Agarophyton chilense (formerly Gracilaria chilensis) has not been explored. Red seaweeds are enriched in polyunsaturated fatty acids and eicosanoids, which are known natural ligands of the PPARγ nuclear receptor. PPARγ is the molecular target of thiazolidinediones (TZDs), drugs used as insulin sensitizers to treat type 2 diabetes mellitus. Medical use of TZDs is limited due to undesired side effects, a problem that has triggered the search for selective PPARγ modulators (SPPARMs) without the TZD side effects. We produced Agarophyton chilense oleoresin (Gracilex®), which induces PPARγ activation without inducing adipocyte differentiation, similar to SPPARMs. In a diet-induced obesity model of male mice, we showed that treatment with Gracilex® improves insulin sensitivity by normalizing altered glucose and insulin parameters. Gracilex® is enriched in palmitic acid, arachidonic acid, oleic acid, and lipophilic antioxidants such as tocopherols and β-carotene. Accordingly, Gracilex® possesses antioxidant activity in vitro and increased antioxidant capacity in vivo in Caenorhabditis elegans. These findings support the idea that Gracilex® represents a good source of natural PPARγ ligands and antioxidants with the potential to mitigate metabolic disorders. Thus, its nutraceutical value in humans warrants further investigation.The biomedical potential of the edible red seaweed  Agarophyton chilense (formerly Gracilaria chilensis) [Agarophyton chilense (A. chilense) is distributed in New Zealand (including Chatham Island) and South America [The edible Chilean red macroalgae ilensis) , commonlilensis) . Agaroph America ,4,5,6, a America ,8.The reported bioactivity of seaweed is varied and includes antidiabetic, anti-inflammatory, antioxidant, and anti-neurodegenerative activity ,10. ThusA. chilense are an excellent source of healthy essential fatty acids such as polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid, arachidonic acid, and oleic, linoleic, and α-linolenic fatty acids. Particularly in the Gracilaria genus, lipid extraction has revealed a complex lipid composition of glycerolipids and omega-3 and omega-6 PUFAs and their oxidized derivatives, known as oxylipins [Red algae such as xylipins ,13,14,15xylipins ,17,18. Ixylipins , which axylipins ,21,22.The PPAR nuclear receptor subfamily comprises three receptor subtypes  encoded by distinct genes, and they are obligate heterodimers of the RXR receptors . PPARs hT2DM is a chronic endocrine disease characterized by hyperglycemia in the blood and resistance to the action of insulin, leading to severe neurological and cardiovascular lesions. Although there are genetic risk factors, obesity is considered the explaination for 60–80% of diabetes cases ,39,40.There are various classes of oral drugs for glycemic management, such as metformin, sulfonylureas, and TZDs. Currently, the first-line oral antihyperglycemic is metformin. However, the majority of patients with T2DM show a deterioration in blood glucose levels over time that cannot be controlled with metformin alone; consequently, a second-line drug to maintain glycemic control is included in the treatment ,42. ReceA. chilense has not been addressed. The principal objective of our work was to study the biomedical potential of A. chilense oleoresin by evaluating its capacity to activate PPARγ and modulate the metabolic dysfunction induced by obesity. We also characterized some of the lipids contained in the oleoresin, such as fatty acids, tocopherols, and β-carotene, and the antioxidant properties in vitro and in vivo using Caenorhabditis elegans (C. elegans) as a model. The oleoresin was produced by organic solvent extraction of dried algae following the procedure published in the patent application WO/2014/186913, and its trademark registered name is Gracilex®. Our results indicate that Gracilex® contains PPARγ activators acting as partial agonists, since they do not induce adipocyte differentiation like the TZD rosiglitazone. Consistently, treatment of male mice fed a high-fat diet (HFD) with Gracilex® reduced glucose and insulin levels increased by the HFD. Moreover, Gracilex® has an antioxidant capacity correlating with high levels of tocopherols and β-carotene. Our study is the first to examine whether lipids derived from A. chilense have nutraceutical or biomedical applications, emphasizing the idea that A. chilense can be used as a source of other molecules in addition to agar-agar.Despite the rich diversity of lipids and the unique phytochemical composition of red macroalgae lipids, the biomedical potential of lipids derived from TM I supplement, Tripsin-EDTA 0.5%, and penicillin/streptomycin, were purchased from Gibco BRL . Fetal bovine serum and horse serum (HS) were obtained from HyClone Laboratories, Inc.  and LipofectamineTM 2000 from Gibco BRL . Drugs and chemicals were purchased from Sigma Chemical Co. , including FMOC-Leu, 3--2,5-diphenyl tetrazolium bromide (MTT), ortho-nitrophenyl-β-galactoside (ONPG), N-acetylcysteine (NAC), cell culture grade dimethyl sulfoxide (DMSO), NP-40, Oil red O, dexamethasone, isobutylmethylxanthine (IBMX), and insulin. Rosiglitazone and T0070907 were purchased from Cayman Chemicals  and INT131 was obtained from MolPort . FMOC-Leu and INT131 are SPPARMs and rosiglitazone and T0070907 are full-agonist and antagonist of the PPARγ, respectively.HPLC-grade dichloromethane, cyclohexane, and water were purchased from Merck . Cell culture reagents, namely Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640, opti-PRO™, GlutaMAXPlasmids used to measure the transcriptional activity of PPARγ (see below) were donated by R.M. Evans’s laboratory .A. chilense individuals, approximately 60 cm in length, were collected from two areas along the Chilean coast in the southern zone: Niebla  and Coliumo . The total biomass (20–30 kg of fresh tissue) was transported to the laboratory at 4 °C and washed with fresh water posteriorly with 1 µm filtered seawater and cleaned before freezing.Vegetative A. chilense, whose trademark registered name is Gracilex®. The detailed protocol is published in the patent application WO/2014/186913 [Harvested seaweed was frozen at −20 °C for two weeks and then chopped into small pieces (3–5 mm in length) with a knife for the production of oxylipins induced by algal tissue after mechanical damage . Chopped4/186913 .® production. The oleoresins were resuspended in DMSO, and 50 µL of sample was taken to measure the antioxidant capacity using an OxiSelect™ TAC Assay Kit according to the manufacturer’s instructions , which is based on the reduction of copper (II) to copper (I), also known as a cupric ion reduction antioxidant capacity (CUPRAC) assay [Oleoresins obtained from dichloromethane extraction of lyophilized spirulina (AquaSolar™) and maqui (Isla Natura™) were prepared as described above for GracilexC) assay . Uric ac® were used to measure tocopherols  and β-carotene by HPLC-electrochemical methods. The fatty acid profile of Gracilex® was measured as fatty acid methyl esters by GC-FID. Both analyses were performed at the Center of Molecular Nutrition and Chronic Disease of Pontificia Universidad Católica de Chile according to the Chilean normative procedure NCh2550-ISO5508.Six preparations of Gracilex® CCL-2™), PC12 (ATCC® CRL-1721™), and 3T3-L1 (ATCC® CL-173™) cell lines  were used for our studies. All cells were cultured, following ATTC’s recommendations, in a 5% CO2 incubator at 37 °C and 95% humidity.HeLa (ATCCPC12 cells were grown and cultured in RPMI 1640 medium supplemented with 10% horse serum (HS), 5% fetal bovine serum (FBS), and penicillin/streptomycin (P/S). HeLa and 3T3-L1 cells were grown and cultured in high-glucose DMEM with 10% and P/S. The 3T3-L1 cells were grown under low passages at a maximum of 70% confluence.TM 2000  and 0.3 μg of PPRE-tk-LUC vector, 0.26 μg of pCMX-PPARγ vector, and 0.1 μg of β-galactosidase expression plasmid  vector  for normalization.A PPAR reporter activity assay was performed by transient transfection of PC12 and HeLa cells with a reporter plasmid containing three tandem repeats of the peroxisomal proliferator response element (PPRE) fused to the herpesvirus thymidine kinase promoter upstream of the coding sequence for luciferase (PPRE-tk-LUC) and a pCMX vector containing full-length murine PPARγ1 . Cells w5 cells/well in 250 µL of RPMI 1640 medium  grown for 24 h and transfected with LipofectamineTM 2000  with 0.8 µg of reporter plasmid , 0.8 µg of fusion protein plasmid (PPARγGAL4), and 0.053 µg of CMV-β-Gal as an internal control [A PPARγ-GAL4 transactivation assay was performed in PC12 cells seeded in 48-well plates at a density of 1 × 10 control ,50. Tran5 cells/35 mm plate for lipid staining or 2 × 105 cells/12-well plate for RNA extraction. After 48 h, the differentiation medium was used, containing freshly prepared solutions of 1 μg/mL insulin, 0.5 mM isobutylmethylxanthine (IBMX), and 0.1 µg/mL dexamethasone in the presence or absence of the test compound RGZ (1 µM), FMOC-Leu (25 µM), or Gracilex® (60 µg/mL). After 48 h, the differentiation medium was replaced with DMEM containing 1 μg/mL insulin, then the medium was changed to DMEM supplemented with 10% FBS and P/S. Every 2 days the medium was changed. After 7 days of differentiation, the cells were harvested for RNA extraction using a HiBind column  following the manufacturer’s instructions. On the tenth day of differentiation, the cells were stained with Oil Red O, as described previously. Briefly, the cells were washed twice with PBS at 37 °C and then fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature. The plate was washed with PBS and treated with 60% isopropyl alcohol for 6 min. After the plates were dried, Oil Red O (6:4) was added for 2 h at room temperature. Subsequently, the plates were washed extensively, dried, and photographed. For quantification of Oil Red O incorporation in cells, cells fixed in the plates were solubilized with pure isopropyl alcohol, and the absorbance of the solution was measured at 490 nm [The 3T3-L1 cells were differentiated as described previously . Brieflyt 490 nm .5 cells/well in 250 µL of RPMI 1640 medium  and grown for 24 h. Then, the medium was replaced with a low-serum RPMI 1640 medium .A 3--2,5-diphenyl tetrazolium bromide (MTT) assay was performed in PC12 cells as described previously, with minor modifications . Briefly4 cells/well in 250 µL of DMEM (10% CS and Ab-Am) and grown for 24 h. The cells were treated with the indicated extracts and corresponding vehicle for 16 h. The cells were washed with PBS and then incubated with 0.5 mg/mL MTT dissolved in a medium without phenol red or serum for 2 h at 37 °C. The MTT solution was discarded, and the cells were lysed with pure DMSO. The formazan product was measured in a spectrophotometer at 570 and 620 nm. The difference between the two measurements was used to calculate the percentage of cell survival with respect to the control.The 3T3-L1 cells were seeded in 48-well plates at a density of 9 × 10The RNA quality was evaluated by agarose gel integrity. Any contaminant genomic DNA was degraded by DNase treatment . The cDNA was generated using M-MLV reverse transcriptase  and random primers, while the qPCR was performed using SYBR Green  in a Stratagene MX-3000P qPCR System (Agilent Technologies). Fatty acid binding 4 (Fabp4) and lipoprotein lipase (Lpl) genes were amplified together with 3 reference genes in each run: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NoNo, and β-actin. The murine primer sequences used for Fabp4 were F: 5′-GCGTGGATTTCGATGAAATCA-3′ and R: 5′-CCCGCCATCTAGGGTTATGA-3′; for Lpl, F: 5′-ATTTGCCCTAAGGACCCCTG-3′ and R: 5′-GCACCCAACTCTCATACATTCC-3′; for GAPDH, F: 5′-TGCACCACCAACTGCTTAGC-3′ and R: 5′-GGATGCAGGGATGATGTTCT-3′; for NoNo, F: 5′-TGCTCCTGTGCCACCTGGTACTC-3′, R: 5′-CCGGAGCTGGACGGTTGAATGC-3′; and for β-actin, F: 5′-CCTGTGCTGCTCACCGAGGC-3′ and R: 5′-GACCCCGTCTCCGGAGTCCATC-3′.C57BL/6J mice were maintained with water and food supplied ad libitum at the Pontificia Universidad Católica de Chile Catholic University animal facility. The mice were housed in a room with controlled temperature (24 °C) and humidity (55–60%) for a 12 h light and dark regime. All procedures were performed during the dark phase. Experimental protocols were approved by the biosafety and bioethics committee of the Pontificia Universidad Católica de Chile (project FONDEF#DO811031). All animal procedures were conducted according to the National Agency of Research and Development (ANID) guidelines.