. Vegetable oils and fats are natural biomaterials explored since the Ancient times, due to their therapeutic properties. Nanotechnology has promoted the miniaturization of materials, improving bioavailability and efficacy, while reducing the toxicity of loaded active molecules. In this work, a screening of 28 vegetable oils was firstly performed, in order to select anti-CJ candidates by the disc diffusion test. Thus, the selected liquid lipids were used as active molecules in nanostructured lipid carriers (NLC) formulations. The three resultant systems were characterized in terms of particle size (~200 nm), polydispersity index (~0.15), and zeta potential (~-35mV), and its physicochemical stability was confirmed for a year, at 25°C. The structural properties of NLC were assessed by infrared (FTIR-ATR) and differential scanning calorimetry (DSC) analyses. The spherical nanoparticle morphology and narrow size distribution was observed by transmission electron microscopy (TEM) and field emission scanning electron (FE-SEM) analyses, respectively. Then, the in vitro antimicrobial activity test determined the minimum inhibitory concentration (MIC) of each formulation against CJ strains, in both free (1–3 mg/ml−1) and sessile (0.78 mg/ml−1) forms. Finally, the in vitro biocompatibility of NLC was demonstrated through cell viability using VERO cell line, in which F6 was found twice less cytotoxic than pure olibanum oil. Considering the abovementioned achieved, F6 formulation is able to be evaluated in the in vivo anti-CJ efficacy assays. Campylobacter jejuni (CJ) is a Gram-negative commensal pathogen prevalent in the intestinal tract of chickens and other animals. Human infection is characterized by inflammatory bowel response, followed by bloody diarrhea and painful symptoms to the affected people and pets. In addition, Guillain-Barre syndrome can be a secondary sequelae, characterized by a potential fatal autoimmune disorder in humans  and their molecular structure was clarified. ®), totalizing 28 samples . The samples that exhibited some anti-CJ activity , for 2 min with an Ultra-Turrax blender . Then, the systems were ultrasonicated for 12 min in a Vibracell tip sonicator  operated at 500 W and 20 kHz, in alternating 30 s (on/off) cycles. Finally, the resultant formulations were cooled to room temperature. The resultant NLC and the concentration of each excipient used were displayed below . The parameters analyzed were nanoparticle size (nm), polydispersity index (PDI), and zeta potential (mV), evaluated by dynamic light scattering (DLS) using in a ZetaSizer ZS90 . The measurements were performed in triplicate (25°C) and ANOVA/Tukey tests were employed (p < 0.05) for statistical analysis, calculated by R software.in vitro antimicrobial activity tests were performed as follows: i) preliminarily disc diffusion test using IAL 2383 CJ strain; ii) determination of minimum inhibitory concentration (MIC) from free forms, through IAL 2383 and 10 (isolated from chicken meat) CJ strains; iii) determination of MIC from sessile forms organized in biofilms, using IAL 2383, IAL 468, and IAL 520 (isolated from chicken meat) CJ strains.The ®) separately, being incubated in microaerophilic for 48 h. Typical colonies were collected and diluted in 10 ml of sterile saline solution (0.9%), adjusted according 0.5 McFarland scale, and confirmed by the plate count . Each bacterial suspension was then diluted in 96-well plate wells reaching the final concentration of 1 × 105 CFU·ml−1 per well , were inoculated with CJ IAL 2383 suspensions adjusted to 0.5 McFarland, as already described. After 10 min, the impregnated discs were positioned over the agar. The plates were incubated in microaerophilic at 37°C for 48 h. After this period, the diameters of CJ growth inhibition zone (mm) was measured for each sample (n = 2)  before NLC preparation. Then, NLC were prepared and F6, F14, and F19 formulations were also tested in here. The experiment was carried out according to the standard M2-A8 from Clinical Laboratory Standard Institute . Briefly (n = 2) .®), supplemented with cation-adjusted  solution plus 5% lysed sheep blood (Laborclin®), in accordance with ISO 20776-121 (®), supplemented with cation-adjusted solution plus 5% lysed sheep blood (Laborclin®), as detailed above. Finally, the plates were incubated at 37°C, for 48 h in conditions of microaerophilia. After this period, the MIC values was determined based on the lowest concentration of formulation that prevented the bacterial growth. The minimum inhibitory concentration (MIC) was obtained for each formulation (n = 3). ANOVA/Tukey tests were the statistical methods adopted, determined by R software (p < 0.05).The minimum inhibitory concentration (MIC) of F6, F14, and F19 formulations were determined against free forms of IAL 2383 and 10 CJ strains, through microdilution method . IAL 2380776-121 . Then, d®) and transferred to 20 ml of Mueller Hinton broth (Difco®) supplemented with 5% chicken juice, being incubated at 37°C for 48 h under microaerophilic condition. Then, the bacterial suspension was standardized to an OD600 = 0.22 to 0.28 absorbance and centrifuged at 5,000 rpm for 10 min. After supernatant removal, the cells were washed twice, and the pellet was re-suspended in 0.9% NaCl solution. Thus, it was diluted in 10 ml of supplemented MH broth with 5% of chicken juice, obtaining a final count of 104 CFU/ml. CJ suspension (200 µl) was added to 96-well plates and incubated for 48 h at 37°C, under microaerophilic conditions. Afterwards, the non-adherent bacteria were washed twice with 0.9% sterile NaCl solution.The formation of biofilms was carried out according Sulaeman and co-workers , with moCampylobacter spp. broth microdilution method .®), supplemented with 10% fetal bovine serum, 10 mg/ml streptomycin (Sigma), 100 U/ml penicillin (Sigma®), and 40 mg/ml gentamycin, (Sigma®) at 37°C in a humidified atmosphere containing 5% CO2. VERO cells were seeded at a density of 5 × 104 cells/well in a 96-well tissue culture microplate. After incubation for adhesion, the cells were treated with different concentrations of F6, F14, and F19 and with previously emulsified O6, O14, and O19 oils as controls, at 37°C, 5% CO2 for 48 h. The cytotoxicity of the samples was assessed by resazurin assay (Sigma–Aldrich®). Then, 10 µl  of resazurin was added per well and incubated for 16–18 h at 37°C in a 5% CO2 incubator. The measurements were performed by a spectrophotometer (GloMax®) at 594 nm, and the cell viability was expressed as a percentage of viable cells at the end of the experiment (n = 3). Unpaired t-test was the statistical method used to compare significant differences between the formulation and its respective pure oil (p < 0.05).VERO cells (monkey kidney fibroblasts) were grown with Dulbecco’s modified eagle medium  were positioned in aluminum pans and the thermal transitions were assessed in the temperature range from 0 to 100°C, at a heating rate of 10°C/min, under nitrogen flow.In the ATR-FTIR technique, the spectra were recorded with FTIR spectrophotometer with ATR cell (BRUKER IFS 66 v/S or Perkin Elmer SPECTRUM 65) equipment. The spectra were operated in reflectance mode in the range of 4500-500 cmFor FE-SEM, the sample was adhered to a stub. After, the stubs were sputtered with gold bath for 120 s at 30 kV. The nanoparticles were visualized in a field JEOL electron scanning microscope (model JSM 5800LV), operating under a variable voltage from 0.3 to 30 kV, with tungsten filament, through the SemAfore 5.21 image capture system software. For TEM, the sample was added to a copper grid. Then, the sample dried with filter paper. The (2% w/w) uranyl acetate was added to provide contrast, and the excess of liquid was removed. Subsequently, deionized water was dropped to the grid, and the excess was removed. The nanoparticles were visualized in a Zeiss–LEO 906 TEM, operating at 60 kV and equipped with an Olympus iTEM CCD camera and image capture software.® and Pluronic 188®, used as surfactant and co-surfactant, respectively.A preliminary screening of 28 vegetable oils Table 1 in vitro antimicrobial activity tests against CJ strains, through the disc diffusion (free form) and MIC tests for free and sessile forms of CJ  than its respective controls Table 2.