Coordinated metabolic processes, such as biosynthesis and catabolism of SGs, ensure constitutive presence of these bitter tasting compounds in plant tissues, which plays a decisive role in the defense against pathogens and herbivores. These compounds are susceptible to hydrolysis mediated by enzymes β-glucosidases, and the resulting aglycones are subsequently directed toward different metabolic pathways in plants. Function of two β-D-glucosidases (named CeBGlu1 and CeBGlu2) from centaury , belonging to the glycoside hydrolase 1 (GH1) family, was confirmed using in vitro assays with recombinant proteins, following their heterologous expression in E. coli and His-tag affinity purification. Although they show slightly differential substrate preference, both isoforms display high specificity toward SGs and the organ-specific distribution of transcripts was positively correlated with the content of SGs in diploid and tetraploid C. erythraea plants. Transient overexpression of CeBGlu1 and CeBGlu2 in C. erythraea leaves induced changes in metabolite profiles. The effectiveness of transgene overexpression has been altered by plant ploidy. UHPLC/DAD/(±)HESI − MS2 profiling of leaves of diploid and tetraploid C. erythraea genotypes revealed that the amounts of major SGs; sweroside, swertiamarin, and gentiopicrin was decreased in agroinfiltrated leaves, especially when CeBGlu1 and CeBGlu2 were co-expressed with transgene silencing suppressor p19. The work demonstrates that in planta metabolic engineering adopting transient overexpression of CeBGlu1 and CeBGlu2 is a suitable tool for the modulation of SGs content and glucosides/aglycones ratio, which might have substantial effects on overall phytochemistry of C. erythraea.Secoiridoid glucosides (SGs) are monoterpenoids derived from the iridoid cyclopentane-C-pyran skeleton with  Centaurium Hill (fam. Gentianaceae) are a rich source of SGs, among which sweroside (SW), swertiamarin (SWM), and gentiopicrin (GP) predominate. Leaves are the major site of SGs biosynthesis and accumulation in C. erythraea (Centaurium species are presumed to share a part of the secoiridoid biosynthetic pathway up to SEC with MIAs-producing Catharanthus roseus. The biosynthetic pathway of SEC starts with the synthesis of geranyl diphosphate (GPP), which is converted to geraniol and continues via a number of intermediates such are 8-hydroxygeraniol, 8-oxogeraniol, nepetalactol, iridotrial, 7-deoxyloganetic acid, 7-deoxyloganic acid, loganic acid and loganin  such as strictosidine, while in C. erythraea SEC is metabolized into secoiridoid glucosides SW, SWM and GP, as well as their derivatives. Enzymes responsible for the conversion of SEC to SW, and further to SWM and GP are not elucidated yet (Secoiridoid glucosides (SGs) are a group of plant-derived natural compounds widely present in species belonging to orders Gentianales, Dipsacales, Cornales, and Lamiales (family Oleaceae) . Speciesrythraea . Secoirirythraea , but is  loganin . The bioated yet .C. erythraea tissues is a net result of the two dynamic metabolic processes, their biosynthesis and their catabolism (β-glucosidases. Cleavage of the glucose (Glu) residue from SW, SWM, and GP results in the formation of aglycones , categorized into the glycoside hydrolase family 1 (GH1), the largest GH family in plants. GH1 β-glucosidases are enzymes that hydrolyze glycosidic bonds to release non-reducing terminal glucosyl residues from various compounds – benzoxazinoid, cyanogenic, iridoid and phenolic glucosides – as well as glucosinolates (β-glucosidase (β-Glu) enzymes have been previously characterized in some members of the order Gentianales, including C. roseus and Rauvolfia serpentina , in 2007 and 2016, respectively, were maintained in vitro on half–strength MS medium . Two-month-old seedlings were individually transferred into pots with Floragard growth medium , and grown under greenhouse conditions at 50–85% humidity. Five-month-old diploid and tetraploid plants, displaying rosette phenotype, were used in agroinfiltration experiments. Seeds of tetraploid C. erythraea, collected in 2006, were obtained from Ecological-Botanical Garden of the University of Bayreuth (Germany). Diploid individuals were of the same origin as those used in in vitro experiments. All the C. erythraea accessions used in the present study are deposited within the seed collection at the Department of Plant Physiology, Institute for Biological Research “Siniša Stanković” – National Institute of the Republic of Serbia, University of Belgrade (Serbia).Agroinfiltration experiments were performed under greenhouse conditions. C. erythraea shoots and roots was isolated using modified CTAB method , in 1.5 ml microtube. A total of 30 μl reaction mixture was incubated for 1 h at room temperature to let the ligation reaction take place.The full length amplification of the candidate gene was performed using Q5 Hot Start High-Fidelity DNA Polymerase  and gene specific forward and reverse primers  followinE. coli competent cells, incubated on ice for 20 min and then heat-shocked for 45 s in water bath at 42°C. The mixtures were immediately placed on ice and subsequently cultivated with 250 μl liquid Luria-Bertani (LB) media followed by 1 h incubation at 37°C. The transformed cells were cultivated on LB agar plate containing ampicillin (100 μg ml–1), which was followed by overnight incubation at 37°C. Positive transformants were verified by colony PCR with CeBGlu gene-specific primers . Subsequent sequencing has confirmed the isolation of two highly similar gene variants named CeBGlu1 and CeBGlu2.For transformation, 5 μl of the ligation product was added into 100 μl of Mach1  primers . Cells hCeBGlu1 and CeBGlu2 sequences, it was possible to design only one pair of primers common to both isoforms  was used. Amplification was conducted in Light cycler QuantStudio 3 , according to the manufacturer’s instructions. General thermocycler conditions were 95°C for 10 min; 40 cycles of 95°C for 15 s; 60°C for 30 s; 72°C for 30 s and final extension at 72°C for 10 min. Expression levels of targeted genes were calculated according to the 2–ΔΔCt method  supplemented with phenylmethylsulfonyl fluoride (PMSF) and 5 mM ascorbate. Protein content was determined according to Shoots and roots of diploid and tetraploid Activity of protein extracts to reduce the content of secoiridoid glycosides was evaluated using standards of SW, SWM and GP as substrates. The reduction in quantity of the mentioned substrates is the net result of combined enzymatic activities present in the extract including glucosyl hydrolases which produce the respective aglycones and unknown biosynthetic enzymes involved in transformations of secoiridoids .g for 10 min. Supernatants were filtered through 0.2 μm cellulose filters and subsequently analyzed for the content of SW, SWM and GP.All reference compounds were diluted in dH2O (w:v = 1:1) and kept as a stock solutions. Aliquots of 30 μl were vacuum evaporated and diluted in 300 μl 50 mM citrate phosphate buffer pH 5.5 containing 10 μg of protein extracts. Reactions were incubated for 24 h at 37°C. Subsequently, 700 μl of methanol was added, and samples were centrifuged at 10,000 CeBGlu1 and CeBGlu2 functionally characterized within the present study.For phylogenetic tree creation, multiple sequence alignment-s were generated using the Muscle algorithm. A neighbor-joining tree was constructed using MEGA X, Version 10.2.6 . ClusterMultiple sequence alignments were generated with the DECIPHER R package  using 5 2 and the jFATCAT rigid model , SW (PubChem id: 161036) and GP (PubChem id: 88708). Conformers for these compounds were generated using the ETKDG version 3 method with imposing small ring torsion angle preferences ; ON060691 (CeBGlu2).Sequence data from this article can be found in the NCBI database under the following accession numbers: CeBGlu1 and CeBGlu2 were cloned into bacterial expression vector pRSETA, yielding the final plasmids pRSETA:CeBGlu1 and pRSETA:CeBGlu2  CodonPlus-RIL cells . Cultures were grown overnight in 20 ml 2 × Yeast Extract Tryptone (2 × YT) media broth supplemented with ampicillin (100 μg ml–1), 50 μg ml–1 kanamycin and 17 μg ml–1 chloramphenicol at 37°C with shaking at 220 rpm. On the next day 200 ml culture was initiated by inoculating above mentioned overnight cultures, with the initial OD600 set to 0.1. Cultures were incubated at 37°C with shaking at 230 rpm. After reaching an OD600 of 0.4–0.5, protein expression was induced using 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Subsequently, incubation continued for 4 h at 21°C. Following centrifugation for 10 min at 5,000 g and removal of supernatant, the cell pellets were harvested. All of the purification steps were performed at 4°C. For each purification batch, Ni-NTA agarose  beads were equilibrated with equal volumes of lysis buffer . Bacterial cells were collected by centrifugation and re-suspended in 500 μl of lysis buffer. Following lysozyme  addition , samples were incubated for 30 min on ice. After 10 min of centrifugation  supernatants were loaded into columns containing Ni–NTA resin and incubated at 4°C using FALC F205 rotary tube mixer . After 1 h, samples were spun at 12,000 g for 5 min at 4°C. Supernatant was discarded and resin was rinsed for 3 times using washing buffer . The recombinant protein was eluted with increased imidazole concentration in elution buffer .pRSETA vector contains a N-terminal polyhistidine (6×His) tag, which can be used as an affinity ligand for protein purification. The pRSETA:p-coumaric acid, 1.25 mM 3-aminophthalhydrazide and 1.7 μl of 30% H2O2. Detection was performed by exposure to radiographic film  for 10 min.Eluted fractions were analyzed using 5–10% SDS-PAGE using Mini-PROTEAN II Electrophoresis Cell  followed by Coomassie blue staining and immunoblotting. Proteins were electro-transferred to PVDF membrane , using Mini Trans-Blot Module electric transfer system . Transfer on the membrane was performed at 4°C for 90 min at a constant voltage of 60 V. Membrane was blocked with 10% (w/v) non-fat dry milk  in phosphate-buffered saline containing 0.05% Tween-20 overnight at 4°C. The presence of 6×His labeled proteins was confirmed using His-probe antibody in 1:100 dilution , which was followed by incubation with goat anti-mouse IgG-HPR . The bound antibodies were visualized by enhanced chemiluminescence (ECL). The membrane was incubated for 5 min at room temperature using ECL solution containing 100 mM Tris–HCl pH 8.5, 0.2 mM CeBGlu1 and CeBGlu2 was determined in a reaction with 20 μg of recombinant proteins and 10 mM 