® toxicity, 12 adult C57BL/6J mice (12 weeks old) from our animal facility were fed with a standard chow diet and corn oil (control) or Gracilex® (300 mg/kg) for 15 days .To study GracilexFor high-fat diet experiments, 50 adult C57BL/6J mice (6 weeks old) were imported from the Jackson Laboratory (USA). At 6 weeks of age, the mice were separated into 5 groups of 10 animals: 4 groups were fed the high-fat diet , and 1 group was fed the low-fat diet  ,55.®, 90 and 300 mg/kg. The treatment was administered once a day by oral gavage for 30 days. The daily volume administered was approximately 50 µL.The animals were weighed twice a week throughout the study. At 12 weeks of age, the mice were treated as follows: control diet (LFD) mice were treated with corn oil, control HFD mice were treated with corn oil, positive control HFD mice were treated with rosiglitazone (5 mg/kg), and HFD mice were treated with two doses of GracilexBlood samples were obtained at the beginning and end of the treatment . Before sampling, the mice were fasted for 15 h. Samples were obtained by puncture of the submandibular sinus, collected in heparinized tubes, and immediately centrifuged (3500 rpm/10 min at 4 °C) to obtain blood plasma.® R&D Systems, MN, USA) and mouse insulin  were detected by ELISAs. Plasma levels of glucose and cholesterol were determined by enzymatic chemical kits .Mouse adiponectin (DuoSet® (300 mg/kg). The treatment was administered once a day by oral gavage for 15 days. The daily volume administered was approximately 50 µL. At the end of the treatment, the mice were euthanized by isoflurane overdose. The liver, kidney, esophagus, and stomach were extracted and maintained in 9.25% formalin (4 °C) for 48 h. For histopathological evaluation, samples were embedded in paraffin, processed under standard conditions, and stained with hematoxylin-eosin. Liver fat accumulation was determined by Sudan black staining, and liver glycogen deposition and kidney basal membrane were evaluated by Schiff’s periodic acid (SPA) staining. The histopathological findings were classified according to magnitude .Twelve C57BL/6J mice (12 weeks old) per experimental group were randomly assigned to treatment with either corn oil (control) or GracilexEscherichia coli (OP50) as a food source [msra-1(tm1421) mutant strain [® or its vehicle DMSO. For administration to nematodes, plates were prepared as follows: Gracilex® was dissolved in DMSO at a concentration of 100 mg/mL. A stock of dead bacteria was prepared by growing the E. coli OP50 strain in lithium borate (LB) buffer overnight. After centrifugation, the pellet was resuspended to obtain a stock concentrated six times. This stock was heated at 70 °C for 30 min and stored at −20 °C until use. Then, 25 µg of Gracilex® was mixed with 50 µL of dead bacteria and NP40 to a final concentration of 0.1% and a final volume of 52 µL. This mix was seeded on 2 mL of nematode growth medium (NGM) agar plates (35 mm). The plates were left to dry in the dark overnight in the laminar flow chamber and stored at 4 °C in the dark for no more than 1 week before use. For the oxidative stress test, L4 stage msra-1 nematodes were grown for 24 h on plates with Gracilex® or DMSO (vehicle). On the day of the test, the nematodes were transferred to a 24-well plate containing 600 µL of 9 mM hydrogen peroxide dissolved in M9 medium . Ten nematodes were placed per well, and the movement was monitored every 10 min as described in [Nematodes were cultivated at 20 °C under standard laboratory conditions on agar plates fed with d source . The oxit strain  was usedribed in  until thribed in .t-test was used to compare the low-fat and high-fat diet groups. For analysis of the differences between subgroups of HFD-fed and LFD-fed mice, a one-way ANOVA and Tukey’s multiple comparison test were applied. Finally, in C. elegans in vivo experiments, a two-way ANOVA with repeated measures was used to compare differences in the survival curves of the nematodes fed Gracilex® and those under control conditions. Fisher’s LSD post-test was used to determine significant differences at appropriate times. Significant differences were calculated at significance levels indicated as: * p < 0.5, ** p < 0.01, and *** p < 0.001. Statistical analysis was performed using GraphPad Prism 5.0 for Mac . We analyzed the data for outliers using the Grubbs method (GraphPad Software). We detected just one outlier in the experimental point Gracilex® + T007 of Cell-based assays for PPARγ transactivation were performed in triplicate and at least three independent experiments. The results were expressed as the mean fold change in induction relative to the control condition ±SEM. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, was applied to determine statistical significance between the control and treatment conditions. The same statistical tests were used for the analysis of 3T3-L1 cell differentiation in the Oil Red O assay and in lipogenic gene mRNA induction as determined by qPCR. For the in vivo studies of insulin resistance, an unpaired ® can induce transcriptional activation of PPARγ, we used two cell lines, HeLa and PC12, for transient transfection assays using the PPRE-tk-LUC reporter [® induced significant activity at 40 μg/mL, resulting in up to 65% of the maximal activation achieved by the full agonist rosiglitazone at 1 μM. To further confirm the activation of PPARγ by lipids derived from Gracilex® and to study the contribution of PPARγ to the increased luciferase, we assessed the ability of Gracilex® to activate the chimeric GAL4-DBD-PPAR-LBD protein from a Gal4-dependent MH100-Luc reporter using PC12 cells , we next characterized the effect of Gracilex® on the adipocyte differentiation of 3T3-L1 cells using a well-established protocol [® at 60 μg/mL mimicked the effects of rosiglitazone  and fatty acid binding protein 4 (FABP4). We observed that only rosiglitazone promoted the adipocyte differentiation program in 3T3-L1 cells  in 3T3-L1 cells at the concentration used.SPPARMs are selective modulators of PPARγ activity with partial agonism and reduced side effects, such as adipose tissue gain . Consideprotocol . Incubatprotocol . Quantifprotocol , derivedlitazone A,B. To fL1 cells C. To uncL1 cells . Taken t® on the development of T2DM induced by a high-fat diet. Studies indicate that a diet high in saturated fat  is the most appropriate for the development of metabolic dysfunction in murine models [® as a nutraceutical agent to improve HFD-induced insulin and glucose resistance. Initially, we compared body weight gain between a group of HFD-fed C57BL/6J mice and another group fed a normal diet (LFD). After 12 weeks, a significant difference (p < 0.001) in body weight was observed between the HFD and LFD groups, and was 27% greater in the high-calorie diet group than in the group receiving a normal diet . No major changes in the kidney and stomach were found (data not shown). In the liver of mice fed a normal chow diet, we observed moderate glycogen accumulation by PAS staining and no changes in fat accumulation  were also able to decrease glucose to control levels  mice  mice D, which one does . Finallyted mice E, and the tissue . AccordiGracilaria genus have already been studied in terms of lipid components by several authors, highlighting the rich and unique composition of sulfoglycolipids, polyunsaturated fatty acids, oxidized fatty acids, pigments, and lipophilic antioxidant molecules [® is shown in Gracilaria sp., a red seaweed closely related to A. chilensis. The omega-3 fatty acid content was low, reaching up to 1.22% of total fatty acids analyzed. In contrast, the total content of omega-6 fatty acids was higher , a flavonoid-rich berry, is well known for its antioxidant and insulin-sensitizing effects [® is slightly superior to the other botanical oleoresins, reaching a mean of 430 mg uric acid equivalent per 100 mg of oleoresin. To further explore the antioxidant molecules, we evaluated the content of tocopherols and β-carotene in Gracilex®. As indicated in ®, and the total tocopherol content was 6673 μg/g Gracilex®, which was distributed as follows in the different types of tocopherols: α-tocopherol (527.7 μg/g), γ-tocopherol (5332.8 μg/g), and ∂-tocopherol (2660 μg/g). Thus, the antioxidant capacity of Gracilex® is partly due to the high levels of β-carotene and tocopherols, particularly γ-tocopherol.The nutritional value of red macroalgae is also due to the diverse content of pigments and antioxidants ,71,72. C effects , and maq effects . Table 2® to confer antioxidant resistance in vivo. The oxidative stress-sensitive msra-1 mutant strain of C. elegans [®, we first evaluated whether the nematodes were able to ingest it in a mixture with dead OP50 bacteria. As shown in ® was visualized along the digestive tracts of the nematodes (highlighted by red arrows), indicating that they can ingest it. For antioxidant resistance assays, msra-1 L4 larval stage nematodes were fed for 24 h with DMSO (control), Gracilex®, or N-acetylcysteine (NAC) mixed with dead bacteria. Then, the worms were challenged with a hydrogen peroxide solution and their movement was scored every 10 min. msra-1 mutants were already inactive compared to 40% of the nematodes fed Gracilex®. From 70–110 min, we consistently found more active nematodes, as shown by the right shift in the survival slope, which was significant at 80 min. Similar behavior was observed in the nematodes previously exposed to 5 mM NAC, a well-known potent antioxidant, which was demonstrated to rescue the unbalance in the redox homeostasis induced by hydrogen peroxide in the msra-1 mutant [® at 80 min. This time response is in the range of NAC, between 70 to 90 min after the peroxide challenge  may contribute to the improvement observed in metabolic dysfunction since it is known that the generation of reactive oxygen species contributes to the progression of T2DM [These findings have several interpretations, first, it is possible that natural lipids directly activate PPARγ similar to SPPARMs; however, this possibility was not evaluated in our study and deserves further investigation. Moreover, the anti-oxidant effect of Gracilex of T2DM .