−1 for F6 and F14, and 1.25 mg/ml−1 for F19 formulation. The MIC results for 10 CJ (free form) were 2.60 mg/ml−1 for F6, and 1.56 mg/ml−1 for F14 and F19 formulations. Besides, in the MIC of sessile forms  determination, F14 and F19 were not able to control the bacterial growth of all the evaluated strains. Only F6 formulation showed ability to inhibit CJ growth in biofilm (sessile form), with MIC values of 0.78 mg.ml−1 in all the tested strains  values were determined for each sample. In general, it was not observed statistically significant differences between the IC50 values of F14 (0.2734 mg/ml) and F19 (0.1466 mg/ml) formulations compared with its respective free oils, O14 (0.3478 mg/ml) and O19 (0.1435 mg/ml), respectively. On the other hand, F6 exhibited IC50 value of 0.3529 mg/ml, being statistically significant higher (p < 0.05) than O6 control (0.1424 mg/ml). In addition, F6 exhibited the highest IC50 value among all the tested samples.The  and O19 Figure 2−1 (νO-CH2 and νCH), 1729–1742 cm−1 (νC = O), and 1109–1170 cm−1 (νC = O) in F6, olibanum essential oil (EO), and ucuuba butter spectra (−1 (νO-H) was not evident in F6 spectrum. DSC analysis showed the thermodynamic transitions of excipients and F6 formulation  from Somalia that has been applied as antimicrobial agent with success, exhibiting MIC values around 4.0–16.0, 1.5–8.3, 4.0–12.0, and 2.0–12.8 mg.ml−1 for Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Proteus vulgaris, respectively  matrix . In addiin vivo efficacy assays.These abovementioned results confirmed that NLC composed of ucuuba butter and EO is able to be evaluated as anti-CJ agent in specific in vivo assays.The use of natural lipids against CJ strains in free and sessile forms is a versatile approach to control and treat this virulent pathogen, especially those strains having antimicrobial resistance. However, the use of these lipids is limited by their physicochemical instability and toxicity. In this work, different NLC composed of natural lipids were prepared and they were shown to exhibit excellent long-term stability over a year at 25°C. Among all NLC, F6 formulation composed of ucuuba butter and olibanum essential oil, was selected as the system that combined the best anti-CJ activity and safety. Supramolecular organization of F6 was detailed, as shown by FTIR-ATR, DSC, TEM, and FE-SEM analyses, confirming an expected NLC typical structural profile. This promising system will be tested The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LR is the main author. She had the idea of developing the project and carried out the development of NLC in the laboratory. LR, GM, DR, BF, RM, and LG developed the MIC analysis and disk diffusion test. FM, LR, and BF performed the cell culture tests. LR, EP, and MB carried out the characterization, in the coordination of the development of the NLC. LR, BF, and FM contributed to the statistical analysis and writing of the article, LR, BF, LG contributed to the coordination of the project as a whole. All authors contributed to the article and approved the submitted version.This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–CAPES-(#88887.336865/2019-00) and National Institute of Science and Technology in Theranostics and Nanobiotechnology –INCT-Teranano .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.


Neurospora and Fusarium. NX biosynthetic genes and their regulation have been thoroughly investigated in Fusarium fujikuroi, an industrial fungus used for gibberellin production. In this species, carotenoid-overproducing mutants, affected in the regulatory gene carS, exhibit an upregulated expression of the NX pathway. Based on former data on a stimulatory effect of nitrogen starvation on carotenoid biosynthesis, we developed culture conditions with carS mutants allowing the production of deep-pigmented mycelia. With this method, we obtained samples with ca. 8 mg NX/g dry mass, in turn the highest concentration for this carotenoid described so far. NX-rich extracts obtained from these samples were used in parallel with carS-complemented NX-poor extracts obtained under the same conditions, to check the antioxidant properties of this carotenoid in in vitro assays. NX-rich extracts exhibited higher antioxidant capacity than NX-poor extracts, either when considering their quenching activity against [O2(1Δg)] in organic solvent (singlet oxygen absorption capacity (SOAC) assays) or their scavenging activity against different free radicals in aqueous solution and in liposomes. These results make NX a promising carotenoid as a possible feed or food additive, and encourage further studies on its chemical properties.Neurosporaxanthin (NX) is a carboxylic carotenoid produced by some filamentous fungi, including species of the genera  Blakeslea trispora and Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) are biotechnological sources for the production of β-carotene and astaxanthin, respectively  [3+)-ligand complex to ferrous (Fe2+) complex in acidic media [2(1Δg)] quenching capacity of an extract or a pure compound, and the high activity of carotenoids or carotenoids-enriched extracts capturing this reactive oxidizing intermediate was reported  . Among oic media . In the reported ,33,34,35reported . Within carS mutant of F. fujikuroi, and we have studied, by using different in vitro assays and liposomes, the antioxidant activity of cell extracts from the NX-enriched mutant, compared with those from the same strain to which the wild carS gene has been introduced, resulting in a very low NX content.This study aims to investigate the antioxidant properties of NX. For this purpose, we have settled suitable overproduction conditions of this xanthophyll by a Fusarium fujikuroi come from the Commonwealth Mycological Institute  and the Kansas State University Collection , respectively. The carS mutants SG22 and SG39 were obtained from IMI58289 by chemical mutagenesis  generated in organic solvent (SOAC assay); and (iv) scavenging activity against hydroxyl radicals (HO•) in liposomes.The antioxidant activity of carotenoid extracts from SG256  and SG39 (NX-enriched) strains was evaluated in this study by their: (i) reaction with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) (DPPH assay); (ii) ferric-reducing activity in aqueous solution (FRAP assay); (iii) quenching activity against singlet oxygen  quenching capacity, whereas EP generates [O2(1Δg)] by thermal decomposition in organic solvent. Absorbance changes of DPBF at 413 nm were monitored for 60 min at 35 °C in a microplate SPECTROstar Omega reader. A standard solution of α-tocopherol (αtoc) was used to calculate relative SOAC values as:The quenching activity of fungal carotenoid extracts was determined by the Singlet Oxygen Absorption Capacity Assay (SOAC), with some modifications ,33. BrieThe estimated concentration of NX in the final reaction mixture of SG39 samples was 21 µM, while in SG256 it was not detectable. For comparative purposes, a standard solution of β-carotene at final concentration of 26 µM in the reaction mixture was evaluated.2 atmosphere at −80 °C and protected from light to avoid oxidation. All solutions and extracts were prepared in chloroform (CHCl3). NX concentration in the extracts of SG39 and SG256, determined spectrophotometrically, were 25.2 µg/mL and undetectable, respectively.Standard solutions of β-carotene (βC 273.3 µg/mL) and β-apo-8′-carotenal (ApoC 283.3 µg/mL), chemical structures included in the scheme depicted in 3 were previously mixed with EYPC solutions  to reach a final concentration of 10 µM in the reaction mixture . Control liposomes lack carotenoids. Chloroform was subsequently removed from lipid-carotenoid solutions by vacuum in a round-bottom flask adapted to a rotatory evaporator apparatus working at a low speed and moderate heating (≤30 °C), to allow the formation of a homogeneous dried lipid film. The lipid-carotenoid film was stored overnight at −20 °C, in the dark, after flushing N2 to avoid oxidation.For preparation of unilamellar liposomes, egg-yolk phosphatidylcholine (EYPC) was selected for 1.5 mM liposome preparation, since EYPC is a major phospholipid component of biological membranes and contains a significant percentage of polyunsaturated fatty acids (PUFAs), which are the main targets for oxidative damage by free radicals. Standard and fungal carotenoids in CHCl11-BODIPY581/591 stock solution, in methanol, were added to multilamellar liposomes, followed by additional 5 min vortex and 10 min ultrasound bath, to reach a final concentration of 10 µM. After that, unilamellar liposomes were prepared by extrusion through 100 nm-pore polycarbonate membranes , at 37 °C, in a mini-extruder device . After 15 passes, a clean, transparent and homogeneous unilamellar 1.5 mM EYPC liposomes suspension was obtained and immediately used in the oxidation assays.In the next day, multilamellar liposomes were prepared by adding 50 mM phosphate saline buffer (PBS), pH 7.5, then agitated in vortex for 5 min, and exposed for 10 min in an ultrasound bath, at room temperature. After lipid-carotenoid film hydration, 25 µL of the free radical-sensitive probe C2O2 with 1.5 mM Fe2+:6 mM EDTA (Fenton reaction) in 50 mM PBS buffer, pH 7.5, 37 °C. Liposome oxidation was monitored for 170 min by the fluorescence decay at 600 nm of the pre-loaded C11-BODIPY581/591 probe in liposomes, as aforementioned   in the absence of Fe2+ to trigger the Fenton reaction (data not shown).Control experiments were carried out to characterize the oxidation pattern of Creatment . As show11-BODIPY581/591 probe (2O2 + 1.5 mM Fe2+:6 mM EDTA) in 50 mM PBS buffer, pH 7.5. The extension of membrane oxidation was quantified from t0 to t170, as the area above curves of The index of membrane oxidation was determined by the fluorescence decay of C91 probe a in 1.5 F. fujikuroi  in organic solvent, and