Ketamine, a racemic mixture of esketamine (S-ketamine) and arketamine (R-ketamine), has received particular attention for its rapid antidepressant and antisuicidal effects. NMDA receptor inhibition has been indicated as one of the main mechanisms of action of the racemic mixture, but other pharmacological targets have also been proposed. This study aimed to explore the possible multiple targets of ketamine enantiomers related to their antidepressant and antisuicidal effects. To this end, targets were predicted using Swiss Target Prediction software for each ketamine enantiomer. Targets related to depression and suicide were collected by the Gene Cards database. The intersections of targets were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Network pharmacology analysis was performed using Gene Mania and Cytoscape software. Molecular docking was used to predict the main targets of the network. The results indicated that esketamine and arketamine share some biological targets, particularly NMDA receptor and phosphodiesterases 3A, 7A, and 5A but have specific molecular targets. While esketamine is predicted to interact with the GABAergic system, arketamine may interact with macrophage migration inhibitory factor (MIF). Both ketamine enantiomers activate neuroplasticity-related signaling pathways and show addiction potential. Our results identified novel, poorly explored molecular targets that may be related to the beneficial effects of esketamine and arketamine against depression and suicide.  Major depression disorder (MDD) is the leading cause of disability and the largest contributor to suicide deaths in the world . AccordiKetamine is a racemic mixture of S-ketamine (esketamine) and R-ketamine (arketamine) that has been used due to its anesthetic and analgesic properties since the 1970s. Recently, esketamine was approved by the FDA for the treatment of refractory depression and suicidal ideation because of its rapid, potent, and sustained effect, which differs substantially from the antidepressant profile of conventional antidepressants. The pharmacological properties of ketamine are mainly attributed to the inhibition of N-methyl-D-aspartate receptors (NMDAr). However, ketamine has multiple pharmacological targets, such as gamma aminobutyric acid (GABA), dopamine, serotonin, opioids, and cholinergic receptors . A singlOne possibility to study the multitarget potential of ketamine is through bioinformatics tools, which allow screening of depression and suicide molecular targets rapidly and in an early approach that can guide future higher cost experimental studies. Bioinformatics is an area with great potential applications for the study of diseases and drug design and discovery. Pharmacological target network analysis, molecular docking, and gene ontology (GO) enrichment analyses may allow the understanding of the polypharmacological action of molecules, investigating their potential targets, and allowing a more rational use of animal models in scientific research ,14,15,16In this study, we used some of these bioinformatics tools to investigate the potential molecular targets of ketamine enantiomers in depression and suicide. Ketamine has been reported to exhibit polypharmacological action, and there may be differences between esketamine and arketamine in terms of their potency and biological effects, which may be due to their ability to interact with different molecular targets ,21. TherAccording to the SwissTargetPrediction database, 105 potential targets of arketamine and 105 potential targets of esketamine were found. Importantly, although most of the target genes were shared by the enantiomers (91 genes), there were differential genes that were unique targets for arketamine (14 genes), and others unique to esketamine (14 genes).As a result of the search for depression-related genes and suicide-related genes in the GeneCards database, we obtained a total of 13,914 genes and 2409 genes, respectively. Of note, suicide and depression shared 1983 genes, according to the prediction. PDE4B, KDM4C, MIF, NOS2, CREBBP, HMOX1), and 6 genes intersected by depression, suicide, and esketamine . According to the Venn diagram, no overlapping genes were observed between arketamine and suicide or between esketamine and suicide. However, the CA12 gene that codes for carbonic anhydrase 12 was shared by esketamine, arketamine, and suicide. Considering that ketamine has been used for treating severe depression with suicide ideation [The Venn diagram illustrated in ideation , we focuHomo sapiens was performed in GeneMANIA. The obtained network was imported and analyzed in Cytoscape V. 3.9.1. software . As a result, a network with 73 nodes, 891 connections, and a clustering coefficient equal to 0.349 was obtained. The colors of the edges (connections) reflected the type of interaction analyzed. The network had 891 connections, of which 34.85% were physical interactions (green), 28.20% were co-expressed (yellow), 25.94% represented shared protein domains (pink), 9.09% were co-localized (blue), 7.20% were predictions (gray), 2.84% were genetic interactions (purple), and 1.88% were metabolic pathways (black). The network construction of esketamine, arketamine, suicide, and depression overlapping genes is shown in KCNN4, NQO1, AOC2, VEGFD, VEGFC, CA3, AOC1, CA12, GABRA3, PDE5A, MTNR1B, GABRB2, CA13, GRIN2C, CA8, GABRA6, GABRA4, GRIN3A, PDE4B, and PDE3B. Subsequently, the network was filtered considering degree > 30 and betweenness centrality between 0.017 and 0.044. From these data, 11 genes were found to have the highest number of connections (Degree > 30) and the shortest paths  in the network. The genes found were GRIN2B, GRIN2A, GRM4, EGFR, KDR, GABRB3, GABRB2, GABRA3, KCNN1, PDE5A, and MTNR1B. The proteins encoding for these genes were as follows: NMDA receptor (GRIN2B and GRIN2A), metabotropic glutamate receptor (GRM4), GABA receptor subunits β2, β3 and α3 subunits , epithelial growth factor receptor (EGFR), melatonin receptor 1β (MTNR1B), kinase insertion domain receptor (KDR), member 1 of the N subfamily of calcium-potassium activated channel (KCNN1), and the enzyme phosphodiesterase 5A (PDE5). The filtered network of the targets obtained is illustrated in The search for the ketamine enantiomers, suicide, and depression overlapping genes in SIRT1, PIK3CA, I-CAM1, GABRA1, GABRB2, and PRF1. As a result, we obtained a network with 26 nodes, 242 connections, and a clustering coefficient equal to 0.714. GABRA1, GABRB1, GABRA4, GABRG2, GABRB2), but genes encoding sirtuin 1 (SIRT1), integrin subunit alpha L (ITGAL), phosphatase (FOSPI3K) and kinase (PI3K) subunits, thrombopoietin receptor (MPL), transcription factor (BCL11A), and perforin 1 (PRF1) were also identified.In the second step of the network analysis, we obtained potential targets shared by esketamine, suicide, and depression. Therefore, the green diagram was built by searching for the genes CREBBP, NOS2, HMOX1, KDM4C, PDE4B, and MIF. As a result, we obtained a network with 26 nodes, 189 connections, and a clustering coefficient equal to 0.293. When we performed the pharmacological network analysis of only esketamine, the result was a cluster of GABAA receptors , highlighting the genes coding for some specific subunits, such as GABRA1, GABRA4, GABRB1, GABR2, and GABRG2. Most interactions were physical interactions (80.30%), followed by predicted interactions (9.48%), genetic interactions (4.28%), co-expression (3.72%), metabolic pathways (1.12%), co-localization (0.74%), and shared protein domains (0.37%). In the network construction, we retrieved 20 genes. After applying degree (DC > 16) and betweenness centrality (BC) filters between 0.039 and 0.157, we obtained the 10 most important genes of the arketamine, depression, and suicide network, as illustrated in CREB1, CREBBP, and COPS5), enzyme-related genes , genes related to immune responses (MIF and CD74), and cytoskeleton (ACTN4). The third network, represented in ROCK1, AKR1B1, JAK2, DHODH, TYK2, PDE4C, TGFBR1, TNKS2, GABRA4, CCND1, PDE4D, OPRD1, RPS6KA1, F13A1, MTNR1B, PDE5A, IGF1R, BRD4, PDE3B, DYRK1A, JAK1, ADORA1, SYK, CSNK1G1, ADORA2B, TNKS, AURKB, EPHB3, CA13, GRK6, and