® is not due to the reported capacity of PPARγ agonists to increase adiponectin levels [® to increase adipocyte differentiation and accumulation of triglycerides measured by Oil Red O staining in 3T3-L1 cells correlates with the decreased adiponectin levels in vivo induced by Gracilex® in HFD-fed mice. Further experiments studying the genetic programs and transcriptomes in 3T3-L1 cells and adipose tissue from HFD-fed mice treated with the vehicle and Gracilex® will help elucidate this issue [On the other hand, this finding indicates that the beneficial effect of Gracilexn levels . Adiponen levels . Nonethen levels . Thus, tis issue .® showed antioxidant activity, as was previously reported in diverse algae-derived extracts. While aqueous extracts have been characterized in depth for either their in vivo activity or their antioxidant components , scarce information exists for the apolar organic-produced fraction, like the one we produced in this study. In the red seaweed Gracilaria blodgetti, a chloroform extract could decrease the oxidative stress induced in leukocytes [As a source of multiple types of chemical molecules, Gracilexukocytes ,95,96,97® using a mutant strain of C. elegans that is sensitive to oxidative stress [® were protected against exposure to hydrogen peroxide at a similar magnitude as that given by N-acetylcysteine treatment, reflected by an increase in nematode survival. N-acetylcysteine is a source of glutathione production, and has been shown to prevent reactive oxygen species generation [C. elegans [® [Depending on the type of alga, solvent used, and season of harvest, a majority or minority of antioxidant components and their activities can be redistributed between polar or apolar fractions ,98. Diffe stress . We founneration  and incr elegans . Therefoegans [® ,102,103.®, an oleoresin produced using organic solvent extraction from the red macroalgae A. chilense, contains natural PPARγ ligands that increase PPARγ transcriptional activity. Like SPPARMs, Gracilex® did not induce preadipocyte differentiation like the PPARγ receptor full agonist rosiglitazone. Furthermore, HFD mice fed Gracilex® showed normalization of glucose and insulin parameters altered by the diet without increased adiponectin. This result was consistent with the idea that the natural lipids present in Gracilex® act as SPPARM-like agonists of PPARγ. However, direct evidence that Gracilex® exerts its biological effects through PPARγ activation warrants future research. Moreover, Gracilex® showed high concentrations of tocopherols and β-carotene, which correlated with the antioxidant effect in vivo using a C. elegans model of oxidative stress. Therefore, antioxidant activities may also contribute to the normalization of insulin and glucose parameters altered by the HFD. Altogether, our results indicate that Gracilex® represents a good source of natural PPARγ ligands and antioxidants with the potential to mitigate metabolic disorders. Nevertheless, its validation in humans is required to ensure its high nutritional value.Altogether, our results indicate that Gracilex


Three sessions per week were performed: (1) 1 h of exercise, (2) exercise until 2% or (3) 4% of body mass has been lost or replaced. Limited to the first and fourth training week, a 12 min walking/running time-trial was completed following the 2 and 4% exercise bouts. Trail making, the Wisconsin card sort, the Stop signal task, Simple visual reaction time and Corsi block-tapping tests were performed immediately following the time-trials. Body mass loss was maintained < 1% with EUH and reached 2.7 and 4.7% with DEH following the time-trials. Except for a lower percentage of correct responses (% accuracy) during the Wisconsin card sort test (p < 0.05) with DEH compared to EUH, no statistically significant decline in cognitive performance was induced by low and moderate levels of hypohydration. Compared to week 1, no statistical differences in cognitive responses were observed after repeated exposures to hypohydration . From a practical perspective, the gains in cognitive performance following training to DEH were mostly unclear, but under certain circumstances, were greater than when EUH was maintained. Based on the battery of cognitive tests used in the current study, we conclude that whether physically active individuals are habituated or not to its effect, exercise-induced hypohydration of 2 and 4% has, in general, no or unclear impact on cognitive performance immediately following exercise. These results encourage further research in this area.We investigated the effect of repeated exposures to hypohydration upon cognitive performance. In a randomized crossover design, ten physically active adults completed two 4-week training blocks, one where they maintained euhydration (EUH) and the other where they were water-restricted (DEH) during walking/running at 55%  Heat stress challenges physiological functions ,2, increHumans have developed coping mechanisms for environmental stressors such as heat stress. Heat acclimation/acclimatization is a strategy that has been shown to mitigate, but not completely prevent, heat-induced cognitive performance decrements ,6. MoreoSweat losses are accelerated by heat stress exposure and, therefore, if not adequately balanced by fluid intake, will progressively lead to hypohydration. Although conflicting findings have been observed about the impact of hypohydration on cognitive performance ,8, reseaFew studies provide answers as to whether humans can adapt to repeated exposures to hypohydration. Garrett et al.  demonstrHigh-order cognitive processing, including executive functions, is essential, especially when tactical and vital decisions must be taken. Indeed, individuals such as military personnel, medical services, firefighters and, in some cases, athletes must maintain a high level of cognitive performance while exposed to hot environments, where access to water is restricted, predisposing them to hypohydration. Therefore, understanding the factors or strategies that could help reduce the effect of hypohydration on cognitive performance under heat-stressful conditions is important from a health and security standpoint .Therefore, the goal of this study was to determine the extent to which low (~2%) and moderate (~4% body mass) hypohydration levels impact cognitive performance following exercise conducted in a hot environment while not habituated to hypohydration, and whether repeated exposures to the effect of hypohydration over 4 weeks moderate the baseline response, compared with a situation where hypohydration is prevented during exercise. It was hypothesized that cognitive performance would be marginally but significantly attenuated by hypohydration while not habituated to its effect, but that after several training sessions with repeated exposures to hypohydration, cognitive performance would be similar whether adequate hydration is being maintained or not during exercise.n = 1), fear of exercising while hypohydrated (n = 1), health problems (n = 1), lack of fitness (n = 1) and personal reason (n = 1). Women underwent cognitive assessment during the follicular phase of their menstrual cycle . Before obtaining their informed written consent, each participant received explanations of the study protocol and associated risks and benefits. The CIUSSS Estrie-CHUS Ethics Committee (#2019-3081) approved all procedures.Fifteen (5 women) non-heat-acclimated physically active  participBoth the experimental design of the study and the methods have already been thoroughly detailed elsewhere . Therefo2max) test using an expired gas analysis system  that had been calibrated with gases of known concentration. After post-void body mass was taken , height, resting blood pressure, heart rate  and body composition  were measured. Then, participants underwent a treadmill maximal oxygen consumption  or not (DEH) water ad libitum . The third familiarization session was performed in the fourth week of each training block immediately following the 1 h exercise bout. Overall, participants underwent three familiarization sessions per training block. Two were performed on separate days the week prior to starting the training blocks. During the first, participants came to the laboratory and performed all cognitive tests twice. During the second, participants completed a 1 h walking/light running exercise bout at 55% TM, Olathe, KS, USA) and inserted a rectal probe . They then entered the environmental chamber  and, after an equilibration period of 2 (exercise sessions only) or 10 min (testing sessions only), started walking/light running at an intensity of 55% 2max. The required intensity was adjusted in the first 10 min of exercise, where O2 consumption was continuously measured. Except for the 1 h exercise bout (2 × 30 min interspersed by 3 min rest inside the chamber), participants completed exercise/recovery cycles of 8/2 min until a sweat-induced body mass loss of either 2 or 4% was reached (DEH) or replaced (EUH). Participants were requested to drink water served at room temperature . Participants could rinse their mouths with water at any time and could pour cold water on their bodies and heads at a rate of 100 mL·h−1. After 1 h of exercise and every 30 min thereafter, energy gels  were consumed. Heart rate, rectal temperature, perceived exertion  and osmolality , participants were nude weighted , dressed themselves, installed a heart rate chest electrode  and thirg scale ) were me® 10) with a 19″ diagonal screen  and with participants wearing headphones  when needed.Cognitive tasks were selected to assess executive functions and consisted of the (1) Trail making test (TMT), (2) Wisconsin card sort test (WCST), (3) Stop signal task (SST), (4) Simple visual reaction time test (SVRT), and (5) Corsi block-tapping test (CBT). The tests were always administered in the same order. The cognitive tests  were performed in a closed and quiet room using a desktop computer  ,23. PartThe SVRT test measures the reaction time of participants to respond to a visual target. A fixed black cross is placed on the center of a white screen background. A visual stimulus covers the fixed cross after varying time intervals. The participants have to press the spacebar as soon as the target stimulus appears. Two blocks of 20 trials were completed. The mean reaction time was taken as the measure of performance. The CBT measures the visuospatial short-term working memory, which represents the capacity of storage and the temporary management of information ,26. Partp ≤ 0.05. This study reports cognitive-related data collected during a larger scale study whose goals were to determine the physiological, perceptual and performance adaptations associated with repeated exposures to exercise-induced hypohydration. Because cognitive-related data were taken as secondary outcomes, no a priori power analysis had been performed. However, to better understand and interpret the results from a practical perspective, data from the second to the third familiarization were used to determine the day-to-day variability for each cognitive