their scavenging activity against different free radicals in aqueous solution and in liposomes ]. Therefore, the observed quenching activity here is directly related to their peculiar electronic-conjugated polyene structure, as all carotenoids are loosely diffusing in organic solvent, according to SOAC methodology [2(1Δg)] [2(1Δg)] quencher in biological systems [The observed quenching activity of SG39 extracts in organic solvent (SOAC assay) is a result of the inherent capacity of NX and other minor carotenoids (including molecular interactions between them) to suppress excitatory energy from [Ohodology . For comhodology . It has [2(1Δg)] , about t[2(1Δg)] , catechi[2(1Δg)] . Therefo[2(1Δg)] . There a[2(1Δg)] . Based o systems .2+ ions , affecting free radical formation in aqueous solution  in organic solvent assays, (SOAC), which were at least as efficient as β-carotene. Similarly, the NX-rich extracts showed high scavenging activity against HO• radicals in liposomal systems. Although more tests are needed to confirm NX efficacy, in overall, these data are very promising regarding a future use of NX as a carotenoid with beneficial properties as a feed or food additive.NX is an unusual C35 carboxylic xanthophyll found only in some filamentous fungi. Since it is absent in human diet, its antioxidant properties have received no attention. For this purpose, it is convenient to have a biotechnologically appropriate NX source. Here, we show that carotenoid overproducing mutants available in the fungus


The consumption of contaminated shellfish with marine toxins causes adverse socioeconomical, environmental and health impacts. The marine toxin okadaic acid (OA) provokes diarrhetic shellfish poisoning (DSP) syndrome characterized by severe gastrointestinal symptoms. Therefore, there is increasing interest in removing these toxins from the marine environment to protect shellfish harvesting sites. Photocatalysis is proposed as an efficient method to detoxify the marine environment.Prorocentrum lima was used to produce high purity DSP toxins, in particular OA, for degradation studies. The profiling, characterization and quantification of DSP toxins in the culture of P. lima were achieved by ultrahigh performance liquid chromatography coupled to quadrupole-time of flight mass spectrometry (UPLC-QTOF-MSE) for accurate-mass full spectrum acquisition data. The effectiveness of UV/TiO2 system to degrade OA in seawater was assessed in lab-scale experiments and identification of transformation products was proposed based on the data obtained during analysis by UPLC-QTOF-MSE. The detoxification potential of the UV/TiO2 system was investigated using the phosphatase inhibition assay.In this study, 2 > 0.96). High-resolution mass spectrometry analysis of the photocatalyzed OA allowed tentative identification of four transformation products. Detoxification was achieved in parallel with the degradation of OA in deionized water and artificial ocean water (≤20 min) but not for seawater. Overall, results suggest that UV/TiO2 photocatalysis can be an effective approach for degrading OA and their TPs in the marine environment.Sufficient amount of high-purity OA  was produced in-house for use in photocatalysis experiments by simple reversed-phase flash chromatography. Complete degradation of OA was observed in seawater after 30 min and 7.5 min in deionized water. The rate constants fitted with the pseudo-first order kinetic model (RTo the best of our knowledge, this is the first report on the use of photocatalysis to degrade marine toxins and its promising potential to protect shellfish harvesting sites.  •P. lima by simple reversed-phase flash chromatography.High purity okadaic acid produced in-house from •2 applied to the degradation of the marine toxin, okadaic acid.UV/TiO•2 in 30 min in seawater.Okadaic acid was degraded by UV/TiO•E.Transformation products were characterized by UPLC-QTOF-MS•2 was demonstrated by protein phosphatase inhibition assay.Reduction in toxicity after UV/TiO Marine toxins are responsible for >60,000 intoxications per year, with overall mortality of around 1.5% . Based oProrocentrum  and Dinophysis  are the main producers of DSP toxins  (There has been an increased interest in finding suitable techniques to remove these toxins from the marine environment. Advanced oxidation processes (AOPs) are widely used to degrade organic pollutants through free radicals as reviewed by . Of diffotoxins) . Major iotoxins) , the useotoxins) , the addotoxins)  or the fotoxins) . Howeverotoxins) .P. lima for photocatalysis experiments; ii) to evaluate the performance of a UV/TiO2 system to degrade/mineralize OA in seawater; iii) to identify possible transformation products (TPs) generated during photocatalysis; iv) to evaluate the detoxification efficiency of UV/TiO2 system for OA in seawater.This work aimed i) to produce cost effective amounts of high-purity DSP toxins from 22.12-P25 from Degussa (Germany) was used as catalyst. It is a mixed phase containing ≈90% anatase and ≈10% rutile with a specific surface area of 50 m2/g. OA (4.2 μg in 0.5 mL ampoule) and DTX1 (4.2 μg in 0.5 mL ampoule) standards for confirmation purposes were obtained from National Research Council Canada  . Instant ocean salt was obtained from Aquarium systems (France). SNAP C18 120 g cartridges were bought from Biotage . Cellulose acetate centrifuge tube filters (0.45 μm), p-nitrophenyl phosphate (pNPP) and the protein phosphatase-1 catalytic subunit (PP1c) from rabbit  were supplied by Sigma-Aldrich .LC-MS grade acetonitrile, methanol and formic acid were purchased from Sigma-Aldrich . Deionized water (DW) was provided by a Milli-Q system . TiO2.2P. lima CCAP 1136/11  was grown on seawater enriched with f2-Si medium  (culture-methanol-water) and loaded onto a SNAP C18 120 g cartridge (Biotage) to concentrate the toxins after tangential flow filtration . This process was repeated twice with the cell residue. Toxins were fractionated using a Biotage Selekt Flash Purification System . The mobile phase had a flow rate of 30 mL/min and a linear gradient from 10 to 100% MeOH over 90 min. Fractions (100 mL) were automatically collected using UV detection (220–254 nm). DSP content in each fraction was analysed by ultrahigh performance liquid chromatography coupled to quadrupole-time of flight mass spectrometry (UPLC-QTOF-MSE). OA containing fractions were pooled, dried down in a centrifugal evaporator  and stored at −20 °C. 35 days . Harvest2.3P. lima culture, were prepared in methanol and in methanol/water  and kept at −20 °C, 4 °C and room temperature (20–22 °C). Effect of high temperature (45 °C) and evaporation was also tested in an additional aliquot of OA (10 μg/mL). Samples were analysed by UPLC-QTOF-MSE at day 0, 7, 14, 21, 28, 35 and 42.Aliquots of OA (10 μg/mL), isolated from the 2.4P. lima culture, were maintained under magnetic stirring at 20–22 °C for 60 min. Experiments were run in triplicate in the dark and under UV irradiation in the presence and absence of the catalyst TiO2 . The experiments were performed at pH 6.9 in DW, 7.3 in AOW and 7.8 in SW. The suspension was kept in the dark under stirring during 5 min (adsorption-desorption phase) and afterwards the UV lamps were switched on. At selected time intervals  samples (0.5 mL) were collected and filtered through a 0.45 μm filter before UPLC-QTOF-MSE analysis.Photocatalysis experiments were conducted in a lab-scale photoreactor  equipped2.5E  equipped with an electrospray ionization (ESI) source. Separation was carried out on a C18 BEH column  at 40 °C. Mobile phase was acetonitrile with 0.1% FA (B) and water with 0.1% FA (A) at a flow rate of 0.2 mL/min. Gradient elution was as follows: 20% B initial conditions rising to 70% B at 9.50 min, increasing further to 100% B at 10 min, holding until 11 min, dropping back to 20% B at 12 min and holding until 15 min. Injection volume was 5 μL. Samples were kept at 5 °C.OA and related compounds were quantified by UPLC-QTOF-MSE acquisition, that allows both precursor and product ions to be acquired simultaneously. Parameters were: 3.2 kV capillary voltage, 40 V cone voltage, 80 °C source temperature, 350 °C desolvation temperature, 50 L/h cone gas flow, 600 L/h desolvation gas flow. Argon was used as the collision gas. MSE consisted in 3 functions: the first function used low collision energy 6 V, the second function used a collision energy ramp of 20–50 eV and the third function acquired the lock mass data for online mass calibration. Sodium formate was used as calibration solution over a mass range of m/z 50–2000. Leucine enkephalin (m/z 556.2771 for positive electrospray mode) was infused at a flow rate of 20 μL/min at 10 s intervals to correct for mass drifts. Acquisition and processing of MS data was done using MassLynx v 4.1 software (Waters).The Q-TOF was operated in positive ESI mode using MS2.62 and 0.2 mM MnCl2. The PP1c from rabbit  was prepared in a buffer (pH 7.4) containing 50 mM Tris-HCl, 1 mg/mL bovine serum albumin (BSA), 1 mM MnCl2 and 2 mM dithiothreitol. One unit hydrolyses 1 