Nigella sativa is plant that is endowed with various pharmacological activities including antioxidant, anticancer, anti-inflammatory, antibacterial, antidiabetic, and immunostimulant. This study aims to investigate the antidiabetic activity of the N. sativa essential oil on two key enzymes the α-amylase and hemoglobin glycation. After the extraction procedure, the N. sativa essential oil, were subject to qualitative and semi-quantitative analysis using GC/MS, for the identification of the different bioactive compounds. This was followed by an evaluation of the in vitro inhibition capacity of the α-amylase and the hemoglobin glycation. Finally, a molecular docking study was conducted to determine the bioactive compounds responsible for the antidiabetic activity. The extracted essential oil showed the presence of different bioactive compounds including α-phellandrene (29.6%), β-cymene (23.8%), 4-caranol (9.7%), thymol (7%). The N. sativa essential oil was found to be endowed with an antiradical scavenging activity with an IC50 of (7.81 ± 0.08 mg/ml), and to have a ferric reducing activity with an IC50 value of (7.53 ± 0.11 mg/ml). The IC50 value for the α-amylase inhibitory activity was 0.809 mg/ml, indicating an inhibitory impact of the enzyme. The IC50 value for the N. sativa essential oil’s hemoglobin antiglycation activity was 0.093 mg/ml. For most predominating phytochemicals present in the N. sativa essential oil, molecular docking studies against human pancreatic α-amylase and human hemoglobin enzymes revealed that these compounds can serve as lead molecules to develop new antidiabetic compounds. Chronic hyperglycemia is a symptom of diabetes, which is a metabolic condition. . AccordiN. sativa (NS) or black cumin is a plant native to the Mediterranean region , the essential oil was recovered and stored in a dark glass container for further use.The The analysis of the essential oil from hydrodistillation was performed in a Shimadzu GC-2010 gas chromatography (GC) that is equipped with a column . The gas chromatography is coupled to GC-MS-QP2010 mass detector. The helium carrier gas is attached to the gas chromatography with a steady pressure (100 KPa). The oven of the GC was put on 50°C for about 1 min. Then, the T was increased with a rate of 10°C per minute to reach a temperature of 250°C. A 1 µl solution of the essential oil already prepared in n-hexane and injected. The GC-MS system was in scan mode when used. By comparing the constituents’ mass spectra to information stored in the National Institute of Standards and Technology’s computer library (NIST147), the constituents’ identities were ascertained.Data was gathered and handled utilizing LabSolutions (version 2.5).The antiradical scavenging potential of NS extracted Eo was investigated with the method proposed by Manzocco et al. (1998) . From eaThe percentage of radical scavenging activity was calculated according to the formula:3Fe (CN)6) and 1.25 ml of phosphate buffer solution (PBS) . Afterward, The resulting mixture was given a 20-min incubation period in a water bath at 50°C, 1.25 ml of trichloroacetic acid (TCA) was added after cooling to stop the reaction. For 10 min, the various tubes were centrifuged at 3,000 rpm. From each tube, 1.25 ml was taken and mixed with 0.25 ml of ferric chloride (FeCl3). Finally, the absorbance was measured at 700 nm against the blank. The positive control used in our study was the ascorbic acid.The ferric reducing power assay was evaluated following the protocol elaborated by  with som2O. At 540 nm, the absorbance was then measured.The evaluation of the capacity of the NS Eo to inhibit the action of the α-amylase enzyme was measured following the protocol proposed by The formula below was used to compute the percentage of α-amylase enzyme inhibition:N. sativa essential oil hemoglobin antiglycation effect, the method described by In order to evaluate the The reaction mixture was then kept at room temperature for 72 h while being kept in a dark area. At 443 nm, the mixture’s absorbance was measured.The following formula was used to calculate the inhibition percentage:https://pubchem.ncbi.nlm.nih.gov/): α-Phellandrene (CID: 7460), β-Cymene (CID: 10812), 4-Caranol (CID: 86056), Thymol (CID: 6989), α-Pinene (CID: 6654), β-Pinene (CID: 14896). Acarbose (CID: 41774) and Gallic acid (CID: 370) were used as standard drugs.Based on the GC-MS analysis, the most dominant compounds identified in the essential oil of NS seeds (>6%) were chosen for molecular docking calculations. 3D structures as SDF format of following selected biocompounds were retrieved from PubChem database (www.rcsb.org). The target receptors were prepared before docking by deleting all water molecules, inhibitors, co-crystallized molecules, and adding missing hydrogen atoms (polar only) with the aid of BIOVIA Discovery Studio Visualizer 2020.The crystal structures of the α-amylase from the human pancreas in complex with nitrite and acarbose (PDB code: 2qv4) at 1.97 Å resolution  and the To investigate the binding method of the selected compounds of NS essential oil with α-amylase (αA) enzyme and human hemoglobin (HbA) protein, molecular modeling studies were carried out using virtual screening software PyRx 8.0 . Briefly50 of the sample and the drug.ANOVA was used to do statistical analysis on the data, which included multiple-group comparisons . The unpaired T student was used to compare the ICThe NS Eo was extracted using hydrodistillation with a modified-Clevenger apparatus, yielding 1.16 percent. The chemical compounds of the essential oil were examined using a gas chromatography linked to mass spectrometry. The components’ identities were discovered through a comparison of their mass spectra with data held at the (NIST). The chemical composition of the Eo was performed in a previous study. The results obtained are drafted in 50 of about 7.809 mg/ml. While it was also found that the NS essential oil is endowed with a ferric reducing power with an IC50 of 7.531 mg/ml. A highly significant difference was observed when compared to ascorbic acid and this was for both antiradical scavenging activity against DPPH, and the ferric reducing power assay (p < 0.0001)  . Our essactivity . On the activity . Finallyactivity .p < 0.0001). The IC50 value recorded for the black cumin essential oil was about 0.809 ± 0.11 mg/ml. Acarbose used as positive control showed a lower IC50 value equal to 0.35 ± 0.02 mg/ml. The statistical analysis performed showed the existence of highly significant difference between the IC50 of the acarbose used as positive control and the IC50 of the NS essential oil (p < 0.001). Our essential oil performed a moderate activity when comparing the IC50 recorded in our Essential oil that was higher than that found when testing the volatile compounds from Sabina chinensis (IC50 187.08 μg/ml), and from Eruca vesicaria   , (Hichri leaves) . Also, t14 mg/ml . Sahin e14 mg/ml  mentioneectively . Also thectively .in vitro using the hemoglobin antiglycation method. The results obtained indicate that the inhibition of the hemoglobin glycation was in a dose-dependent manner (p > 0.05). The IC50 value registered was 0.072 ± 0.017 mg/ml for gallic acid and 0.093 ± 0.002 mg/ml for NS essential oil (p > 0.05). On the other hand, different studies assessed on the essential oil of Juniperus oblonga and Juniperus sabina branches were able to inhibit hemoglobin glycation in dose dependent manner but using high concentrations   .The knowledge of the binding mode and the probable binding position of a given drug within the protein scaffolds plays a vital role to determine and understand the effectiveness of the molecule to serve as a therapeutic agent . In thisvia Van der Waals forces. According to the obtained results, the re-docked acarbose derivative showed similar conformational poses and was found to have almost interactions with the similar amino acids where the co-crystallized acarbose interacted. The RMSD value which gives the average deviation between the crystal structure pose and the re-docked pose of the acarbose derivative was 1.298 Å (The docking procedure was initiated by re-docking the native ligand (acarbose derivative), to the binding site of the crystal structure of the human pancreatic α-amylase protein (2qv4) for validating the docking protocol. In the crystal structure of α-amylase, the co-crystallized ligand showed conventional hydrogen bonds with Thr163, Ala106, Gly164, Val107, Gln63, Tyr62, His299, Arg195, Asp300, and Glu233 amino acids. Moreover, this ligand also interacts with several other residues of protein such as Ile51, Asn105, Gly104, Leu165, Glu60, His101, Leu162, Tyr151, Ile235, Lys200, His201, Asp197, Ala198, Asn298, Trp58, Trp59, and His305  1.298 Å . This vain vitro α-amylase inhibition assay.Analysis of the docking outcomes summarized in via hydrogen bonds with seven amino acids, namely, Asp300, Arg195, Glu233, Asp353, Asp356, His305, and Thr163; and also showed hydrophobic interactions (VDW) with Tyr62, His101, His299, Ile235, Asn352, Asp197, Ala198, Leu162, Leu165, Trp58, Trp59, Trp357, Val98, Val354, and Ile235. For all abundant phytoconstituents, predominantly hydrophobic interactions were responsible for the binding of these molecules to the active site of α-amylase. Also, it was observed that these molecules formed interactions with similar residues with slight differences. The most stable complex  thymol—α-amylase was stabilized by one Pi-Sigma, one Pi-Pi stacked, one Pi-Alkyl, and seven VDW interactions involving the amino acids Tyr62, His299, Asp197, Trp58, Trp59, Asp300, Arg195, His101, and Leu165. For the other complexes, analysis of the best-docked poses revealed that they mainly exhibited numerous hydrophobic interactions including Pi-Sigma, Pi-Alkyl, Pi-Pi stacked, and VDW, except 4-Caranol which in addition to these interactions form a hydrogen bond with Asp197 residue.The chemical bonding mode of the complexes formed between studied compounds and the binding pocket residues of α-amylase are displayed in On the other hand, the study of the X-ray crystal structure of human pancreatic α-amylase demonstrated the presence of three characteristic amino acids namely Asp197, Asp300, and Glu233 which contribute to the overall catalytic action in starch-hydrolyzing enzyme . Asp197 Interestingly, acarbose was found to form strong hydrogen bonds with all these three enzyme key residues, which may explain its lower binding potential value in comparison with other studied compounds. Meanwhile, the docking data also showed that all the selected phytochemicals interact with two of these enzyme key residues (Asp197 and Asp300), except α-pinene which forms interaction only with Asp300 amino acid. Overall, the natural metabolites studied in this work as well as acarbose were found to interact at the active site of α-amylase with similar amino acid residues as the native ligand did, especially the enzyme key residues.Based on the findings, these identified compounds in the NS essential oil could be considered promising metabolites playing a major role in the inhibition of the human pancreatic α-amylase enzyme.To explore the putative binding mode of our compounds to the active site residues of human hemoglobin protein, molecular docking studies were performed. The results of the study have been depicted in As it can be seen in 3) form six Pi-Alkyl interactions. Moreover, the compound also binds with the three amino acids Leu29, Met32, and Asn97 via VDW forces. Thymol was surrounded by Leu106, Leu110, Val134, Val137, Phe71, Phe103, Val67, Leu68, Leu141, Ala70a, and Ala138 via Pi-Alkyl , Pi-Pi stacked (with Phe71) and VDW  interactions.The representation of the complex formed between gallic acid and HbA, indicating the presence of four hydrogen bonds with Val62, His87, Phe98, and Ser133; two Pi-Alkyl interactions with Leu101 and Leu136; in addition to seven VDW interactions with Leu105, Leu83, Leu129, Leu66, Ala65, Val132, and Ser102 residues. β-cymene -HbA complex was stabilized by Pi-Alkyl bonds with Leu106, Val134, Val137 and Phe71; and VDW forces involving the amino acids Phe103, Phe85, Leu68, Leu110, Leu141, Val67 and Val138. α-phellandrene showed similar interactions like β-cymene with slight modifications. Surprisingly, the two analog α-pinene and β-pinene were found to interact with the same residues (with more residues for α-pinene) despite the fact that they were bound to the different chains of HbA. The hydrogen atom of the hydroxyl group (OH) of 4-caranol forms a conventional hydrogen bond with His87, while the 6-ring and the methyl groups (CHN. sativa are endowed with an abundance of a variety of chemical compounds including α-Phellandrene, β-Cymene, and 4-caranol, among others. Our findings indicate that the volatile components of black cumin have a powerful ability to impede the pancreatic α-amylase and hemoglobin glycation. On the other hand, In order to determine the potential binding mode of the most prevalent chemicals found in NS essential oil, molecular docking studies were conducted, on human pancreatic α-amylase and human hemoglobin. The docking findings suggest these compounds as lead molecules against α-amylase and human hemoglobin. However, appropriate experiments using these phytochemicals should be performed to determine their in vitro and in vivo antidiabetic effects.The essential oil of