test. The practical significance of findings was then determined using the second-generation p-value technique, as previously described [All statistical analyses were performed using IBM SPSS Statistics software . Shapiro–Wilk tests were used to analyze data normality. Dependent t-tests or Wilcoxon signed-rank tests were used to compare the characteristics of the participants before each training block. Three factors (hydration*condition*training) were analyzed via ANOVAs and, for variables with missing cases, linear mixed models  were used to determine the impact of, and interaction effect between, hydration (EUH vs. DEH), conditions (2 vs. 4%) and training (pre vs. post). Data are presented as mean ± standard deviation (SD). Statistical significance was set at escribed . To obtain the day-to-day variability for each cognitive test, first, the performance data collected during the second familiarization session were subtracted from that obtained during the third familiarization session and, then, the means and associated standard deviations were computed. The smallest worthwhile change in performance for any given cognitive tests was taken as the product of the day-to-day SD × 0.2 . A 95% −2, 55 ± 7 mL·kg−1·min−1, 193 ± 7 beats·min−1, 82.4 ± 6.5% and 14.0 ± 6.6%. A comparison of the baseline values shows that body mass (∆block2-block1 = −0.4 ± 1.2 kg), body mass index (∆ = −0.15 ± 0.4 kg·m−2), 2max (∆ = −1.0 ± 2.6 mL∙kg−1∙min−1), fat-free mass (∆ = −0.15 ± 1.8%) and fat mass (∆ = 0.2 ± 1.6%) were not significantly different before the first and second training block . At baseline, mean age, height, body mass, body mass index, maximal oxygen consumption, maximal heart rate, fat-free mass and fat mass were, respectively, 23 ± 5 years, 176 ± 8 cm, 71.6 ± 11.8 kg, 23.1 ± 2.9 kg·mp < 0.01) effect for pre-exercise urine specific gravity and urine osmolality, with no other main or interaction effects . Moreover, pre-exercise body mass  and plasma osmolality (average of 288.5 ± 5.0 mOsm·kg−1) did not differ among trials and were suggestive of adequate hydration. Participants started all testing sessions in a well-hydrated state. Indeed, as demonstrated in p = 0.0001), without a difference between EUH and DEH (p = 0.45) or a hydration*condition effect (p = 0.08). Total exercise duration decreased through training, in both conditions, and was significantly lower during the second testing session . No hydration*condition*training effect was observed (p = 0.15). The overall mean exercise intensity during the fixed-intensity exercise bout was 56 ± 2% 2max .Total exercise duration  reached 75.8 ± 9.5 and 141.4 ± 16.9 min with the 2 and the 4% conditions, respectively (p = 0.0001), with a hydration*condition (p = 0.001) but no other effects . Post hoc analyses revealed that a hydration effect was present for both conditions (both p < 0.01), but that a condition effect was only present with DEH (p = 0.0008) but not EUH (p = 0.17). Similarly, at the end of the 12 min self-paced time-trial, urine osmolality  and specific gravity  increased more with DEH than EUH (both p = 0.0001), with a condition effect (p = 0.04 and 0.006) but no other main or interaction effects .At the end of the 12 min time-trial, body mass losses remained <1% for EUH (0.8 ± 0.2%) and reached 2.7 ± 0.2% and 4.7 ± 0.2% for DEH with the 2 and the 4% conditions, respectively. −1, representing 93 ± 7% of maximal heart rate. The end of performance rectal temperature, perceived exertion and perceived thirst , but not heart rate (p = 0.11), were significantly higher with DEH compared to EUH (p = 0.03) and DEH (p = 0.0001) for rectal temperature, but only with DEH for perceived thirst (p = 0.001). Values were higher with DEH compared to EUH in the 2% condition  as well as in the 4% condition . The end of performance heart rate (+4 ± 13 beats·min−1) and rectal temperature (−0.1 ± 0.3 °C), respectively, increased and decreased through training (both p = 0.04), similarly between EUH and DEH, as suggested by the absence of a hydration*training effect . No hydration*training effect was observed for perceived thirst (p = 0.051). However, post hoc analyses revealed that thirst perception was higher with DEH compared to EUH pre- and post-training (p < 0.05). Interestingly, perceived thirst at the end of performance increased through training with EUH , but remained stable with DEH . No other effects were observed .Physiological and perceptual responses have been published elsewhere . The oved to EUH . Hydratip > 0.05).Overall, cognitive performance was not affected by low and moderate levels of hypohydration. Compared to the baseline, no differences were observed after repeated exposures to hypohydration. The mean duration to complete the five cognitive tests remained constant throughout trials , without condition (p = 0.73) or hydration*condition (p = 0.97) effects. The average time to complete the TMT decreased with training (p = 0.008), without hydration*training (p = 0.56) or condition*training (p = 0.74) effects. No hydration*condition*training effect was observed (p = 0.54). While no hydration effect was observed overall, interpreting the data from part A and B depicts a different conclusion. No main interaction effects  were observed in part A . However, the time to complete part B was significantly lower with DEH compared to EUH , with training (p = 0.007) but no other effects . For the SVRT, a slightly but non-significantly longer mean reaction time was observed with DEH compared to EUH . No other main or interaction effects were observed . The mean SSRT during the SST slightly decreased with training (p = 0.004) from 230.5 ± 32.8 (pre) to 213.4 ± 24.7 ms (post), without hydration (p = 0.70), condition (p = 0.56) or any interaction effects . p > 0.05) were observed for percentage of perseverative errors during the WCST . However, hydration (p = 0.001) and hydration*training (p = 0.03), but no other main or interaction effects , were observed for accuracy (% correct responses). Post hoc analyses revealed that a hydration effect was only observed in post-  but not in pre-training , without a training effect either with EUH or with DEH (both p > 0.05). For the CBT, the lengths of the last correctly recalled sequence were 7.3 ± 0.7 (EUH pre), 7.2 ± 0.9 (DEH pre), 7.3 ± 0.7 (EUH post) and 7.3 ± 1.1 (DEH post) with the 2% condition. These values reached 7.3 ± 0.8, 7.4 ± 1.1, 7.3 ± 0.9 and 7.4 ± 0.8, respectively, with the 4% condition. No main and no interaction effects were observed .No main or interaction effects , a 64% chance of worthwhile effect with DEH compared to EUH was observed in the 4% condition for the SVRT. Similarly, a 54% (2% condition) and 57% (4% condition) chance of worthwhile effect with DEH compared to EUH was observed for the WCST and the TMT, respectively. In other words, such results highlight that, for these tests, the gains in cognitive performance following training to hypohydration have been observed to be possibly greater than when EUH was maintained. Conversely, regarding % accuracy during the WCST, a practical advantage with EUH has been observed, with improved % accuracy from pre- to post-training with EUH training , while it decreased with DEH. Other effects were trivial or unclear.The aims of this study were to determine (1) the impact of low and moderate hypohydration levels on cognitive performance while not habituated to hypohydration and (2) whether repeated exposures to the effect of hypohydration moderate the baseline response. While previous studies were specifically designed to verify whether repeated exposures to hypohydration could lessen its negative impact upon endurance performance or physiological functions, this is the first one to investigate whether habituation to hypohydration could mitigate the potential cognitive impairment associated with hypohydration immediately following exercise. The main findings of this study were: in physically active individuals, immediately following exercise and compared with euhydration, (1) low and moderate exercise-induced hypohydration do not impair cognitive performance, (2) repeated exposure to exercise-induced hypohydration over a 4-week period did not change these outcomes from a statistical perspective and (3) from a practical perspective, the impact of repeated exposure to exercise-induced hypohydration is mainly unclear, highlighting that more studies in this area are necessary. Contrary to our first hypothesis, our results indicate that exercise-induced hypohydration ranging from 2 to 4% of body mass does not impair cognitive performance following exercise when unhabituated to being exposed to its multifaceted effects. Therefore, the current findings do not support the reported claim that fluid loss ≥2% body mass ,31 negatIt is frequently argued that symptoms associated with hypohydration, including but not limited to thirst, mental fatigue and impaired mood ,36,37, mOur second hypothesis was that repeated exposures to low and moderate exercise-induced hypohydration would moderate, in a positive manner, the baseline response. More specifically, we proposed that there would be no difference in cognitive efficiency between EUH and DEH at post-testing, implying that the degree of improvement in cognitive performance through training would be more important with DEH than EUH. Because no hypohydration-related cognitive decrements were observed at baseline between EUH and DEH following exercise, while participants were not familiar with the effect of hypohydration, unfortunately, our results prevent us from answering the question of whether or not the brain can develop while submitted to repeated bouts of hypohydration, specific adaptations to reestablish cognitive functions to a euhydrated-associated baseline level. That being said, we could have observed that the cumulative effect of repeated exposures to low and moderate hypohydration induces mental fatigue over time and, as a result, lessen cognitive performance at post-testing. However, such a trend was not observed, and repeated exposures to hypohydration did not worsen the baseline response. In other words, no cognitive impairments were observed with repeated exposures to low and moderate hypohydration beyond exercise-induced EUH. This observation is particularly important for firefighters , militarp-value. The latter aimed to provide a clearer perspective on the practical implication of the present findings. Regarding the changes in cognitive performance from pre- to post- training, at 2% hypohydration, the overall effect seems uncertain since three of the five results were unclear and a contradiction was observed between the other two  and moderate (4%) exercise-induced hypohydration under hot ambient conditions, based on the battery of cognitive tests we used, our results indicate that, immediately following exercise, it does not impact his or her ability to provide a cognitive performance that is similar to when these body losses are fully replaced through water consumption. From a practical perspective, although the gains in cognitive performance following training to DEH were mostly unclear, under certain circumstances, they were greater than when euhydration was maintained. Further studies in this research area are required.