nmol of pNPP per minute at pH 7.4 at 30 °C according to the manufacturer's instructions . The assay was conducted in flat-bottom 96-well plates. Ten μL of OA  or 10 μL of each sample was placed in each well in triplicate and 10 μL of PP1 solution was added. After 4 min incubation time at room temperature the reaction was initiated by addition of 180 uL of pNPP stock solution. The plate was incubated at 37 °C immediately. PP1 activity was determined by measuring the absorbance at 405 nm at 4 min intervals for 40 min using a microplate reader  and the Gen5 software for data acquisition . IC50 determinations were calculated using four-parameter, variable slope, non-linear regression analysis .Phosphatase enzymes are able to hydrolyze pNPP into p-nitrophenol that can be measured by absorbance detection. The colorimetric assay was performed as described in 2.750), the 48 h Daphnia magna LC50, the bioaccumulation factor and the developmental toxicity were chosen as toxicological endpoints. Consensus method was selected as toxicity is estimated by taking an average of the predicted toxicities from five different QSAR methodologies  and Nearest Neighbor).The toxicity of OA and its TPs was predicted by using the Toxicity Estimation software Tool  from the US Environmental Protection Agency . This so33.1Prorocentrum have been confirmed to produce DSPs. Therefore, the culture P. lima was assessed as a viable source of DSP toxins, in particular OA, for the photocatalysis experiments.Many marine toxins have very complex structures that cannot or are prohibitively expensive to be synthesised. On the other hand, the high cost of commercially available toxin standards  make lab-scale studies requiring high amounts (≈3–5 mg) of toxin unfeasible. Several benthic species of the genus P. lima culture was analysed using high resolution mass spectrometry in positive mode (E fragmentation showed the presence of OA (Rt 9.20 min) and DTX1 (Rt 11.15 min) in the culture of P. lima. Identification was done by comparison of retention time and fragmentation patterns of commercially available standards of OA and DTX1  with the samples. The low energy (LE) spectrum (Rt 9.20 min) of OA in the P. lima culture  spectrum (Rt 9.21 min) shows an abundant ion at m/z 827.4575 [M + Na]+, the characteristics losses of water at m/z 787.4630, 769.4552, 751.4434, 733.4316 and 715.4200 and the specific fragments at m/z 429.2278, 305.2069 and 287.1950 (t 11.15 min) shows the protonated ion m/z 819.4935, the Na+ adduct at m/z 841.4717, the first loss of H2O at m/z 801.4843 and the multimer Na+ adduct at m/z 1660.9813. The HE (Rt 11.16 min) spectrum shows the Na+ adduct the consecutives losses of H2O at m/z 801.4865, 783.4685, 765.4592 and 747.4487, and the fragments 267.1255, 301.2088, 401.2854, 445.3135, 489.3397, 533.3653, 577.3853, 621.4148. In addition, a peak coeluted 0.3 min later than DTX1. The LE spectrum (Rt 11.20 min) of the coeluting peak showed a signal at m/z 1057.7203 and 819.4976. No standard was available for comparison but the mass spectra suggested that the coeluting compound is an analogue derived from DTX-1 and maybe a novel diol ester. The LE and HE spectrum showed the following ions m/z: 819.4938 [DTX1 + H]+, 841.4723 [DTX1 + Na]+, 801.4844 [DTX1 + H-H2O]+, 783.4653 [DTX1 + H-2H2O]+, 765.4743 [DTX1 + H-3H2O]+, 1659.9741 [2DTX1 + Na]+ and the fragments at m/z 401.2881, 445.3150, 489.3403, 533.3678, 577.3889 (corresponding to losses of -CO2)  .Fig. 2IdE in fractions 16–19 (60–70% MeOH content), DTX1 in fractions 18–21 (67–77% MeOH content) and DTX1 analogue in fractions 21–23 (77–83% MeOH content). Fraction with the highest purity and content of OA was 17 , for DTX1 fraction 19  and for DTX1 analogue fraction 22 . Prorocentrum species produce different DSP content depending on the species and/or strains  that can be easily isolated and purified (>90%) to be used in photocatalysis studies using a more simple extraction and purification process.Overall, 2 L of concentrated cells contained ≈84 mg of OA, ≈23 mg of DTX1 and ≈17 mg of the analogue derived from DTX1. Toxins were fractionated using a Biotage Selekt Flash Purification System and 28 fractions were obtained. OA was identified by UPLC-QTOF-MS strains . Previou3.23.2.1Dinophysis bloom.OA showed little change in methanolic solutions or deionized water at different temperatures  over time (42 days) . Heating to 45 °C also resulted in no losses of the toxin. OA was found to be very stable. A similar outcome was reported in previous studies. 2) experiments were performed to explore the effectiveness of this technique to degrade the toxin and subsequently its potential application in the marine environment.As a result of the persistence of the free OA toxin in water bodies, lab-scale photocatalysis (UV-TiO2 which result in a higher or lower adsorption capacity. Within the pH range of the samples here used (6.9–7.8) both OA (pKa 3.87) and the TiO2 surface (point of zero charge is pH 6.5) carry negative charges and adsorption on the catalyst surface is suppressed. On the other hand, it was found that OA was completely removed after 7.5 min, 12.5 min and 30 min in DW, AOW and SW, respectively using UV/TiO2 0.1% w/v  (o/Ct) = k t; where Co and Ct are the concentrations of OA in the solution at time 0 and t (min), respectively, k is the rate constant (min−1). The rate constant was 0.437 min−1 in AOW and five times lower in SW (0.088 min−1). In the case of DW, due to the fast degradation of OA, there was only three time points to calculate the rate constant so the uncertainty of the results is high. The rate constant in DW was 0.498 min−1 at with this pseudo-first-order model but the R2 was 0.70 . Interestingly DW and AOW showed similar rate constants, despite the amount of salts present in AOW. In photocatalytic processes the presence of ions has been reported as a negative effect, at concentrations as low as 10−3 M, due to competitive adsorption at the active sites of the catalyst  D: ln. on water  as well on water .3.2.22. Overall, four peaks were observed at tR 7.2, 7.5, 8.1 and 10.2 min. Time-dependent evolution profiles of TPs during UV/TiO2 processes are shown in Transformation products (TPs) were formed and removed during the photocatalytic degradation of OA using UV/TiOE fragmentation . The peak at 10.2 min with a protonated m/z of 759.4781 differs in 45 Da with OA (m/z 805.4775). The tentative identification points to norokadanone generated by the decarboxylation of OA. The spectrum  shows the characteristic losses of water , the Na+ adduct (m/z 781.4587) and the multimer 2M + NH4+ (m/z 1534.9861). The peaks eluting at 7.2, 7.5 and 8.2 min are minor compounds and share the same spectra (m/z 819.4547 [M + H]+). Although OA methyl ester and 35S DTX1 are isomers the LE and HE spectra show characteristic ions of DTX1  that indicates that they could be isomers of DTX1   .3.2.3Finally, it is important to determine if the photocatalytic system was able to detoxify the seawater. OA and DTxs are potent serine/threonine protein phosphatase inhibitor, for that purpose, the phosphatase inhibition assay was carried out.50 value of OA was 1183 nM, which is higher than the ones reported earlier (IC50 3.6–315 nM)  is shown in Fig. S5. The optical density (OD) increased linearly with increasing phosphatase concentrations  and with time at a fixed concentration of PP1 (5 μg/mL) . The concentration-inhibition curve of OA  in the presence of PP1 (5 μg/mL) is shown in Fig. S5C. Under these conditions, the IC–315 nM) . IC50 va0 could not be ascribed to the formation of toxic intermediates which appeared afterwards. At 10 and 20 min, complete detoxification of the irradiated solution was achieved in DW and AOW, respectively, demonstrating the efficiency of heterogeneous photocatalytic oxidation in the elimination of acute toxicity of OA under the investigated conditions. On the other hand, at 60 min, PP1 activity was 64% in SW when the investigated compounds were removed after 30 min.The potential toxicity of the samples before and during the heterogeneous photocatalytic degradation of OA was evaluated by the phosphatase inhibition assay. Results are presented as percentage of activity of PP1 in Findings from It is important to note that not all the DSP toxins, even when they are structurally related, show the same biological negative effects and the mode of action is still not clear. For example, it was reported that phosphatase inhibition was responsible for the diarrhetic events . However4Prorocentrum lima proved to be a viable source of DSP toxins, in particular OA. Two liters of concentrated cells contained 84 mg of OA, 23 mg of DTX1 and 17 mg of the analogue derived from DTX1. OA could be easily isolated and purified. This is the first study that assessed the effectiveness of UV/TiO2 system to degrade OA. The marine toxin was completely degraded after 30 min in seawater and even faster in deionized water (7.5 min). The degradation follows a pseudo-first order kinetic type. Four TPs, norokadanone and three possible isomers of DTX1, were identified in the photocatalytic UV/TiO2 system. Detoxification was parallel to OA degradation in deionized and artificial ocean water but not for seawater.2 photocatalysis can be an effective approach for degrading OA and their TPs in the marine environment. Further research should be conducted to look for less energy demanding system that could be easily scaled up. It would be worthy to explore: (i) UV-LED as they can provide energy saving, long lifetime, and environment-friendliness (Hg free); (ii) doped TiO2; (iii) different supporters for the catalyst; (iv) solar light activated materials; (v) degradation of intracellular and extracellular toxins.Overall, results suggest that UV/TiODolores Camacho-Muñoz: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing. Linda Ann Lawton: Conceptualization, Methodology, Validation, Resources, Writing - review & editing, Funding acquisition. Christine Edwards: Conceptualization, Methodology, Validation, Resources, Writing - review & editing, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