4-nitrophenyl β-D-glucopyranoside (pNPG) as a substrate, in 50 mM citric phosphate buffer pH 5.5 at 40°C for 48 h. The reaction was stopped by the addition of ice cold 1 M Na2CO3 (1:1 = v:v) and colorimetric detection of p-nitrophenol, the product of pNPG hydrolysis, was spectrophotometrically measured at 410 nm (Beta-glucosidase activity of t 410 nm .C. erythraea (100 mg) were ground into homogeneous powder using liquid nitrogen and extracted with 1 ml of 99.8% methanol  by vortexing for 30 s and subsequent sonication for 10 min using an ultrasonic bath . After centrifugation at 10,000 g for 10 min supernatants were filtered using 0.2 μm syringe filters . C. erythraea methanol extract (10 μl) was evaporated in a Vacuum Rotor Evaporator  at room temperature. Subsequently, dried extracts were dissolved in 300 μl 50 mM citrate phosphate buffer (pH = 5.5) containing 20 μg of purified recombinant enzyme (CeBGlu1 or CeBGlu2) and incubated for 48 h at 37°C. Following incubation, 700 μl of methanol was added to stop the reaction, and reaction mixtures were centrifuged for 10 min at 10,000 g. Supernatants were filtered through 0.2 μm cellulose filters  and subsequently subjected to UHPLC/DAD/(±)HESI−MS2 quantification of SGs. Control samples were prepared by replacing recombinant protein with the elution buffer used for protein purification.Aerial parts of CeBGlu1 and CeBGlu2 was further tested using standards of epideoxyloganic acid, loganin, secologanin, sweroside, swertiamarin, gentiopicrin, apigetrin, isoquercitrin, and vitexin as substrates. All reference compounds were diluted in dH2O (w:v = 1:1) and kept as a stock solutions. Aliquots of 30 μl were dried in vacuum evaporator and diluted in 300 μl 50 mM citrate phosphate buffer pH = 5.5. Reaction mixture containing 30 μl of previously diluted standard compound and 20 μg of purified recombinant enzyme in a final volume of 300 μl 50 mM citrate phosphate buffer pH = 5.5 was incubated for 48 h at 37°C. Subsequently, 700 μl of methanol was added, and samples were centrifuged at 10,000 g for 10 min. Control samples contained elution buffer instead of purified recombinant protein, and the final concentration of standards was 3 μg ml–1. Supernatants were filtered through 0.2 μm cellulose filters and injected into UHPLC/DAD/(±)HESI−MS2 instrument.Hydrolytic activity of CeBGlu1 and CeBGlu2 sequences were PCR-amplified from pTZ57R/T plasmids with the addition of PacI/AvrII restriction sites. Amplicons were ligated to transient expression vector pJL-TRBO (CeBGlu1/CeBGlu2 gene specific primers (CeBGlu1 and pJL-TRBO:CeBGlu2 (E. coli competent cells were cultured overnight in LB liquid medium containing kanamycin (50 μg ml–1) at 37°C in a shaker incubator at 230 rpm . GeneJET Plasmid Miniprep Kit  was used to purify plasmids from 5 ml of Mach1 E. coli cultures according to the manufacturer’s instructions. After plasmid isolation, each CeBGlu sequence was confirmed by sequencing.pJL-TRBO , and pos primers . After h:CeBGlu2 , Mach1 ECeBGlu1 and pJL-TRBO:CeBGlu2 plasmids extracted from Mach1 cells were transferred to A. tumefaciens GV3101 strain by electroporation using “Gene Pulser”  and subsequently cultured in LB plates containing kanamycin (50 μg ml–1), gentamicine (25 μg ml–1) and rifampicin (10 μg ml–1). A single colony of recombinant bacteria was inoculated into 5 ml liquid LB media containing antibiotics and incubated overnight at 28°C with shaking at 230 rpm. On the next day, 1 ml of bacterial suspension was sub-cultivated in 10 ml of liquid LB media containing 10 mM MES-KOH (pH 5.5) and 20 μM acetosyringone. Cultures were incubated overnight at 28°C with shaking at 230 rpm. The agrobacterial cells were harvested by centrifugation for 20 min at 3,000 g, and the pellet was resuspended in the infiltration medium  to a final OD600 of 1.0. After an incubation at room temperature for 4 h, cultures were introduced into abaxial surface of leaves of five-month-old C. erythraea plantlets using a blunt tipped plastic syringe and applying gentle pressure. Additionally, to prevent the silencing of transgene expression in C. erythraea, pJL-TRBO:CeBGlu1 and pJL-TRBO:CeBGlu2 were co-infiltrated in a 1:1 ratio with pBIN vector expressing the p19 silencing-suppressor gene from TBSV (pBIN:p19). After agroinfiltration, plants continued to grow for 5 days in the greenhouse. Leaves were harvested from diploid and tetraploid C. erythraea plants and immediately frozen at liquid nitrogen. Samples were stored at −80°C until further use.The recombinant pJL-TRBO:C. erythraea was manually ground in liquid nitrogen into fine powder and diluted in 96% methanol (w:v = 10:1). Following vortexing for 1 min, extraction was performed overnight at 4°C. The next day, extraction was continued for 20 min in an ultrasonic bath  maintained at room temperature. Samples were centrifuged at 8,000 g for 20 min and supernatants were filtered using 15 mm RC filters with 0.22 μm pore size . Samples were stored at 4°C until use. All extractions were performed in biological triplicates.Plant material (shoots and roots) of –1 according to 2) pressure 27 AU, ion sweep gas (N2) pressure 1.0 AU and auxiliary gas (N2) pressure at 10 AU, capillary temperature 275°C, skimmer offset 0 V. Argon was used as the collision gas in the collision-induced fragmentation of the selected reaction monitoring (SRM) mode of the instrument, and collision energies (cE) were set as shown in r = 0.999, p < 0.001. Total concentrations of targeted secoiridoids were obtained by calculating their peak areas, and were expressed as μg per 100 mg of plant fresh weight (μg 100 mg–1 FW). Xcalibur software (version 2.2) was used for the instrument control, data acquisition and analysis. All analyses were performed using three biological replicates.Samples were analyzed using Dionex Ultimate 3000 UHPLC system  equipped with a DAD detector and connected to a triple quadrupole mass spectrometer . Samples were chromatographically separated on a Hypersil gold C18 column (50 × 2.1 mm) with 1.9 μm particle size  thermostated at 40°C. Mobile phase, consisting of water + 0.01% acetic acid (A) and MS grade acetonitrile (B), was eluted at flow rate of 0.4 ml minp < 0.05). For comparison of hydrolytic activities, as well as for comparing SGs content in leaves after agroinfiltration with CeBGlu1 and CeBGlu2 cloned into pJL-TRBO expression vector (without or with silencing inhibitor p19) Student’s t-tests were used for data analysis (p < 0.05).Statistical significance was determined by using Minitab Statistical Software . For statistical analysis of relative gene expression and compound quantification in shoots and roots of diploid and tetraploid plants one-way ANOVA was performed followed by Fisher LSD test  from O. europea is reported to have a defensive role in young organs and meristem tissues as well as in mature tissues, where it activates oleuropein into a potent protein cross-linking agent during de-compartmentalization caused by pests (β-glucosidase (OeGlu) is highlighted as a molecular target of high biotechnology interest to regulate qualitative and quantitative content of bioactive secoiridoids in olive oils, and thus their organoleptic properties (C. roseus and R. serpentina (fam. Apocynaceae), strictosidine is hydrolyzed by β-glucosidases (β-D-glucosidase (SGD) compartmentalization in C. roseus, initiated by cellular breakup after tissue wounding, the highly reactive strictosidine aglycone with prominent feeding-deterrent/toxic properties is released, and further conducted toward the production of cytotoxic MIAs iridoid glucosides), which often defines their dual-defense system against herbivores. Highly active and unstable aglycones released from iridoid and secoiridoid glycosides and monoterpenoid indole alkaloids usually display more prominent biological activities toward herbivores and pathogens, by adversely affecting their enzymatic machinery. Aglycones react with nucleophilic side chains of amino acids to form covalent protein complexes , and actglycones , 2015. Oosidases . Oleuropby pests . Oleuropoperties . Likewisosidases . Followixic MIAs , 2011.C. erythraea are defense compounds constitutively present in tissues that, due to their extremely bitter taste, pre-ingestively deter feeding and thus reduce consumption rates of various non-adapted herbivores. The presumption of the present study was that C. erythraea might also possess β-glucosidases displaying high specificity toward SGs. By activating aglycones of SEC, SW, SWM, and GP, these hydrolytic enzymes could be involved in initial steps of their catabolism. Furthermore, resulting aglycones might offer multi-level protective features to plants, and, as previously shown, shape their antioxidant properties  displaying high homology with strictosidine β-D-glucosidase previously characterized from C. roseus  were revealed. Full lengths of isolated CeBGlu1 and CeBGlu2 comprised open reading frames of 1,659 bp encoding polypeptides of 552 amino acids with calculated molecular masses of 62.06 kDa. The two CeBGlu gene candidates shared a high level of similarity (99.3%) and differed at 12 nucleotide sites, i.e., 4 amino acids  (O-β-D-glucosidase (RsRG) (β-D-glucosidase (RsSGR1) from Rauvolfia serpentina (β-D-glucosidase from R. verticillata (RvSGD) (β-D-glucosidase from C. roseus (CrSTR) (Carapichea ipecacuanha (CiIpeglu1)  (Amino acid sequences of es order : stricto (GsSGD) , raucaffe (RsRG)  and strirpentina , stricto (RvSGD) , stricto (CrSTR) , and betIpeglu1) . The higctively) .β-D-glucosidase amino acid sequences of diverse plant species to narrow the prediction of the function of both CeBGlu candidates  showed the highest homology with β-D-glucosidases of the order Gentianales that prefer alkaloid glucosides as substrates  (Phylogenetic tree was constructed incorporating ndidates . The neiIpeglu1) .