ADORA3, which are the 31 target genes shared by esketamine, arketamine, and depression. As a result, a network with 51 nodes, 698 connections, and a clustering coefficient equal to 0.584 was obtained. This high degree of clustering was composed of several types of phosphodiesterases. Most interactions in this network were shared protein domains (42.12%), followed by co-expression (26.24%), metabolic pathways (17.70%), physical interactions (6.43%), co-localization (5.77%), genetic interactions (0.98%), and predicted interactions (0.76%). In the network construction, we retrieved 20 genes. After applying degree (DC > 11) and betweenness centrality (BC) filters between 0.038 and 0.079, we obtained the 8 most important genes, as illustrated in The purple network represented in We used GO and KEGG for a global evaluation of ketamine enantiomers, suicide, and depression overlapping genes. According to biological process (BP) analysis of GO, the top five terms were chemical synaptic transmission, anterograde trans-synaptic signaling, trans-synaptic signaling, synaptic signaling, and regulation of membrane potential A. FurtheWe used GO and KEGG enrichment analysis to investigate the functions and biochemical pathways of esketamine, suicide, and depression overlapping genes. According to BP analysis of GO, the top five terms were GABA signaling pathway, chloride transmembrane transport, inorganic anion transmembrane transport, GABAergic synaptic transmission, and regulation of postsynaptic membrane potential A. MoreovTo investigate the biological functions and biochemical pathways involved in the action of arketamine in depression and suicide, GO and KEGG analyses were performed. According to BP, the top five terms were positive regulation of cytokine production, cytokine production, regulation of cytokine production, positive regulation of gene expression, and positive regulation of multicellular organism processes A. AdditiWe used GO and KEGG for a global evaluation of overlapping ketamine enantiomers and depression genes. The top five BP terms were cyclic nucleotide catabolic process, ribonucleotide catabolic process, purine-containing compound catabolic process, purine ribonucleotide catabolic process, and cyclic nucleotide metabolic process A. MoreovGRIN2B and GRIN2A genes, respectively), which were consolidated pharmacological targets for both ketamine enantiomers, being considered our positive control in this docking study. Therefore, the main pharmacological targets of esketamine and the respective affinity values were GluN2B , PDE7A , PDE5A , JAK1 , PDE3A , GluN2A , and GABRG2 . The main pharmacological targets of arketamine and the respective affinity values were as follows: PDE3A , MIF , GluN2B , PDE7A , GABRB2  PDE5A , JAK1 , and GluN2A . To perform docking, the targets of each filtered network were selected by the parameters DC and BC, plus the top five highest values of closeness centrality (CC). After selecting the top 5 biological targets from each network, molecular docking was performed according to the structures and parameters described in In the computational prediction, MIF (1GCZ structure) had one of the highest docking scores for arketamine  and interacted in the center of the protein, as illustrated in According to docking scores, arketamine was predicted to have a higher affinity for PDE3A and PDE5A  than esketamine , while the affinity of esketamine and arketamine was similar for PDE7A . In molecular docking simulations, we found hydrogen bond interactions between PDE3A Leu910 and esketamine B, PDE7A GABRB2, GABRB3, and GABRA3 genes were potential targets shared by both enantiomers of ketamine. In the molecular docking analyses, the proteins obtained from PDB were 6D6T (GABAAα1β2γ2 receptor), 6DW1 (GABAAα1β1γ2 receptor), and 7QNA (GABAAα4β3γ2 receptor). We also found that the Aα1β2γ2 receptor, showing the neurosteroid (left) and benzodiazepine (right) binding sites. In molecular docking simulations, we found two hydrogen bond interactions between GABAA receptor Arg141 and esketamine in the neurosteroid binding site , the benzodiazepine site , and the neurosteroid site  were potential targets for interaction with ketamine. ing site I, and a ing site J.The main purpose of this study was to explore the potential molecular targets of ketamine enantiomers in suicide and MDD by employing network pharmacology and molecular docking simulations. Our workflow involved a sequential analysis composed of the following steps: (a) identification of molecular targets of ketamine enantiomers in SwissTargetPrediction; (b) collection of genes targets of MDD and suicide in GeneCards; (c) creation of a Venn diagram to obtain the overlapping genes; (d) construction of the network of overlapped genes; (e) implementation of network filter parameters to obtain the highest closeness, betweenness, and degree centrality; and (f) running molecular docking simulations. Our bioinformatics analysis uncovered that both enantiomers of ketamine, esketamine, and arketamine share biological targets that may be implicated in their antidepressant and antisuicide properties. However, due to the large number of pharmacological targets found in the network analyses, we limited the molecular docking analyses to the nodes that had the best topological parameters. The main targets obtained are NMDA receptors (GluN2 subunits), GABA receptors , macrophage migration inhibitory factor (MIF), and various subtypes of phosphodiesterases . However, it is important to consider that other targets not analyzed may be relevant to the antidepressant and antisuicidal effects of ketamine enantiomers.GRIN1), glutamate-binding GluN2 , and glycine-binding GluN3 subunit (GRIN3A and GRIN3B). Regarding GluN2, four different subunits (GluN2A-D) are present throughout the central nervous system [GO and KEGG functional analyses pointed out relevant targets for both ketamine enantiomers and were useful in directing subsequent studies. In GO enrichment analysis related to molecular function, NMDA receptor activity was pointed out as one of the main targets. In agreement, ketamine is a well-known, noncompetitive open-channel NMDAr antagonist . The GRIs system . Of notes system .We found that GluN2 subunits of NMDAr are potential targets for the action of both ketamine enantiomers, particularly GluN2B, using docking analyses. Interestingly, a study by Zhang and co-workers  reportedAlthough NMDAr is the key target of esketamine and arketamine, other targets may be implicated in their antidepressant properties. Indeed, it has been discussed whether the inhibition of NMDAR is critical for the mood-enhancing effects of ketamine enantiomers, and a hypothesis that has been raised is that their antidepressant and antisuicidal effects may be dependent on their interaction with multiple molecular targets . RegardiMIF) as a central node. Subsequently, our study identified MIF, to our knowledge, for the first time, as a potential molecular target of arketamine by molecular docking. MIF is a cytokine originally characterized by its ability to prevent migration of macrophages, which is now recognized as a key mediator of the innate immune system and adaptive immunity [MIF gene polymorphism may be related to attempted suicide in patients with bipolar disorder, other studies failed to indicate a relationship between MIF levels or MIF gene polymorphism and suicide [MIF-794CATT5–8 microsatellite or MIF-173G/C single-nucleotide polymorphism (SNP) on the MIF gene promoter. In addition, no significant alterations in MIF levels in the dorsolateral prefrontal cortex were observed in suicide completers as compared to control individuals [Network construction of overlapping arketamine, suicide, and depression genes identified macrophage migration inhibitory factor  may be molecular targets of ketamine enantiomers. PDEs are classified into 11 subtypes based on sequence homology and structural and enzymatic features mainly related to sensitivity to modulators . These ePDE3 occurs as two groups of isoenzymes (PDE3A