To investigate associations between a high genetic disease risk and disease severity in patients with systemic lupus erythematosus (SLE).Patients with SLE  and healthy controls (n=2802 and n=9859) were genotyped using a 200K Immunochip single nucleotide polymorphism array. A genetic risk score (GRS) was assigned to each individual based on 57 SLE risk loci.–86 and OR 7.48 (6.73 to 8.32), p=2.2×10–304 for the discovery and the replication cohorts, respectively). In the discovery cohort, patients in the high GRS-quartile had a 6-year earlier mean disease onset (HR 1.47 (1.22 to 1.75), p=4.3×10–5), displayed higher prevalence of damage accrual (OR 1.47 (1.06 to 2.04), p=2.0×10–2), renal disorder (OR 2.22 (1.50 to 3.27), p=5.9×10–5), anti-dsDNA (OR 1.83 (1.19 to 2.81), p=6.1×10–3), end-stage renal disease (ESRD) (OR 5.58 (1.50 to 20.79), p=1.0×10–2), proliferative nephritis (OR 2.42 (1.30 to 4.49), p=5.1×10–3), anti-cardiolipin-IgG (OR 1.89 (1.13 to 3.18), p=1.6×10–2), anti-β2-glycoprotein-I-IgG (OR 2.29 (1.29 to 4.06), p=4.8×10–3) and positive lupus anticoagulant test (OR 2.12 (1.16 to 3.89), p=1.5×10–2) compared with patients in the low GRS-quartile. Survival analysis showed earlier onset of the first organ damage (HR 1.51 (1.04 to 2.25), p=3.7×10–2), first cardiovascular event (HR 1.65 (1.03 to 2.64), p=2.6×10–2), nephritis (HR 2.53 (1.72 to 3.71), p=9.6×10–7), ESRD (HR 6.78 (1.78 to 26.86), p=6.5×10–3) and decreased overall survival (HR 1.83 (1.02 to 3.30), p=4.3×10–2) in high to low quartile comparison.SLE was more prevalent in the high, compared with the low, GRS-quartile (OR 12.32 (9.53 to 15.71), p=7.9×10A high GRS is associated with increased risk of organ damage, renal dysfunction and all-cause mortality. Our results indicate that genetic profiling may be useful for predicting outcomes in patients with SLE. The field of genetics has been revolutionised by genome-wide association studies, with over 100 genetic loci associated with systemic lupus erythematosus (SLE) discovered.Genetic risk scores have shown promise for understanding the polygenic contribution to many complex diseases but have been scarcely investigated in SLE.In the present study, we demonstrate that a high genetic risk is associated with an early onset of SLE, increased organ damage, cardiovascular disease and end-stage renal disease, as well as impaired survival.Our results suggest that genetic profiling of patients with SLE may be useful for predicting outcome of the disease.Systemic lupus erythematosus (SLE) is a chronic disease characterised by loss of tolerance to self-antigens, formation of immune complexes and an activated type I interferon system.STAT4), have displayed association with nephritis, ischaemic stroke, severe renal insufficiency and a younger age at disease onsetFamilial aggregation and twin studies provide compelling evidence of genetic predisposition in SLE, with a more than 10-fold higher concordance rate for monozygotic than for dizygotic twins.Comprehension of the genetic contribution to permanent organ damage is important for understanding the pathogenesis of SLE. Additionally, prediction of disease outcome is essential for optimising monitoring and treatment strategies, to reduce both unnecessary side-effects and long-term disease complications. Genetic risk scores (GRSs) have been applied in several fields of medicine, and studies have demonstrated their ability to predict matters like cardiovascular disease, prostate cancer risk and body mass index scores.et al.The discovery cohort included 1001 patients from the University clinics in Uppsala, Linköping, Karolinska Institute (Stockholm), Lund, and from the four northern-most counties in Sweden. All subjects fulfilled ≥4 ACR-82 classification criteria for SLE and were of European descent.10.1136/annrheumdis-2019-216227.supp1Supplementary data10.1136/annrheumdis-2019-216227.supp2Supplementary dataGenotyping of the discovery cohort was performed using the Illumina 200K Immunochip SNP array by the SNP&SEQ Technology platform at Science for Life Laboratory in Uppsala, Sweden. For quality control (QC) procedures, see online supplementary file.et al.Cumulative GRSs were assigned to each individual based on SNPs with previous association with SLE at genome wide significance in the European population from the publication by Chen https://www.ebi.ac.uk/gwas/. A RAC and GRS was assigned to each patient and control using the same 57 SNPs and OR as in the discovery cohort analysis, see et al.Individuals in the replication cohort were independently genotyped using the Illumina 200K Immunochip SNP array, available at We used ordinal or logistic regression to assess differences in prevalences between groups. Age was included as a covariate in all analyses, and significant results were subsequently analysed in a second model, with the age at SLE diagnosis as an additional covariate. The generalised Wilcoxon test was employed to assess differences in survival. For more information on statistical analysis, see online supplementary file. Statistical analyses were performed using R.–67). To test whether the difference between groups would increase when considering the contribution to SLE by each SNP, a weighted GRS was constructed. Similar to the RAC, the GRS followed a Gaussian distribution with higher mean scores in patients than in controls (mean (SD) 8.52 (1.20) compared with 7.45 (1.20)) (–86). Moreover, patients with a GRS in the high quartile received their SLE diagnosis significantly earlier in life, with a mean age at SLE onset in the high and low quartiles of 33 and 39 years, respectively ) . In the  (1.20))  and was ectively .comparison=1.4×10–14). In addition, the prediction accuracy of the GRS was higher in patients<20 years at SLE onset  (We subsequently employed receiver operating characteristic (ROC) curve analysis to compare prediction accuracies of the scores. The GRS was significantly better than the RAC at discriminating between patients and controls (area under the ROC curve (AUC) 0.78 compared with 0.71, pt onset) .–213) and the GRS (OR 7.48 (6.73 to 8.32), p=2.2×10–304). comparison=2.2×10–16) , p=5.47×102×10–16) .10.1136/annrheumdis-2019-216227.supp3Supplementary data–1).Because the GRS was superior to the RAC in discriminating between patients and controls, subsequent analyses focused on this score. The GRS was analysed as a continuous variable in all regression analyses, with –2). –2), with affected individuals acquiring their first damage at a mean age of 43 years, compared with 51 years in the low GRS-quartile  (–2)   with the–3) . Patient–3) (–2) .–2), with a mean age at the first event in affected individuals of 45 years, compared with 51 years in the low GRS-quartile (–3), but not their first VTE (p=3.0×10–1)  .–5 and p=3.6×10–4, respectively), with doubled odds of each manifestation in the high-to-low GRS-quartile comparison (–7), with a mean age at nephritis onset of 31 years, compared with 39 years in the low GRS-quartile (–2)  . In addiile (–2) . We subsile (–2) .