βC. erythraea -glucosidase candidates, in parallel with the content of major SGs, in both diploid and tetraploid genotypes. The presence of CeBGlu1 and CeBGlu2 transcripts in C. erythraea was validated by amplifying their specific fragments using qRT-PCR. Due to the high sequence similarity between the two gene candidates, a combination of primers common to both transcripts were employed  was significantly higher in shoots than in roots of both diploid and tetraploid enotypes . It is wenotypes . Similarenotypes , the domenotypes , and higC. erythraea organs, pure SW, SWM and GP were subjected to an in vitro enzymatic assay using crude protein extract of shoots or roots, and the decrease in SG content was analyzed using UHPLC/DAD/(±)HESI−MS2 analysis. SWM and GP were efficiently hydrolyzed, while no significant decrease in SW content was recorded  competent cells. To achieve simple and efficient purification of the enzyme, CeBGlu1 and CeBGlu2 were expressed in fusion with the His-tag.Isolated BGlu candidates were sub-cloned into the pRSETA vector; pRSETA:E. coli, they were isolated using His-tag affinity purification and subsequently resolved in SDS-PAGE (CeBGlu1 and CeBGlu2 molecular masses of ∼70 kDa than expected (62.06 kDa) . Purified preparations of recombinant CeBGlu1 and CeBGlu2 were also analyzed by western blot using the anti-6×His antibody , are most commonly used artificial substrates in enzymatic assays for confirmation of β-glucosidase enzymes function  was analyzed. Both enzymes efficiently hydrolyze synthetic glucoside pNPG, which confirms their glucosidase activity  compared to CeBGlu1 (2.267 μmol pNPG min–1 mg–1 protein). Similarly, enzymes isolated from Lamium galeobdolon (LgGlu2 and LgGlu4) hydrolyze pNPG, but the efficiency of the two tested enzymes differs (β-glucosidases of Lotus japonicus (LjBGD2 and LjBGD4) hydrolyze pNPG, but show higher affinity for plant-synthesized substrates, such as prunasin, lotaustralin, and rhodiocyanoside  does not show hydrolytic activity against tested pNPG substrate, and its function was assigned based on a reaction in which OeGlu efficiently hydrolyzes a specific substrate available in planta – secoiridoid oleuropein  hydrolyze the tropan alkaloid scopolin, but do not exhibit hydrolytic activity against a synthetic substrate  were recorded , SWM (3.5 μg ml–1 extract) and GP (2.1 μg ml–1 extract) in C. erythraea methanol extract were not equal, it was not possible to make conclusions about the preferable substrates of the two CeBGlu.recorded . CeBGlu2β-D-glucosidase activity of recombinant CeBGlu1 and CeBGlu2 enzymes was determined against several plant-derived glucosides of the β-D-type , secoiridoid glucosides , and flavonoid glucosides . Pure 1,5,9-eDLA was isolated from methanol extracts of Nepeta rtanjensis as described by β-D-glucosidase activity of recombinant proteins was evaluated by UHPLC/DAD/(±)HESI−MS2 quantification of targeted glucosides . The highest hydrolytic activity of CeBGlu2 was recorded for GP (99.7%) and SW (98.3%), followed by AP (63.5%) and SWM (52.2%). Generally, both CeBGlu1 and CeBGlu2 display higher affinity for GP, than for SW and SWM. Although significant activity of CeBGlu1 and CeBGlu2 was recorded when using AP (apigenin 7-O-glucoside) as a substrate, one should bear in mind that, up to the best of our knowledge, this compound was not previously detected in C. erythraea methanol extracts  based viral vectors have been well documented . TMV-basl system .CeBGlu gene, Agrobacterium-mediated transient expression in centaury leaves was carried out. To that end, the CeBGlu coding region was subcloned into the pJL-TRBO vector. The resultant plasmids designated pJL-TRBO:CeBGlu1 and pJL-TRBO:CeBGlu2 were used to transform A. tumefaciens GV3101 for the transient in planta expression. The two CeBGLU candidates were overexpressed in leaves of five-month-old diploid and tetraploid C. erythraea plants, alone or in combination with p19 , which is believed to prevent the onset of post-transcriptional gene silencing in the infiltrated tissues  8-barrel  8-barrel structure is a sheet formed from two antiparallel strands βa2 and βa3. The regions with low pLDDT are on the termini, between β4 and β5, as well as between β6 and βa1, matching closely the regions with missing density in the experimental structure of raucaffricine β-D-glucosidase from R. serpentina which strengthens the conclusion these regions are disordered.To gain insights into sequence-structure relations of the two centaury 8-barrel  fold chand loops . Strandse barrel . Based orel  and the content of SGs in analyzed C. erythraea tissues. Thus, it provides the first evidence of SGs-related β-glucosidases in centaury plants, and advances our understanding of SGs deglycosylation, as a part of their catabolism. These enzymes could serve as a molecular target of high biotechnological interest, in order to produce centaury plants with an optimal composition of secoiridoids, which are essential defense compounds in plants with beneficial effects in human and animal health. One of the major challenges ahead is to gain better understanding of how plants regulate the function of CeBGlu1 and CeBGlu2 in distinct tissues and developmental stages, and in response to biotic and abiotic stress stimuli, which is the course of our further work. Furthermore, it is imperative to assess their biological function and confirm their localization at the cellular and organelle level and define conditions under which these enzymes come into contact with their physiological substrates. Alternatively, gene silencing of the CeBGlu1 and CeBGlu2 combined with metabolic profiling of silenced plants might provide more information on the role and function of these genes, and could also result in elevated amounts of SGs in tissues. In parallel, unrevealing the remaining unknown steps of SGs biosynthetic pathway in C. erythraea is of essential importance for establishing the biotechnology-based production of these valuable bioactive compounds. Accumulating knowledge will, in the future, enable manipulation of SGs biosynthesis and catabolism through multi-target metabolic engineering, which will further enable large-scale production of desired secoiridoids in C. erythraea.The present study highlights the direct link between nih.gov) repository  under accession numbers ON060690 and ON060691.The data presented in this study are deposited in the National Center for Biotechnology Information (JB, DMa, SŽ, MM, MS, and DMi conceived and designed the experiments. JB, MM, NA, MS, DMa, SŽ, BF, and TB performed the experiments. DMi performed the phytochemical characterization of samples. MD was responsible for the 3D modeling and ligand docking analysis. JB, MM, DMa, and DMi organized and wrote the manuscript with editing from all the authors. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Notably, a similar behaviour was shown by SH-SY5Y neuroblastoma cells plated onto LMN. Interestingly, we did not observe this phenomenon when plating the cells on substrates such as Bovine Serum Albumin (BSA) or Polylysine (PL), thus suggesting a crucial involvement of ECM proteins as well as of β1 integrin activation.Ion channels are implicated in various diseases, including cancer, in which they modulate different aspects of cancer progression. In particular, potassium channels are often aberrantly expressed in cancers, a major example being provided by hERG1. The latter is generally complexed with β1 integrin in tumour cells, and such a molecular complex represents a new druggable hub. The present study focuses on the characterization of the functional consequences of the interaction between hERG1 and β1 integrins on different substrates over time. To this purpose, we studied the interplay alteration on the plasma membrane through patch clamp techniques in a cellular model consisting of human embryonic kidney (HEK) cells stably transfected with hERG1 and in a cancer cell model consisting of SH-SY5Y neuroblastoma cells, endogenously expressing the channel. Cells were seeded on different substrates known to stimulate β1 integrins, such as fibronectin (FN) for HEK-hERG1 and laminin (LMN) for SH-SY5Y. In HEK cells stably overexpressing hERG1, we observed a hERG1 current density increase accompanied by V Thankd values .Potassium channels are functionally expressed in several somatic cancer cell lines and primary tumours ,8,9 and It has already been shown  that hal+ currents endogenously expressed were increased after cell adhesion in these cells [Regarding neuroblastoma cells, our group previously reported that a long-lasting hERG1 activation occurs after integrin-mediated adhesion, which is associated with the induction of neurite extensions and differentiation ,19. No ose cells . Subsequse cells .To further examine these interactions over time, we studied and characterized through patch clamp experiments the hERG1-β1 integrin interplay on different coatings  at different intervals of time from the cells. In particular, we tested a cellular model consisting of the human embryonic kidney (HEK) cells stably transfected with hERG1 on FN, BSA and PL. We then validated the role of this interplay in the tumour cell line SH-SY5Y on LMN as a pathophysiological model . We used2 in a humidified atmosphere, in Dulbecco’s modified Eagle’s Medium  supplemented with 4% L-Glut and 10% fetal bovine serum . We certify that all the cells used in the present study were routinely screened for Mycoplasma contamination, and only Mycoplasma negative cells were used. HEK293 cells expressing the hERG1 construct (HEK hERG1) were prepared as previously described [HEK293 cells were obtained from the American Type Culture Collection (ATCC). SH-SY5Y cells were a kind gift from Prof. P. Defilippi . Cells were routinely cultured at 37 °C with 5% CO2 concentration. The culture surface was coated with a minimal volume (1 mL for 35 mm Petri dishes). The dishes were left air-drying for 1 h at room temperature before introducing cells and medium.Fibronectin  coating was performed following the standard protocol provided with the product. In particular, FN was diluted in sterile PBS (Euroclone) at 5 µg/cmLaminin  coating was performed in accordance with the information provided with the product. More in detail, LMN was diluted in sterile PBS at 0.02% concentration to coat the culture surface (1 mL for 35 mm Petri dishes) and left air-drying for 1 h before plating cells.Heat-inactivated BSA was prepared by heating a BSA (Sigma-Aldrich) solution (25 mg/mL) in sterile PBS (Euroclone) at 70 °C for 1 h. Coating of culture dishes was performed by adding the BSA at 0.25 mg/mL in DMEM at 37 °C for 1 h (1 mL for 35 mm Petri dishes) before seeding cells.2 and rocked gently to ensure an even coating. After 5 min the solution was removed by aspiration and the surface was washed with sterile water. The dishes were left to air-dry at least 2 h before introducing cells and medium.Polylysine  coating was performed following the standard protocol for the product. The culture surface was coated with 1 mL/25 cm5–15, T30–45, T60–90), during which cells were maintained in an incubator at 37 °C, 5% CO2. The beforementioned time points are the intervals of time (expressed in minutes) ranging from the cell seeding to the time in which the recordings were acquired. The patch pipettes were pulled from borosilicate glass capillary tubes to a resistance