and 3B) that are involved in the hydrolysis of cGMP, mainly cAMP ,43. AlthPDE7 comprises 2 subclass members: PDE7A and B. This enzyme, abundant in the brain, is responsible for the hydrolysis of cAMP. The highest levels of PDE7A have been reported in the olfactory bulb, olfactory tubercle, hippocampus, cerebellum, medial habenula nucleus, pineal gland, area postrema, and choroid plexus . NotablyPDE5A gene), namely PDE5A1, PDE5A2, and PDE5A3 [PDE5A single nucleotide polymorphism (SNP) may be associated with MDD [PDE5 hydrolyzes cGMP, which is a critical component of the cGMP-protein kinase G (PKG) intracellular signaling in neurons. There are three isoforms of PDE5A, encoded by a unique gene  family. These receptors have R1 subunits where orthostatic ligands bind (encoded by the GABBR1 gene) and R2 coupled to G protein (GABBR2) [Ar are pentamer chloride channels and have 19 genes that encode for subunits of the GABAA receptor, which is organized into classes  and isoforms  [GABRA1 gene encodes the GABA Type A Receptor Subunit Alpha1(GABAAα1) protein, the GABRB3 gene has GABA Type A Receptor Subunit Beta3 (GABAAβ3) as its product, and the GABRG2 gene encodes the GABA Type A Receptor Subunit Gamma2 (GABAAG2) protein. The assembly of GABAAr as heteropentamers produces complex heterogeneity in their structure, and different subunit compositions are possible. A typical GABAA receptors are made up of two alpha subunits, two beta subunits, and one gamma subunit, and the interaction sites lie at the connections between these subunits. The GABA interaction site is located between the interfaces of the alpha and beta subunits, the neurosteroid site is located in the transmembrane domain between the alpha and beta subunits, and the benzodiazepine site is located at the interface between the alpha and gamma subunits [GABRB2, GABRB3, and GABRA3 genes are potential targets shared by both enantiomers of ketamine, while GABAAG2 coded by GABRG2 is predicted to mainly interact with esketamine. Network pharmacology, GO, KEGG analysis, and molecular docking pointed out that GABA genes are also potential targets for both enantiomers but mainly for esketamine. The GABA receptor (GABAr) family is divided into metabotropic and ionotropic receptors. GABA(GABBR2) . GABAAr nd ρ1–3) . Thus, tsubunits . In our Ar) reported that this compound recovered hedonic behavior in mice and that this behavior was prevented using the benzodiazepine antagonist flumazenil, suggesting the involvement of the benzodiazepine site of GABAAr in anhedonia symptoms [Ar may be involved in the mechanism of action of ketamine in improving anhedonic symptoms in MDD.The benzodiazepine site of GABAr is closely implicated in anhedonic behavior. Anhedonia is one of the symptoms of major depressive disorders that is closely related to suicide risk . Of notesymptoms . A clinisymptoms . These dAr, have potential use as antidepressants and anesthetics [A receptor to rapid antidepressant responses, brexanolone and zuranolone, positive allosteric modulators of the neurosteroid site of this receptor elicit rapid and sustained antidepressant effects in postpartum depression and MDD [Neurosteroid analogs, molecules that bind to the allosteric site of neurosteroids on GABAsthetics . Reinfor and MDD ,59,60. Ketamine is known for its dissociative properties and abuse potential , used evOne of the limitations of our study is that we cannot predict the effect of gender on the findings for each ketamine enantiomer. The availability of software able to predict sex differences between esketamine and arketamine enantiomers in depression and suicide would be welcome to provide information on this issue. However, it has been reported in preclinical studies that females are more sensitive to the antidepressant effect of ketamine ,65,66, bAlthough our study suggests novel and poorly explored proteins as possible targets for the antidepressant and antisuicidal effects of ketamine enantiomers, future experimental studies are needed to confirm the role of these targets in the effects of ketamine enantiomers. The list of biological targets of arketamine and esketamine was obtained from SwissTargetPrediction   by searcHomo sapiens. The network was imported and analyzed in Cytoscape V. 3.9.1 [The list of genes obtained from the crossover was searched in GeneMANIA  using default settings , and a nV. 3.9.1 . TopologV. 3.9.1 . It is cV. 3.9.1 ,16,77. TGO and KEGG enrichment analyses were performed in ShinyGO v.0.76.3   for eachThe molecular docking analysis was carried out to predict the binding affinity between arketamine or esketamine and the potential biological targets using the DockThor 2.0 software   in a simIn summary, our study uncovered that ketamine had a polypharmacological profile, with many biological targets involved in suicide and MDD. The findings indicated GRIN2A, GRIN2B, PDE7A, PDE5A, and PDE3A as the most likely targets predicted for both ketamine enantiomers. Regarding S-ketamine, it was predicted that the main targets were GABRB2 and GABRG2. In the case of arketamine, it was predicted that MIF was the main target. These new targets pointed out in our study have not yet been explored with regard to their involvement in the antidepressant and antisuicidal effects of ketamine and therefore deserve attention in future experimental studies.


A novel electrocatalytic sensing strategy was built foruric acid(UA) determination with an exceptionally developed poly(tartrazine)-modifiedactivated pencil graphite electrode (pTRT/aPGE) in human serum andartificial urine. The oxidation signal of UA at 275 mV in pH 7.5 phosphatebuffer solution served as the analytical response. Cyclic voltammetry,electrochemical impedance spectroscopy, scanning electron microscopy,energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopywere used to characterize the sensing platform, which was able todetect 0.10 μM of UA in the ranges of 0.34–60 and 70–140μM. The samples of human serum and artificial urine were analyzedby both the pTRT/aPGE and the uricase-modified screen-printed electrode.The results were statistically evaluated and compared with each otherwithin the confidence level of 95%, and no significant differencebetween the results was found. It also binds iron, preventing iron-dependent ascorbate oxidation.Due to these properties, UA hinders the destruction of human tissuesand cells.4 High level of UA may lead toLesch-Nyhan syndrome, hyperuricemia, obesity, gout, diabetes, kidneyfailure, and cardiovascular disease, while low level of UA could belinked to Alzheimer, polyarthritis, Parkinson, acromegaly, multiplesclerosis, and yellow fever atrophy.5 In addition, a low levelof UA might be related to a deficiency of molybdenum and toxicityof copper, and an anomalous low UA level may indicate Wilson’sdisease and Fanconi’s disease.6 Therefore,monitoring the level of UA in human urine or blood is of great importanceand pivotal indicator in various fields involving clinical diagnostics,health assessment and monitoring, and biological analysis.Uric acid  is the end product of purinemetabolism in humans, formed by the oxidation of xanthine and hypoxanthine,but is more toxic than these7 chemiluminescence,9 UV–visible spectrophotometry,11 fluorescence spectroscopy,13 chromatography,16 and electroanalytical methods.27 The method based on capillary electrophoresis has a high limit ofdetection  and a long analysis time .7 In chemiluminescence-based methods,9 there are disadvantages including complex procedures, time-consumingsteps for system preparation, and analysis time, and the usage ofuricase enzyme8 and porcine liver.9 In UV–visible spectrophotometric methods,both of them have complex procedures and LODs at the high magnitudesof nM 10 and μM ,11 one of them requiresa column for preconcentration and purification at sample preparationstep,10 and the other needs a long time, high temperature , and enzyme  for probe synthesis.11 The methodsbased on fluorescence spectroscopy have long probe preparation  and analysis time  andrequire some enzymessuch as uricase and horseradish peroxidase.13 When the methods based