–3), with more than doubled odds of being triple positive . The GRS was not significantly associated with APS; however, patients in the high GRS-quartile were more likely to have a positive aPL test  for our most important findings . The GRS2GP-I-IgG as well as presence of ≥3 aPLs  and PRDM–ATG5 (rs6568431) risk variants were associated with increased SDI scores (OR 1.29 (1.10 to 1.52), p=2.9×10–3 and OR 1.31 (1.11 to 1.55), p=1.4×10–3, respectively) whereas TMEM39A (rs1132200) displayed an association with lower SDI scores (OR 0.70 (0.55 to 0.90), p=1.4×10–3).Finally, all SNPs included in the GRS were analysed individually for association with the SDI. The Our study is the first to demonstrate an association between high cumulative genetic risk and survival, organ damage, cardiovascular disease, proliferative nephritis, ESRD and antiphospholipid antibodies in patients with SLE, introducing GRSs as a potential tool for prediction of disease severity. We employed both a weighted GRS and an unweighted RAC for our analyses, and their similar prediction accuracies regarding most outcomes—including organ damage and mortality—suggest that the added effect of multiple loci plays a more central role in the contribution to disease severity than the individual contribution by any high risk SNP.The present study confers three important findings that may aid in explaining the association of the cumulative genetic risk with organ damage. First, we demonstrate that a high GRS is associated to an earlier onset of CVE, which is an important component of the SDI.et alet al.Our demonstration of a 6-year difference in SLE onset between the high and low GRS-quartiles supports previous findings by both Taylor STAT4 variant has previously been associated with a more severe disease phenotype including ischaemic stroke and increased SDI scores.ATG5 gene encodes a protein involved in autophagy.We found two individual variants positively associated with increased organ damage. The In analysis of the HLA-GRS, we found a negative association with aPLs and clinical APS. The reason for this may be that the DRB1*03:01 tag SNP rs1269852, due to its high prevalence and OR for SLE in our cohort, made a substantial contribution to the total score. Patients carrying this SLE-HLA allele are less likely to carry the DRB1*04 and *13 alleles, which are associated with secondary APS.The strength of our study is the large population including more than 1000 well-characterised patients with SLE, the comprehensive collection of clinical data and the long mean disease duration, allowing for long time follow-up of damage accrual. The validation of the GRS in a population including more than 15 000 patients and controls also confirms the significance of the cumulative genetic score. There are, however, some limitations. The retrospective approach of our study may confer a falsely low difference in overall survival between patients with high and low GRS, as only patients deceased after year 2000 are included in our study population. In addition, we lacked data regarding cumulative prednisolone dose and cumulative disease activity, which are important risk factors for the development of organ damage.Despite displaying moderate accuracy in the prediction of the examined manifestations, the combination of their relatively high prevalence, their severity and the benefit of early detection indicates a clinical relevance to the GRS. For example, an ESRD screening test with a GRS cut-off level of 9.5 would generate 22% positive samples, of which 31% would develop the complication compared with 5% of negative cases. Importantly however, the present study explores a GRS weighted by ORs for SLE rather than for renal manifestations. As there are several SNPs associated specifically with lupus nephritis,In conclusion, a high GRS is associated with a more severe SLE phenotype involving an earlier onset of the disease, more organ damage and renal dysfunction, as well as impaired survival. Our results indicate that genetic profiling may provide a tool for predicting disease outcome and thus aid in the clinical decision process.


Codium tomentosum is a green macroalgae with high biotechnological potential, due to its rich lipidome, although few studies have addressed it. This study aimed to investigate the seasonal changes in lipid and pigment profiles of C. tomentosum, as well as to screen its antioxidant activity, in order to evaluate its natural plasticity. Samples of C. tomentosum were collected in two different seasons, early-autumn (September/October) and spring (May), in the Portuguese coast (wild samples), and in a land-based integrated multitrophic aquaculture (IMTA) system (IMTA samples). Total lipid extracts were analysed by LC–MS, GC–MS, and HPLC, and antioxidant activity was screened through free radical scavenging potential against DPPH and 2,20-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals. Wild samples showed a high seasonal variability, modifying their lipidome and pigment profiles according to environmental shifts, while IMTA samples showed a relatively stable composition due to early-stage culturing in controlled conditions. The lipids that contributed the most to seasonal discrimination were glycolipids  and the lyso forms of phospholipids and glycolipids. Lipid extracts showed antioxidant activity ranging from 61 ± 2 to 115 ± 35 µmol Trolox g−1 of lipid extract in DPPH assay and from 532 ± 73 to 927 ± 92 µmol Trolox g−1 of lipid extract in ABTS assay, with a more intense antioxidant activity in wild spring samples. This study revealed that wild specimens of C. tomentosum presented a higher plasticity to cope with seasonal environmental changes, adjusting their lipid, pigment, and bioactivity profiles, while IMTA samples, cultured under controlled conditions, displayed more stable lipidome and pigment compositions.In the last decades, the use of algae in biotechnology and food industries has experienced an exponential growth.  Edible seaweeds are known to be rich in bioactive metabolites, some of them not present in land plants [Oceans host an impressive wealth of life forms that harbor unique biochemical diversity. Several studies have recently confirmed the high potential of marine organisms as source of bioactive compounds, with interest for the food, fodder, pharmaceutical, biomedical and cosmetic industries ,2,3,4. Ad plants . These md plants ,2, antimd plants , and antd plants , holdingd plants  and for d plants , among od plants ,11,12, bd plants . Metabold plants ,15,16,17d plants . The undIn occidental societies, edible seaweeds do not constitute a significant portion of the human diet, although its consumption has increased in the last years due to the high valorization of oriental cultures and the recognition of the functional benefits of these "plants" from the sea. Progressively, seaweeds are being associated with functional and fortified foods due to the presence of natural bioactive compounds, trace elements, and balanced fatty acid profiles ,19,20,21Codium tomentosum  is a green marine macroalgae (Phylum Chlorophyta) native to the north east Atlantic coast. This macroalga inhabits rock pools and lower seashores and is persistent throughout the year [Codium make these macroalgae appreciated in the gourmet cuisine and cosmetic industry, with the latest using its extracts as a skin protecting agent in commercial formulations [C. tomentosum has a high lipid content (~10%) in comparison to other seaweeds, which usually display between 1% and 2% of lipid content [the year ,23. It pthe year , and orgthe year . Organolulations ,24,25. L content ,20. Addi content . HoweverC. tomentosum is shaped by different growth conditions and habitats, analyzing and characterizing its lipid extracts and antioxidant activity. Total lipid extracts, of algae collected from three different sites and in two seasons, were used to identify spatial and temporal changes in lipid, fatty acid and pigment profiles and antioxidant activity. High-resolution hydrophilic interaction liquid chromatography–mass spectrometry and tandem mass spectrometry (HILIC–MS and MS/MS) were used to study the profile of polar lipids, gas chromatography–mass spectrometry (GC–MS) to study the profile of total esterified fatty acids and high performance liquid chromatography (HPLC) to study pigment profiles. Furthermore, antioxidant activity of lipid extracts was screened through the free radical scavenging potential against α,α-diphenyl-β-picrylhydrazyl (DPPH) and 2,20-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals.Thus, the aim of this study is to ascertain how the biochemistry of C. tomentosum samples presented significantly higher amounts of lipids in autumn (Aguda 57.34 ± 6.86 and Barra 66.15 ± 11.57 µg lipid mg DW−1) than in spring . On the contrary, these seasonal differences were not identified in domesticated samples collected at an integrated multitrophic aquaculture (IMTA) system (autumn 48.33 ± 2.06 and spring 46.77 ± 2.52 µg lipid mg DW−1)  a.Lipidomic analysis of total lipid extracts by HILIC–LC MS and MS/MS allowed the identification of 296 molecular ions, belonging to three main groups of polar lipids, namely glycolipids , phospholipids (151 molecular ions), and betaine lipids (44 molecular ions) . A complPeak areas obtained after integration of extracted ion chromatograms (XIC) of each lipid ion were normalized by the internal standard of each lipid class. The analysis of normalized XIC areas showed different results depending on the sampling site. In Barra, significant increments in most lipid classes were recorded in spring samples, while in the other two sites the differences were not always significant . RegardiC. tomentosum were used for the analysis of hierarchical clustering and principal component analyses (PCA). Both analyses showed an association of wild samples according to sampling season, with this being closer for wild spring samples , trans- and cis-neoxanthin (t-Neo and c-Neo), violaxanthin (Viola), siphonaxanthin dodecenoate (Siph-do), and β,ε-carotenes  and the chlorophylls a and b (Chl a and Chl