Chenopodium formosanum (CF), also known as Djulis, is a traditional cereal in Taiwan. This study investigated the protection mechanisms of CF extract against ultraviolet (UV) radiation and advanced glycation end products (AGEs)-induced stress. The results indicated that CF extract had strong antioxidant and free radical scavenging effects. It could reduce UV-induced intracellular ROS generation and initiate the antioxidant defense system by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in human skin fibroblasts. CF extract modulated mitogen-activated protein kinase (MAPK) and transformed growth factor-beta (TGF-β) signaling pathways to alleviate oxidative stress-induced skin aging. Moreover, the results revealed that CF extract not only promoted collagen synthesis but also improved aging-induced collagen degradation. CF extract attenuated AGEs-induced ROS production and the upregulation of receptor for AGEs (RAGE). The overall results suggest that CF extract provides an effective anti-aging strategy by preventing skin damage from oxidative stress and collagen loss with potent antioxidant, anti-photoaging, and antiglycation activities.Skin aging is a complex process involving photoaging and glycation stress, which share some fundamental pathways and have common mediators. They can cause skin damage and collagen degradation by inducing oxidative stress and the accumulation of reactive oxygen species (ROS).  Aging is defined as the result of a progressive decline in the physiological function of cells and tissues of the body. During aging, the accumulation of damaged products gradually affects the organization and repair capacity of tissue. Skin, being the protective barrier for the body, is often subjected to intrinsic and extrinsic factors of aging . The preBoth environment ROS produced by UV light and endogenous ROS formed by oxidative metabolism regulate cellular oxidative stress and destroy the collagen-rich extracellular matrix (ECM), which is not only the hallmark of skin connective tissue aging but also the major cause of skin deterioration . Damage UVB rays are the most damaging waveband of solar radiation reaching the earth, which results in oxidative stress by stimulating ROS formation and is involved in hyperpigmentation, skin inflammation, immunosuppression, photoaging, and carcinogenesis ,7. It isε-(1-Carboxymethyl)-L-lysine (CML) produced by oxidative modification is the major glycated protein in humans [AGEs are a heterogeneous group of molecules formed as a result of glycation, a spontaneous non-enzymatic reaction between the reducing sugars and amino groups of proteins . The intn humans ,15. Durin humans . Addition humans .Chenopodium formosanum (CF), also known as Djulis, is an edible plant in Taiwan and is usually cooked with rice or millet as a staple food. CF is considered a high nutritional value food and a source of bioactive compounds to human health [n health . Besidesn health ,20, CF an health ,22,23,24n health , antihypn health ,26, hepan health ,28,29, an health ,31 and an health ,32. Nevem/z 481 to the product ion at m/z 371 for 20-hydroxyecdysone (m/z 611 to the product ion at m/z 303 for rutin (m/z 481) and rutin (m/z 611) in CF extract. The content of rutin and 20-hydroxyecdysone in CF extract were 14.5 ± 0.4 mg/g and 19.5 ± 0.6 mg/g, respectively.The content analysis of target bioactive compounds in CF extract was conducted by high-performance liquid chromatography (HPLC)-Mass/MS and the multiple reaction monitoring (MRM) method. The MRM ion chromatogram and mass spectrum on electrospray ionization (ESI) positive mode of 20-hydroxyecdysone and rutin standard were shown in ecdysone  and the or rutin  are show50) of CF extract on DPPH free radical scavenging was 84.7 ± 13.0 µg/mL.DPPH is a commonly used reagent to estimate the total free radical scavenging activity of antioxidants by detecting the ability of extracts to provide hydrogen protons. The result showed that CF extract exhibited strong scavenging activity of 40.2% ± 3.3% at 50 µg/mL and 89.1% ± 2.6% at 500 µg/mL, while the scavenging activity of ascorbic acid was 96.6% ± 2.3% at 10 µg/mL a. The haThe reducing capability of the CF extract observed in the present study is shown in The superoxide anion radical scavenging activity was 82.2% ± 6.1% for 250 µg/mL BHA (comparative control) and ranged from 59.6% ± 3.5% to 73.1% ± 2.6% for 100–1000 µg/mL CF extract c.50 of CF extract was 956.5 ± 59.2 µg/mL for hydrogen peroxide scavenging activity.The hydrogen peroxide scavenging activity ranged from 10.3% ± 1.1% to 61.1% ± 2.3% for 200–1500 µg/mL CF extract and was 96.3% ± 2.0% for BHA (250 µg/mL) d. The IC50 of CF for hydroxyl radical scavenging activity was 240.7 ± 24.3 µg/mL.The hydroxyl radical scavenging activities of CF extract (100–1000 µg/mL) and the comparative control mannitol (µg/mL) are shown in 50 of CF extract was 625.5 ± 46.0 µg/mL for metal chelation.To evaluate the cytotoxic effect of CF extract in human skin fibroblasts (Hs68 cells), the viability of cells treated with CF extract was assessed by MTT assay. As shown in 2) radiation induced cytotoxicity in Hs68 cells in a dose-dependent manner (2) and subsequently treated with CF extract  for 24 h; CF extract significantly alleviated UV-induced cytotoxicity  for 2 h, ROS production in Hs68 cells was induced 2.16-fold compared with that in the non-irradiated group. However, treatment with 100–250 µg/mL CF extract significantly decreased ROS generation. ROS were downregulated 1.64-fold compared with the radiation group by CF extract at 250 µg/mL  radiation induced Nrf2 expression and decreased Keap1 expression, whereas CF extract (250 µg/mL) significantly induced Nrf2 expression by 2.4-fold compared with the radiation group. After UV radiation and treatment with CF extract at 250 µg/mL the protein expression of HO-1 increased by 2.5-fold. The results indicated that CF extract could initiate the antioxidant defense mechanism through the Nrf2/HO-1 signaling pathway and protect cells from oxidative stress in a biological system.Under normal conditions, Keap1 forms a complex with Nrf2 and promotes the ubiquitination and degradation of Nrf2. However, the Nrf2 signaling pathway is initiated when cells are stimulated by environmental oxidative stress. In this study, Hs68 cells were treated with CF extract to observe its effects on the Nrf2 signaling pathway after UV radiation. As shown in UV radiation promotes Nrf2 activation and translocation into the nucleus of cells. In this study, the Nrf2 immunofluorescence staining assay in human skin fibroblasts was performed to determine the level of Nrf2 activation. The translocation of Nrf2 from the cytoplasm to the nucleus increased after UVB radiation, whereas the translocation was also enhanced after CF extract treatment . The resUV radiation induces the phosphorylation of MAPK, which triggers downstream signal transduction to activate transcription factor AP-1 and enhance MMP expression. When the skin is exposed to oxidative stress, MMPs are upregulated, which then induce collagen degradation and wrinkle formation. As shown in Tissue inhibitors of metalloproteinases (TIMPs) are inhibitors of MMPs expressed in the skin connective tissue. TIMP-1 expression was decreased after UVB radiation and enhanced by CF extract (100–250 µg/mL) . These rp-c-Jun and c-Fos (2.4- and 1.2-fold compared with the control group), whereas they were downregulated by CF extract (100–250 µg/mL). Treatment with CF extract significantly decreased the expression of p-c-Jun and c-Fos at 250 µg/mL  significantly restored protein expression .To investigate the effect of CF extract on glycation stress, Hs68 cells were treated with CML. After treating the cells with CML (100 µg/mL) for 6 h, the amount of ROS generated was 1.92-fold higher than that in the non-treated group. However, treatment with CF extract (100–250 µg/mL) significantly decreased ROS production. ROS was downregulated 1.30-fold compared with the CML-treated group by CF extract at 250 µg/mL . FurtherHs68 cells were treated with CF extract (50–250 µg/mL) for 24 h, and type I procollagen expression was measured using western blotting. After treatment with CF extract, type I procollagen expression was significantly upregulated (1.7-fold compared with the control group) a. The to2) radiation or treated with CML (100 µg/mL) were treated with CF extract for 24 h. As shown in Hs68 cells exposed to UVB  is a native grain plant grown in Taiwan, mainly cultivated in aboriginal residential areas. Apart from cereal use, the application of CF based on antioxidant properties has attracted increasing attention [ttention ,29,30,35ttention , reducinttention ,38. Rutittention ,22; sevettention  and suggttention . CF has ttention . In thisttention . This stChenopodium formosanum [Microsorum grossum (Polypodiaceae) is one of the most commonly used ferns in Polynesian traditional medicine, and its frond and rhizome extracts contain 20-hydroxyecdysone as the main bioactive components. Microsorum grossum was known to have UVB-protective effects on human dermal fibroblasts by upregulating the antioxidant enzyme HO-1 and inhibiting stress-induced premature senescence [Chenopodium quinoa (Amaranthaceae) is the seed crop known as the “golden grain” by the native Andean people in South America, including 20-hydroxyecdysone, which has been shown to inhibit intracellular ROS production and MMPs activity [C. quinoa seeds possess strong inhibition activity against collagenase and the DPPH free radicals, and a potent ability to chelate iron ions [20-Hydroxyecdysone is a bioactive compound belonging to phytoecdysteroids, commonly found in rmosanum ,22,23,24nescence . Chenopoactivity . In addiron ions . Many prron ions . Plant eROS are byproducts of regular oxidative cell function in the electron transport chain of aerobic metabolism reaction. Excessive UV exposure and other environmental stressors overwhelm the cutaneous antioxidant capacity further inducing ROS production, DNA fragmentation, lipid peroxidation, and apoptosis ,46. HighThe skin possesses an endogenous antioxidant defense system to deal with oxidative stress induced by aging. Nrf2 is a transcription factor that acts against oxidative stress, and Keap1 is a pressure sensor that regulates Nrf2 expression . When thPhotoaging is caused by an imbalance between the accumulation and degradation of ECM after repetitive UV absorption. UV radiation damages alternations of cutaneous collagen and hastens the generation of ROS, which in turn act on cells and matrix components to mediate further turnover ,47. ROS One of the factors of intrinsic skin aging is the progressive accumulation of AGEs throughout the lifespan. RAGE belongs to the immunoglobulin superfamily of cell surface receptors, which interacts with several ligands, especially CML, one of the glycation products of the Maillard reaction . RAGE isGlycation-induced biological products are known to be associated with skin aging, diabetes, and the progression of some tumors. During the aging process, the accumulation of AGEs changes the mechanical properties of skin by increasing stiffness . Therefo2PO4-KOH) were purchased from USB Corporation . 