on the chromatography technique are examined,using expensive instruments and large amounts of chemicals28 are among the common disadvantages.On the other hand, there are also method-specific disadvantages suchas high LOD value , presence of high-temperature process ,14 and long analysis time .16 However, owing to several noticeable features of electrochemicalmethods involving ease of fabrication and miniaturization, low cost,portability, quick response, notable sensitivity and selectivity,they have been identified to be more potent than other techniquesfor determining UA.Numerousmethods of determining UA have been reported, includingones based on capillary electrophoresis,1 β-cyclodextrin/reduced graphene oxide/screenprinted electrode,2 (platinum nanoparticles)-grapheneflakes-flavin mononucleotide/gold interdigitated microelectrode,3 multiwalled carbon nanotubes/poly/glassy carbon electrode,4 acrylic acid-ethylene glycol dimethacrylate-2,2′-azobis(2-isobutyro)nitrile/carbon paste electrode,5 nickelferrite/glassy carbon electrode,6 platinumnanosheets/fullerene/glassy carbon electrode,21 nickel ferrite nanorods/sulfur-doped carbon nanoparticles/glassycarbon electrode,22 poly(solid red a)/carbonnanotube paste electrode,23 N,P-doped hollowmesoporous carbon nanospheres-phytic acid/glassy carbon electrode,24 SnO2/graphene/glassy carbon electrode,25 three-dimensional porous graphene/glassy carbonelectrode,26 poly)/glassycarbon electrode,27 nickel hydroxide/solargraphene/glassy carbon electrode,17 platinumnanoparticles/multiwalled carbon nanotubes/glassy carbon electrode,18 copper nanoparticles/polypyrrole/glassy carbonelectrode,19 poly(p-aminophenol)/glassycarbon electrode,20 gold clusters/N-acetyl-l-cysteine–multiwalled carbon nanotubes/glassycarbon electrode,29 gold-silver bimetallicnanoparticles/graphene oxide/thionine/glassy carbon electrode,30 urate oxidase/metal organic frameworks (goldnanocages)/glassy carbon electrode,31 andurate oxidase-cobalt-based metal–organic framework/boron nanosheets-doxorubicin/glassycarbon electrode32 have been reported fordetermining UA and are summarized in Table S1.A wide variety of chemically modified electrodesincluding methylcellulose/grapheneoxide/iron oxide nano hydrogel/glassy carbon electrode,34 Among them, polymerfilm-modified electrodes provide advantages such as good adhesionto the surface, improved peak current, large effective surface area,worthy ionic and electronic conductivity, catalytic effect, and goodselectivity and sensitivity.100 Due to these properties, polymer film electrodesare widely used in the development and application of electrochemicalsensors and for the determination of many biologically active species.38Chemically modified electrodes are known to improve electron-transferkinetics over bare electrodes.Our research motivationis to create an electrochemical sensingplatform that addresses the limitations found in the literature, suchas the reliance on costly supporting materials like glassy carbonelectrodes, the time-consuming and multi-step sensor preparation processes,and the limited availability of disposable platforms. To achieve thisgoal, a cost-effective and disposable pencil lead was employed, enablingthe development of a polymer film-modified electrode in a single stepfor the determination of UA. In parallel with addressing these issues,our aim is also to provide sensitivity, accuracy, and selectivitythat are on par with the methods described in the literature.To the best of our knowledge, this is the first example of electrochemicalpolymerization of tartrazine onto the pencil graphite electrode (PGE).The poly(tartrazine) film was characterized by cyclic voltammetry(CV), electrochemical impedance spectroscopy (EIS), scanning electronmicroscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), andX-ray photoelectron spectroscopy (XPS) techniques, and the producedplatform was successfully used for determining UA in human serum andartificial urine samples.22.1d-glucose , dopamine hydrochloride, glycine , citricacid , sodium chloride , sodium nitrite ,sodium perchlorate ,calcium chloride , l-cysteine , iron(III) chloride (Sigma-Aldrich 8.03945), aluminumchloride , magnesium chloride , l-ascorbic acid , sodium sulfate ,and sodium nitrate were used as analytical reagent grade. All solutions were preparedin ultrapure water and stored in high-density polyethylene falcontubes, and the solutions of tartrazine and UA were prepared daily.Tartrazine, UA , acetic acid, sodium hydroxide , potassium dihydrogen phosphate , potassium chloride , sucrose , 2, immersiondepth: 1 cm), an Ag/AgCl electrode , and a platinum wire  as theworking, reference, and auxiliary electrodes, respectively. All graphitegrades, H, HB, B, and 2B, were purchased from a stationary in Kocaeli.Voltammetric measurements were carried out with an EIS module combinedDropSens potentiostat/galvanostat using a standard three-electrodesystem consisting of a poly(tartrazine)-modified activated pencilgraphite electrode  that serve the purpose of automaticallycalibrating the XPS-binding energy (BE) scale. The calibration processinvolved using reference lines from Au 4f7/2 (84.1 eV),Cu 2p3/2 (932.2 eV), and Ag 3d5/2 (368.2 eV)to ensure accurate measurement of BEs.An uricase-modified screen-printed electrode (DropSens Ref. UA10)was used for comparison measurements in human serum and artificialurine samples. A Mettler Toledo Seven Compact pH meter with InLabRoutine Pro-ISM probe was used to prepare buffer solutions. Ultrapurewater was obtained by the Merck Millipore Milli-Q Direct 8 system.SEM analyses were performed with a Hitachi Schottky SU5000 field emission-scanningelectron microscope with 15 kV of voltage, 30 of spot intensity (relativeamount), and a SE(L) detector. EDX analyses were carried out witha FEI Oxford Instruments model 7260 EDX with 15 kV of voltage and102.239 in 0.1 M of pH 7 phosphatebuffer solution (PBS) and 0.1 M KCl via cycling five times between−0.6 and 2.0 V with 50 mV/s of scan rate, and the final volumeof the solution was set to 10 mL in both cases. The increase and decreaseof the peak current values in the respective regions proportionalto the number of cycles indicate that the electro-polymerization hasoccurred, as shown in The processof preparing pTRT/aPGE, electro-polymerization voltammograms of thepolymer film-modified electrode, and possible electrochemical detectionmechanism of UA are illustrated in 2.33[Fe(CN)6], 1 mM of K4[Fe(CN)6], and 0.1 M ofKCl) with a step amplitude of 3 mV and a proper scan rate. The solutionconsisting of 0.05 M pH 7.5 PBS with or without real samples  was set to 10 mL with ultrapurewater and deaerated with argon for 3 min.The potential was scanned from −0.4 to 1.0 V using the differentialpulse voltammetry (DPV) mode with 5 mV of step amplitude, 25 mV ofpulse amplitude, and a scan rate of 25 mV/s. CV measurements wereapplied in the range of −1 to 1 V and human serum  was used to determine either the addedor the content of UA with the proposed and the amperometric method.Humanserum samples were obtained from healthy volunteer individuals andthose were tested for anti-HCV, anti-HIV-1/2, HBsAg, HBV DNA, HCVRNA, HIV RNA, and syphilis and were found to be negative. An artificialurine sample was prepared in accordance with the literature.In addition, recovery experiments were performed for human serum(50-fold diluted sample) and artificial human urine (10-fold dilutedsample) samples spiked with 10, 25, 50, 80, 100, and 130 μMof UA.33.13[Fe(CN)6], 1 mM of K4[Fe(CN)6], and 0.1 M ofKCl  foraPGE, [Rs(CPEfRf)(CPEdlRct)W] for pTRT/aPGE electro-polymerizedin pH 5 acetic acid/acetate buffer solution, and [Rs(CPEh[Rh(CPEfRf)(CPEdlRct)])] for pTRT/aPGE electro-polymerized in pH 7 PBS andNaOH solution (Table S2). Rs is the resistance from solution, electrode, wires, andconnectors, Rct is the charge-transferresistance, Cdl and CPEdl arethe capacitance of the double-layer, W is Warburgimpedance; Rh and CPEh arethe resistance and capacitance of the incomplete capacitive semi-circleat the high-frequency region, and Rf andCPEf are the resistance and capacitance