of 4–5 MΩ. Capacitances were manually compensated after the reaching of a stable gigaseal. The cell capacitances of cells were 34.8 ± 0.6 pF. Experimental protocols and data acquisition were performed with the Multiclamp 700 A or Multiclamp 1D amplifiers and pCLAMP 9.2 software  was used for data analysis. The hERG1 inward tail currents were recorded with a 25 KHz sampling rate and a 0.2 kHz low-pass filter. Cells’ identification and patch were performed at 40× magnification with a Nikon Eclipse TE300 microscope , equipped with a Photometrics CoolSNAP CF camera . Cell membrane potentials were held at −80 mV, and hERG1 inward tail currents were elicited using preconditioning holding potential ranging from 0 mV to −100 mV (10 mV step increment) followed by 1 s hyperpolarizing step (−120 mV) with an intersweep interval of 15 s. The internal pipette solution contained (in mM): 130 K+ aspartate, 10 NaCl, 4 CaCl2, 2 MgCl2, 10 Hepes–NaOH, 10 EGTA, pH 7.3. The external solution contained (in mM): 130 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose (EK = −80 mV), pH of 7.4. Resting membrane potential (Vrest) values were measured in I-0 mode.On the experimental day, HEK293 cells were detached, resuspended in DMEM + HI BSA and seeded on FN-coated, BSA-coated, PL-coated or LMN-coated Petri dishes for patch clamp experiments. Electrophysiological recordings were performed at room temperature (~25 °C) in the whole-cell configuration of the patch clamp technique, at different time intervals (minutes) after cell seeding  when tested with the D’Agostino and Pearson omnibus normality test . Two-way ANOVA test with Tukey’s multiple comparison post hoc test was used for comparisons among different coatings over time . Data are reported as mean ± SEM. Statistical analysis was performed using Prism 9 software .Parametric tests were used for statistical analysis, i.e., unpaired hERG1) and the resting potential (Vrest) values. We observed different behaviours related to the coating over time. First, we monitored the CDhERG1 at different intervals of time, the latter expressed in minutes, ranging from the cell seeding to the time in which the recordings were acquired.To investigate the hERG1/β1 integrin interplay over time, HEK-hERG1 cells were seeded onto FN, BSA and PL, and electrophysiological recordings were performed at different time intervals after cell seeding to monitor the maximal hERG1 current density . We then compared the FN coating with the BSA and the PL, in two different intervals of time, T0–30 and T30–60, and it clearly emerged that only the adhesion onto FN determined a significant increase in the CDhERG1 at T60–90 as shown in hERG1 increase that was not observed in cells seeded onto BSA or PL . In parallel with the CDhERG1 increase, the average Vrest of FN-seeded HEK-hERG1 cells was hyperpolarized from the initial values  and it was significantly different from what we observed in the cells seeded onto BSA and PL for all the time intervals monitored, as shown in T0–T5-15 = 0.113; pT0–T30–45 = 0.0002; pT0–T60–90 < 0.0001; 1e: pT0–30 = 0.0005; pT30–60 < 0.0001; T0–30FNvsBSA = 0.008; pT0–30FNvsPL = 0.130; pT0–30BSAvsPL > 0.999; pT30–60FNvsBSA < 0.001; pT30–60FNvsPL = 0.011; pT30–60BSAvsPL = 0.912). Moreover, the different coatings did not affect the cell capacitance over time (p = 0.476).As shown in rest hyperpolarization were due to biophysical or kinetic modifications of the channel. In particular, we analysed whether the steady-state activation curves of hERG1 were modified by cell adhesion onto FN when compared to the one seeded onto BSA and PL. In both cases, we did not observe any significant modification of V1/2 related to the coating . However, in agreement with previous work [1/2 of activation was around −40/−50 mV. Representative patch clamp traces and I/V curves obtained in HEK-hERG1 cells seeded onto either FN or BSA at different time intervals are reported in hERG1 increase only occurs in the first condition and that it is not accompanied by a V1/2 modification.To better characterize this phenomenon, we investigated whether or not the current density increase and the VhERG1, Vrest and V1/2 dynamics over time in SH-SY5Y neuroblastoma cells, in which we previously demonstrated that integrin stimulation determines a long-lasting activation of IhERG1 and that this is the only endogenously expressed potassium current [5–15, T30–45, T60–90, T120–150, T180–240 and T300–360 min after plating) showed a CDhERG1 3.4-fold increase (at T60–90: 52.1 ± 13.0 pA/pF) from the initial value (15.5 ± 3.7 pA/pF). Afterwards, CDhERG1 decreased, attaining values around 30–35 pA/pF (p = 0.002). As we observed in HEK cells stably overexpressing hERG1, the current density increase was accompanied by a Vrest hyperpolarization, reaching the peak after 90 min from cell seeding (∆ = 10 mV) and then diminishing to the initial values around −30 mV .Next, we further investigated CD current . Neurobl current  but its  current ,22,23. Ed −30 mV c. BecausThe regulatory interaction between cell–cell or cell–substrate adhesion receptors and ion transport was identified several decades ago. It is now known that integrins mediate adhesion but are also deeply involved in ion fluxes’ modulation . The dynβ1 integrin activation, triggered by cell adhesion onto ECM proteins (FN and LMN), induces a double effect on hERG1 channels. In particular, we observed an increase in the hERG1 current amplitude together with the Vrest hyperpolarization. Interestingly, no such phenomenon was observed when cells were plated onto BSA and PL. However, our hypothesis of a biophysical alteration induced by the hERG1-β1 complex formation was proved wrong. In fact, no significant differences were observed regarding the activation V1/2, thus excluding a biophysical modification of hERG1. Therefore, our work is in accordance with what is described above, but it also highlights the crucial role of ECM proteins in the β1 integrin activation and in the macromolecular complex formation.Although understanding of macromolecular complexes and their clinical relevance has increased significantly in recent years, numerous topics for further research remain. Importantly, our results showed the functional response of hERG1 to different coatings, such as FN, PL, BSA and LMN in both a reconstituted model (HEK-hERG1) and in a physiological one (SH-SY5Y). Our electrophysiological recordings showed that rest, found in cancer cells, could be regarded as a “sustaining proliferative signal” that instructs cells to rapidly advance in the cell cycle [rest, such as human neuroblastoma cells [+ concentration influence the membrane potential setting during the cell cycle. A transient decrease in K+ efflux before entering the G2 phase and a relatively high level of K+ efflux during the M phase, indeed, has been found in both mouse neuroblastoma and Ehrlich ascites cells [In addition to this, it is well known that the depolarized Vll cycle . Howeverll cycle , mouse nll cycle , MCF-7 hll cycle  and lympll cycle ,33,34. Sll cycle ,31, whiles cells ,35.+ current (IKir) later in development [hERG1 itself is cell cycle-dependent [hERG1, Vrest and proliferation. The mechanisms underlying ion channel-dependent proliferation of cancer cells include possible non-conducting, direct interactions between ion channels and other pro-proliferative signalling mechanisms [Voltage-gated potassium channels, then, are involved in the regulation of proliferation and membrane potential. It is well known that hERG channels are expressed at early developmental stages in the neural crest, central nervous system, dorsal root ganglion (DRG) and skeletal muscle, and are replaced by a classic inward rectifier Kelopment ,37. Moreelopment  and theielopment ,12,38,39chanisms . For insIt is evident that in such a scenario, unveiling of the dynamics of the interaction of the macromolecular complexes between ion channels and receptors and their effect on ion currents and molecular signalling is urgently needed. Such findings, indeed, would give us important opportunities for new pharmacological targeting. The latter will need versatile tools, such as bispecific antibodies, as they offer the unique advantage of simultaneously binding two or more proteins, impacting the downstream signalling ,40,41,42hERG1 increase together with the Vrest hyperpolarization in response to cell adhesion onto ECM proteins, such as fibronectin for HEK-hERG1 cells and laminin for SH-SY5Y cells. Our first hypothesis of a biophysical modification of the channel was proved wrong, opening the way for further investigations regarding the pathways involved and the mechanisms hiding behind crucial phenomenon, which could open the way to new pharmacological strategies for targeting.In conclusion, our study aimed to functionally characterize the hERG1/β1 interplay over time. Our findings showed a CD


Pseudomonas aeruginosa AS_23 was isolated from petroleum-contaminated soil in Nigeria. Here, we present the draft genome sequence, consisting of an 6,760,157-bp genome with 6,348 protein-coding sequences, 6,253 functionally assigned genes, and 61 RNA genes. Average nucleotide identity-based whole-genome comparisons suggest that this is a new strain of Pseudomonas aeruginosa. Pseudomonas aeruginosa is ubiquitous, and the strains have a suitable genetic repertoire for this lifestyle (Pseudomonas aeruginosa AS_23 from Nigeria. The organism was isolated from soil (pH 5.53) that had been collected  at a petroleum mining site in Delta State, Nigeria , put into sterile sample bottles, kept in an airtight zipped bag, and transported to the laboratory. The soil was dried for 5 days, and 1 g was suspended in 9 mL sterile water and vigorously shaken (−1 to 10−10) were prepared. An aliquot (0.1 mL) from each dilution was plated on a sterile nutrient agar plate and incubated at 37°C for 48 h. Bacterial growth was recorded only from the 10−1, 10−2, 10−3, and 10−4 dilutions. The plate with the 10−4 dilution was selected for the study. Based on morphological observations, four colonies were identified, and each was plated on a nutrient agar plate by streaking and incubated at 37°C for 24 h. To obtain pure colonies, each colony was streaked on two other nutrient agar plates and incubated at 37°C for 24 h in succession. The isolates were grown overnight in nutrient broth at 37°C. A 2-mL portion was centrifuged at 14,000 × g for 3 min, and the supernatant was discarded. This procedure was repeated with a fresh 2-mL portion. To wash the cell pellets, 500 μl of phosphate-buffered saline was added, and the mixture was centrifuged at 14,000 × g for 3 min. The