2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide(III) (K3Fe(CN)6), ferric chloride (FeCl3), ascorbic acid, phenazine methosulfate (PMS), β-nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH), nitrotetrazolium blue chloride (NBT), butylated hydroxyanisole (BHA), 2-deoxy-D-ribose, thiobarbituric acid (TBA), trichloroacetic acid (TCA), mannitol, ferrous chloride (FeCl2), ethylenediaminetetraacetic acid (EDTA), 2′, 7′-dichlorofluorescin-diacetate (H2DCFDA), 2-propanol and 20-hydroxyecdysone were purchased from Sigma-Aldrich . Rutin was obtained from ACROS Organics . HPLC-grade acetonitrile and formic acid were obtained from J.T. Baker, Thermo Fisher Scientific, Inc. . All other chemicals used were of analytical grade or higher grade.Reagents used in the cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA, were obtained from Gibco, Thermo Fisher Scientific, Inc. . CML was purchased from Cayman Chemical . Sodium dodecyl sulfate (SDS), 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), and potassium phosphate-potassium hydroxide (KHChenopodium formosanum was added to water at the ratio of 1:10 (w/v) and extracted in three different temperatures, first at 25 °C, then at 50 °C, and finally at 70 °C. The mixture was centrifuged at 4600× g for 20 min and the supernatant was collected. The powder of CF extract was obtained after removing solvent from the supernatant by freeze-drying and stored at room temperature.CF used in this study was purchased from Pingtung County, Taiwan, and the CF extract was supplied by TCI Co., Ltd. . The preparation method of CF extract has been reported previously . BrieflyThe HPLC-ESI mass spectrometric analysis was performed using Dionex Ultimate 3000 liquid chromatography  coupled with an ion trap mass spectrometer . The separation method was performed as described by Chen et al., with minor modification . CF extrm/z 481 to the product ion at m/z 371 for 20-hydroxyecdysone and the transition from the precursor ion at m/z 611 to the product ion at m/z 303 for rutin. Ion isolation width was set to 4 m/z with a fragmentation amplitude of 0.5 V. The compound was identified and quantified by comparing the mass spectra obtained by ESI-MS/MS with commercial reference standards.Mass spectrometry was carried out by the program Esquire Control (V6.2) and LC/MS was controlled by Hystar (V3.2) . The mass spectrometer operating parameters were set as follows: nebulizer pressure 30 psi, dry gas 12 L/min, dry temperature 300 °C, capillary voltage of 3600 V with an end plate of −500 V. MS/MS was applied to all major ions in the selected mass range. Multiple reaction monitoring (MRM)-profiling was performed by analyzing the following ion transitions: the transition from the precursor ion at Reaction mixtures containing a methanol solution of DPPH and serial CF extract dilutions of 50–500 µg/mL were placed in a 96-well microplate and incubated for 30 min. The absorbance was determined at 517 nm using a microplate reader . Ascorbic acid (10 µg/mL) was used as a comparative control .3Fe(CN)6 and then incubated at 50 °C for 20 min. TCA was added, and the reaction mixture was centrifuged at 3000 rpm for 10 min. FeCl3 solution was mixed with supernatant and the absorbance was measured at 700 nm using a multi-mode reader . Ascorbic acid (100 µg/mL) was used as a comparative control [Serial concentrations of CF extract (100–1000 µg/mL) were mixed with phosphate buffer and K control .Reaction mixtures containing PMS, NADH, and NBT were prepared in phosphate buffer and serial CF extract dilutions of 100–1000 µg/mL. The mixture was reacted for 5 min, and the absorbance was read at 560 nm using a microplate reader . BHA (250 µg/mL) was used as a comparative control .2O2 was performed as previously described [2O2 solution and reacted in the dark for 10 min. The absorbance of the reaction mixture was recorded at 230 nm using a multi-mode reader , and BHA (250 µg/mL) was used as a comparative control.The activity of CF extract to scavenge Hescribed . Various2PO4-KOH buffer, 2-deoxy-D-ribose, FeCl3, ascorbic acid, EDTA, H2O2, and deionized water. After incubation at 37 °C for 1 h, TBA and TCA were added to the mixture, and the mixture was incubated at 100 °C for 15 min and then centrifuged at 4000 rpm and 25 °C for 10 min. Subsequently, the absorbance of the supernatant was measured at 532 nm using a microplate reader . Mannitol (2500 µg/mL) was used as a comparative control [The assay was conducted by mixing CF extract dilutions (100–1000 µg/mL), KH control .2 solution and the reaction was initiated by adding ferrozine. After the mixture had reached equilibrium, the Fe2+-chelating activity of CF extract was monitored by measuring the absorbance of the Fe2+-ferrozine complex at 562 nm using a multi-mode reader . EDTA (100 µM) was used as a comparative control.The chelation of ferrous ions by CF extract was carried out according to the procedure described previously . CF extr2. At near confluence (80–90%), cells were disaggregated in trypsin-EDTA and subcultured [Hs68 cells were purchased from the Bioresource Collection and Research Center . Cells were maintained in 10-cm cell culture dishes and grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in an incubator containing 5% COcultured .2). The UVB energy spectrum (280–320 nm) was supplied by UV Crosslinker CL-1000M  with an emission peak at 302 nm and the exposure time was 15–30 s. The UVB radiation doses in cell viability assay were 20–100 mJ/cm2 to evaluate the appropriate UVB exposure dose to induce cytotoxicity. Hs68 cells were radiated with a high dose UVB (80 mJ/cm2) for 2 h to investigate the rapid radical scavenging ability of CF extract against short-term overproduced intracellular ROS. Other experiments use UVB radiation (40 mJ/cm2) in Hs68 cells for 24 h to mimic cells under long-term oxidative stress conditions. CML was supplied as a crystalline solid and aqueous solutions of CML were prepared by dissolving the compound in PBS buffer. The cells were treated with 100 µg/mL CML.The UV exposure dose selection for Hs68 cells referred to previous studies ,56. Prio4 cells/well), allowed to attach overnight, and were treated with 1 mL of various concentrations of CF extract (50–250 µg/mL) dissolved in DMEM for 24 h. MTT solution was added to the plates followed by incubation at 37 °C for 4 h. Then, 2-propanol was added to dissolve the MTT formazan crystals and the absorbance was measured at 570 nm using a microplate reader  [Hs68 cells were seeded into 24-well plates (4 × 10Austria) .2DCFDA)/fluorescent (DCF) system that measures ROS activity within the cell. Hs68 cells were seeded into 24-well plates (4 × 104 cells/well) and allowed to attach overnight. Cells were incubated in a serum-free medium in the presence of various concentrations of CF extract (100–250 µg/mL) after UVB radiation or CML treatment. The cells were washed with PBS, and the ROS detection reagent DCFDA was added to each well for 30 min. Images were captured using a fluorescence microscope , and the fluorescence (excitation/emission: 488 nm/520 nm) were measured using a microplate reader  [The assay is based on the use of an established non-fluorescent  .5 cells/dish) were collected and homogenized with protein extraction buffer on ice. The cell lysates were vortexed for 30 min at 4 °C and centrifuged to obtain the supernatant as an intracellular protein. Prepared samples containing equal amounts of total protein were separated by electrophoresis on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride blotting membranes. Nonspecific binding was blocked with non-fat milk in TBST buffer. The membrane was incubated with primary antibodies against, c-Jun, c-Fos, MMP-1, MMP-3, TGF-β, Smad3, and pro-COL1A1 , MMP-9, TIMP-1, and HO-1 . Nrf2, Keap1, RAGE, p-c-Jun, p-p38, p-ERK and β-actin  at 4 °C overnight. Subsequently, the membranes were washed with TBST buffer several times, and the corresponding anti-immunoglobulin G-horseradish peroxidase was used as the secondary antibody. The blots were incubated with a chemiluminescence detection reagent. Immunoreactive bands were detected with the ECL western blotting detection system , and the signal intensity was quantified using ImageJ software  [After the indicated treatments, Hs68 cells  .5 cells/well) and allowed to attach overnight. Cells were irradiated with UVB (40 mJ/cm2) and treated with 1 mL CF extract dissolved in DMEM. The cells were washed with PBS, fixed with paraformaldehyde for 30 min, and blocked using non-fat milk with Triton X-100/PBS-buffer  for 60 min. Samples were incubated with primary rabbit anti-Nrf2 antibody overnight at 4 °C. Slides were washed with PBS and then incubated with an anti-rabbit secondary antibody for 2 h at room temperature. Slides were mounted using ProLong® Gold Antifade Mountant with DAPI . All samples were analyzed using a confocal spectral microscope imaging system  [The cells were seeded on slides in a 6-well plate (Hs68 cells: 1 × 10Germany) .The total collagen content was quantified using the Sirius Red Collagen Detection kit . The cell culture medium was collected and mixed with the concentration reagent. The mixture was vortexed and incubated at 4 °C for 24 h. After incubation, the mixture was centrifuged at 10,000 rpm, and the supernatant was discarded. Following this, acetic acid was added to the microtube to dissolve the pellet, and the obtained solution was used as the test sample. Sirius Red solution, for collagen staining, was added to each tube, vortexed, incubated for 20 min at room temperature, and centrifuged. The supernatant was removed by carefully pipetting without disturbing the pellet. Washing solution was added to each tube, and the above steps were repeated once. Finally, an extraction buffer was added and the absorbance was measured at 540 nm using a microplate reader .p < 0.05 were considered statistically significant.Data are presented as mean ± standard deviation at least three independent experiments. All statistical analyses were performed using GraphPad Prism 5 . Significant differences between groups in the experiments were analyzed using one-way ANOVA, followed by Tukey’s post-hoc test. Differences with The present study reveals the molecular mechanisms responsible for the protective effect of CF against UV and AGEs-induced intracellular ROS generation and collagen degradation in skin fibroblasts. As a consequence, our study provides current understanding and information on the role of CF extract in the cellular defense system against oxidative stress via activation of the Nrf2/HO-1 signaling pathway. Additionally, CF not only modulates MAPK/AP-1/MMPs and TGF-β signaling pathways to prevent oxidative stress-induced collagen loss but also attenuates the upregulation of receptors for AGEs. CF extract can be developed into healthy foods or applied to skincare products and cosmetics as an antioxidant, anti-photoaging, and antiglycation agent to delay aging in the future. The regulatory mechanisms of CF extract in Hs68 cells are shown in