of the polymerfilm.41 A well-known circuit, Rs(Cdl[RctW]), consists of a semi-circle correspondingto Rs(CdlRct) at a high-frequency region and a linearregion related to Warburg impedance (W) at a low-frequencyregion for aPGE.28 Another semi-circleemerged at a high-frequency region for pTRT/aPGE electro-polymerizedin pH 5 acetic acid/acetate buffer solution shows a uniform polymerfilm layer where current must first flow through this layer and thenthe inner part of the electrode .42 Therefore, circuit (CPEfRf), representing the pTRT film, is added to the Rs(Cdl[RctW]) circuit for aPGE. After the electro-polymerizationof tartrazine in the presence of pH 7 PBS or NaOH solution, the incompletecapacitive semi-circles at a high-frequency region arise due to theirregularity of the current flow at the formed polymer film layer.41 Therefore, in addition to the (CPEfRf) circuit representing the polymerfilm, the (CPEhRh) circuitis added owing to the current flow abnormality. In these circuits,the reason for removing the W is that the tangentangle of the linear part differs from 45° due to the currentflow irregularity. Therefore, when the W is addedto the circuit, a fitted-curve compatible with the experimental datacannot be obtained. These abnormalities are due to the disruptionof coordination between the azo group and the adjacent hydroxyl groupin the tartrazine molecule at neutral and basic pH values. At acidicpH values between 4.2 and 6.5, polymerization takes place uniformlysince the azo group and the adjacent hydroxyl group are in coordination.44 All EIS data and findings were found to be compatible with the literature.42EIS data were fit with differentcircuits of Rs values. Besides, due to the non-uniformity of the polymer,the resistance of the polymer film (Rf) is higher in the coating carried out in NaOH than that in the presenceof pH 7 PBS. This is due to the presence of tartrazine in the anionicform in the basic solution and the partial anionic character in theresulting polymer. Therefore, the Rf valueis greater due to the repulsive force between this anionic polymerand the redox pair [Fe(CN)63–/Fe(CN)64–].44The conductivity valuesof each polymer film-modified electrodegreatly increase compared to aPGE from the obtained W values show that pTRT/aPGE is moreefficient in diffusion-induced substance transport than aPGE, as listedin Table S2.In addition, Figure S1. Similar to our previous study,45 thebare PGE does not show a uniform distribution, while it becomes more uniformand has a channeled structure when activated . As expected, these channels fill with polymer when the TRT is coated onto the aPGE .SEM images for barePGE, aPGE, and pTRT/aPGE appear in Figure S2. It is observedthat bare PGE containscarbon (98.1%) and oxygen (1.9%),45 and afteractivation, the oxygen ratio (15.7%) increases considerably.45 With the electro-polymerization of TRT, nitrogen(4.0%) due to the azo group in the TRT structure, a small amount ofsulfur (0.2%) due to the sulfo groups of TRT, and a small amount ofsodium (0.1%) possibly diffused into the polymer structure due tothe salt form of TRT are observed in the EDX spectrum.EDX spectra of the bare PGE, aPGE, andpTRT/aPGE appear in 45 An approximately two-foldincrease in the C 1s signal at pTRT/aPGE obviously signifies a carbon-basedformation on the surface .45 The BEs at 531.6 and 531.8 eV belong to the C=Ogroups, and the BEs of O 1S at 532.9 and 533.1 eV are due to the partialaromatic C–O–C structures of graphite46 and/or organic C–O–C structures of the obtainedpolymer,47 respectively .XPS measurementswere performed to further explain the electro-polymerizationthat occurred onto the aPGE, as shown in ectively 3AI,BI.II).45 The two most important pieces of evidence ofelectro-polymerization are the emergence of N 1S and S 2p chemicalenvironments in XPS measurements.48 N 1S spectrashow the presence of −N–C structures at 399.7 eV andimine (−C=N) structures at 402.5 eV (III).49 The BEs of S 2p1/2 and S 2p3/2 at 169.5 and 168.2 eV correspond to the SO3 structures(IV).50The formal oxidation state at 1071.6 eV may beattributed to thediffused sodium ions into the pTRT from the salt of TRT 3BII.45 T402.5 eV 3BIII.49 ructures3BIV.50As a consequence, greatly harmoniccharacterization data obtainedfrom CV, EIS, SEM, EDX, and XPS measurements demonstrate that thepTRT/aPGE is properly manufactured for determining the UA in syntheticand real samples.53Considering the characterization data andthe oxidation reactionof TRT, the possible electro-polymerization mechanism of TRT and thevoltammetric oxidation of UA are proposed in accordance with the literature,as depicted in 3.2Ip – v1/2)of the equationIp, n, D, C, A, and v refer to the peak current (ampere), thenumber of transferred electrons, the diffusion coefficient (cm2/s), and the concentration (mol/cm3) of the redoxcouple, the effective surface area (cm2) and the scan rate(V/s), respectively. Of these, n and D are 1 and 7.6 × 10–6 cm2/s forthe redox couple ([Fe(CN)6]3–/[Fe(CN)6]4–), respectively. The effective surfaceareas were found as 15.5 mm2 for aPGE and 33.4 mm2 for pTRT/aPGE, depicting a 115% increase.The Randles–Sevcik equation is used to calculatethe effective surface areas of aPGE and pTRT/aPGE from the slope (Ip – v) of the equationF, R, T, and Γ are the Faraday constant , the gas constant (8.314 J/mol·K), temperature (K),and the surface coverage (mol/cm2), respectively, and itwas found as 4.52 nmol/cm2.The Brown–Ansonequation is applied to reckon the surface coverage of pTRT layer fromthe slope . The logarithm of peak height ) was plotted against the logarithm of the scan rate ), and the related equation with a slope of 0.788,log(Ip) = 0.788 log(v) + 0.515 (R2: 0.994), depicted thatthe electrode reaction was on the basis of a joint diffusion- andadsorption-controlled system . The reason why adsorption accompanies diffusion is due to thefact that UA, an organic substance, tends to be close to the pTRT/aPGE,which has also an organic structure.54The electrode reactionmechanism belonging to the translocationof UA was examined by recording CV voltammograms at increasing scanrates varied from 10 to 1000 mV/s (RT/[(1 – α)nF]) of the Ep(mV) –log curve, Ep = 81.505 log(v) + 147.340 (R2: 0.984), as 0.64 .It is concluded that the transition state acts asymmetrically betweenthe responses of UA and UA-4,5-diol against the applied potential.56The apparent charge-transfer coefficient (α) was calculatedfrom the slope .51The slope of the 45Furthermore, CV measurementswere performed with bare PGE, aPGE,pTRT/PGE, and pTRT/aPGE in a solution containing 50 μM of UAto exhibit the electrocatalytic effect of the produced platform, asshown in 3.3Iph) against the examined parameter and determined to be2B, 0.5 mM, 0.1 M, 100 mV/s, 15, 7.5, 0.05 M, and 0, as appeared in Figure S6, respectively.Significant parameters for the electro-polymerization involvingpencil graphite grade, the concentration of TRT and pH 5 acetic acid-acetatebuffer solution, scan rate, and the number of CV cycles, and for theanalysis including pH and the concentration of PBS solution and ionicstrength were investigated with pTRT/aPGE using 30 μM of UAin the relevant ranges by plotting the peak height  and m is the slope of the calibrationcurve], 0.34–60, and 70–140 μM, respectively.The sensitivity of the polymer film-modified electrode was obtainedas 0.415 μA·μM–1·cm–2 depicting a remarkably sensitive platform for UA detection. Furthermore,the chronoamperomograms and calibration curve of amperometric measurementsappear in Figure S7.The DPV resultsand calibration curves of electrochemical UA oxidation are shown in The reproducibility and repeatability of the producedplatform,pTRT/aPGE, were investigated at 10, 30, 60, and 100 μM UA, andthe relative standard deviation (RSD %) values were found to be 2.7–5.1and 1.3–3.3%, respectively (the number of replicates: 6). Theresults show that the produced platform has remarkable reproducibilityand repeatability.The stability of the pTRT/aPGE was investigatedin UA-spiked samplesas well as 12 d following the first measurement. The peak heightsdecreased