Plantago coronopus can be used in biosaline agriculture as it tolerates salt concentrations of 100 mM NaCl. To increase the salt tolerance of this plant, plant growth regulators such as polyamine spermidine, salicylic acid, gibberellins, cytokinins, and auxins were added in a hydroponic culture before the irrigation of NaCl (200 mM). In 45-day-old plants, dry weight, water content, osmolyte (sorbitol), antioxidants , polyamines ) and ethylene were determined. In non-saline conditions, all plant regulators improved growth while in plants treated with salt, spermidine application was the most effective in improving growth, osmolyte accumulation (43%) and an increase of antioxidants (24%) in P. coronopus. The pretreatments that increase the sorbitol content, endogenous amines (bound spermine fraction), phenols and flavonoids may be the most effective in protecting to P. coronopus against stress and, therefore, could contribute to improving the tolerance to salinity and increase nutritional quality of P. coronopus.Climate change, soil salinisation and desertification, intensive agriculture and the poor quality of irrigation water all create serious problems for the agriculture that supplies the world with food. Halophyte cultivation could constitute an alternative to glycophytic cultures and help resolve these issues.  We wanted to identify which PGRs produced the best results to investigate: (1) ways to improve its tolerance to salinity, (2) boost its growth, and (3) increase the nutritional quality of this species. The results could provide technical guidance for increasing the cultivation of this halophyte and the benefits that it provides.The following PGRs were added to a hydroponic culture of P. coronopus seeds collected from Brujuelo saltmarsh in Jaén (Spain) and cultivated hydroponically showed similar dry weight at 0 and 100 mM NaCl and a decrease at 200 mM NaCl (whole plant) [p ≤ 0.05) whose having the highest values, above all in RDW (A in Previous works by our research group showed that e plant) . We deciRDW A in . In the RDW A in , KinetinPlantaginaceae. For this reason, in the leaves of P. coronopus this osmolyte was analyzed at 45 days of culture. In pretreatments without salt (p ≤ 0.05) of osmolyte accumulation (It is well known that soluble carbohydrates (sorbitol) are plentiful in the family out salt A no signmulation B, (increP. coronopus at 45 days of culture to observe whether any pretreatment PGRs increased phenols and flavonoids content. The results shown in p ≤ 0.01) increased the content of phenols and flavonoids by 24% compared to untreated plants . The values of phenols and flavonoids under non-saline conditions did not have relevant results or show any significant differences between pretreatments (data not shown).We studied the effect of PGRs application with salt on the antioxidant content  in the leaves of P. coronopus. Therefore, we considered it necessary to analyze PGRs application on the endogenous PA content  in the absence or presence of salt. The data are shown in P. coronopus. This increase mainly occurs in bound and free PA fractions. However, under saline conditions, PA levels are modulated by salt. We detected a decreased of endogenous Put  in pretreatments Kinetin + salt and Spd + salt , the total PAs and Spd , the total PAs and Spm  were always positive. However, the negative correlation between ethylene (C2H2) and Spm  and ethylene and total PAs  indicated that these metabolites (sorbitol. Spd and Spm) are necessary, especially under saline conditions, for enhancing salt tolerance and mitigating the adverse effect of stress .Parameters studied: SLDW (stem + leaf dry weight); RDW (root dry weight); SLWC (stem + leaf water content); RWC (root water content); SOR (sorbitol); PUT (Free + Bound + Conjugated); SPD (Free + Bound + Conjugated); SPM (Free + Bound + Conjugated); Total PAs ; CThe benefits that PGRs application have on growth and abiotic stress are well known ,43,44. UP. coronopus [Frankenia pulverulenta and Atriplex prostrata that also grow in the Brujuelo saltmarsh  [P. coronopus, especially under pretreatments Spd, SA, and Kinetin in aerial parts and Spd, SA, and IAA in roots , PAs  may be useful markers of salt-tolerant genotypes [Cymodocea nodosa [Solanum chilense [C. nodosa improving chlorophyll fluorescence levels under different saline treatments, maintaining the photosynthetic apparatus functional, under long-term hypo-osmotic stress [Previous studies showed a fall in dry weight at 200 mM NaCl in oronopus . This sp, Spain) . In saltots A in . More sporonopus ,47,48. Ooronopus . Regardie effect ,50. Withe effect ,51,52,53e effect . A posit applied . Few stuenotypes ,56 and ma nodosa  and Solachilense . Specific stress . Neverthc stress ,60, and +, Cl−) in their aerial parts and excrete excess salt through saline glands, bladder hairs or by developing succulence in their leaves [P. coronopus leaves. In previous studies, we detected a high concentration of ions  related to a certain degree of succulence [Plantaginaceae have a “constitutive mechanism” of tolerance in which the transport of Na+ and Cl− ions (inorganic osmolytes) to the leaves and compartmentalization in the vacuole, contribute to cellular osmotic balance, and increase antioxidant metabolism under saline stress [2+ efflux, net H+ fluxes, and activated H+-ATPase pump under stress, but all these experiments were realized in the roots of glycophyte seedlings [P. coronopus (therefore ion data were not included). On the other hand, the osmoprotective compounds  favor water uptake, act as chaperons to molecular stabilized proteins and membranes, scavenge ROS, and/or protect antioxidant enzymes [Plantago species [P. coronopus, sorbitol was found in high concentrations in both saline and non-saline PGR pretreatments  accumulate inorganic ions  (an enzyme that synthesizes Spd and increase the content of endogenous Spd and Spm) was cloned and characterized in the obligate halophyte Eutrema salsugineum and inserted into transgenic tobacco plant subjected to water and salinity stress. The results showed lower malondialdehyde  levels, less ion leakage and ROS levels, which indicates better protection in biomembranes, higher water content and more antioxidant enzymes than in non-transformed plants [P. coronopus.The best treatment under both non-saline and saline conditions was the PA Spermidine. Thus, we decided to analyze the endogenous PA content in this halophyte. In the biosynthetic pathway precursors of diamine Put are ornithine and arginine, while the triamine Spd and tetramine Spm are produced by addition of aminopropyl groups from S-adenosyl methionine (SAM) that are sequentially incorporated to Put and Spd by enzymatic reactions catalyzed, respectively, by Spd synthase and Spm synthase. The SAM is decarboxylated by SAMDC (S-adenosyl-methionine decarboxylase) ,47,53. Coronopus  made it oronopus . In the thmoides . In addithmoides ,74. Therthmoides ,76. We cd plants . ClearlyArabidopsis plants  under abiotic stress revealed better growth, maintaining higher photosynthetic activity, higher Fv/Fm and an increase in the PIABS (Performance Index Based on Absorption). The enhancement in PIABS caused a higher efficiency of quantum yield and specific energy fluxes of PSII, and also higher activities of antioxidant enzymes were found in the transformed plant [Ethylene and PAs have a common precursor, SAM. The increase of total PAs was accompanied by a decrease in ethylene production under different PGR treatments, which could contribute to PA accumulation , therebyed plant .P. coronopus were randomly collected in September 2016 from Brujuelo saltmarsh  . Seeds were kept dry at 4 °C before being washed with sterile distilled water and sown in Petri dishes at 25 ± 1 °C and a photoperiod of 16 h [−2 s−1, 400–700 nm, provided by Sylvania Inc., Danvers, MA, USA, lamps, photoperiod 16h/8h in a day/night cycle, temperature (day) 25 °C ± 1 °C and (night) 16 °C ± 1 °C, and relative humidity of 55–75%.Seeds of  of 16 h . After 1 of 16 h . Plants 3; polyamine: Spd; and salicylic acid: SA.In a growth chamber, plants were acclimated (in hydroponic conditions) for two weeks. Subsequently, these plants were treated for 10 days with different growth regulators applied to the nutrient solution when watered. The growth regulators used were the following: auxin: IAA; cytokinin: kinetin; gibberellins: GASix treatments were established with six pots for each treatment PGRs No PGRsIAA (1 µM)Kinetin (1 µM)3 (1 µM)GASpd (0.5 mM)SA (0.5 mM)Six treatments were established with six pots for each treatment PGRs + salt7.NaCl (200 mM)8.IAA (1 µM) + NaCl (200 mM)9.Kinetin (1 µM) + NaCl (200 mM)10.3 (1 µM) + NaCl (200 mM)GA11.Spd (0.5 mM) + NaCl (200 mM)12.SA (0.5 mM) + NaCl (200 mM)Subsequently, these pots were irrigated with two concentrations of NaCl: 0 mM (treatment 1–6) and 200 mM (treatment 7–12). NaCl levels were selected according to previous experiments realized by us . To avoiThe following parameters were determined: fresh weight (FW) , dry weight (DW) (leaves + stems and roots), and water content (WC) (leaves + stems and roots). To obtain DW, plants were placed in a forced-air oven at 70v °C for 72–96 h until a constant weight was obtained. This material was used to determine sorbitol, phenols, flavonoids, and endogenous PAs . Water content was calculated following the formula: WC (%) = (FW − DW/FW) × 100, where SL = stem and leaves, and R = Roots . In fresP. coronopus leaves (mature plants). For 10 min, dry leaves (45-day-old) were boiled in milliQ water and subsequently filtered with filters (0.22 µm). Afterwards, all samples  were injecting (20 µL) in a Waters 717 autosampler into a Prontosil 120-3-amino column . The conditions of isocratic flux were: (1 mL/min) of 85% acetonitrile for 25 min in each run. Sor integration peaks were obtained in the Waters Empower software and the quantification was realized compared with the standard calibration curve. A Waters 1525 HPLC (high-performance liquid chromatography) coupled with a 2424 evaporative light scattering (ELS) detector  were used to determinate Sor content. The source parameters of ELSD were gain 75, data rate 1 point per second, nebulizer heating 60%, drift tube 50 °C, and gas pressure 2.8 Kg/cm2. All experiments were conducted at room temperature.Sorbitol (Sor) was analyzed following Hassan et al.  for P. cg and the supernatant was collected.The method of Boestfleisch et al.  was foll−1) or 10 µL of methanolic extract. The reaction is completed with Folin–Ciocalteu reagent (10 µL). After waiting 8 min sodium carbonate (7%) (100 µL) was added. In the dark and room temperature, tubes were incubated for approximately 90–100 min. Total phenols were calculated using a standard curve. The samples at wavelength of 765 nm were measured in a spectrophotometer VARIAN Cary 4000 UV-VIS .The quantification of total phenols was performed following the protocols by Dudonné et al. . One