Adverse prognosis in Ewing sarcoma (ES) is associated with the presence of metastases, particularly in bone, tumor hypoxia and chromosomal instability (CIN). Yet, a mechanistic link between these factors remains unknown. We demonstrate that in ES, tumor hypoxia selectively exacerbates bone metastasis. This process is triggered by hypoxia-induced stimulation of the neuropeptide Y (NPY)/Y5 receptor (Y5R) pathway, which leads to RhoA over-activation and cytokinesis failure. These mitotic defects result in the formation of polyploid ES cells, the progeny of which exhibit high CIN, an ability to invade and colonize bone, and a resistance to chemotherapy. Blocking Y5R in hypoxic ES tumors prevents polyploidization and bone metastasis. Our findings provide evidence for the role of the hypoxia-inducible NPY/Y5R/RhoA axis in promoting genomic changes and subsequent osseous dissemination in ES, and suggest that targeting this pathway may prevent CIN and disease progression in ES and other cancers rich in NPY and Y5R. Ewing sarcoma tumour cells frequently metastasize to the bone but the molecular mechanisms governing this process are not well understood. Here, the authors show that neuropeptide Y/Y5 receptor pathway is activated in the hypoxic tumour microenvironment, which results in cytokinesis defects and chromosomal instability, leading to bone invasion. While patients with localized disease have a 5-year event-free survival (EFS) of 65-80%, a 3-year EFS for those with metastatic tumors remains below 30%, and decreases to 8–14% in patients with osseous dissemination3. This lack of effective therapies reflects a poor understanding of the mechanisms underlying ES metastasis. Although ES is triggered by chromosomal translocations that lead to the formation of EWS-ETS fusion proteins, the etiology of ES progression is unclear, as the same translocations are present in localized and metastatic tumors and the mutation rate of ES is low5. In addition, ES dissemination has been associated with decreased transcriptional activity of EWS-FLI1, suggesting that metastasis in these tumors is driven by mechanisms distinct from their initiation8.The presence of metastases is the most adverse prognostic factor in Ewing sarcoma (ES), an aggressive bone and soft tissue tumor affecting children and adolescents12. Such aberrations commonly arise due to mitotic defects leading to the formation of polyploid cells, which subsequently undergo atypical cell divisions with chromosome losses14. This, in turn, drives chromosomal instability (CIN), which facilitates cancer cell adaptation to stressors, such as therapies, hypoxia, or a metastatic niche microenvironment14.One of the few genetic markers of worse survival in ES is the presence of secondary chromosomal changes, including chromosome gains and complex karyotypes17. Additionally, concurrent TP53 and STAG2 mutations, which may promote CIN, are associated with a dismal prognosis in ES, while STAG2 mutations alone correlate with metastasis18. Thus, even though ES is triggered by a single chromosomal translocation and is considered a genomically quiet tumor, CIN appears to contribute to its progression. Yet, the factors triggering CIN in ES remain unknown.In ES, the chromosomal alterations consistent with CIN triggered by polyploidy correlate with chemoresistance, and their frequency increases in metastases and relapsing tumors20. In vitro, hypoxia increases the invasiveness of ES cells and up-regulates metastatic genes, while activation of HIF-1α promotes metastasis25. Yet, the mechanisms by which hypoxia exerts such effects are not fully understood.Hypoxia is another factor implicated in worse outcomes of ES. In ES patients, the presence of non-perfused tumor areas or necrosis correlates with adverse outcomes, multiple metastases and frequent bone dissemination33. We have shown that ES cells express high levels of NPY and its receptors, Y1R and Y5R, which is dependent on EWS-FLI1 transcriptional activity35. We have also established that hypoxia increases the expression of NPY and Y5R, as well as induces the expression of Y2R23. Moreover, hypoxia up-regulates dipeptidyl peptidase IV (DPPIV), a protease which cleaves NPY, converting it to a selective Y2R/Y5R agonist36. These changes in the NPY system shift its activity in ES cells to the Y2R/Y5R axis, which promotes their pro-metastatic features, such as cell motility23. However, since high constitutive expression of Y5R is driven by EWS-FLI1 and further increased by hypoxia, the levels of this receptor exceed those of inducible Y2R27. Hence, it is plausible that Y5R can also be activated independently of Y2R in hypoxic ES.Neuropeptide Y (NPY) and its receptors are expressed in various tumors and have multifaceted actions relevant to cancer biology, such as regulation of angiogenesis, cell proliferation, differentiation, survival, motility, and invasivenessThe goals of the current study were to test the effect of hypoxia on ES dissemination and determine the contribution of the NPY axis to this process. Our results identified hypoxia-induced over-activation of the NPY/Y5R/RhoA pathway as a mechanism leading to CIN and osseous dissemination. We have also shown that blocking Y5R in hypoxic ES tumors prevented hypoxia-induced genomic changes and bone metastasis. These findings provide the rationale for targeting Y5R in ES to prevent the disease progression.37. ES cells were injected into gastrocnemius muscles of SCID/bg mice, in close proximity to the tibial crest to allow for investigation of their local bone invasiveness. Primary tumors were allowed to grow to a volume of 150–250 mm3. Then, the tumor-bearing leg was either amputated (control) or subjected to FAL followed by amputation 3 days later. After amputation, metastasis was monitored by longitudinal MRI, followed by histopathological analysis. As previously described, this approach recapitulates ischemia and reoxygenation seen in solid tumors37. Control tumors at the size of 150–250 mm3 had low endogenous hypoxia  to trigger tumor ischemia Fig. 37. ES ceia Fig. S, while tia Fig. S. In thesia Fig. S. The inc38. SK-ES-1 xenografts in FAL-treated mice developed multiple metastases originating in bone, as seen by MRI and confirmed by histopathology  control SK-ES-1 primary tumors had nuclear sizes comparable to those in the original SK-ES-1 cell line, cells isolated from primary tumors of FAL-treated mice exhibited an increased frequency of hypertrophic cells with enlarged nuclei  control tumors, which exhibit high endogenous hypoxia   revealed an increased frequency of numerical chromosome aberrations in cells derived from primary tumors of FAL-treated mice and their corresponding metastases  and at euthanasia (metastases). While cells from small  Fig. . Moreoveses Fig. . Similarses Fig. . Lastly,ses Fig. .Fig. 2HySince genomic changes in hypoxic ES tumors can be triggered by a variety of factors activated by ischemia, we tested whether hypoxia in vitro recapitulates this effect. Culture of SK-ES-1 cells in 0.1% oxygen for 72 h increased the occurrence of cells with enlarged nuclei Fig. . This wa38. ES cells located in the invaded niches within bone were positive for the ES marker, CD99, and a hypoxia marker, carbonic anhydrase IX (CAIX)  Figs.  and S4a3one Fig. . FISH wione Fig. . Collectne Figs. , indicat40. The subsequent propagation of these cell fractions allowed for obtaining their progeny: diploid populations from normoxic and hypoxic conditions termed NOR-2n and HYP-2n, respectively, and near-tetraploid cells arising due to aberrant divisions of 4n cells, termed NOR~4n and HYP~4n , and then tested in the xenograft model with or without FAL. In primary tumors, in vivo Dox administration (+Dox diet) induced NPY5R gene editing with approximately 65% efficiency, which resulted in a heterogeneous ES cell population -inducible Cas9 nuclease and sgRNA targeting on Fig. S. These con Fig. S. FAL-indon Fig. S and the on Fig. S. Converson Fig. S. Out of nes Fig. . An insunes Fig. . Hence, To provide direct evidence for the presence of hypertrophic cells in hypoxic tumors, we performed histopathological analysis of tissues from ES xenografts and human tumors, as well as other malignancies. In solid tumors, hypoxic cells are most commonly found on the border of necrotic tissues Fig. . Our anaA similar analysis of human ES samples is challenging, since available tissues are typically limited to pre-treatment biopsies, which contain insufficient material for detailed topographical analysis, and post-treatment surgical specimen that often contain mainly necrotic tissues. To overcome this problem, we focused our analysis on unique large ES specimens derived from tumors not exposed to treatment. In these cases, tumor cells adjacent to necrotic areas and accumulating around blood lakes had nuclei enlarged by ~1.5-fold, as compared to the main tumor mass, similar to ES xenografts Fig. . The sam25. Hence, we created an in vivo hypoxia model to test the impact of ischemia on ES metastatic pattern and uncover mechanisms underlying these effects37. Using this model, we have shown that hypoxia preferentially promotes bone metastasis. These data are in agreement with clinical observations indicating a high prevalence of bone metastases in ES patients with underperfused tumors19.Growing evidence indicates the role of tumor hypoxia in ES metastasis38. Recent reports implicated transcriptional changes shifting the balance away from EWS-FLI1-driven gene expression toward Wnt signaling, as well as the translational activation of pro-metastatic genes induced by Y-box binding protein 1 (YB-1) under cellular stress as metastatic triggers in ES43. However, these studies focused on lung metastases and the mechanisms of osseous dissemination remain understudied. Dickkopf-related protein 2 (DKK2) is one of the few factors implicated in ES bone invasiveness, yet its role in distant bone metastasis has not been shown44. Furthermore, none of the mechanisms proposed in the aforementioned studies address the role for the acquired CIN in ES metastases. Our data implicate the formation of polyploid cells in the hypoxic tumor environment as a central mechanism promoting ES bone metastasis, since their progeny had an enhanced capability to initiate local bone invasion and distant bone metastases. We were not able to test the metastatic properties of the 2n population from hypoxic ES cells, as they converted to the 4n-like phenotype with passaging. Hence, hypoxia triggers long-term effects, and the subsequent formation of polyploid cells occurs even upon re-oxygenation, as suggested by previous data indicating that tumor cells preserve the cellular memory of hypoxia45. Notably, NOR~4n cells did not metastasize, proving that polyploidy alone is not sufficient to trigger metastasis and suggesting that HYP~4n cells have additional features that facilitate this process.Our findings highlight the differences in mechanisms driving metastases to various niches, as previously suggested by the divergence in metastatic patterns of ES xenografts49. Other mechanisms are likely to include an increased affinity of HYP~4n cells to bone and their ability to invade this tissue. Investigations into these processes may reveal vulnerabilities of the metastatic cells that could be therapeutically targeted39.Mechanisms underlying the preferential metastasis of HYP~4n cells to bone remain to be determined. Since low oxygen tension is an important feature of osseous pre-metastatic niche, the high frequency of bone metastases from hypoxic tumors may reflect metabolic adaptation of tumor cells to low oxygen11. Such chromosome changes associate with hypoxia in various malignancies and commonly arise due to mitotic defects that lead to tetraploidy and initiate a characteristic genome evolution, as divisions of these tetraploid intermediates result in abnormal chromosome segregation leading to their losses and CIN50. With time, this process results in monosomies that hinder cell growth and trigger a subsequent round of polyploidization. These changes confer an advantage to tumor cells and allow them to overcome selective pressures, such as hypoxia or cytotoxic therapy52. In line with this, our data indicate that polyploid cells survive hypoxia in ES xenografts and that their progeny initiate bone metastases. Notably, with prolonged passaging in vitro, HYP~4n cells