b)  but, unlike wild samples, these pigments were present in higher levels during spring.Total lipid extracts of wild A system c. Pigmend Chl b) . Pigment50) was 134.2 ± 46.0 µg mL−1 with a Trolox Equivalent (TE) of 115.1 ± 35.2 µmol Trolox g−1 of lipid of C. tomentosum from Barra–S (50) was 22.2 ± 2.2 µg mL−1 with a TE of 927.2 ± 92.1 μmol Trolox g−1 of lipid from domesticated samples collected in spring (DPPH assays showed that lipid extracts from samples collected during spring presented a lower inhibition concentration (IC) than that displayed by samples collected during autumn . The low Barra–S . Results Barra–S . The lown spring .In general, marine organisms exhibit a high plasticity to environmental changes in order to optimize their metabolism and compensate to seasonal variations in their home habitats ,15,26. IC. tomentosum populations identified the highest lipid content in samples collected in September (early-autumn), while lower lipid amounts were recorded in May (spring). Sea water temperature in the study areas is usually higher in the early autumn [C. tomentosum [C. tomentosum in early autumn. An annual sampling will allow to survey lipid dynamics along seasons and estimate upwelling influence in wild population lipidomes.Wild macroalgal populations exposed to environmental fluctuations showed a dominant seasonal pattern on lipid content characterized by an increase during winter, likely as a response to shorter days, lower irradiance levels, and temperature ,15,27,28y autumn , thus rey autumn ,31 . IMTA samples showed two different groups, associated with wild samples from the opposite season. While wild samples were expose to tidal and more severe seasonal fluctuations, IMTA samples were cultivated in tanks under more stable conditions as referred above. This may also explain the low seasonal variability in the total lipid amount of IMTA samples.The PCA analysis performed with lipid profiles showed a very close grouping in wild samples collected during spring, indicating the importance of environmental conditions in the biochemical composition of Palmaria palmata. Although, a lower amount of glycolipids in autumn could be related with a degradation of chloroplast membranes [C. tometosum samples from the wild collected in September. Additionally, sample discrimination by season was supported by an increase in normalized peak areas of several lyso forms of galactolipids (MGMG and DGMG) and phospholipids  in samples collected from the wild during spring. Curiously, wild samples presented an increase on the relative abundance of all lysolipids in spring. However, it is worth considering that in the IMTA samples the trend was the opposite, with a decrease of lysolipids in spring samples. The increase of lysolipids in spring recorded in samples from the wild may be related with a protective mechanism against photodamage caused by an increased in irradiance during spring months. As already confirmed in other photosynthetic organisms, exposure to high irradiance impairs photosynthesis due to an excessive production of reactive oxygen species (ROS) [At a molecular level, galactolipids exhibited a major variation. These glycolipids are the main lipid constituent of thylakoid membranes and it has already been described that under low irradiance conditions, such as those occurring during days with a short photoperiod, these lipids increase their relative abundance as an adaptation of the photosynthetic machinery . Consideembranes , this waes (ROS) . The acces (ROS)  when stuC. tomentosum in Barra beach is small and vulnerable, thus could be more vulnerable under more severe environmental changes.It should be highlighted the significant increase in almost all lipid classes recorded in Barra samples from autumn to spring. The population of n-3 and 18:3 n-3, is associated with galactolipids to increase fluidity and activity of thylakoid membranes [n-3 (SDA) and 20:5 n-3 (EPA), and Chl a, suggesting that under low light conditions the increase of this photosynthetic pigment is accompanied by accumulation of these fatty acids [n-3 PUFA, in autumn versus spring samples, mostly significant in algal samples from the wild, can be associated with physiological adaptations triggered by natural shifts in irradiance [The main lipids in membranes that enclose photosynthetic machinery  are characterized by being rich in PUFA . These lembranes ,28,40. Fty acids ,42. The radiance .n-6 and 20:4 n-6 in spring.Water temperature is another abiotic parameter that plays a major role on fatty acid regulation of marine organisms. Several studies have reported that low temperatures rise levels of PUFA in cells membranes in order to maintain membrane fluidity ,43. Nevet-Neo, c-Neo and Viola). Furthermore, the high content in Chl a and b recorded for IMTA samples in both seasons may be linked with the increased availability of dissolved nutrients that favor a higher algal Chl content [C. fragile [Irradiance is a significant factor influencing the profile of photosynthetic pigments. The significantly lower content in photosynthetic pigments displayed during spring versus autumn for samples of wild algae can be related with the increase of the light period and irradiance levels. Spring samples were collected close to the summer solstice (May), when day length is nearly maximal and irradiance levels are higher than in autumn. Photosynthetic organisms adapt their pigment content to prevailing light conditions, with their concentrations increasing under lower irradiance levels as a compensatory response to lower photon availability ,38,44. O content . The pre content . Ganesan fragile ,46.C. tomentosum lipid extracts was more efficient against ABTS●+ than DPPH● radicals. Although antioxidant activity of this macroalga has been previously tested, these tests were performed with aqueous [C. tomentosum enriched in antioxidant components, such as glycolipids, during this period. In this sense, studies addressing seasonal shifts in bioactivity of natural products detected maximal bioactivity and antimicrobial potential in the late spring and summer months [Fucus vesiculosus harvested in a IMTA system revealed that a greater amount of bioactive components were present in this alga during winter than during spring [Antioxidant scavenging activity of  aqueous  and etha aqueous , but notr months ,48,49. Tg spring .n-6), oxylipins, or pigments [C. tomentosum. Additionally, it must be expressed that C. tomentosum belongs to the Bryopsidales order, where photoprotective mechanisms are unclear and require further investigations [Algae present multiple mechanisms of chemical defense against biotic  and abiotic  stressors ,51. Intepigments ,46,53. Ppigments . Carotenpigments . Both fapigments , chloropigations ,58.l-serine (dMPS), 10,30-bis(1–dimyristoyl-sn-glycero-3-phospho)-glycerol (tMCL), 1,2-dimyristoyl-sn-glycero-3-phosphate (dMPA), N-heptadecanoyl-d-erythro-sphingosylphosphorylcholine (SM d18:1/17:0) , and 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) were purchased from Avanti Polar Lipids, Inc. .Phospholipid standards 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dMPC), 1,2-dimyristoyl-snglycero-3-phosphoethanolamine (dMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol (dPPI), 1,2-dimyristoyl-sn-glycero-3-phospho-(10-rac-)glycerol (dMPG), 1,2-dimyristoyl-sn-glycero-3-phospho-● was purchased from Aldrich . 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS●+) was obtained from Fluka . Ammonium acetate and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich . All the other reagents and chemicals used were of the highest grade of purity commercially available. Milli-Q water was used as ultrapure water .Chloroform (CHCl3), methanol (MeOH), ethanol, and acetonitrile were purchased from Fisher scientific ; all the solvents were of high-performance liquid chromatography (HPLC) grade and were used without further purification. DPPHCodium tomentosum samples were collected in three different locations of the Portuguese coast, Aguda beach (Aguda samples) , Paredão da Barra beach (Barra samples) , and on a land-based integrated multitrophic aquaculture (IMTA) in Aveiro  (IMTA samples). Samples were collected along two different periods Autumn 2017 (September 2017 for Aguda and Barra samples/October 2017 for IMTA samples) and Spring 2018 . ALGAplus farms seaweeds integrated with fin fish production (seabream and seabass) using an IMTA system. Five samples (n = 5) were used for each local and season . After collection, C. tomentosum samples were rinsed with distilled freshwater, cleaned to remove epiphytic organisms, frozen, freeze-dried, and stored individually at −80 °C for biochemical analysis.C. tomentosum samples. A total biomass of 250 mg of macroalga was mixed with 2.5 mL of methanol and 1.25 mL of chloroform in glass centrifuge tubes. Following vigorous homogenization and a small step of sonication (1 min), samples were incubated on ice for 2 h and 30 min on an orbital shaker . Samples were then centrifuged at 626 g for 10 min at room temperature and the organic