by 7.2 and 4.6% for solutions containing 10 and 90 μMof UA, respectively. Those results indicate the exceptional stabilityof pTRT/aPGE as well.Table S3. From the results obtained, it was foundthat even dopamine, which has the most effect on UA determination,changed the peak height by less than 5% at 40 μM. Accordingly,the results show that the produced sensor works selectively for determiningUA.Interference effects of dopamine, ascorbicacid, urea, glucose,sucrose, potassium chloride, sodium chloride, sodium sulfate, sodiumcarbonate, magnesium chloride, calcium chloride, and sodium nitratewere examined in the presence of 20 μM UA using pTRT/aPGE basedon a criterion of ±5% change in peak height, as detailed in 3.5Figures S8 and S9 and 58Human serum andartificial human urine samples were analyzed by both the developedmethod and the amperometric method. The voltammograms, chronoamperomograms,and the results for these samples are given in Table S4, the measured levels of UA exhibit strong concordancewith the spiked concentrations, demonstrating high detection recoveriesranging from 96.76 to 104.76%. The repeated electrochemical DPV measurementsshow low RSD values and a narrow distribution ranging from 1.44 to4.36%. These findings affirm the high reliability and suitabilityof using pTRT/aPGE for UA detection in both human serum and artificialhuman urine samples, with a substantial agreement between measuredand spiked UA concentrations, along with notable detection recoveryand low RSD.In addition to the aforementioned measurements, standardadditionof UA was applied to both human serum and artificial human urine samplesat various concentration levels. The accuracy of the method was furtherevaluated through recovery values, while precision was assessed usingRSD % values. According to the data presented in F-test and t-test. The fact that the experimental F  and t  values aresmaller than the critical F  and t  values indicates that the developed method gives an accurate responseat the 95% confidence interval (In order to interpret the precision and truenessof the proposedmethod, the results were evaluated statistically by comparing theamperometric and the suggested method by applying interval 1.4A unique, disposable, andcheap electrochemical sensor based ona polymer film of tartrazine prepared in a single step was designedand developed for determining UA in human serum and artificial humanurine samples. The synthesized polymer film was thoroughly characterizedby numerous techniques such as CV, EIS, SEM, EDX, and XPS. Collectively,the increase in the effective surface area of the produced electrode,the improvement of the electron conduction rate and therefore theconductivity, and the formation of a uniform polymer layer exhibitedan electrocatalytic oxidation of UA by raising the peak height ofabout 130% over bare PGE.–1·cm–2, 0.34–60, and70–140 μM, respectively. Furthermore, the analyticalperformance of the pTRT/aPGE was enhanced with sample applicationof human serum and artificial human urine. The results of the proposedsensor were compared to the results of the amperometric method, andthe significant statistical difference between the methods was notfound at 95% confidence interval implying the good accuracy of thedeveloped method.The produced platform, pTRT/aPGE,reached an outstanding LOD andsensitivity, and wide linear ranges as 0.10 μM, 0.415 μA·μM


Cell surface localized pattern recognition (PRR) and intracellular nucleotide-binding leucine-rich repeat (NLR) receptors detect pathogen-associated molecular patterns or pathogen-derived effector molecules, respectively, and induce a range of common immune responses. These include Cativation . Recent tivation . Based oectively . In Arabimmunity ,6. RNLs iosperms . The Araimmunity –9. Althoimmunity ,10, theyArabidopsis, ADR1 and its 2 paralogs ADR1-LIKE 1 and ADR1-LIKE 2 act redundantly downstream of multiple CNLs and TNLs [Arabidopsis NRG1.1 and NRG1.2 serve as redundant signaling components specifically required for TNL-induced immunity and contribute to basal resistance in the absence of ADR1s [Arabidopsis and Nicotiana benthamiana ADR1s and NRG1s contribute to some sensor NLR-triggered immune responses in an unequally redundant manner. In Arabidopsis, the ADR1 subfamily is predominantly involved in mediating resistance  and the NRG1s in triggering cell death [N. benthamiana NRG1 is sufficient to mediate resistance against tobacco mosaic virus (trigger of the TNL N) or Pseudomonas syringeae (trigger of the TNL Roq1) infections, since knocking out NbADR1 does not affect these resistance responses [In and TNLs ,9,11,12,and TNLs ,6) and band TNLs . Arabidoof ADR1s ,9. Interof ADR1s . It is responses –16. ThisWhy did plants evolve 2 RNL subfamilies that act more or less redundantly? RNLs represent downstream signaling hubs of at least 2 pathogen-sensing receptor networks. Consequently, genetic loss of RNLs or pathogen-mediated disturbance of their function would impair immune signaling by multiple immune receptors and thus, lead to enhanced disease susceptibility against various pathogens ,15. PlanThe genetic requirement of RNLs by TNLs is well documented. Until recently, it was not clear how exactly effector recognition by TNLs leads to RNL activation. Immune induction by most TNLs depends on the presence of genes encoding for the structurally unique plant-specific lipase-like proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and SENESCENCE-ASSOCIATED GENE 101 (SAG101) . EDS1 foFollowing effector recognition by TNLs, the TIR domain embedded enzymatic NADase and ADPR polymerase-like activity produces small signaling molecules that initiate a physical association of EDS1-PAD4 with ADR1 family members and EDS1-SAG101 with NRG1s ,21,23–26Arabidopsis ADR1s, EDS1, and PAD4 are not only required for immune signaling initiated by TNLs but also for some plasma membrane (PM)-localized PRRs [Arabidopsis [zed PRRs ,6. The Pzed PRRs ,6. RLP23zed PRRs . This hized PRRs ,11,19,28bidopsis . This mabidopsis .Arabidopsis RNLs is mediated by the interaction of positively charged residues in the CCR domain with phosphatidylinositol-4-phosphate at the inner leaflet of the PM and is required for RNL cell death function [R domain of RNLs is structurally similar to the N-terminal cell death inducing domains of the RNL-independent Arabidopsis CNL HOPZ-ACTIVATED RESISTANCE 1 (ZAR1), the wheat CNL Sr35 and the mammalian and plant MIXED-LINEAGE KINASE LIKE (MLKL) proteins [2+ permeable cation channels that are essential to trigger cell death and immunity, probably by disturbing ion homeostasis and/or activating other cell death/immunity inducing components [The PM localization of function ,31. Non-function ,12,27,32proteins ,32–36. Emponents ,38. Likemponents ,21,27,31Transcriptional analyses revealed that RNLs induce changes that are reminiscent of the transcriptional changes induced by CNLs during pathogen infection . Thus, iDespite their importance for plant innate immunity –7,11, thAnother interesting mechanistic question that remains to be solved is whether RNLs trigger cell death and disease resistance by directly disturbing ion homeostasis and if so, how exactly cation influxes induce cell death and transcriptional reprogramming. Upon infection, RNL function and channel formation may be regulated by effector- or PRR-mediated TNL activation, and by PRR kinase activity . This woMembrane pores formed in mammalian cells during necroptosis, pyroptosis, and apoptosis have been shown to mediate not only cation fluxes but also the release of peptide hormones and pro-inflammatory signals that trigger cell death and activate immunity . Thus, iArabidopsis and Nicotiana species. Therefore, it will be of great importance to analyze RNL contributions to immunity in other plants that do not belong to the Brassicaceae or Solanaceae families. In this regard, research on grasses, including important crop plants like rice, corn, or wheat, would be of high interest, since these monocotyledonous plants have lost TNLs and NRG1s [So far, RNL importance for immunity was only demonstrated for nd NRG1s ,41.