lost their metastatic properties, which is in line with acquiring unfavorable monosomies and a need for the next polyploidization to regain fitness14. Hence, the enhanced metastatic potential of HYP~4n cells is a transient state. This phenomenon may explain why bone-derived TC71 cells required another hypoxic stimulus to trigger osseous metastasis, despite their near-triploid karyotype53. In addition to their metastatic properties, HYP~4n cells were chemoresistant, as shown in other tumor types52. Thus, hypoxia-induced changes may contribute to the disease recurrence and secondary ES dissemination, providing a rationale for targeting this cell population.Both HYP~4n tumors and their corresponding metastases exhibited high CIN that mimicked chromosome gains described in aggressive ES54. Under these conditions, NPY stimulated the migration of ES cells with a cancer stem cell phenotype via the Y2R/Y5R axis23. We have now shown that in addition to this Y2/Y5R-induced cell motility, hypoxia can trigger selective stimulation of Y5R, which over-activates the RhoA pathway, leading to cytokinesis defects and polyploidization. As a central cytoskeleton regulator, RhoA is tightly controlled during cytokinesis. Initially, high RhoA activity in the cleavage furrow drives its ingression, with subsequent inhibition of the RhoA being crucial for detachment of the daughter cells42. Our data indicated a defect in this last stage of cytokinesis in CHO-K1/Y5R transfectants, leading to the formation of multinucleated and polyploid cells. A similar phenotype was observed in hypoxic ES, in association with a Y5R-dependent increase in RhoA activity. In addition to inducing polyploidy, RhoA activation may overcome the p53-mediated tetraploidy checkpoint by inhibiting its negative regulator, LATS255. The survival of the polyploid cells and their subsequent genome evolution may also be facilitated by TP53 and STAG2 gene mutations, respectively, both of which associate with worse prognosis in ES patients18. While the metastatic effect of STAG2 loss has been recently associated with transcriptional reprogramming, previous reports indicated increased copy number aberrations in ES tumors with TP53 and STAG2 mutations56. Moreover, the impact of STAG2 loss on chromosomal segregation in polyploid cells has never been addressed.We have previously established that despite a high expression of NPY and Y5R in ES cells, selective activation of this pathway requires hypoxia8. As a LATS2 inhibitor, RhoA can also activate downstream targets repressed by this factor, i.e. YAP/TAZ transcription regulators, which promote ES invasiveness57. It remains to be determined whether the hypoxia-driven NPY/Y5R signaling provides an alternative mechanism of RhoA activation in the presence of high EWS-FLI1, or if it is associated with its down-regulation, as shown for lung metastases8. Earlier reports indicating an increase in EWS-FLI1 expression in hypoxic ES cells favor the former hypothesis21.The role of the RhoA pathway in ES progression has been implicated by previous studies. EWS-FLI1 represses Rho-actin signaling, while metastatic cells with low EWS-FLI1 have an increased activity of this pathway58. Likewise, expression of the NPY system was elevated in tissues from bone ES. In an ES orthotopic model, high endogenous NPY expression resulted in increased bone invasion and osseous dissemination, while NPY knock-down reduced the bone degradation induced by the tumor38. The current data provide a mechanism for the role of the NPY system in osseous metastasis. However, its direct impact on bone remodeling in the context of ES needs to be investigated. Importantly, NPY regulates bone homeostasis via direct effects on osteoblasts and its central activity in the brain59.The role of the NPY/Y5R pathway in ES osseous dissemination is supported by our previous clinical and preclinical data. NPY serum levels were higher in ES patients with pelvic and axial tumors originating in bone, as compared to those with extra-osseous lesions37. Moreover, the dose of the antagonist had to be limited due to its anti-orexigenic activity60. It is therefore possible that a longer and more effective Y5R inhibition (e.g. using antagonists that do not cross blood brain barrier) may have an effect on the overall metastatic potential of ES cells, particularly given the inhibitory effect of the Y5R antagonist on ES cancer stem cell motility23. Notably, Y5R has been implicated in chemoresistance26. Hence, our data warrants further investigation into its role in ES dissemination and recurrence.In the current study, a Y5R antagonist was administered for only five days, during the time when xenografts develop endogenous hypoxia, yet it successfully reduced hypoxia-induced polyploidization and prevented dissemination to bone62. Here, we have shown that like ES, neuroblastoma tumors contain hypertrophic cells, which arise in hypoxia. In breast and hepatic cancers, Y5R promotes cell proliferation and migration64. High systemic levels of NPY are also associated with advanced stage prostate cancer66. Notably, patients with the above cancers frequently present with treatment-resistant osseous metastases, suggesting that the mechanisms uncovered in ES may be relevant to other malignancies.In addition to ES, the expression of NPY and its Y5R has been shown in other cancers. In neuroblastoma, high NPY release associates with metastases and relapse, while Y5R expression is elevated in chemoresistant tumors and cells with an angioinvasive phenotypeIn summary, we have provided evidence for the role of hypoxia in promoting ES bone metastases and identified a specific mechanism leading to their development, which is different from those responsible for extra-osseous dissemination. We have shown that tumor hypoxia stimulates the NPY/Y5R axis, which leads to RhoA over-activation, cytokinesis defects and polyploidy Fig. . The pro13-36, were obtained from Tocris . Y1R agonist, NPY, was provided by Dr. Annette Beck-Sickinger, University of Leipzig, Germany. Rho inhibitor I, C3 transferase, was purchased from Cytoskeleton, Inc. .NPY was purchased from Bachem . Y5R agonist, BWX 46, Y5R antagonist, CGP71683, and Y2R agonist, NPY67. All media were supplemented with penicillin (200 units/mL), streptomycin (200 µg/mL), and fungizone (1 µg/mL). Primary cultures of ES cell lines were developed from tissue fragments of ES xenografts – primary tumors and metastases - in their corresponding culture media37. Hypoxia was created in an O2 Control InVitro Cabinet  equipped in oxygen sensor regulating a flow of 95% N2 and 5% CO2 gas mixture to maintain 0.1% O2 levels through the duration of the experiments.Human ES cell line, SK-N-MC (HTB-10), human neuroblastoma cell line, SK-N-BE(2) (CRL-2271), and Chinese hamster ovary cells, CHO-K1 (CCL-61), were obtained from American Type Culture Collection  and cultured according to the supplier’s recommendation. Other ES cell lines were obtained from Dr. Jeffrey Toretsky . SK-ES-1 cells were cultured in McCoy’s 5A modified medium supplemented with 15% fetal bovine serum (FBS) in collagen-coated flasks, while all other ES cells were maintained in RPMI medium with 10% FBSNPY5R gene . The specificity of the selected Y5R sgRNA was confirmed using the Cas-OFFinder tool68. The stably transfected cells (SK-ES-1/Dox-Cas9/Y5R sgRNA) were selected based on blasticidin (5ug/ml) and puromycin (1ug/ml) resistance. Then, the heterogenous pool of SK-ES-1/Dox-Cas9/Y5R sgRNA cells with edited NPY5R gene was obtained by Dox treatment . The profile of Cas9-targeted NPY5R gene disruption in the tumor cell population was evaluated with next-generation amplicon sequencing using the Illumina MiniSeq System. Gene editing efficiency in the heterogeneous population of tumor cells was analyzed with Crispresso2 software69.SK-ES-1 cells were transduced with lentiviral vectors encoding doxycycline (Dox)-inducible Cas9 nuclease (hEF1α-Blast-Cas9 Nuclease) and single-guide RNA (sgRNA) against human 38. In all, 2 × 106 (SK-ES-1 and TC32) or 1 × 106 (TC71) of ES cells suspended in 0.1 ml of saline were injected into gastrocnemius muscles of 3-4 weeks old female SCID/beige mice , adjacent to the left proximal tibial crest. The growth of primary tumors was monitored by periodical measurements of the limb and the volume of tumors was calculated according to the formula (D × d2/6) × π, where D was the longer diameter and d was the shorter diameter. Once tumors reached a volume of 150 mm3 (SK-ES-1) or 250 mm3 (TC71), tumor-bearing limbs from control mice were amputated. To assess hypoxia, 3 h before amputation, the mice were injected intraperitoneally with hypoxyprobe-1 , 1.5 mg/mouse. In the hypoxia groups, mice were subjected to FAL proximal to the bifurcation of the profunda femoris and femoral artery and the amputation was performed 3 days later. Metastasis formation was monitored by periodic MRI for 30–50 days, depending on the cell line. Mice were euthanized at the end of the experiment or when significant metastasis burden was detected. Macroscopic metastases, organs and bones were harvested and fixed in 10% buffered formalin for histopathological analyses. To test the effect of Y5R inhibition on ES metastases, once primary tumors reached a volume of 150 mm3, mice were randomized into two groups and treated with vehicle or Y5R antagonist, CGP71683, 20 mg/kg, administered for 5 days by daily intraperitoneal injections, followed by limb amputation and subsequent metastasis monitoring. Alternatively, mice were injected with SK-ES-1 or SK-ES-1/Dox-Cas9/Y5R sgRNA cells and fed with the control diet or pellets supplemented with 625 mg/kg doxycycline , starting 7 days before injection. Once tumors reached a volume of 150 mm3, mice on either the Dox- or Dox+ diet were randomized into control and FAL-treated groups. Metastases were monitored as above.All procedures involved in animal experiments were approved by the Georgetown University Institutional Animal Care and Use Committee. Orthotopic ES xenograft and FAL were performed as previously describedMYCN oncogene under a rat tyrosine hydroxylase promoter (TH-MYCN mice) were obtained from the National Cancer Institute 70. Both male and female mice at the age of 6–12 weeks were used for tumor tissue analyses. In the orthotopic neuroblastoma xenograft model, 2 × 105 of SK-N-BE(2) cells suspended in 10 µl of Matrigel  were injected into adrenal fat pad of 3–4-weeks-old female SCID/beige mice (Charles River Laboratories)71. In both models, tumor growth and metastasis were monitored by MRI, and the animals were euthanized once primary tumors reached a volume of 1000 mm3. Tissues from primary tumors and metastases were fixed and subjected to histopathological analyses.129×1/SvJ mice expressing the human 76. During imaging mice were anesthetized with 2% isoflurane, 30% oxygen, and 70% nitrous oxide, placed on a holder with respiration monitoring and imaged either in a 40 or 23 mm Bruker mouse volume coil for whole body and brain imaging. The anatomical imaging protocols used were two-dimensional, T2-weighted RARE sequences that included the following parameters: TR = 3000 ms, TE = 24 ms, matrix = 256 × 256, FOV = 4.35 × 3.0 cm, slice thickness = 0.5 mm, RARE factor = 4, and averages = 4.MRI was performed in the Georgetown University Lombardi Cancer Center Preclinical Imaging Research Laboratory on a 7 Tesla Bruker horizontal spectrometer run by Paravision 5.1 software, as previously describedEWS-FLI1 gene fusion were assessed and three surgical specimens excised from tumors not subjected to treatment were selected for further analyses.The human ES samples were obtained by Dr. Iżycka-Świeszewska  in compliance with institutional ethical regulations provided by Medical University of Gdańsk Bioethics Committee, which approved their use for research (protocol# NKBBN/448/2015). Use of these samples was further reviewed and approved by Georgetown University Institutional Review Board. The study cohort contained archival formalin-fixed paraffin-embedded (FFPE) tissue sections from human ES, which were collected for clinical diagnostic purposes in years 2002–2011 and fully de-identified. Hence, according to the definition provided by the U.S. Department of Health & Human Services, this study was not considered human subjects research and no informed consent or a waiver was required. 