hun−1), or 25 µL of methanolic extract, and NaNO3 (3.75%) (10 µL). After waiting 6 min, the reaction was completed with AlCl3 (10%) (15 µL). After 5 min of incubation, NaOH (1 M) (50 µL) was added. Total flavonoids were calculated from a standard curve. The samples and the curve standard at wavelength 510 nm. were measured in a spectrophotometer VARIAN Cary 4000 UVA-VIS .The quantification of the total flavonoids was performed following Dewanto et al. . In thisv/v), then centrifuged at 23,000× g at 4 °C for 20 min. The supernatant was used to determine free polyamines by dansylation method [g for 15 min. Later, these samples needed to be filtered using microfilters . Twenty µL were injected into a Bio-Rad HPLC system  equipped with a Nucleosil 100-5 C18MN 250/04 column . The conditions of HPLC to quantify the integration peaks were the following: a methanol/water stepped gradient program changing from 60% to 100% methanol over 25 min, flow rate 1 mL min−1, and temperature of 35 °C. A Shimadzu RF-10Axl fluorimeter detector (excitation wavelength 320 nm and emission wavelength 510 nm) was used to determine dansylated free polyamines.For PAs extraction the method followed by Ghabriche et al.  was usedn method . The samg at 4 °C for 20 min., and the supernatant was hydrolysed and dansylated under the same conditions described above. Dansylated free PAs (supernatant), dansylated conjugated PAs (supernatant hydrolysed) and dansylated bound PAs (pellet hydrolysed) were injected (20 µL) in the HPLC, in addition to PA standards , for quantification.Bound forms  and conjugated forms  were also analyzed. We added 200 µL of HCL (12 N) to the same amount of supernatant (200 µL) and transferred to dark tightly capped glass tubes. These tubes were placed in a heater and heated at 110 °C for 24 h to realize sample hydrolysis. After HCl was evaporated, the residue was resuspended in 200 µL of perchloric acid (10%) and used for dansylation. The pellet was used to extract bound PAs. This was dissolved in 5 mL of NaOH (1N). The mixture was centrifuged at 23,000× P. coronopus (45-day-old) were immediately transferred into a 5 mL flask (containing at the bottom filter paper and 50 µL of distilled water). All flasks were sealed with a silicone-rubber stopper (to prevent gas leakage). Flasks were incubated on a stove for 1 h incubation period, at 30 °C in darkness. Later, a 1 mL gas sample was injected into a HP 5890 series II, Hewlett Packard  gas chromatograph fitted with a flame ionization detector and a 2 m × 4 mm stainless-steel column packed with 50–80 mesh Poropack-R. The conditions of chromatograph were: N2, H2 and synthetic air flow rates 50, 86, and 400 mL min−1, respectively. To analyze and quantify ethylene production, peaks integration was compared with the retention time of ethylene (C2H4) standard, (purity 99.9%).The method of Bueno et al.  was follp ≤ 0.05 and p ≤ 0.01). All parameters in the absence or presence of salt were compared using Pearson’s correlation coefficients.A randomized block design was used in our experiments. Data are presented as mean ± standard error (SE). A Statgraphics Centurion v. 17 (University of Jaén) was used to perform analyses of variance (ANOVA). Significant differences between means were determined using Tukey’s multiple range test  to the nutritive solution can improve growth and increase salt-stress tolerance, as well as increasing the osmolyte (sorbitol) and antioxidant compounds (phenols and flavonoids) under saline conditions. The increase in the endogenous PA pool, especially Spd and Spm (bound forms), is probably related to the protection of subcellular structures, the maintenance of photosynthetic activity, osmotic adjustment, ionic homeostasis and the improvement of antioxidant activity. In addition, the increase in Spd levels showed a negative correlation with ethylene, indicating than the decrease in ethylene also can contribute to PA accumulation. Auxins, CKs, GAs and SA pretreatments stimulated growth under non-saline conditions, but these PGRs were unable to mitigate the adverse effects of stress. Therefore, Spd application is the best pretreatment for P. coronopus cultivation and can contribute to improving the tolerance to salinity and nutritional quality of this halophyte, although it will be necessary to research in each halophyte which is the better treatment to apply.Halophyte cultivation as a part of biosaline agriculture could help improve productivity and crop quality and be used to restore saline and degraded land. In


NAD+ also serves as a substrate for enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs) and ADP-ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD+ metabolism has become essential to monitor genetic- and/or drug-induced perturbations related to metabolic status and diseases , and its possible therapies. Here, we present a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of the NAD+ metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to separate a total of 18 metabolites belonging to pathways leading to NAD+ biosynthesis, including precursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids . For clinical applications, quantitative LC-MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD+ biology from mechanistic to clinical applications.Nicotinamide adenine dinucleotide (NAD NAD(H) and NADP(H) both serve as hydride carriers in the cell (donors and acceptors), but they act differently. In general terms, NAD(H) plays a crucial role in catabolism, energy metabolism , while NADP(H) is involved in anabolic reactions  as well as in antioxidant response and detoxification processes +, 381.01 [M+2Na–H]+.Reduced nicotinamide riboside (or 1-(beta-D-ribofuranosyl)-1,4-dihydronicotinamide) was obtained by organic synthesis, as reported previously ,44. A so NMR see  and MS: + precursors on the NAD+ metabolome. Cells were grown in a 6-well format (9 cm2 of surface area/well) under a humidified atmosphere with 5% CO2 at 37 °C in high glucose DMEM medium without glutamine and without pyruvate (Thermo Fisher), supplemented with 1% penicillin-streptomycin (Thermo Fisher), 10% FBS (Thermo Fisher), 1% glutamine , 1% sodium pyruvate  and 1% MEM non-essential amino acids (Thermo Fisher). For the analysis of endogenous NAD+ metabolism intermediates, 500,000 cells were seeded per well in 6-well plates and harvested after overnight culture. For the analysis of NAD+ metabolism intermediates upon treatment with different precursors, 300,000 cells were seeded per well in 6-well plates and after 1-day culture, a medium change with the different precursors was performed. After another 20 h, cells were harvested. For harvest, cells were washed with 0.9% (m/v) sodium chloride and after discarding the washing solution, the 6-well plate was transferred into liquid nitrogen. After this quenching step, plates were stored at −80 °C until extraction.The human liver cell line Hep G2 (purchased from Sigma-Aldrich) was used as a cell model to test extraction performance and perturbation effects of NADC57BL/6 mice were kept in a temperature and humidity-controlled environment with a 12:12 h light–dark cycle, having access to nesting materials and were provided with ad libitum access to water and food. Further details on animal care were described elsewhere . All tisDonors were anonymously recruited in fasting conditions by the Metabolic Unit at Nestlé Research . For the first pilot study, plasma, serum and whole blood were collected from the same blood draw from 9 volunteers. Whole blood (~5 mL) was collected in vacutainer tubes and aliquots of 60 µL of whole blood (flash-frozen in liquid nitrogen within 10 min of collection), plasma  and serum . All samples were flash-frozen in liquid nitrogen and stored at −80 °C prior analysis. For the second pilot study, whole blood was collected from 3 donors to assess effect of aliquot volume and freeze–thaw cycles. From each blood draw, five aliquots of different volumes were rapidly transferred in cold conditions, (4 °C) to individual tubes  in a maximum of 10 min time and subsequently flash-frozen in liquid nitrogen and stored at -80°C. To assess the effect of freeze–thaw cycles, the same 1 mL aliquot was thawed three times for analysis .Urine (Seronorm) from at least 2 donors was purchased from Sero  and kept at −80 °C before analysis.+ metabolome. The first two methods (80% cold methanol and 40:40:20 with 0.1 M formic acid) were based on Lu et al. -biomass, as internal standards. The cells were scraped, transferred into tubes, vortexed for 10 s and incubated on ice for 3 min. An aliquot (69.6 µL) of ice-cold 15% (m/v) ammonium bicarbonate was added to the extracts for neutralization, followed by 20 min incubation on dry ice. The extracts were centrifuged, the supernatants recovered and dried, and reconstituted for LC-MS/MS analysis as described in the 80% cold methanol procedure.Previously quenched and frozen Hep G2 cells were extracted with 800 µL of pre-cooled on ice solution of 40:40:20 (v/v) while adding 10 µM Nam-d4, NR-d4, and Trig-d3 and 60 µL of [U-13C]-biomass, keeping the samples cold throughout the procedure. For biofluids, 60 µL were taken for extraction . For tissues, 2–10 mg of tissue were homogenized using 3 mm tungsten carbide beads using a tissue grinder  for 3 min at 20 Hz. Mammalian cells were scraped with extraction solution with a 1 mL-pipet tip, well by well, and transferred into tubes. All samples followed 10 min of agitation at 4 °C in a thermo-shaker , followed by 10 min centrifugation at 21,130× g. After extraction, the upper polar phase was recovered, as well as the protein layer taken for total protein quantification  by bicinchoninic acid (BCA) assay (ThermoFisher Scientific). The recovered extract was dried overnight in a vacuum centrifuge at 4 °C and 5 mbar, and then stored at −80 °C, before analysis. Dried samples were reconstituted in either 25 µL (cells) or 60 µL  in 60% (v/v) acetonitrile/water, and the supernatants transferred into glass vials for LC-MS/MS analysis.Frozen biological materials  were extracted using an adapted protocol from Bligh and Dyer, with 1300 µL cold methanol–water–chloroform 5:3:5 (®-Fusion(P)), guarded by a pre-column  Guard Kit) operating at 35 °C. The mobile phase  was pumped at a flow rate of 0.25 mL/min flow over a linear gradient of decreasing organic solvent , followed by washing (17–21 min at 25% B) and re-equilibration (21.1–30 min at 92.5% B). Retention time of individual analytes was confirmed using authentic standards. MS conditions were obtained by single compound optimization, via infusion of standard compounds into the MS source, to obtain specific parent and product ion pairs  per metabolite. In the final method, the MS operated in positive mode ionization at 3500 V on MRM. The scan width was 1 m/z for each scan and scan time was 0.05 s, the peak width for Q1 was 0.25 FWHM and for Q3 0.70 FWHM. The sheath gas was set to 20 arbitrary units, and