Renal cell carcinoma (RCC) accounts for approximately 2–3% of all adult malignancies. Clear cell renal cell carcinoma (ccRCC), which comprises 70–80% of all RCC cases, is the most common histological subtype.ccRCC transcriptome data and clinical information were downloaded from the TCGA database. We used the TCGA and GEPIA databases to analyze relative expression of BMP1 in various types of human cancer. GEPIA was used to perform survival analysis for BMP1 in various cancer types. Upstream binding miRNAs of BMP1 were obtained through several important target gene prediction tools. StarBase was used to predict candidate miRNAs that may bind to BMP1 and candidate lncRNAs that may bind to hsa-miR-532-3p. We analyzed the association between expression of BMP1 and immune cell infiltration levels in ccRCC using the TIMER website. The relationship between BMP1 expression levels and immune checkpoint expression levels was also investigated.BMP1 was upregulated in GBM, HNSC, KIRC, KIRP and STAD and downregulated in KICH and PRAD. Combined with OS and DFS, BMP1 can be used as a biomarker for poor prognosis among patients with KIRC. Through expression analysis, survival analysis and correlation analysis, LINC00685, SLC16A1-AS1, PVT1, VPS9D1-AS1, SNHG15 and the CCDC18-AS1/hsa-miR-532-3p/BMP1 axis were established as the most potential upstream ncRNA-related pathways of BMP1 in ccRCC. Furthermore, we found that BMP1 levels correlated significantly positively with tumor immune cell infiltration, biomarkers of immune cells, and immune checkpoint expression.Our results demonstrate that ncRNA-mediated high expression of BMP1 is associated with poor prognosis and tumor immune infiltration in ccRCC. Renal cell carcinoma (RCC) remains a major type of cancer, with a significant increase in incidence over the past years . In 2019Bone morphogenic proteins (BMPs) participate in cartilage and bone formation. BMP1 is a metalloendopeptidase belonging to the astacin superfamily and is a splice variant of mammalian tolloid protein, whereas BMP2 to BMP16 belong to the transforming growth factor-β (TGF-β) superfamily . BMP is To address these questions, we first investigated expression of BMP1 and its prognostic significance in a variety of human cancers. Then, the mechanism of noncoding RNA (ncRNA)-associated regulation of BMP1 in ccRCC was examined. Finally, we explored the relationship of BMP1 expression with biomarkers of immune cells, immune cell infiltration, and immune checkpoints in ccRCC. Overall, our results reveal that high expression of BMP1 in ccRCC is associated with poor prognosis and tumor immune infiltration through ncRNAs.U test with the R package ggplot2.The TCGA database, including clinical data, miRNA expression, mRNA expression, genome variation and methylation data of various human cancers, is a very useful cancer database . In thishttp://gepia.cancer-pku.cn/), a web tool including TCGA and Genotype-Tissue Expression (GTEx) data, was adopted to research BMP1 expression in various types of human cancer [GEPIA  is an important database for performing miRNA-related research [StarBase (research . StarBashttps://cistrome.shinyapps.io/timer/) is a very important website that is often used to analyze tumor-infiltrating immune cells [TIMER . Statistical significance was determined when the p-value or the log-rank p-value was less than 0.05.We first detected expression of BMP1 in 18 types of human cancer to illuminate the roles of BMP1 in carcinogenesis using the TCGA database. We found that BMP1 was highly expressed in 10 cancer types compared to normal samples, including BRCA, CHOL, GBM, HNSC, ESCA, KIRC, KIRP, LUSC, STAD and THCA, but was expressed at low levels in 2 cancer types, KICH and PRAD. However, no significant difference in BMP1 in BLCA, COAD, LIHC, LUAD, READ and UCEC was detected  and KIRC  was associated with poor prognosis  among all cancer types  and overall survival (OS), for BMP1 in GBM, HNSC, KIRC, KIRP, STAD, KICH and PRAD. For OS, high expression of BMP1 in GBM  hypothesis, lncRNAs can competitively bind shared miRNAs to increase mRNA expression. Hence, there should be a negative correlation between lncRNAs and miRNAs or a positive correlation between lncRNAs and mRNAs. In total, 6 lncRNAs were selected for further analysis , CD8+ T-cell biomarkers (CD8B), neutrophil biomarkers (ITGAM and CCR7), M1 macrophage biomarkers , M2 macrophage biomarkers  and dendritic cell biomarkers  in ccRCC.To further study the role of BMP1 in tumor immunity, we investigated relationships between expression of BMP1 and biomarkers of immune cells in ccRCC. As presented in Table CTLA-4 and PD1/PD-L1 are important immune checkpoints that are related to the effect of tumor immunotherapy. Considering the potential oncogenic role of BMP1 in ccRCC, we evaluated the association of BMP1 with CTLA-4, PD1 or PD-L1. Based on TIMER, expression of BMP1 correlated significantly positively with CTLA-4 and PD1 in ccRCC, which was adjusted by purity. However, expression of BMP1 correlated significantly negatively with that of PD-L1 in ccRCC Fig. A–C. ThroIn this study, we identified BMP1 as being upregulated in GBM, HNSC, KIRC, KIRP, and STAD. Additionally, we found that BMP1 can serve as a biomarker for poor prognosis in patients with KIRC. We also established the LINC00685, SLC16A1-AS1, PVT1, VPS9D1-AS1, SNHG15, and CCDC18-AS1/hsa-miR-532-3p/BMP1 axis as the most potential upstream ncRNA-related pathway of BMP1 in ccRCC. Moreover, our findings revealed a significant positive correlation between BMP1 levels and tumor immune cell infiltration, biomarkers of immune cells, and immune checkpoint expression.We first investigated expression of BMP1 across cancers using TCGA data, after which the GEPIA database was also used to confirm BMP1 expression. Survival analysis for BMP1 in cancer types of interest indicated that ccRCC patients with high expression of BMP1 had poor prognosis. The preliminary results of Xiao et al.  also demLncRNAs sponge miRNAs and regulate miRNA-targeted mRNAs at the posttranscriptional level in the cytoplasm , 20. To Based on the ceRNA hypothesis , the pot+ T cells, B cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells, in ccRCC. Furthermore, BMP1 is significantly positively associated with biomarkers of these infiltrated immune cells. These findings indicate that BMP1 might regulate development of ccRCC through tumor immune infiltration. In addition, the effectiveness of immunotherapy depends not only on adequate infiltration of immune cells into the tumor microenvironment but also on adequate expression of immune checkpoints [In recent years, the tumor microenvironment (TME) has gained much attention and is considered to be a key factor affecting treatment resistance, tumor development and prognosis –32. Our ckpoints . Thus, wIn general, we demonstrate that BMP1 is highly expressed in multiple types of human cancer (including ccRCC) and strongly correlates with unfavorable prognosis in ccRCC. We explored the upstream regulatory mechanism of BMP1 in ccRCC, namely, LINC00685, SLC16A1-AS1, PVT1, VPS9D1-AS1, SNHG15 and the CCDC18-AS1/hsa-miR-532-3p/BMP1 axis (Fig. Although our study has some advantages, it also has some limitations. First, the results were obtained from the TCGA database only, and verification in other databases is needed. Second, all the results were obtained by analyzing public datasets. In addition, further experiments should be performed in vivo and in vitro to verify these results.