19 ES cases with confirmed t chromosomal translocation corresponding to the TM-1 kit; HPI, Inc. Burlington, MA; 1:300), anti-CAIX , anti-Y5R  and anti-Ki67  antibody. Tissue sections from xenografts and human ES were assessed by two independent pathologists.For the metastasis detection in animal models, all organs, long bones and vertebral columns, as well as other macroscopic bone and soft tissue metastases detected by MRI or during necropsy, were collected, sectioned, and stained with H&E. The bones were longitudinally cut to expose bone marrow cavity at the entire bone length. The presence of metastases was assessed based on H&E staining. The metastases were classified as osseous when MRI at early stages of their development and/or histopathological analysis indicated their bone origin, in contrast to the secondary lesions that originated in soft tissues and exhibited local bone invasion at their late stages. Immunostaining was performed on FFPE tissues using mouse monoclonal anti-CD99  and anti-CD68  antibody, as well as rabbit polyclonal anti-pimonidazole , clear hyperchromatic nucleus with the regular chromatin pattern, often visible nucleolus or multiple nucleoli with clear borders, and narrow bright cytoplasm with an intact nuclear membrane. The cells with pyknotic nuclei were not taken into consideration. Moreover, immunostaining with anti-CD99 and anti-CD68 antibody was performed to confirm the tumoral origin of these cells and exclude the macrophages, respectively. Furthermore, Ki67 staining was performed to detect actively proliferating cells.Cells were cultured on glass cover slips, stained with wheat germ agglutinin  at a concentration of 1.2 µg/ml and then fixed in 4% paraformaldehyde and stained with DAPI (Thermo Fisher Scientific) at a concentration of 2 µg/ml. Nuclear sizes were measured using ImageJ software  based on DAPI staining, while WGA staining allowed for identification of multinucleated cells. Parallel analysis was performed on H&E stained tumor tissues. The nuclear size of the cells in the main tumor mass, in bone invasion areas and on the borders of necrosis were measured using ImageJ software. Mitotic segregation errors and abnormalities in the morphology of interphase nuclei were assessed based on DAPI staining under Zeiss Axioskop fluorescence microscope .Cells were fixed in 75% ethanol, and stained with propidium iodide (PI) according to the standard procedures. Flow cytometric analysis was done on LSR-Fortessa  and data analyzed using FCS Express and ModFit LT software packages . Gating strategy is depicted in Fig. S77. At least 100 intact nuclei per case were captured and evaluated using the BioView Duet automated imaging system .Fluorescence in situ hybridization (FISH) assays were performed to evaluate gene copy number and chromosome ploidy in the primary and metastatic FFPE tissue samples and in the ES cell lines. DeepSee Quad probe , containing probes for the CDKN2A locus at 9p21 and centromeres of chromosomes 3, 7 and 17 (CDKN2A/CEN3/7/17), were hybridized to harvested interphase cells from cell lines fixed in methanol-acetic acid fixative and 5μm FFPE tumor sections using a laboratory standardized protocol with slight modificationsNPY5R gene (mapped at 4q32.2) in ES cells, a FISH probe was constructed, using a BAC clone (RP11-638L1) containing sequences of the NPY5R gene . For the centromere 4 probe , a directly labeled ‘SureFISH’ orange red probe was hybridized according to the manufacturer’s protocol . BAC clone DNA was prepared and labeled with biotin-16-dUTP  using nick translation78. Probes were hybridized to interphase ES cells. The biotin-labeled probe was visualized with avidin conjugated to FITC  and nuclei were counterstained with DAPI. Digital image acquisition was performed using a 100X objective mounted on a Leica DMRBE microscope  equipped with optical filters for DAPI Fluorescein, FITC, and TRITC  and a cooled charge-coupled device (CCD) camera . The IPLab software package  was used for image acquisition and processing. Slides were scored by two independent observers. A minimum of 50 nuclei were evaluated in each case. Only intact non-overlapping nuclei were scored. The detection of two FISH signals was considered as normal copy number, and three or more signals as gain or amplification.For FISH analysis of the A volume of 20 µL of the media was mixed with 100 µL of MeOH containing the internal standard (4-Nitrobenzoic acid) which was prepared in MeOH at a concentration of 30 µg/mL and homogenized for 60 s. The samples were transferred to the GC vials then dried under vacuum at 35 °C. Dry samples were derivatized by adding 10 μL of methoxyamine (20 mg/mL) then heated in an agitator at 60 °C for 30 min. This was followed by 100 μL of MSTFA. The vials were transferred to an agitator to heat at 60 °C for 30 more minutes. Finally, the vials were capped and a volume of 2 μL was injected directly to the GCMS in a splitless mode into an Agilent 7890B GC system  that was coupled with a Pegasus HT TOF-MS . Separation was achieved on a Rtx-5 w/Integra-Guard capillary column , with helium as the carrier gas at a constant flow rate of 0.8 mL/min. The temperature of injection, transfer interface, and ion source was set to 150, 270, and 320 °C, respectively. The GC temperature programming was set to 0.2 min. of isothermal heating at 70 °C, followed by 6 °C/min oven temperature ramping to 300 °C, a 1.0 min. isothermal heating of 300 °C, 20 °C/min to 320 °C, and a 2.0 min. isothermal heating of 320 °C. Electron impact ionization (70 eV) at full scan mode (40–600 m/z) was used, with an acquisition rate of 20 spectra per second in the TOF/MS setting. The collected spectra were processed, aligned, and deconvoluted with the software ChromaTOF v. 4.72.0.79. Chromosome counts from at least 3 independently developed clones were compared between the experimental groups.CHO-K1 cells were grown to 80% confluency and incubated with colcemid at a concentration of 1 µg/ml for 2 h and the metaphase chromosome spreads were prepared according to the standard proceduresES cells were transduced with Premo™ FUCCI Cell Cycle Sensor BacMam 2.0 vectors encoding geminin-GFP and Cdt1-RFP (Thermo Fisher Scientific), according to the manufacturer’s protocol, and incubated overnight. Transduced cells were subsequently incubated in normoxia or hypoxia for 24 h and stained with Hoechst 33342 (Thermo Fisher Scientific) at a concentration of 2 µg/ml. Cells were then sorted into 2c (2n) and 4c/GFP-/RFP + (4n) populations using FACSAria (Becton Dickinson).Cells were cultured on glass cover slips, fixed in 4% (w/v) paraformaldehyde and stained with rabbit polyclonal anti-Y5R  and mouse monoclonal anti-RhoA-GTP  antibodies. DNA was stained using DAPI at 0.5 µg/ml in PBS.80. To this end, cells were lysed in the buffer containing 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 3 mM EGTA, 250 mM sucrose and the protease inhibitor cocktail . The lysate was incubated for 15 min on ice and sonicated, followed by centrifugation at 500 g for 5 min at 4 °C. The supernatant was then collected and centrifuged at 20,000 × g for 30 min at 4 °C. The cell membrane pellet was incubated in the buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 4 mg/ml β-dodecylmaltoside and protease inhibitors for 1 h on ice and centrifuged at 20,000 × g for 30 min at 4 °C. The resulting membrane protein solution was subsequently used for Western blot with goat polyclonal anti-Y5R antibody , while mouse monoclonal antibody against alpha 1 sodium potassium ATPase  served as a loading control. Alternatively, whole cell extracts were used with a mouse monoclonal anti-β-actin antibody  as a control. Densitometry was conducted using ImageJ software.Western blot for Y5R was performed on membrane proteins, isolated as previously described41. cDNAs of human Y1R, Y2R and Y5R were cloned into pEGFP-N1 vector at NheI and BamHI restriction sites. Transfection was performed using Lipofectamine 2000 (Invitrogen). Stable transfectants carrying EGFP-fused NPY receptors were selected with geneticin (Sigma) at a concentration of 1 mg/ml followed by single cell clone isolation. The membrane localization of the Y5R-EGFP fusion protein in the selected clones was confirmed by co-staining with WGA , followed by fixation with 4% paraformaldehyde and confocal microscopy using Olympus Fluoview BX61 microscope .SK-ES-1 cells were transfected with pEGFP-N1 or pmCherry-N1 vectors  using Lipofectamine 2000 . Stable transfectants were selected with geneticin  at a concentration of 0.25 mg/ml. Transfection of CHO-K1 cells with NPY receptors was performed as previously describedCHO-K1 cells were transfected with vectors encoding EGFP fused to NPY receptors. 4 h later, several fields with EGFP-positive cells were selected and imaged with Nikon TE300 microscope  for 96 h. Imaging was performed using both bright field and fluorescence settings to identify EGFP-positive cells. Data was acquired using Metamorph software .−7M for the desired time (1-90 min). Cells were lysed immediately after treatment. Total and phosphorylated p44/42 MAPK were detected in cell lysates using mouse monoclonal anti-phospho p44/42 E10 antibody  and rabbit polyclonal anti-p44/42 MAPK antibodies . The band intensities were quantified by densitometry using ImageJ software.Stable Y5R transfectants were treated with NPY at a concentration of 10®AQueous One Solution Cell Proliferation Assay . Additionally, 3H-thymidine uptake under the same conditions was measured. To this end, 0.5 μCi of [3H]thymidine per well was added for the last 6 h of incubation and the cells were harvested in a 96-well harvester  and counted in a Wallac 1205 Betaplate Liquid Scintillation Counter 83. The effect of NPY (10−7M) or Y5R antagonist (10−6M) on cell number was determined by cell count 48 h after culture in various serum concentrations . To assess DNA synthesis in situ, CHO-K1/Y5R transfectants were cultured on glass cover slips, incubated with 10 µM EdU (Thermo Fisher Scientific) for 1 h and stained according to the manufacturer’s protocol. To assess resistance to chemotherapy, ES cells were plated in 96-well or 24-well plates in the standard growth medium, treated with a range of doxorubicin concentrations (0.05–0.4 µg/ml) and cultured for 72 h. The number of viable cells was assessed by MTS assay or cell count upon trypan blue exclusion.CHO-K1 cells stably transfected with Y5R cDNA were plated in 96-well plates, cultured in 1% FBS media for 24 h and treated with a range of NPY concentrations. After 24 h, the number of viable cells was assessed using MTS-based CellTiter 96−7M NPY for 20 min or 10−6M Y5R antagonist, CGP71683, for 30 min. Subsequently, cells were lysed and levels of RhoA-GTP were assessed using RhoA Pull-down Activation Assay Biochem Kit (Cytoskeleton Inc.) according to the manufacturer’s protocol. SK-ES-1 cells were either pre-incubated in serum-free media for 24 h and then treated with selective NPY receptor agonists at a concentration of 10−7M for 20 min, or cultured in media containing 10% FBS and incubated in normoxia or hypoxia for the desired time, in the presence or absence of 10−6M Y5R antagonist or Rho inhibitor I (0.01 µg/ml). Subsequently, cells were lysed and the levels of active RhoA were assessed as above.CHO-K1 cells, wild type or stably transfected with Y5R-EGFP, were incubated for 24 h in serum-free media, followed by stimulation with 10χ2 or Mann–Whitney tests were used, as appropriate. The generalized estimating equation (GEE) models were used to compare the treatment effect and time trend on the number of metastases  developed over time between control and treatment groups. Significant associations were assessed at an alpha level of 0.05. All in vitro experiments were repeated at least three times. Animal experiments were performed twice and the combined results from both independent experiments are shown. Data is presented as mean ± standard errors.Statistical analyses were performed using SAS 9.4  and GraphPad Prism 6 software. Between-group comparisons were assessed using one-way repeated measures ANOVA with post-hoc t-test, independent-samples t-tests or paired-samples t-tests. For count data, Fisher’s exact, Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary


Five hundred 8-d old male broilers Cobb500 were randomly allotted into 10 treatments in factorial arrangement with 5 Cu levels , and 2 sources  for a 10-d-experiment.P < 0.05) in CuPro fed chicks compared with CuSO4.5H2O group. Average daily feed intake (ADFI) decreased linearly (P < 0.05) as dietary Cu increased. A quadratic response (P < 0.05) to Cu levels was found for FCR, being optimized at 9.87 and 8.84 mg Cu/kg in CuPro and CuSO4.5H2O diets, respectively. Copper supplementation linearly increased liver Cu content (P < 0.05) and tended to linearly increase (P = 0.07) phosphorus (P) and copper in tibia. Manganese and zinc were higher (P < 0.05) in tibia of CuPro fed birds. Broilers fed CuPro exhibited lower liver iron (P < 0.05) content, lower activities of Cu, Zn superoxide dismutase (CuZnSOD) in breast muscle and liver, and glutathione peroxidase in liver. Glutathione peroxidase reduced linearly (P < 0.05) with CuPro levels and increased linearly (P < 0.05) with CuSO4.5H2O levels and were lower (P < 0.05) in all CuPro levels in breast muscle. Breast muscle malondialdehyde concentration tended to be higher (P = 0.08) in broilers fed CuSO4.5H2O. Copper levels linearly increased (P < 0.05) metallothionein (MT) and malate dehydrogenase (MDH) expression in liver, and six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) in the intestine. Copper elicited a quadratic response (P < 0.050) in AKT-1 and mammalian target of rapamycin (mTOR) in breast muscle, CuZnSOD in liver and antioxidant 1 copper chaperone (ATOX 1) in intestine. Broilers fed CuPro exhibited higher mRNA expression of mTOR in muscle breast and lower CuZnSOD in liver and ATOX 1 in intestine. Interaction (P < 0.05) between levels and sources was found in mRNA expression for GSK-3β, MT, and CuZnSOD in breast muscle, FAS and LPL in liver and MT and CTR1 in intestine.Feed conversion ratio (FCR) was better (CuPro showed beneficial effects on feed conversion and bone mineralization. Organic and inorganic Cu requirements are 9.87 and 8.84 mg Cu/kg, respectively.The online version contains supplementary material available at 10.1186/s12917-022-03286-5. Copper (Cu) is an indispensable trace element for poultry maintenance, growth, health, and survival. As co-factor for several metalloenzymes, Cu is involved in a plethora of biological processes, which include mitochondrial respiration, erythropoiesis, connective tissue maturation, free radical scavenging, hormone secretion pathways and immune system defenses, among others –5. DeficIn the last revised edition of Nutrient Requirements of Poultry , the Pou4.5H2O) or, to a lesser extent, other inorganic salts such as oxides and carbonate. These inorganic sources offer low cost, but they are less bioavailable than some organic sources [Supplemental Cu has been conventionally provided as either copper sulfate pentahydrate , manganese (Mn), iron (Fe), and selenium (Se) have been provided as inorganic salts. We have two main concerns about this practice. Firstly, commercial flocks are typically fed diets containing trace minerals supplied entirely as inorganic salts or as organic chelates, so responses to dietary levels of those minerals are most appropriately measured in the typical environment. Secondly, considering the interactions among trace minerals regarding intestinal absorption and metabolic pathways, physiological responses and gene expression could be also affected by the source of the trace minerals and supplemental copper levels. We hypothesized that trace mineral source and copper levels may provide different responses in animal growth, mineral concentration, antioxidant status and gene expression depending on the source of the other trace minerals added to the diet. Due to Cu antimicrobial effects, researchers have put efforts towards investigating dietary Cu supplementation at levels that exceed requirements , 32, 33,The objectives of this research were: 1) to assess the effects of different dietary supplemental Cu levels as organic or inorganic in the same source of trace mineral supplement on growth, tissue mineralization, antioxidant status and gene expression linked with absorption, lipid, and protein metabolism, and 2) to estimate the optimal supplemental organic and inorganic Cu levels for chick performance.4.5H2O, and 1.18, 3.42, 8.97, 11.9 and 15.5 mg/kg for diets containing CuPro as the Cu source. These values were near the expected content of 0, 4, 8, 12, and 16 mg/kg  between the supplemental levels and sources investigated on broiler performance responses were found (Table P < 0.05) in chicks fed CuPro and organic trace minerals than in those fed inorganic trace minerals. There was a tendency (P = 0.053) for greater ADFI in broilers fed CuSO4.5H2O and inorganic than organic trace minerals. Graded supplemental Cu levels elicited a linear increase in (P < 0.05) ADFI and a quadratic response (P < 0.05) in BW, ADG, and FCR. According to the polynomial quadratic regression model, the supplemental dietary Cu levels which optimized broiler responses considering both sources combined and each one separately, where the quadratic term significant, is presented in Table 4.5H2O supplemented diets, respectively.No interaction effects (nd Table . Broiler P = 0.05 for grea P = 0.05 for greaP > 0.05) mineral content in breast muscle (Table P > 0.05) between broilers fed diets supplemented with inorganic versus organic trace minerals. Increasing levels of dietary Cu supplementation linearly increased (P < 0.05) Cu concentration in liver. Broilers fed CuPro and organic trace minerals had lower concentrations of Fe (P < 0.05) in liver compared with birds fed inorganic trace minerals. As dietary Cu supplementation increased, there was a tendency for a linear increase (P < 0.10) in the concentration of Cu and P and a tendency for a quadratic increase (P < 0.10) in the concentration of Mn in broiler tibia. The concentrations of Mn and Zn in the tibia of broilers fed CuPro and organic trace minerals were greater (P < 0.05) compared with broilers fed CuSO4.5H2O and inorganic trace minerals.Overall, neither trace mineral sources nor dietary Cu supplementation, nor their interaction, affected (le Table . No diffP < 0.05) of SOD in breast muscle and liver and GSH-Px in liver compared with birds fed diets containing CuSO4.5H2O and inorganic trace minerals (Table P < 0.05). As CuPro supplementation increased in diets with organic trace minerals, breast muscle GSH-Px activity decreased linearly (P < 0.05), whereas a linear increase occurred in chicks fed CuSO4.5H2O and inorganic trace minerals diets (P < 0.05). Regardless of the level of Cu supplementation, GSH-Px activity in breast muscle of chicks fed CuSO4.5H2O and inorganic trace minerals was higher (P < 0.01) than in birds fed CuPro and organic trace minerals. Malondialdehyde concentration in breast muscle tended to be slightly lower (P = 0.08) in chicks fed diets with CuPro and organic trace minerals.Chicks fed CuPro and organic trace minerals exhibited a lower activity P < 0.0 of SOD iP-values from analyses of variance of mRNA expression of genes in breast muscle, liver and jejunum of chicks fed supplemental Cu levels and different sources of trace minerals are detailed in Table The AKT-1, mTOR, GSK-3β, MT, and CuZnSOD mRNA expression in the breast muscleP < 0.05) than at other levels. Dietary Cu supplementation level elicited a quadratic response (P < 0.05) on the mRNA expression of mechanistic target of rapamycin (mTOR) in chick breast muscle, and chicks fed CuPro and organic trace minerals exhibited a higher (P < 0.05) mTOR expression compared with inorganic trace minerals fed chicks . There was a quadratic effect (P < 0.05) of the supplemental Cu levels on the mRNA expression of GSK-3β (P < 0.05) when Cu was provided as CuSO4.5H2O with inorganic trace minerals, whereas no Cu-level effects (P > 0.05) were found in chicks fed CuPro with organic trace minerals diets  but no effects of Cu levels (P > 0.05) were detected in chicks fed CuPro  MT mRNA expression compared with CuPro and organic trace minerals  of the supplemental Cu levels on mRNA expression of CuZnSOD occurred in chicks fed either CuPro or CuSO4.5H2O  in chicks fed diets supplemented with CuPro at 12 mg Cu/kg and organic trace minerals compared with chicks fed the same level provided as CuSO4.5H2O and inorganic trace minerals  in MT mRNA expression in chick liver, but no effect (P > 0.05) of trace minerals source occurred , and chicks fed CuPro and organic trace minerals exhibited a higher value (P < 0.05) compared with birds fed CuSO4.5H2O and inorganic trace minerals  were found for fatty acid synthase (FAS) and lipoprotein lipase (LPL) mRNA expression , and at the levels of 8 and 12 mg Cu/kg diet, chicks fed CuPro diets exhibited a lower (P < 0.05) FAS mRNA expression compared with chicks fed CuSO4.5H2O  on the mRNA expression of LPL in chick liver was identified in birds fed increasing levels of CuSO4.5H2O  in the liver of chicks fed CuPro at 12 mg Cu/diet, but lower at 16 mg Cu/ kg diet (P < 0.05) compared with chicks fed CuSO4.5H2O and inorganic minerals supplemented diets  the malate dehydrogenase (MDH) mRNA expression in chick liver, but there was no effect (P > 0.05) of mineral source  for MT and Copper transporter 1 (CTR1) mRNA expression  in MT mRNA expression in jejunum as Cu supplementation increased, whereas in CuSO4.5H2O fed chicks, jejunal MT mRNA expression changed quadratically (P < 0.05) with the maximum value at 12 mg Cu/kg diet. mRNA expression of MT in jejunum was greater (P < 0.05) in chicks fed CuPro diets at 4 mg Cu/kg diet compared with birds fed CuSO4.5H2O supplemented diets; however, at 8 and 12 mg Cu/kg diet, chicks fed CuSO4.5H2O diets exhibited a higher MT mRNA expression (P < 0.05) compared to the CuPro group  in the jejunum of chicks fed CuSO4.5H2O compared with the CuPro group  and quadratic increase (P < 0.05), respectively in transmembrane epithelial antigen of the prostate (STEAP-1)  in the jejunum of chicks fed CuSO4.5H2O and inorganic trace minerals compared with CuPro and organic trace minerals  Fig.  and anti-1) Fig.  in chickals Fig. .Fig. 11C4.5H2O. Additionally, based on genetic improvements, it was theorized that the requirements of dietary Cu to support optimum growth in broilers would be higher than the 8 mg/kg described by NRC [We designed the current assay to investigate broiler responses to dietary supplemental Cu levels in inorganic or organic trace mineral supplements and establish the concentrations of Cu required to optimize performance responses with supplemental trace minerals as organic or inorganic sources. We hypothesized that when provided as CuPro, lower supplemental Cu levels would produce similar responses to higher amounts of Cu provided as CuSOd by NRC .4.5H2O and organic Cu do not differ with regards to growth rate in broilers [To illustrate the extent to which growth, tissue mineralization and physiological responses are affected by dietary Cu and estimate the optimum dietary supplemental Cu for such responses with regression analysis, Cu supplemental levels ranged from 0 to 2 times the level recommended by NRC , i.e., 1broilers  and Whitbroilers . Althougbroilers , who foubroilers  reportedbroilers  found a broilers  that WhiSimilarly, Wu et al.  did not As dietary Cu supplementation increased, retention of P in chick