washing was repeated.ifestyle , 2. Theiifestyle . Here, wy shaken . From thN50 value of 114,588 bp, a total assembled size of 6,760,157 bp, genome coverage of 66×, and a GC content of 66.07%. The completeness evaluation using CheckM (v1.1.2) (Pseudomonas aeruginosa AS_23 and Pseudomonas aeruginosa UCBPP-PA14 (GenBank assembly accession number GCF_000014625.1), Pseudomonas aeruginosa B136-33 (GenBank assembly accession number GCF_000359505.1), and Pseudomonas aeruginosa VRFPA04 (GenBank assembly accession number GCF_000473745.2).The genomic DNA was extracted using a ZymoBIOMICS DNA miniprep kit. Default parameters were used for all software unless otherwise specified. DNA libraries were prepared using the Nextera XT kit (Illumina) and the Nextera Index kit (Illumina) and fragmented using Illumina Nextera XT fragmentation enzyme. Twelve cycles of PCR were performed to construct DNA libraries, which were purified using AMPure magnetic beads, eluted in Qiagen EB buffer, and quantified using a Qubit 4 fluorometer and the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit. Genome sequencing was performed with the Illumina HiSeq X Ten platform (2 × 150-bp paired-end sequencing). Trimming of raw single-end reads was done using BBDuk v37.78) , giving .78 , giv(v1.1.2)  indicateJANAWV000000000. The version described in this paper is version JANAWV010000000. The raw reads were deposited in the Sequence Read Archive (SRA) under BioProject accession number PRJNA815837 and BioSample accession number SAMN29499814.This genome has been deposited in DDBJ/EMBL/GenBank under accession number


The implementation of coronavirus disease of 2019 (COVID-19) lockdown has affected the daily practices of subjects with chronic diseases such as diabetes and caused negative impact on their lifestyle and habits such as physical activity, dietary habits and accessibility to medications. Diabetic people are considered the most vulnerable groups to COVID-19, and the lockdown measure has disturbed the diabetes self-management. In our study, we aimed to assess, for the first time at the regional level (Souss Massa Region), the COVID-19 lockdown impact on HbA1c levels in patients with type 1 diabetes (T1D) and type 2 diabetes (T2D). We carried out a cross-sectional quantitative analysis at the health center of the industrial district in Agadir City.p = 0.005). Our analysis revealed a significant association of HbA1c deviation with educational level and medical coverage (p = 0.01). No significant association was detected between HbA1c deviation and age, gender, weight, height, current BMI status, fasting blood sugar, family history, urban or rural areas, marital status, professional activity, socioeconomic income, type of diabetes, dietary, comorbidities, diabetic complications, housing, adherence to the dietary recommendations, physical activity, medical appointments, stopping medication, self-monitoring, fasting and anxiety about getting COVID-19.We found a significant improvement in post-lockdown mean ± SD HbA1c in 150 subjects suffering from T1D and T2D; COVID-19 lockdown had no deleterious effect on HbA1c levels in Moroccan patients with T1D and T2D. In December 2019, a new beta coronavirus of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had emerged in Wuhan City, China. The transmission rate of the infection, termed COVID-19 by World Health Organization (WHO), was extremely high and it spread rapidly worldwide leading to a global pandemic. On March 11, 2020, WHO had declared COVID-19 as a pandemic. Globally, 216,229,741 coronavirus-confirmed cases and 4,496,681 deaths have been reported in August 2021 according to WHO declarations. In Morocco, COVID-19 led to 853,373 cases and claimed 12,437 lives in August 2021, according to WHO Coronavirus (COVID-19) Dashboard (WHO 2021). In order to mitigate and slow the spread of the disease, public health authorities applied exceptional measures in many parts of the world; social distancing and lockdowns. On March 20, 2020, Morocco established a highly strict lockdown until June 10, 2020, and citizens were advised to remain indoors except for limited necessities such as emergency medical help or alimentary shopping , the COVID-19 lockdown impact on HbA1c levels in patients with T1D and T2D.Our cross-sectional quantitative analysis was carried out from April 9, 2021, to June 6, 2021, at the health center of the industrial district in Agadir City. According to eligibility criteria, 150 patients were included in this study.Anonymized data were generated from a structured questionnaire created in Sphinx software by taking into consideration the education level of the interviewed patients.Part 1: socio-demographic characteristics including twelve questions .Part 2: disease characteristics including seventeen questions .Part 3: patients’ attitudes comprising twenty questions .The validated questionnaire consisted of four parts:Confirmed T1D or T2D based on medical records; fasting blood sugar > 126 mg/dl; HbA1C > 7%Aged ≥ 18 years with a ≥ 1 year T1D and T2D diagnosisRecruited and monitored at the Health Center of the industrial district in Agadir City.Incomplete medical recordsNo HBA1c records in the last 3 monthsChange of antidiabetic treatment at least 6 months before the lockdown and during the lockdownPregnancyActive oncological illness Comorbidity with psychiatric, neurological disorders and non-communicable diseasesp value ≤ 0.05 was considered statistically significant.Anonymized data were divided according to two different periods: pre-lockdown and post-lockdown. Statistical analysis was performed using IBM SPSS Statistics 23 software. The degree of association was assessed using odds ratio (OR) . A Dependent variables: glycemic balance Independent variables: diabetes, COVID-19, confinement, socio-demographic factors and attitudes.HbA1c deviation = HbA1c post-COVID-19-lockdown − HbA1c pre-COVID-19-lockdown The study was conducted in accordance with Helsinki Declaration. Authorization was obtained by Souss Massa Regional Direction of Health before starting the study while respecting the confidentiality of the data and the respect of anonymity in the processing of the data. All participants gave verbal informed consent to answer the anonymous questionnaire.2) 27.08 ± 5.97. Most of them lived in urban areas (90%), 42% were illiterate, 63.3% were married, 93.3% lived with their families, and 46% were unemployed. A low percentage had medical coverage (28.7%) and 56.7% of the patients had a low annually family income < 36,000 (Dhs). The interviewed participants (T1D and T2D) had suffered from diabetes for an average of 11.29 ± 8.73 years with a dominance of T2D for 53.3%  completed the survey questionnaire, 52 men (34.7%) and 98 women (65.3%) with a mean age of 54.93 ± 17.15 (years). The majority of participants had BMI  was observed: 72.43 ± 14.83 (pre-lockdown) versus 72.24 ± 14.49 (post-lockdown). On the other hand, we observed a slight decrease in adherence to dietary guidelines from 75.3% (pre-lockdown) to 70.7% (post-lockdown). The majority respected the dietary recommendations during the lockdown period. According to the recommendations of the Francophone Diabetes Society, (minimum level ≥ three times 30 min per week) .No significant association was detected between HbA1c deviation and adherence to the dietary recommendations, physical activity, medical appointments, stopping medication, self-monitoring, fasting, and anxiety about getting COVID-19. However, a significant association was observed of HbA1c deviation with educational level and medical coverage (n = 205 T2D): post-lockdown HbA1c = 7.7 ± 1.7 versus 8.2 ± 1.9%, p < 0.001 : pre-lockdown HbA1c (%) 6.9 ± 1.3 versus post-lockdown HbA1c (%) 6.7 ± 0.9; p = 0.015 . Fernández et al. (n = 307), and their HbA1c decreased from 7.4 ± 1.0 to 7.1 ± 1.0% . In contrary, other studies described a deleterious effect of lockdowns on glycemic control. Recently, Khare and Jindal found a 0.51% increase in mean HbA1c values after home confinement (Khare and Jindal In our original study, we aimed to assess the COVID-19 lockdown impact on HbA1c levels in Moroccan patients with T1D and T2D. We detected an overall improvement in HbA1c value by the end of lockdown time compared to pre-lockdown period. The pre-lockdown mean ± SD HbA1c was 8.35% ± 1.97 versus 7.97% ± 1.57 after the lockdown. The HbA1c deviation was independent of the age, gender, weight, height, current BMI status, fasting blood sugar, family history, urban or rural areas, marital status, professional activity, socioeconomic income, type of diabetes, dietary, comorbidities, diabetic complications, housing, adherence to the dietary recommendations, physical activity, medical appointments, stopping medication, self-monitoring, fasting and anxiety about getting COVID-19. Our findings are consistent with several studies that demonstrated no effect of the COVID-19 lockdown on HbA1c. A study carried out by Anjana et al. in 2020 revealed an improvement in HbA1c (Possible reasons cited for the decrease in HbA1c levels in our study during lockdown were a more regular lifestyle: stability at home, spending more time with family, less stress due to teleworking conditions, less exposure to pollution, more time to prepare healthy meals, meals on time, increased contribution to household chores, better sleep and awareness of more serious risks complications of COVID-19 and comorbidities.Difficulties in accessing exercise centers or outdoor exercise could explain the slight decrease in physical activity offset by household chores. The majority of participants maintained self-monitoring and compliance with dietary recommendations during the confinement period, which compensated for the difficulties in traveling to health centers for medical appointments.Our finding reported a significant association in HbA1c deviation according to educational level and medical coverage. Moderate and high level of education could improve the glycemic control. Several studies have recorded the negative impact of low educational level on diabetic state (Larsson et al. To conclude, the confinement due to COVID-19 did not cause a main disturbance in glycemic balance. Short duration of home confinement might be sufficient to affect significantly the glycemic control and other risk factors. Further work with larger study samples is needed to identify the metabolic changes that occurred after the strict lock-in period. Nonetheless, the slight improvement recorded implies an improvement in public health policies aiming at encouraging telemedicine and raising awareness about maintaining a healthy lifestyle.