phase, containing the lipids, was collected. A re-extraction step was performed in the original biomass, and the resulting organic phase was combined with the first. Ultrapure water was added to the organic phase in order to resolve a two-phase system. Samples were centrifuged at 626 g for 10 min at room temperature, and the lipid-containing organic lower phase was collected. The upper aqueous phase containing no lipid compounds was discharged. Lipid extracts, recovered from the organic phases, were dried under a nitrogen stream and preserved at −20 °C for further analysis [The Bligh and Dyer method  was modi−1 at 35 °C. Acquisition in the Orbitrap® mass spectrometer was performed in both positive (electrospray voltage 3.0 kV) and negative (electrospray voltage −2.7 kV) modes, with high resolution with 70,000 and automatic gain control (AGC) target of 1 × 106. Capillary temperature was 250 °C, and the sheath gas flow was 15 U. For MS/MS determinations, a resolution of 17,500 and AGC target of 1 × 105 was used, and the cycles consisted in one full scan mass spectrum, and ten data-dependent MS/MS scans were repeated continuously throughout the experiments, with the dynamic exclusion of 60 s and an intensity threshold of 2 × 104. Normalized collision energy™ (CE) ranged between 20, 25, and 30 eV. Data acquisition was performed using the Xcalibur data system . Two analytical replicas were acquired by sample.Total lipid extracts were analyzed by hydrophilic interaction liquid chromatography on a HPLC Ultimate 3000 Dionex  with an autosampler coupled online to a Q-Exactive hybrid quadrupole mass spectrometer . The analyses performed involved the use of a solvent system consisting of two mobile phases. Mobile phase A consisted of water, acetonitrile and methanol , with 2.5 mM ammonium acetate, and mobile phase B was a mixture of acetonitrile and methanol (60% and 40%), with 2.5 mM ammonium acetate. Elution started with 10% of mobile phase A, which was held isocratically for 2 min, followed by a linear increase to 90% of mobile phase A within 13 min and maintained for 2 min. After this procedure, conditions were returned to the initial settings in 13 min (3 min to decrease to 10% of phase A and a re-equilibration period of 10 min prior next injection). To perform HILIC–LC–MS analyses, 10 μg of total lipid extract, 2 µL of phospholipid standards mix —0.01 µg, and dMPA—0.04 µg) and 88 µL of eluent (10% of mobile phase A and 90% of mobile phase B) were mixed and a 5 µL of this mixture was injected into the Ascentis Si column HPLC Pore column , with a flow rate of 50 µL minn-hexane containing a C19:0 internal standard  and 200 μL of a methanolic solution of potassium hydroxide (2 mol L−1) and vortexed for 2 min. A 2 mL of a saturated solution of sodium chloride was added and the sample was centrifuged at 626× g for 10 min. The organic phase containing the FAMEs was transferred to a microtube and dried under nitrogen [n-hexane and 2 μL of this solution was used for GC-MS analysis on an Agilent Technologies 6890 N Network chromatograph  equipped with a DB-FFAP column with 30 m length, an internal diameter of 0.32 mm and a film thickness of 0.25 μm . The GC was connected to an Agilent 5973 Network Mass Selective Detector operating with an electron impact mode at 70 eV and scanning the mass range m/z 50–550 in a 1 s cycle in a full scan mode acquisition. The oven temperature was programmed from an initial temperature of 80 °C, standing at this temperature for 3 min, followed by three consecutive linear increments to 160 °C at 25 °C min−1, to 210 °C at 2 °C min−1, and to 250 °C at 30 °C min−1. Temperature was maintained at 250 °C for 10 min. The injector and detector temperatures were 220 and 250 °C, respectively. Helium was used as carrier gas at a flow rate of 1.4 mL min−1 [Fatty acids of total lipid extract were transmethylated to be analyzed by GC-MS. In order to obtain fatty acid methyl esters (FAMEs). A 30 μg of lipid was transferred to a Pyrex glass tube and dried under nitrogen. Dry lipids were mixed with 1 mL of nitrogen . FAMEs wmL min−1 . Fatty a−1. Pigments were identified from absorbance spectra and retention times and concentrations were calculated in comparison with pure crystalline standards .Lipid extracts (500 µg) were dissolved in 1 mL of 95% cold buffered methanol (2% ammonium acetate). Samples were sonicated for 30 s and briefly vortexed to ensure complete resuspension. Extracts were filtered through 0.2-µm PTFE membrane filters and immediately injected into a HPLC system  with a photodiode array detector. Chromatographic separation was carried out using a Supelcosil C18 column  for reverse phase chromatography and a 35 min elution program. The solvent gradient followed Kraay et al.  with an ●) was evaluated using a previously described method [● in ethanol (250 µmol L−1) was prepared and diluted to provide a working solution with an absorbance value of ~0.9 measured at 517 nm using a UV-vis spectrophotometer  controlled by the SkanIT software version 3.2 (Thermo Scientific). To evaluate the radical stability, a volume of 150 µL of ethanol was added to 15 microplate wells followed by addition of 150 µL of DPPH● diluted solution during an incubation period of 120 min at room temperature, with absorbance measured at 517 nm every 5 min. For evaluation of the radical scavenging potential, a volume of 150 µL of C. tomentosum lipid extract  and 150 µL of Trolox standard solution  were placed in each well followed by addition of 150 µL of DPPH● diluted solution, and again an incubation period of 120 min, with absorbance measured at 517 nm every 5 min. Control lipid extracts were tested by replacing 150 µL of DPPH● diluted solution by 150 µL of ethanol. Radical reduction was monitored by measuring the decrease in absorbance during the reaction, thereby quantifying radical scavenging, which is accompanied by a radical color change. All measurements were performed in triplicate on two different days. The % of DPPH radical remaining was determined according to: % DPPH remaining = ((Abs samples − Abs control) after 120 min/(Abs samples − Abs control) at the beginning of reaction) × 100.The antioxidant scavenging activity against the α,α-diphenyl-β-picrylhydrazyl radical )/Abs DPPH) × 100.50) were calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity is expressed as Trolox Equivalents , according to:The concentration of samples capable of reducing 50% of DPPH radical after 120 min (IC50 Trolox (µmol L−1) × 1000 /IC50 of samples (µg mL−1).TE = IC●+) was evaluated using a previously described method [−1) was prepared by mixing 10 mL of ABTS stock solution (7 mmol L−1 in water) with 10 mL of potassium persulfate K2S2O8 (2.45 mmol L−1 in water) [●+ diluted solution and an incubation period of 120 min, with absorbance measured at 734 nm every 5 min. For an evaluation of the radical scavenging potential, a volume of 150 µL of lipid extract  and 150 µL of Trolox standard solution  were placed in each well followed by addition of 150 µL of ABTS●+ diluted solution, and a new incubation period of 120 min, with absorbance measurements at 734 nm every 5 min. The control lipid extracts were also assayed by replacing 150 µL of ABTS●+ diluted solution by 150 µL of ethanol. Radical reduction was monitored by measuring the decrease in absorbance during the reaction, thereby quantifying radical scavenging, which is accompanied by a radical color change. The percentage of ABTS radical remaining was determined according to: % ABTS remaining = ((Abs samples − Abs control) after 120 min/(Abs samples − Abs control) at the beginning of reaction) × 100.The antioxidant scavenging activity against the 2,20-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation  . This miThe free radical-scavenging activity of samples was determined as the percentage of inhibition of ABTS radical according to: Inhibition% = ((Abs ABTS − (Abs samples − Abs control))/Abs ABTS) × 100.50) were calculated by linear regression using the concentration of samples and the percentage of the inhibition curve. The activity is expressed as Trolox Equivalents , according to: TE = IC50 Trolox (µmol L−1) × 1000/IC50 of samples (µg mL−1)The concentration of samples capable of reducing 50% of ABTS radical after 120 min  of ≤ 5 ppm .All experimental data are shown as mean ± standard deviation (C. tomentosum, highlighting the relevant role of ecological and environmental conditions in the metabolism of biological samples. These results emphasize the need for a comprehensive understanding of natural plasticity in the metabolism of marine organisms, which must be taken into account when conducting analytical and prospective studies. Understanding the seasonal influence in biochemical composition of organisms with potential in biotechnological applications is crucial to maximize the production of high-value compounds under controlled conditions, mimicking nature.The present study demonstrated the existence of spatial and seasonal adaptations in lipid and photosynthetic pigment profiles, as well as in the antioxidant activity of