tibia tended to increase. A positive correlation between levels of Cu supplementation and the retention of P and Ca were observed by Muszyński et al.  in growiIn the current research, we noticed that by adding organic Cu and trace minerals in chick feeds, Zn and Mn content in bird tibia were increased by approximately 4.5 and 6%, respectively. Both of these trace elements are indispensable for proper bone and cartilage development. Whereas Zn is involved in collagen synthesis, one of the major structural proteins of cartilage and bones , Mn is aInorganic Cu and trace minerals consistently resulted in increased the activities of the antioxidant enzymes SOD and GSH-Px than did organic minerals, which suggests that inorganic minerals have greater pro-oxidant activity compared with organic ones. Such theory may be supported by the trend we found in the MDA concentration, a biomarker of lipid peroxidation. Our findings show that the MDA was approximately 55% higher in breast muscle of chicks fed inorganic Cu compared with organic. Presumably, the greater oxidative stress with inorganic minerals triggers the production of the protective antioxidant enzymes. Koslosky et al.  reported4.5H2O, respectively.The Cu levels and microminerals sources affected the mRNA expression of several genes related to fat and protein metabolism. Those effects may help to understand FCR responses. The AKT-1 is involved in protein metabolism as one of major signaling pathways control protein synthesis , and an 4.5H2O and inorganic minerals, which may contribute to the improved FCR with organic minerals.The mTOR is a serine/threonine protein kinase  which re4.5H2O on the expression of GSK-3β indicating that CuSO4.5H2O affected the expression of this gene and at higher levels causes a down regulation, while CuPro maintained the GSK-3β mRNA expression in the same amount regardless of the level. This result can be correlated to the better FCR observed in broilers receiving diets with CuPro. These data suggest that dietary Cu as an organic trace mineral source can support protein synthesis in broiler breast muscle, benefiting early performance.The GSK-3β mRNA expression is correlated with increased mRNA expression of eIF4E and tends to increase p70S6K; these proteins are involved in protein synthesis . In the 2 + and Hg2 + [4.5H2O had higher mRNA expression of MT in breast muscle compared to CuPro, but in intestine, birds fed CuPro higher mRNA MT expression compared to CuSO4.5H2O. Previous research demonstrated that Cu increases MT [The MT is a low molecular weight protein (6.1 kDa) found in the cell cytosol and produced in response to high levels of Cu and Zn and heavy metals such as Cdnd Hg2 + . Within nd Hg2 + . The MT nd Hg2 + . In the eases MT , 69. Fryeases MT  reported+) by a reductase before Cu is absorbed. Some enzymes involved in this process are cytochrome B reductase 1 and STEAP-1 reductase [Cu absorption in the small intestine is saturable, and some of the molecules responsible for the kinetics have been identified in the last decade. The luminal Cu is reduced to the cuprous form .4.5H2O), resulting in a total dietary Cu concentration of 9 mg/kg, slightly more than the NRC [2. Experimental feeds (mash) and demineralized water were provided ad libitum throughout the 9-day experimental period. Photoperiod was set at 12 h light/12 h darkness. Prior to the experimental period, all chicks were weighed and assigned to treatment groups so initial body weight (167.6 ± 1.58 g) was similar among experimental treatments. Ten replicate cages of 5 chicks were randomly assigned to each of 10 treatment groups. Each cage was considered an experimental unit.A total of five hundred 1-d-old male Cobb 500 chickens were obtained from a local commercial hatchery and used in the current assay. From 1 to 7 d of age, birds were fed a pre-starter diet formulated to meet or exceed nutritional recommendations of Rostagno et al. , except  the NRC  requirem4.5H2O, 24.5% Cu) and Cu proteinate . A semi-purified basal diet, based on casein, albumin, corn, and dextrose (Table 4.5H2O or Bioplex® Cu) and to the trace mineral supplement  as follows: 1) basal diet with organic trace mineral supplement (ORG) without supplemental Cu; 2) ORG + 16 mg Cu/kg feed supplemented as Bioplex® Cu; 3) basal diet supplemented with inorganic trace mineral supplement (INO) without supplemental Cu; and 4) INO + 16 mg Cu/kg of feed supplemented as CuSO4.5H2O. Diets supplemented with organic trace mineral supplement containing 0 and 16 mg Cu/kg as Bioplex® Cu were mixed to produce five dilutions, resulting in five different supplemental organic Cu concentrations: 0, 4, 8, 12, and 16 mg Cu/kg of feed. The same procedure was adopted with diets supplemented with inorganic trace mineral supplement, resulting in the same levels of supplemental inorganic Cu. Organic trace microminerals (ORG) were supplied as Bioplex®Fe (15% Fe), Bioplex®Zn , Bioplex®Mn , and selenium yeast as Selplex® , whereas inorganic trace minerals (INO) sources included iron sulfate , zinc sulfate , Manganese sulfate , and sodium selenite , and I was added as calcium iodide (86% I) in both trace mineral supplements. Except for Cu, all trace minerals were supplied to meet or exceed NRC [A 2 × 5 fractional factorial arrangement was used to investigate the effect of 2 sources of Cu (organic and inorganic), each at 5 supplemental Cu levels . Copper sources were Cu sulfate  and average daily feed intake (ADFI), from which average daily gain (ADG) and feed conversion ratio (FCR) were calculated. Mortality rate was also monitored, to adjust FCR. At the end of the assay, one bird per cage (10 birds/treatment) was randomly selected and sacrificed by cervical dislocation without previous anesthesia. A longitudinal incision was made in the abdominal cavity to collect liver, and the left and right breast muscles. The left tibia was also collected. The liver and muscle tissues were lyophilized for 72 h at − 80ºC under 800 mbar of pressure , ground in a ball mill , and stored for further analysis of mineral content. After appropriate dilution, Cu, Zn, Mn, Fe, Ca, and P contents were estimated by atomic absorption spectrophotometer  in the Animal Nutrition Laboratory  using AOAC official method 968.08 . Tibias The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in tissues were measured according to Walsh et al.  using thwww.idtdna.com/Primerquest/Home/Index) available on the IDT (http://www.idtdna.com) database. The reactions were carried out according to the manufacturer’s protocol using the SYBR Green detection kit with GoTaq qPCR Master Mix . The RT-qPCR reaction conditions were adjusted with initial denaturation temperature at 95 °C for 2 min and 40 cycles of annealing at 60 °C  and denaturation at 95 °C for 15 s. At the end of the amplification cycling reaction, an additional step was added with a gradual increase in temperature from 60 to 95 °C for analysis of the dissociation curve. The amplification of genes was performed in duplicate using the ABI 7300 system . Amplification efficiency ranged from 91 to 109% except for MT, in the intestine (80%), and CuZnSOD in the breast muscle (115%). The values of the coefficient of determination (R2) were equal to or greater than 0.98 for all genes  was randomly selected and sacrificed by cervical dislocation without previous anesthesia. Breast muscle, liver, and intestine  samples were collected immediately, ground, stored in individual tubes containing RNALater  and frozen at -80° C for further analysis of gene expression. Total RNA from snap-frozen tissue samples of breast muscle, liver, and intestine was extracted in Trizol reagent  according to the manufacturer’s protocol. Genomic DNA was removed from extracted RNA with deoxyribonuclease . The purity (260:280 absorbance), concentration, and integrity of the total RNA from each sample were quantified using a Nanovue (GE electronics) spectrophotometer and gel electrophoresis (1% agarose gel and stained with ethidium bromide). Standard cDNA synthesis was achieved by GoScript Reverse Transcription (RT) system  according to the manufacturer’s protocol with annealing temperature of 25 °C for 5 min and extension of 42 °C for 1 h. Three cDNA concentrations  and three primer concentrations  were used. The efficiency of PCR amplification was calculated for each gene, targets and housekeeping, using the following formula: E = (10 (-1/slope) -1) × 100) [The cage served as the experimental unit for growth performance, while the single chick per cage represented the cage as the experimental unit for tissue mineral contents, antioxidant enzyme activity and mRNA gene expression.P < 0.05) of source effect (only two levels) was evaluated through an F-test, whereas orthogonal contrasts of linear and quadratic responses were used to assess effects of Cu levels. Finally, the supplemental level of inorganic and organic Cu which optimized biological responses assessed were estimated by using a polynomial regression model as follows: Y = (β0 × X2) + (β1 × X) + β2, where Y is the dependent variable, X is the dietary supplemental Cu level, β0 is the quadratic coefficient, β1 is the linear coefficient, and β2 is the intercept. Statistical significance was considered as 0.05 and the term “tendency” is used for situations in which the P-value is between 0.05 and 0.1.Data were analyzed as a completely randomized design under an incomplete 2-way (source x levels) factorial assay with factors of inorganic versus organic trace minerals, levels of Cu from CuPro, and levels of Cu from copper sulfate. Several of the possible combinations of these factors were excluded from the experiment so the traditional two-way factorial analysis was generalized to a fractional factorial design, which consists of a carefully chosen subset (fraction) of experimental treatments . This apG. gallus genes, housekeeping genes for chickens and their specific primers used in RT-qPCR analysis, Parameters of gene-specific primers and reference genes obtained from the efficiency curve analysis in RT-qPCR, Growth performance of 17 d broilers chicks fed different copper levels and sources, Optimum supplemental copper (Cu) level for broiler chicks considering sources individually or combined, Mineral concentration (dry matter) on tissues of 17 d old broiler chicks fed different copper levels and sources, Antioxidant enzyme activity and malondialdehyde concentration in 17 d old broiler chicks fed different copper levels and sources, mRNA expression in tissue of broiler fed different copper levels and sources. Figures: Copper levels and sources on mRNA expression of AKT-1 gene in broiler breast muscle, Copper levels and sources on mRNA expression of mTOR gene in broiler breast muscle, Copper levels and sources on mRNA expression of GSK-3β gene in broiler breast muscle, Copper levels and source on mRNA expression of MT gene in broilers breast muscle, Copper levels and sources on mRNA expression of CuZnSOD gene in broiler breast muscle, Copper levels and sources on mRNA expression of MT gene in the broiler liver, Copper levels and sources on mRNA expression of CuZnSOD gene in the broiler liver, Copper levels and sources on mRNA expression of FAS gene in the broiler liver, Copper levels and sources on mRNA expression of LPL gene in the broiler liver, Copper levels and sources on mRNA expression of MDH gene in broiler liver, Copper levels and sources on mRNA expression of MT gene in the broiler intestine, Copper levels and sources on mRNA expression of CTR1 gene in broiler intestine, Copper levels and sources on mRNA expression of STEAP-1 gene in broiler intestine, Copper levels and sources on mRNA expression of ATOX 1 gene in broiler intestine.Tables: Ingredient composition of the semi-purified basal diet (as fed basis), Analyzed concentration of copper and trace minerals in experimental diets (as fed basis); The responses collected in the current research are described as means of treatments in Tables from Additional file 1.