This study assessed the mobility levels among critically ill patients and the association of early mobility with incident proximal lower-limb deep-vein thrombosis and 90-day mortality.This was a post hoc analysis of the multicenter PREVENT trial, which evaluated adjunctive intermittent pneumatic compression in critically ill patients receiving pharmacologic thromboprophylaxis with an expected ICU stay ≥ 72 h and found no effect on the primary outcome of incident proximal lower-limb deep-vein thrombosis. Mobility levels were documented daily up to day 28 in the ICU using a tool with an 8-point ordinal scale. We categorized patients according to mobility levels within the first 3 ICU days into three groups: early mobility level 4–7 (at least active standing), 1–3 (passive transfer from bed to chair or active sitting), and 0 (passive range of motion). We evaluated the association of early mobility and incident lower-limb deep-vein thrombosis and 90-day mortality by Cox proportional models adjusting for randomization and other co-variables.p = 0.87 and 0.91, 95% CI 0.39, 2.12; p = 0.83, respectively). However, early mobility groups 4–7 and 1–3 had lower 90-day mortality .Of 1708 patients, only 85 (5.0%) had early mobility level 4–7 and 356 (20.8%) level 1–3, while 1267 (74.2%) had early mobility level 0. Patients with early mobility levels 4–7 and 1–3 had less illness severity, femoral central venous catheters, and organ support compared to patients with mobility level 0. Incident proximal lower-limb deep-vein thrombosis occurred in 1/85 (1.3%) patients in the early mobility 4–7 group, 7/348 (2.0%) patients in mobility 1–3 group, and 50/1230 (4.1%) patients in mobility 0 group. Compared with early mobility group 0, mobility groups 4–7 and 1–3 were not associated with differences in incident proximal lower-limb deep-vein thrombosis  and Current controlled trials, ID: ISRCTN44653506 (registered on 30 October 2013).The PREVENT trial is registered at ClinicalTrials.gov, ID: The online version contains supplementary material available at 10.1186/s13054-023-04333-9. I2 0%), but not with mortality [Immobility in patients admitted in the intensive care unit (ICU) is common due to the nature of critical illness and the interventions provided for its management, including sedatives, narcotics, and/or paralytic agents. Immobility increases the risk of multiple adversities that include muscle atrophy and weakness , insulinortality .The role of early mobility for VTE prevention is not well studied. This study aimed to assess the association of early mobility with the incidence of proximal lower-limb deep-vein thrombosis and 90-day mortality of critically ill patients.This was a post hoc analysis of the Pneumatic Compression for Preventing Venous Thromboembolism (PREVENT) trial , a multicenter randomized controlled trial that was conducted in 20 sites in Saudi Arabia, Canada, Australia, and India. The Institutional Review Boards of participating centers approved the trial. Informed consent was obtained from all enrolled patients or their surrogate decision-makers. In this trial, adult medical, surgical, or trauma critically ill patients receiving pharmacologic thromboprophylaxis with an expected ICU stay of > 72 h were randomized to receive adjunctive intermittent pneumatic compression with pharmacologic thromboprophylaxis or pharmacologic thromboprophylaxis alone. Twice-weekly lower-limb ultrasonography was performed until ICU discharge, death, full mobility, or 28 days after enrollment, whichever occurred first. The trial found no effect on the primary outcome  .The PREVENT trial enrolled 2003 patients. Except for the study intervention and the required periodic screening with lower-limb ultrasound, routine ICU management, including mobility, was as per the local standard of the participating centers. Mobility levels were documented daily up to day 28 in the ICU using the tool of the Agency for Healthcare Research and Quality Safety Program for Mechanically Ventilated Patients [For this study, we surveyed the participating centers for their mobility practices during the trial period , the use of organ support , depending on normality of distribution. We presented categorical variables as frequencies with percentages and compared them using the chi-square test or Fisher’s exact test, as appropriate.p values were computed. A p value < 0.05 was considered statistically significant. All analyses were conducted using SAS software, version 9.4 .We assessed the association of mobility level with categorical outcomes  using Cox proportional models and with continuous outcomes  using generalized linear mixed models adjusting for the following covariables: randomization group , type of heparin (unfractionated heparin versus low molecular weight heparin), femoral central venous catheters, mechanical ventilation, vasopressor therapy, APACHE II score, and body mass index. The results were presented as adjusted hazard ratios (aHR) or beta estimates with 95% CIs. Kaplan–Meier survival curves were plotted to compare deep-vein-thrombosis-free time and survival time between the different mobility groups. Log-rank The mobility practices of the participating centers during the trial periods are presented in Additional file   had an early mobility level of 4–7 during the first 72 h and 356 patients (20.8%) had an early mobility level of 1–3 while 1267 patients (74.2%) had a mobility level of 0. The baseline characteristics of these patients are shown in Table p < 0.0001). Table During ICU stay, 247/905 (27.3%) patients had mobility level above 0 on day 7, 81/395 (20.5%) patients on day 14, 52/242 (21.5%) patients on day 21, and 30/139 (21.6%) patients on day 28  and in early mobility 1–3 group versus mobility 0 group (p = 0.62). On Cox proportional models, early mobility groups 4–7 and 1–3 compared with early mobility group 0 were associated with similar risk of incident lower-limb deep-vein thrombosis .Incident proximal lower-limb deep-vein thrombosis occurred in only 1/58 patient in the early mobility level 4–7 , 7/348 patients in the early mobility group 1–3  and 50/1230 patients in the early mobility group 0  .Among included patients, the 90-day mortality was 8/85 (9.4%) patients in the early mobility level 4–7, 37/356 (10.4%) patients in the early mobility level 1–3 and 380/1266 (30.0%) patients in the early mobility level 0 (p = 0.63 for early mobility 4–7 versus 0 and p = 0.99 for early mobility 1–3 versus 0). Patients with higher early mobility levels (4–7 and 1–3) had significantly more ventilator-free days  in the mobility level 4–7 group and 28 days  in the 1–3 group versus 21 days  in the 0 group  and ICU-free days  in the mobility level 4–7 group and 22 days  in 1–3 group versus 16 days  in 0 group . However, there were no significant differences in vasopressor-free days between the mobility level 4–7 group versus 0 and between the mobility level 1–3 group versus 0. The other secondary outcomes of this study are presented in Table Pulmonary embolism was diagnosed in 1/85 (1.2%) patient with early mobility level 4–7, 0/356 (0%) patient with early mobility 1–3 and 14/1267 (1.1%) patients with mobility 0  [Critical illness is associated with immobility, often confounded by pre-ICU underlying limitation to mobility, cardiopulmonary instability and ICU therapies such as sedation and neuromuscular blockers. In the current study, we found that mobility more than range of motion was provided to only 441/1708 (25.8%) patients during the first 3 days of ICU stay. This finding is not uncommonly observed in ICUs , 22. A p< 0.001) .p < 0.01; adjusted relative risk 0.67, 95% CI 0.50, 0.90) [p = 0.0004) [Several studies have shown that mobility in critically ill patients was associated with better clinical outcomes, including lower rates of deep-vein thrombosis, ventilator-associated pneumonia, and pressure injury, increased muscle strength and shorter duration of mechanical ventilation, and ICU and hospital stay , 23, 24.0, 0.90) . Another 0.0004) . A syste 0.0004) . This wa 0.0004) . Additio 0.0004) , 18. In  0.0004) . In this 0.0004) . The lim 0.0004) , 27.The strengths of the study include that data came from a multicenter randomized controlled trial wherein twice-weekly structured surveillance for lower-limb deep-vein thrombosis was performed in a large number of patients, thus reducing ascertainment bias. Our study characterized important associations between higher versus lower early mobility levels and important clinical outcomes besides mortality. VTE is rarely studied as an outcome of mobility. Our review of clinicaltrials.gov database showed that none of the 10 ongoing randomized controlled trials that were evaluating mobility in critically ill patients had VTE as a pre-specified outcome (Additional file Only a small proportion of critically ill patients had a high level of mobility early in their ICU stay. There was no association between early mobility levels and lower-limb deep-vein thrombosis. Although early mobility level was independently associated with lower mortality risk, this association does not establish causality and randomized controlled trials are required to assess whether and to what extent this association is modifiable.Additional file 1. Supplementary tables.