the auxiliary gas was kept at 15 arbitrary units. The vaporizer temperature was set to 280 °C and the temperature of the ion transfer tube to 310 °C. The tube lens (S-lens) and collision energy were individually optimized for each analyte, using a 0.05 s scan time.The UHPLC consisted of a binary pump, a cooled autosampler, and a column oven , connected to a triple quadrupole mass spectrometer  equipped with a heated electrospray ionization (H-ESI). Chromatographic conditions were based on reported metabolomics analysis of polar metabolites . In deta+ metabolome in whole blood, calibration curves were constructed from the analysis of 10 standard compound aqueous solutions (range 0.3 to 20 μM), including internal standards, submitted to the same extraction procedure as regular samples. A linear regression was calculated per metabolite. As quantification criteria, a minimum of five points were evenly distributed over the regression line and the coefficient of determination was >0.9. Further statistics were done in R, v. 3.5.0 (R Core Team).The software Xcalibur v. 4.1.31.9 (Thermo Scientific) was used for instrument control, data acquisition and data processing. Positive ion mode extracted chromatograms using the metabolites MRM traces were integrated, normalized to internal standard  and total protein . Retention time and mass detection of metabolites were confirmed with authentic standards. For the quantification of the NAD+ biology in health and disease, from metabolic to signaling phenomena. We developed and presented a method with widespread applicability. The method includes 18 NAD+ metabolites and it was tested on a variety of biological samples, from cell lines to mouse tissues and biofluids, and human samples.Given the pivotal role of NAD(P)(H) to maintain energy and redox balances, great interest has emerged to understand NAD+ metabolome measurements are labile in human whole blood. Thus, pre-analytical and analytical protocols must be rigorously established for the specific biological matrices of interest to prevent potential degradation and ensure robust analytical outcomes.Sample acquisition and handling can alter measurement of redox cofactors since some metabolites in the NAD+ metabolome and the concentrations of the metabolites are dependent on sample type  and treatment/condition/nutritional status. Nevertheless, to better understand the dynamic behavior of the NAD+ subnetwork, a wide metabolite coverage is essential to map pathways and the metabolic fate of specific precursors.The coverage of the NAD+ metabolome at the clinical level becomes attractive not only for assessing NAD+-related metabolic status in populations, but also for interindividual variability and to evaluate therapies with NAD+ precursors.Finally, being able to monitor the NAD


ANGPTL8, an important regulator of lipid metabolism, was recently proven to have additional intracellular and receptor-mediated functions. This study aimed to investigate circulating levels of ANGPTL8 and its potential association with the risk of kidney function decline in a cohort study.2 of body surface area, a decrease in eGFR of ≥ 30% from baseline, chronic kidney disease (CKD)-related hospitalization or death, or end-stage renal disease. The association between baseline ANGPTL8 levels and kidney function decline was assessed using multivariable-adjusted Cox proportional hazards models, and inverse possibility of treatment weight (IPTW) was utilized to prevent overfitting.We analysed 2,311 participants aged 40 years old and older from the China Cardiometabolic Disease and Cancer Cohort (4C) Study. Kidney function decline was defined as an estimated glomerular filtration rate (eGFR) less than 60 mL per minute per 1.73 mThere were 136 (5.9%) cases of kidney function decline over a median of 3.8 years of follow-up. We found that serum ANGPTL8 levels at baseline were elevated in individuals with kidney function decline compared to those without kidney function decline during follow-up . Compared with the first quartile, multivariable-adjusted hazard ratio  for kidney function decline was 2.59  for the fourth ANGPTL8 quartile. Furthermore, compared with patients in the first ANGPTL8 quartile, those in the fourth ANGPTL8 quartile were more likely to report a higher stage of CKD . The conclusions of the regression analyses were not altered in the IPTW models. Multivariable-adjusted restricted cubic spline analyses suggested a linear relationship of ANGPTL8 with kidney function decline .Participants with higher circulating ANGPTL8 levels were at increased risk for kidney function decline, highlighting the importance of future studies addressing the pathophysiological role of ANGPTL8 in CKD.The online version contains supplementary material available at 10.1186/s12933-021-01317-3. ElevateChronic kidney disease (CKD) is a devastating condition that is reaching epidemic levels owing to the increasing prevalence of diabetes mellitus, hypertension and obesity, as well as the ageing of the population . CKD mayPrevious cross-sectional studies revealed that ANGPTL8 levels were increased in T2DM patients with albuminuria , 17. OurParticipants in the present study were recruited from Hubei Province, China, from 2011 to 2012 as part of the Risk Evaluation of Cancer in Chinese Diabetic Individuals: A Longitudinal (REACTION) Study, which was renamed the China Cardiometabolic Disease and Cancer Cohort (4C) Study –22. The 2 or CKD-related hospitalization) (N = 245) or missing serum creatinine values at baseline (N = 979), were not fasting at baseline  or visits (N = 508), were missing body mass index (BMI) values, glucose measurements, glycaemic measures, lipid profiles or information on a history of CVD, diabetes or hypertension , or had missing outcome data (N = 935). Additionally, the highest and lowest 0.5% of ANGPTL8 values were trimmed (N = 23) , leThe Committee on Human Research at Ruijin Hospital, Shanghai Jiaotong University School of Medicine, approved the study protocol , and all of the participants provided written informed consent. All of the methods were performed in accordance with the relevant guidelines and regulations.Blood samples were collected after overnight fasting. Serum was separated by centrifugation, aliquoted and then stored at  − 80 °C. Fasting serum ANGPTL8 levels were assessed using ELISA kits  with an intra-assay coefficient of variation (CV) of ≤ 6.5% and an interassay CV of ≤ 9.2% (provided by the manufacturer). The procedures were performed in accordance with the manufacturer’s instructions. All of the samples were analysed in duplicate.2 according to the CKD-Epidemiology Collaboration (CKD-EPI) formula [2; (2) G2, 60 ≤ eGFR < 90 mL/min/1.73 m2; (3) G3, 30 ≤ eGFR < 60 mL/min/1.73 m2; (4) G4, 15 ≤ eGFR < 30 mL/min/1.73 m2; and (5) G5, eGFR < 15 mL/min/1.73 m2 [Kidney function decline was defined as meeting at least 1 of the following criteria: (1) eGFR < 60 mL/min/1.73 m formula , 25, (2) formula , (3) hos/1.73 m2 . All of 2 test for categorical variables. We used Cox proportional hazards models to investigate the association between ANGPTL8 levels at baseline and the risk of kidney function decline. The relationship between ANGPTL8 and CKD stage was tested using ordinal logistic regression. We adjusted multivariable models for baseline age, sex, BMI, high-density lipoprotein (HDL), low-density lipoprotein (LDL), TC , TGs, aspartate aminotransferase (AST), alanine transaminase (ALT), and history of diabetes, hypertension and CVD. The inverse possibility of treatment weight (IPTW) was utilized to prevent overfitting [Continuous variables are expressed as the means ± standard deviations (SDs)  or medians  . Normal distribution of the data was tested using the Kolmogorov–Smirnov test. We obtained p values using the Kruskal–Wallis test for continuous variables and the χrfitting . Stratifrfitting . A knot Of the 2,311 participants, 136 (5.9%) patients developed kidney function decline during a median follow-up of 3.8 years , FPG (r = 0.07), TGs (r = 0.04), AST (r = 0.08) and creatinine r = 0.13) and inversely correlated with HDL (r =  − 0.05), eGFR at baseline (r =  − 0.13), eGFR at the final visit (r =  − 0.11) and eGFR change , hypertension and no CVD . However, the association was diminished in individuals without hypertension or with CVD .We performed stratified analyses according to age, sex, BMI, diabetes, hypertension, CVD or hyperlipidaemia at baseline. As shown in Fig. This retrospective cohort study found that serum baseline ANGPTL8 levels were elevated and independently associated with kidney function decline. Furthermore, multivariable-adjusted restricted cubic spline suggested a dose–response relationship of ANGPTL8 with the risk for kidney function decline. However, estimates for this association in individuals with CVD or without hypertension were not statistically significant.ANGPTL8, an important regulator of TG metabolism, has recently been proven to have additional intracellular and receptor-mediated functions , 30–32. . Stratified analyses also showed that the association between ANGPTL8 levels and kidney function decline was more significant in older individuals (age ≥ 60 years). Age is related to mitochondrial dysfunction, increased oxidative stress, inflammation, and hormonal changes [The underlying mechanisms for these pathophysiological associations await exposure. Our study found that ANGPTL8 was positively correlated with age, FPG, AST, and TG but inversely correlated with HDL. Previous studies found that ANGPTL8 levels were positively related to age , 18, 35. changes , which p changes . Second, changes . Thus, d changes , 38. ANG changes  and posi changes . However changes –6, ather changes , and NAS changes . Thus, A changes , which m changes , 39, 40. changes  and hype changes . TherefoOn the other hand, ANGPTL8 shares considerable structural similarity with ANGPTL3 and ANGPTL4 . As the There are some limitations of this study. First, all of the participants in our study were Chinese adults aged ≥ 40 years, limiting the generalizability of the findings. Interestingly, the previous 4 cross-sectional studies enrolled populations with a mean age > 50 years and concluded that elevated ANGPTL8 levels were associated with kidney function decline , 33, 34,In conclusion, participants with higher circulating ANPTL8 levels had an increased risk for kidney function decline, highlighting the importance of future studies addressing the pathophysiological role of ANGPTL8 in CKD.Additional file 1: Table S1. Medication for patients with chronic diseases, according to quartile of ANGPTL8 levels. Table S2. Ordinal logistic regression analysis of the association between ANGPTL8 and renal stage. Table S3. Correlations between ANGPTL8 levels and clinical variables. Figure S1. Flow diagram for the study population selection. Figure S2. ROC curves for the performance of ANGPTL8 concentration in detecting kidney function decline.


Hair testing for classic drugs of abuse offers the possibility of longer detection times (for drug substances) when compared to urine analysis. Hair analysis is routinely used to detect drug abuse in forensic cases and clinical toxicology, whereas it is rarely used at addiction clinics to monitor the clinical efficacy of therapeutic interventions. Here, we explore for the first time whether hair analysis might represent a valid tool to track the clinical improvements in a population of patients with cocaine use disorder (CocUD) who underwent a repetitive transcranial magnetic stimulation (rTMS) treatment.In recent years, hair has become an alternative biological specimen for drug testing in the fields of forensic and clinical toxicology. The advantages of hair testing include larger detection windows (months/years), depending on the length of the hair shaft, compared to those of urine/blood (hours to 2–4 days for most drugs). Segmental hair analysis can disclose a month-to-month (considering 1 cm segment cuts) information of drug exposure (single or repeated) and potentially identify patterns of drug use/administration. Repetitive transcranial magnetic stimulation (rTMS) was recently proposed as a valid tool for therapeutic purposes in addictions, including cocaine use disorder (CocUD). Here, we proposed hair testing analyses of classic drugs of abuse in a clinical setting to monitor the clinical changes in treatment-seeker CocUD patients undergoing protocol treatments with rTMS stimulating the left dorsolateral prefrontal cortex (l-DLPFC). We collected hair samples from nine CocUD patients at different stages from the beginning of treatments. Hair sample analyses revealed significant changes in the patterns of cocaine use, according to the negativity of urine screening tests and the clinical reductions of craving. These data, albeit preliminary, suggest that hair testing analysis of classic drugs of abuse could be extended to clinical settings to monitor the clinical efficacy of innovative therapeutic interventions, such as rTMS. In contrast to drug testing in conventional matrices, testing of hair may provide data on long-term exposure to drugs. The typical hair sample  identifies drug use as far as three months back, while body hair may go back even further, as opposed to urine analysis, which typically detects drug use from the past 48 to 72 h ,2,3. Fortert-butyl methyl ether, were obtained from Carlo Erba .Cocaine (COC), benzoylecgonine (BEG), morphine (MOR), 6-monoacetylmorphine (6-MAM), codeine (COD), methadone (MTD), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA) 3,4-Methylenedioxymethamphetamine (MDMA) Delta-9-tetrahydrocannabinol (THC), 11-nor-Delta 9 tetrahydrocannabinol-9-carboxylic acid (THC-COOH), and respective deuterated standards used as internal standards (ISs) were supplied by LGC Standards . N-O-Bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA with 1% TMS) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MBTFA) were obtained from Sigma-Aldrich . All reagents of analytical grade, such as methanol, dichloromethane This research study focused on hair testing analyses of classic drugs of abuse in nine treatment-seeker subjects diagnosed with CocUD, according to the Diagnostic and Statistical Manual of Mental Disorders 5th Edition (DSM–5) , as asseStock standard solutions containing all substances at 1 mg/mL concentrations were prepared in methanol. Working solutions at concentrations 1 μg/mL were immediately prepared by dilution of the stock standards with methanol and stored at −20°C until analysis. The internal standard (IS) working solutions were used at a concentration of 1 μg/mL. The method linearity for each compound was investigated in the range of 0.1–25 ng/mg. Calibration curves were obtained with five calibration levels by adding 0.1, 0.5, 1, 5, 10, and 25 ng analytes under investigation per mg hair. The solutions were prepared daily for each analytical batch by adding suitable amounts of working solutions to 25 mg of a pre-checked drug-free hair pool, donated by laboratory personnel. Quality control (QC) samples of 0.3, 4, and 20 ng analytes under investigation per mg of hair were prepared daily in drug-free hair samples, and used for calculation of validation parameters. Deuterated internal standards (10 ng each) were added, and samples were analyzed following the complete procedure. Dilution integrity was tested for over-the-curve samples with a concentration 5 and 10 times higher than the highest calibrators, with a dilution in methanol before sample treatment, verifying precision and accuracy to be within 15%.Hair samples were cut close to the scalp in the vertex region. Hair strands were then divided in two different segments depending on the length of the hairs. The first was cut at 3 cm from the proximal region, representing hair growth in the last three months, and the second at 3 cm to account approximately for the last 6 months. These cuts were decided according to international literature on the subject , and contert-butyl methyl ether, and shaken with a vortex for 2 min. The organic layer was evaporated to dryness under nitrogen flux at a temperature lower than 40 °C. Trifluoroacetyl derivatives were formed by reaction with 50 µL of MBTFA as a derivatization agent, in a dry bath, at 70 °C for 30 min.The pooled drug-free hair and hair samples (25 mg) were washed with 3 mL dichloromethane and allowed to dry at room temperature. One milliliter of 1 M sodium hydroxide added with ISs was added to every hair sample. The samples were then extracted with 3 mL of v/v) BSTFA/TMCS at 70 °C for 30 min. Finally, 1 μL of the sample was injected into GC/MS.Hair samples (25 mg) fortified with deuterated standards were incubated in a sodium hydroxide solution for 20 min at 95 °C and extracted with 3 mL of n-hexane: ethyl acetate (9:1). The organic extract was evaporated under a nitrogen stream at 40 °C. For extraction of THC-COOH metabolite, the remaining aqueous sample was acidified with 1 mL of 1 N HCl (resulting pH 4–5), extracted with 3 mL n-hexane: ethyl acetate (9:1) and centrifuged again to obtain a second extract. The combined organic phases were evaporated to dryness under nitrogen at 40 °C, and were then derivatized with 50 µL of 99:1 (m/z) to determine retention times and characteristic mass fragments. Retention times and qualifying ions of all drugs monitored in the selected-ion-monitoring (SIM) mode are reported in The GC–MS instrument consisted of a gas chromatographer Agilent 7890 A coupled with 5975 C MSD . Ultra Inert GC column Zebron  was installed. Analytes separation for all drugs of abuse was achieved on a fused silica capillary column  . The oven temperature for classic drugs of abuse was programmed at 80 °C for 1 min, increased to 230 °C at 35 °C/min, and then raised to 290 °C at 10 °C/min, and held for 10 min. To analyze amphetamine, the oven temperature was initially maintained at 70 °C during 2 min and programmed to 200 °C at 40 °C per min, and subsequently to 290 °C at 10 °C per min. Helium was used as a carrier gas at a flow rate of 1.2 mL/min. For cannabinoids determination, the oven temperature was programmed at 140 °C for 2 min, increased to 290 °C at 20 °C/min, and held for 10 min. For all substances, split injection mode (15:1) was used. Helium (purity 99%), with a flow rate of 1 mL/min was used as a carrier gas. The injection port, ion source, quadrupole, and interface temperatures were 260 °C, 230 °C, 150 °C, and 280 °C, respectively. The dwell time was 0.2 msec. The electron-impact (EI) mass spectra of analytes were recorded by the total ion-monitoring mode  or major antidepressants  interfered with the assay. Blank hair samples injected after the highest point of the calibration curve did not present any traces of carryover. Absolute analytical recovery (mean ± SD of 15 replicates) ranged from 90.5% ± 4.2 for 6-MAM, 94.3% ± 2.6 for MOR, 88.9% ± 3.2 for COD, 90.1% ± 5.2 for COC, 91.4% ± 2.8 for BZE, 86.2 ± 4.5 for MTD, 88.2% ± 5.3 for EDDP, 92.2% ± 3.2 for A, 90.1% ± 3.2 for MA, 91.4% ± 2.1 for MDMA, 90.3% ± 3.2 for MDA, 80.6 ± 6.2% for THC, and 81.7 ± 4.3% for THC-COOH. The matrix effect showed less than 10% analytical suppression due to endogenous substances for both the analytesAs shown in In Drug testing in hair is important in clinical and forensic settings, to prove consumption, and help evaluate one’s level of impairment. Hair testing is particularly useful in drug rehabilitation programs, workplace drug testing, and driver’s license renewals. This stable matrix may be used to confirm long-term exposure to drugs over a period of weeks to years, depending on the length of hair collected. To our knowledge, this study proposes, for the first time, the use of a well-established methodology for drug testing in hair in a clinical setting, to monitor the therapeutic effects of an innovative treatment for cocaine addiction. rTMS stimulating l-DLPFC is receiving increasing attention as a potential treatment for addictions, including CocUD ,9. IndeeThe LC–MS/MS method developed for the simultaneous determination of The GC–MS method, developed for the simultaneous determination of 13 drugs of abuse in hair samples, is simple, rapid, and robust. The method was applied to real hair samples collected from CocUD patients undergoing innovative addiction treatments. Despite our results coming from a very small number of patients, we suggest that this methodology be considered as a support measure, to evaluate the decrease in cocaine consumption and changes in the patterns of drug use.