These results showed that the immunomodulatory effects were differently impacted by commercial date seed pills, a finding which was related to the large-scale manufacturing of the pills and the incubation concentrations used. These results also shed light on a new trend of using food byproducts as an innovative supplement.Dates have been demonstrated to display a variety of bioactivities and are rich in polyphenols. In this work, we assessed the underlying immunomodulatory effects of date seed polyphenol extracts that had been industrially encapsulated and fabricated into commercial pills in RAW264.7 macrophages using the NF-κB and Nrf2 signaling pathways. The outcomes showed that in RAW264.7 cells, the date seed pills effectively stimulated nuclear translocation of NF-E2–related factor 2 (Nrf2) and NF-κB, along with downstream cytokines , ROS ratios, and SOD activity. It is interesting to note that the encapsulated pills activated Nrf2 nuclear translocation more effectively than the non-encapsulated ones did. Additionally, pills at 50 µg mL Phoenix dactylifera L., are significant cultivated products in North Africa and the Mediterranean region. Date fruits have long been regarded as the perfect meal supplement by Middle Easterners because of their high sugars, dietary fiber, macronutrients, and micronutrient levels [In addition to various regions, namely Europe (the southern part), America ; date fruits, or t levels . Date frt levels . In addit levels . Numerout levels . Recent t levels .Owing to its extra nutritional content, date fruits are an essential dessert that are marketed as a variety of foods. However, around 12–15% of the date fruits are seeds, which are mostly considered as the fruits’ byproduct and have not been economically valued yet. The region’s Bedouin tribes used to drink infusions made from roasted date seeds, but they have otherwise not been significantly used by humans to date. According to earlier research, date seeds have a high polyphenolic content and exceptional antioxidant activity. Numerous animal studies document biological benefits, including a reduction in oxidative damage, an improvement in memory and comprehension in Alzheimer’s models, and an anti-diabetic effect ,7. More κB) regulates several examples of immune-activating cytokines, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and interleukin-1β (IL-1β); it is an oxidative strain-receptive transcription factor [κB dysregulation, and drugs that control NF-κB may prove to be innovative treatment options. Furthermore, another crucial controller of cellular oxidative anxiety called NF-E2-related factor-2 (Nrf2) is a catalyst for cellular immune reactions [α, IL-6, and IL-1β, as well as mitigate the translocation of NF-κB into the nucleus [κB p65 in BV2 microglial cells [According to epidemiological research, the polyphenols in diets and supplements made from fruit may lower the hazards of cancer, diabetes, and cardiovascular illnesses . It has n factor . Chroniceactions . It has  nucleus . Kim et al cells . Additioal cells .κB and Nrf2 signaling pathways. In this study, we investigated and assessed the various immunomodulatory effects of industrially produced date pills and identified a putative immunoregulatory mechanism involving Nrf2 nuclear translocation and NF-κB on RAW264.7 macrophages. The findings of this study shed light on how date polyphenols behave functionally.These compounds are unstable, though. In this situation, microencapsulation technologies could help stabilize polyphenols for use in industrial applications. The process of microencapsulation is used to turn liquid solutions into powders for simpler handling and to increase the stability of bioactive compounds by shielding them from oxygen, water, light, and other environmental factors . TherefoPVDF layers , dimethyl sulfoxide (DMSO), and 3--2,5-diphenyltetrazolium bromide (MTT) were acquired from Sigma-Aldrich . The following items were acquired from Thermo Fisher Scientific: Penicillin-streptomycin mix, Dulbecco’s altered Eagle’s medium (DMEM), trypsin (0.25% EDTA), fetal bovine serum (FBS), tris-buffered saline (TBS), and phosphate-buffered saline (PBS) . Proteintech Group, Inc. sold anti-NF-κB p65, anti-lamin B1, and anti-Nrf2 antibodies for rabbits. Bioscience Technology provided sodium lauryl sulphate, tris base, acrylamide, glycine, ammonium persulfate, bromophenol blue, and N,N’-methylene acrylamide . Proteintech provided TNF-α, IFN-γ, IL-6, and IL-1β enzyme-allied immunosorbent test (ELISA) implements . Date seeds of the Siwa variety were acquired from the Shalli Company  in March 2022; the seeds were harvested at room temperature 12 h prior to arriving at our lab where they were then prepared immediately without being stored. All the other substances employed in this investigation, including the solvents, were of analytical grade.The date seeds were washed , dried , roasted , milled , seized to 45 mesh for more homogenization and uniformity, and formulated to prepare the end formula of the pills. After that, the pills were formed using a ZP-15E-19E automatic rotary tablet press machine (China) using the production line of Tadawina Company . Gum arabic and maltodextrin were used to fabricate the pills, with each pill containing a ratio of 400, 500, and 100 mg per g of polyphenols, maltodextrin, and gum arabic, respectively. 2O- HCOOH, 99:1, v/v) and phase B  made up the mobile phase (solvent B). The stream ratio was 0.08 mL per min, and the oven was kept at 35 °C. The elution schedule was as follows: isocratic for 1 min with 2% B, 2–18% B (1–7 min), isocratic with 18% B (7–9 min), 18–30% B (9–12 min), isocratic for 2 min with 30% B, 30–75% B (14–27 min), and then cleaning and reconditioning of the column. The infusion had a 2 µL volume. An electrospray basis and an ion trap mass analyzer were included in the Bruker Daltonics Amazon  mass spectrometer that was used for the ESI-MS/MS analysis and connected to the UPLC-PDA system. A negative ion manner  and positive ion mode  were used to  the MS2 experiments’ collision energy for fragmentation, which was adjusted to 1. Retention periods, UV-visible spectra, and MS spectra were used to identify the samples. We compared our data with polyphenols-based data found using standard components that were identified in related references [−1). Phloroglucinolysis retains the stereochemistry and potential substituents of proanthocyanidin terminal units while releasing extension units as the matching phloroglucinol derivatives. Total flavan-3-ol content was computed as the sum of constitutive units liberated during phloroglucinolysis. The data sets examined were collected, aligned, and normalized via MarkerLynx software. Peak width at 5% height of 1 s, peak-to-peak baseline noise of 1, noise elimination of 6, and intensity threshold of 10,000 were utilized to capture metabolite peaks. A retention time window of 0.2 min and a mass window of 0.05 Da were utilized to align the data.The polyphenols of the date seed pills were extracted using our previous method . Chromatferences ,20. The 2 (5%) and cultivated in DMEM accompanied with FBS , penicillin (100 U mL−1), and streptomycin 100 (μg mL−1). The media were changed three times a week and were planted at a previously mentioned thickness (roughly 1.0 × 105 cells per cm2). Before usage, all cells underwent more than three passages.The Chinese Academy of Sciences’ Cell Bank of Form Culture Compilation sold the RAW264.7 murine macrophage cell culture and an Abelson leukemia viral-stimulated polyp cell culture . Cells were hatched in a 37 °C hatchery with CO5 cells mL−1, RAW264.7 cells were sown in 96-well bowls and hatched for 12 h at 37 °C. The cells were later re-incubated in serum-free media that had been supplemented with pill extracts at doses of 50, 250, 500, 750, and 1000 µg mL−1 after the supernatants had been aspirated. The blank control was medium alone. Following a 24-h maturation period, the upper phase was removed, each well was mixed with 100 µL of MTT solution, and all of the cells were incubated at 37 °C for 4 h. A 100 µL solution of DMSO (100%) was subsequently used to lyse each cell at room temperature (25 °C) for 10 min. Utilizing a Varioskan Flash microplate reader, the optical densities of the control cell (Ac) and each of the pill-based cell wells (At) were determined at 570 nm . For all studies, six separate tests were run. As previously mentioned, the equivalence Cv (%) = (At − Ac)/Ac × 100 was used to compute cell viability (Cv) [An MTT test was utilized to measure cell capability. At an intensity of 1 × 10ity (Cv) .β, IL-6, IFN-γ, and TNF-α in the matrix were evaluated. Briefly, the RAW264.7 cells were preserved for 24 h in a 5% CO2 hatchery at 37 °C after being pre-hatched in a 12-well plate at an intensity of 1.0 × 106 cells per well. Culture media were collected and transferred into 96-well plates covered with 100 μL of each of the antibody blends for IL-1β, IFN-γ, TNF-α, and IL-6 and held at 37 °C for 60 min. After four washes, 100 μL of horseradish peroxidase (HRP) blend was inserted, and the bowls were then hatched for 40 min at 37 °C. The bowls were then dark-hatched at 37 °C for 15 min before 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substratum mix was inserted. Finally, five minutes after adding a stop solution, the transmission density at 450 nm was detected using a Varioskan Flash microplate reader . Pill extract-free incubation was used for the control group. Standard curves were used to calculate the cytokine levels. There were three duplicates of each test run. The concentrations of cytokine were presented as picograms each mL (pg mL−1) of sample.Using commercial ELISA kits, the ratios of IL-15 cells per well. For 20 min, the cells were subjected to 50, 250, 500, 750, and 1000 μg mL−1 of pill extract. For the pill-free sample, a medium without polyphenols was employed. The media were then evenly mixed with a fluorescent probe (10 µM), and each cell was hatched at 37 °C for an extra 20 min.ROS generation was evaluated using a modified version of the previously published 2,7-dichlorofluorescein diacetate (DCHF-DA) test . The amoTo eliminate any DCFH-DA that had not penetrated the cells, the cells were then rinsed three times with warm PBS. Using the FACS Aria II flow cytometer, the DCF fluorescence of the cell suspension was quantified . The DCF fluorescence strength represents the quantity of intracellular ROS. The trials were conducted three times and the outcomes are shown as mean estimates ± SD.2O was utilized in place of the 20 μL sample, and as the test control, 20 μL of thinned buffer was utilized in place of the 20 μL enzyme operating blend. At a wavelength of 450 nm, the measurements were performed using an ELISA microplate reader. Each sample’s SOD action was calculated. One unit of SOD activity was defined as the enzyme quantity necessary to block 50% of the lessening interaction between superoxide anions and H2O-soluble tetrazolium salt-1 (WST-1) in a sample blend (20 μL).According to the instructions provided with the kit, SOD rates were measured using ELISA . In a 96-well plate, 20 μL of pills and 200 μL of employed blend were combined uniformly. Then, the enzymatic blend (20 μL) was inserted, and the pill-based samples were hatched at 37 °C for 20 min. As the plain control, 20 μL of ddH2 hatchery at an intensity of 1.0 × 106 cells per well. The cells were then preserved for 24 h in media that had been supplemented with 50, 250, 500, 750, and 1000 μg mL−1 of pill extract. After that, the cells were collected, and a nuclear and cytoplasmic extraction kit was used to make nuclear extracts . The supernatants were collected for further protein analysis after the isolates were cold-centrifugated at 4 °C for 20 min at 11,000× g. A 10% DYY-6C SDS-PAGE  at 80 V for 2 h was utilized to divide the nuclear protein. After that, 12.5 and 7.5 μL of nucleoprotein extract and PageRuler Prestained Protein Ladder, respectively, were added to the gel. A PVDF layer was employed to transport the proteins at 350 mA for 1.5 h with the aid of a BIO-RAD Mini-Trans-Blot Cell . The PVDF layer was hatched with initial antibodies disbanded in 0.5% skim milk blend at 4 °C for an extra 2 h after being clogged with 5% skim milk in TBS covering 0.5% Tween 20 (TBS-T). The anti-Nrf2, anti-NF-κB p65, and anti-lamin B1 primary antibodies were thinned at a rate of 1:500. The PVDF membrane was cleaned with TBS-T solution and then hatched for 1 h at RT with a secondary goat anti-mouse IgG antibody that was HRP-labeled. The PVDF layer was then cleaned with TBS-T for 20 min. After being submerged in an enhanced chemiluminescence (ECL) fluid that had been freshly made before usage, the membrane was then left to sit at RT for 3 min before being imaged using a LAS-4000 MINI biomolecule imager . The band intensity in relation to the internal reference lamin B1 represents the protein expression. Three tests were conducted on each sample, and the findings were portrayed as means ± SD.RAW264.7 cells were pre-hatched in a 6-well bowl for 4 h in a 5% COp-value of < 0.05.The findings were shown as means ± SD. Using SPSS 21.0 Statistics, a one-way analysis of variance (ANOVA) followed by post hoc Tukey’s checks for numerous differences was utilized to evaluate the data . A result was deemed statistically significant if it had a −1 of individual polyphenols ; the concentration of flavones and flavonols was 0.045 ± 0.001 g kg−1 ; and the concentration of hydroxycinnamic acids was 0.06 ± 0.001 g kg−1 . The components that were the most prevalent were flavan-3-ols. With a typical level of polymerization, the total quantity of flavan-3-ols determined after depolymerization was 49.3 ± 1.8 g kg−1. This included 45.9 ± 1.1 g kg−1 of epicatechin and 3.4 ± 0.4 g kg−1 of catechin  with 0.25–0.4 mg mL−1 of purslane extract increased, rather than decreased, cell viability [To examine how the pills affected the viability of RAW264.7 macrophage cells, MTT tests were used . The finiability ., et al. [−1) different concentrations of thinned peach cured by several dehydration approaches significantly increased cell viability. Our results showed that the corresponding Maillard adducts in the date seed samples after roasting (160 °C/15 min) were not cytotoxic. The impacts of low and high doses of date seed pills on the macrophage immune response were clarified in subsequent tests using the same doses of 50–1000 μg mL−1 pill extract polyphenols. The innate immune system’s fight against infections is greatly aided by the cytokines released by macrophages [α, IL-1β, IFN-γ, and IL-6) in various pill-incubated RAW264.7 cells was assessed using ELISA kits. According to −1 of pills boosted IFN-γ, TNF-α, and IL-1β production by 35, 33.3, and 38.8%, respectively. This finding is in line with an earlier study that found that docosahexaenoic acid triggers the manifestation of immune cytokines in RAW264.7 cells at concentrations between 1.2 and 3.0 μM [Guo, et al.  also fourophages . In the d 3.0 μM .β, alongside TNF-α and RIG-I cytokine nodding [−1 pill incubation caused the smallest increase in production of these cytokines. Intriguingly, the accumulation of TNF-α, IFN-γ, and IL-1β in 1000 µg mL−1 pill-incubated cells (RAW264.7) was decreased by 18.18, 116.6, and 175.2%, respectively, with incubation with the pills producing a dose-dependent effect. An excessive level of polyphenols may interact with bioactive proteases, causing them to dissolve and undergo functional modifications that have an inhibitory effect on cytokine production [−1 dramatically restrained the production of TNF-α and IL-6 [−1. In general, at healthy doses, IL-6 has little autocrine impact on immune cells and has no influence on the cell’s ability to release further cytokines. Nevertheless, it has been hypothesized that elevated IL-6 concentrations might cause irritation by regulating the release of other cytokines. In the current investigation, the date seed pills significantly lowered the level of IL-6 in cells (RAW264.7), reducing the cells’ defensive response. The catechins and epicatechins in the pills were mostly responsible for this action, a finding which might be confirmed by a prior discovery that the green tea catechin epigallocatechin gallate prevented IL-6 release in HEK293T cells [Because the levels of catechin, epicatechin, and protocatechuic acid in the date seed pills  were sim nodding . Our res nodding . Notablycells RAW4.7 was doduction ,28. Figuand IL-6 . Fluctua−1 pills, the formation of intracellular ROS was reduced by 8.61%. The pills inhibited ROS by 7.75% less than the polyphenols from thinned peach, which inhibited ROS by 16.36%. Because they contained high concentrations of catechin, epicatechin, and phenolic acids, 50 µg mL−1 pills were found to significantly reduce the production of ROS in this study. According to previous studies, the main ingredients of Moringa oleifera leaf extracts (0.1 mg mL−1) that significantly reduced the formation of ROS in HEK293 cells were cryptochlorogenic acid and isoquercetin [−1 pills led to a notable increase by 61.5% in intracellular ROS generation, in which polyphenols at high concentrations behaved as pro-oxidants [The primary oxidation products in cells are ROS, which are essential actors in cell signaling events. Their buildup triggers macrophages to endure extra apoptosis or autophagy, which increases the onset of cell death . Figure oxidants . Accordioxidants , flavonooxidants , Maillar−1 pills, intracellular SOD activity was increased by 7.8%, mostly due to the substantial amounts of epicatechin present in the date seed pills. The finding by Zhao et al. [−1 pills reduced SOD activity by 60.75%. The findings suggest that polyphenols might function as either antioxidants or pro-oxidants depending on the quantities of the polyphenol components. Redox stress may result from the pro-oxidant effects which can disrupt redox equilibrium [Antioxidant enzymes like SOD are linked to the preservation of cellular redox equilibrium. Increasing the activity of antioxidant enzymes has been proven to lower ROS ratios in cells and thereby preserve intracellular redox equilibrium in several studies . In thiso et al.  that polilibrium .κB pathway on macrophage biological activity. Therefore, to investigate how date seed pills affect the activation of the NF-κB signaling pathway, nuclear NF-κB p65 scores were measured via Western blotting. According to −1 pill incubation initiated NF-κB p65 nuclear translocation, which is similar to the discovery that 50 µg mL−1, 50 µg mL−1, and 2% of polysaccharides, ginsenosides, and fatty acids, respectively, separately upregulated nuclear NF-κB manifestation to employ immunological activity in RAW264.7 cells [κB p65 nuclear translocation by 66%. Interestingly, nuclear NF-κB p65 rates were decreased by 33% after 1000 µg mL−1 pill incubation. Nuclear NF-κB p65 levels were significantly reduced by the pills. It may be inferred that high-dose pill incubation likely had pro-oxidant impacts at first, inducing inflammatory responses, and then acted as an anti-inflammatory agent by lowering the nuclear translocation of NF-κB. Hence, incubating with higher doses of pills might prevent the onset and progression of the cellular inflammatory response in the presence of oxidative stress. An earlier study that found that high-dose (100 µg mL−1) phenolics from grape waste might reduce NF-κB stimulation and mitigate lipopolysaccharide-induced oxidative strain in RAW264.7 cells provides support for this finding [κB to attach to p65 and enter the nucleus, thereby decreasing the affinity of p65 for associated genes and enabling it to separate from the binding site and enter the cytoplasm. As a result, NF-κB p65 nuclear translocation was inhibited by incubation with high-dose pills.Analyzing the nuclear translocation of p65 can help identify the impact of the NF-.7 cells . Additio finding . Additio−1 pills had higher nuclear Nrf2 levels, whereas cells cured with 1000 µg mL−1 pills had lower levels. In particular, incubation with 50 µg mL−1 pills increased Nrf2 nuclear translocation. Additionally, incubation with 1000 µg mL−1 pills reduced Nrf2 nuclear localization. According to Granato, Mocan and Câmara [κB signaling pathways. This is mostly caused by the elevated catechin concentration in the date seed pills. Numerous studies showing that catechin can prevent ketoprofen-stimulated oxidative injury to the gastric mucosa by modulating Nrf2 in vitro and in vivo might be used as evidence to support this finding.In the intracellular environment, Nrf2 activation can improve the level of antioxidation and the maintenance of redox equilibrium. Western blotting was utilized to analyze the effects of date seed pills on nuclear Nrf2 levels and the findings are displayed in d Câmara , high diκB and Nrf2 pathways /Nrf2-ARE pathway. The inhibitory protein Keap1 is typically linked to Nrf2 in the cytosol under normal physiological circumstances. Pill incubation, on the other hand, stimulates the split of Nrf2 and Keap1, allowing Nrf2 to enter the nucleus and collaborate with the tiny Maf centrosome protein with ARE in developers, and ultimately influence the action of antioxidant enzymes and ROS buildup [−1 pills without experiencing any harmful effects. It was discovered that the pills exhibit bioactivity related to the activation and inhibition of NF-κB and Nrf2 when incubated at the chosen doses. This bioactivity was strongly associated with the antioxidant and pro-oxidant actions of the pill models. According to de Camargo et al. [κB stimulation tests, however our data do not support this theory. The bioactivity of a given pill is highly dependent on its dose, which was found to alter the oxidation–deoxidation balance of the cells and cause a range of cytokines and nucleic factors to react to stress in diverse ways.Based on the aforementioned results, we hypothesized that the pills may potentially trigger cellular immunological responses by means of the NF-pathways . By acti buildup . Additioo et al. , it has In the meantime, the bioactive phenolic substances might penetrate the cells and immediately alter or initiate the nucleic factors, followed by the production of enzymes and cytokines. The question then becomes: is consuming a lot more polyphenols better or worse than consuming a lot less? The answer could be directly related to the unique redox environments present in the intracellular and extracellular environments. Therefore, based on our findings, we can assume that the pills may have advantages regarding the regulation of the redox status and inflammatory response that are related to an organism’s immune response.−1. Additionally, incubation with 50 µg mL−1 pills improved NF-κB and Nrf2 nuclear translocation, encouraged the creation of the cytokines TNF-α, IFN-γ, and IL-1β, boosted intracellular SOD activity, and reduced ROS buildup. In contrast, NF-κB and Nrf2 nuclear translocation was inhibited by incubation with 1000 µg mL−1 pills. Overall, it is possible to surmise that the pills controlled the NF-κB and Nrf2 signaling pathways linked to the cellular stress response and redox homeostasis to mediate immunological activation. The findings showed that date seed pills had varying effects on the immunological responses in RAW264.7 macrophages. We are currently studying the adipocyte differentiation of our pills in 3T3-L1 cells. Moreover, we are planning to optimize the encapsulation of the date pills by reducing the carrier ratios and increasing the active ingredients. In addition, the binding between the carriers and active ingredients inside the pills ought to be elucidated, and a full characterization of the pills should also be considered.According to the current investigation, encapsulation significantly enhanced the stability of the polyphenols present in date seed pills and had a variety of impacts on RAW264.7 macrophages. However, Nrf2 nuclear translocation was impacted by pills at 50 and 100 µg mL