GsWRKY23 that is specifically upregulated in salt-tolerant Glycine soja accession BB52 seedlings was identified by transcriptomic analysis under salt stress. How the physiological functions and mechanisms of the GsWRKY23 gene affect salt tolerance was investigated using transformations of soybean hairy roots and Arabidopsis, including wild-type (WT) and atwrky23-mutant plants. The results showed that GsWRKY23 in the roots, stems, and leaves of BB52, along with its promoter in the cotyledons and root tips of GsWRKY23pro::GUS Arabidopsis seedlings, displayed enhanced induction under salt stress. GsWRKY23 localises to the nucleus and shows transcriptional activation ability in yeast cells. Compared to GsWRKY23-RNAi wild soybean hairy-root composite plants under salt stress, obvious improvements, such as superior growth appearance, plant height and fresh weight (FW), and leaf chlorophyll and relative water content (RWC), were displayed by GsWRKY23-overexpressing (OE) composite plants. Moreover, their relative electrolytic leakage (REL) values and malondialdehyde (MDA) contents in the roots and leaves declined significantly. Most of the contents of Na+ and Cl− in the roots, stems, and leaves of GsWRKY23-OE plants decreased significantly, while the content of K+ in the roots increased, and the content of NO3− displayed no obvious change. Ultimately, the Na+/K+ ratios of roots, stems, and leaves, along with the Cl−/NO3− ratios of roots and stems, decreased significantly. In the transgenic WT-GsWRKY23 and atwrky23-GsWRKY23 Arabidopsis seedlings, the salt-induced reduction in seed germination rate and seedling growth was markedly ameliorated; plant FW, leaf chlorophyll content, and RWC increased, and the REL value and MDA content in shoots decreased significantly. In addition, the accumulation of Na+ and Cl− decreased, and the K+ and NO3− levels increased markedly to maintain lower Na+/K+ and Cl−/NO3− ratios in the roots and shoots. Taken together, these results highlight the role of GsWRKY23 in regulating ionic homeostasis in NaCl-stressed overexpressed soybean composite plants and Arabidopsis seedlings to maintain lower Na+/K+ and Cl−/NO3− ratios in the roots and shoots, thus conferring improved salt tolerance.WRKY proteins are a superfamily of transcription factors (TFs) that play multiple roles in plants’ growth, development, and environmental stress response. In this study, a novel WRKY gene called  During their long evolutionary process, plants have formed complex and effective signal networks that can transform extracellular stress factors into intracellular signals and mediate plants’ response processes and adaptation mechanisms to various stresses by activating or inhibiting stress-related gene expression to improve adaptability to adverse environments. In this process, various transcription factors (TFs), which can be regulated as cascade switches, often play the role of upstream connection and coordination [Soil salinization is an increasingly serious problem worldwide, and the global salinized land area has reached approximately 1 billion hectares, accounting for 7% of the total land area . Soil san plants ,6. As andination .cis-acting elements in the promoter of target genes, and they participate in a variety of biological processes in plants, including cell division, growth, development, ageing, and various abiotic or biotic stress responses [cis-acting elements, and then regulate related biochemical reactions and physiological metabolic processes through the expressed substances to reduce or avoid greater damage to plants. Regulating or suppressing the expression of genes encoding these transcription factors will generally improve the adaptability of plants to various stresses, such as salt, drought, low temperature, pathogens, and insects [2H2 or C2HC type), which is involved in protein interaction and assisted DNA binding [cis-acting element in the target gene promoter. According to the number of WRKY domains and the type of zinc-finger motifs, WRKY proteins can be divided into three groups: group Ⅰ proteins contain two WRKY domains and a C2H2-type (or C2HC-type) zinc-finger motif; group Ⅱ proteins contain only one WRKY domain with a C2H2-type zinc-finger motif, which can be subdivided into five subgroups (Ⅱa-Ⅱe) according to the difference in amino acid sequence; group Ⅲ proteins consist of a WRKY domain and a zinc finger of the C2HC type [SPF1, the first member of the WRKY gene family, was isolated from sweet potatoes [WRKY gene family members with multiple roles in plants have been identified. Many studies have shown that these WRKY genes mainly respond to biotic or abiotic stress [AtWRKY23 is controlled by auxin through the ARF7/19 transcriptional response pathway, which can stimulate flavonoid biosynthesis and is needed for proper root growth and embryo development [Cucumis sativus L.), which is homologous to AtWRKY23, may be involved in the positive regulation of fruit peel yellowing [Transcription factors (TFs), also known as trans-acting factors, can activate or inhibit the transcription of downstream target genes by recognising esponses . When pl insects ,11. As o binding ,13. WRKYpotatoes , an incrc stress ,16 and pc stress , seed dec stress , plant dc stress , and bioc stress . For exaelopment . WRKY23 ellowing .Glycine max), as the main source of plant-derived protein, edible oil, and animal feed, play an important role in global agricultural production, sustainable development, and the human diet [G. max was domesticated from its wild relative G. soja in China, and generally, G. soja displays relatively higher salt tolerance than G. max [G. soja species for adaptation to stressful environments have been lost, resulting in obviously decreased genetic diversity in G. max cultivars [G. soja and G. max, it is possible for these stress-tolerance-related genes in G. soja to be reintroduced into the cultivated soybeans through breeding approaches [G. soja for molecular design breeding of salt-tolerant G. max cultivars. In our previous study, transcriptomic analysis was carried out on the roots of halophytic G. soja BB52 seedlings under NaCl treatment to explore the molecular mechanisms of its strong salt tolerance. A large number of differentially expressed genes (DEGs) were enriched in Ca2+-signalling-related pathways in the plant-pathogen interaction pathway [2+ signal action on the MPK4 protein, the transcription factor WRKY gene family was enriched downstream of the MPK4 regulation pathway, and the transcription factor GsWRKY23 gene, which was significantly upregulated in the roots of BB52 seedlings under salt stress, was identified. In this work, the function of the GsWRKY23 gene in the salt tolerance of G. soja accession BB52 was investigated through multiple experimental techniques, including tobacco leaf transient expression, protein transcriptional activation assay and subcellular localisation, overexpression or RNA interference (RNAi) transformation of G. soja hairy roots mediated by Agrobacterium rhizogenes, and transgenic Arabidopsis (including WT and atwrky23 mutant) mediated by Agrobacterium tumefaciens. The changes in the phenotype and physiological parameters of GsWRKY23-overexpressing/RNAi hairy-root soybean composite plants and transgenic Arabidopsis exposed to NaCl treatment were determined. The aims of this study were to reveal the physiological mechanisms of GsWRKY23 mediating the alleviation of ionic toxicity in G. soja plants under salt stress, and to provide a crucial scientific basis and gene resources for molecular design breeding and salt-tolerance improvement in cultivated soybeans or other crops in the future.Cultivated soybeans  gene of the PBI101 vector to obtain the recombinant reporter vector GsWRKY23pro::GUS, and GsWRKY23pro::GUS-transgenic Arabidopsis was then obtained by A. tumefaciens GV3101-mediated transformation  of ormation . When treedlings B. Thus, GsWRKY23 was cloned into the pGBKT7 to construct a pGBKT7-GsWRKY23 plasmid, and then pGBKT7 and pGBKT7-GsWRKY23 were introduced into Y2HGold yeast cells to detect the transcriptional activation ability of the GsWRKY23 protein. As shown in GsWRKY23 plasmids grew well on YPDA medium, but only the yeast strain transformed with the pGBKT7-GsWRKY23 grew on SD/-Trp/-His medium and turned blue when X-α-Gal was added. This indicates that GsWRKY23 activated the expression of the reporter genes (HIS and MEL1) in yeast cells and showed transcriptional activation activity.The CDS of GsWRKY23 without a termination codon was cloned into the pCAMBIA1300-eGFP vector to obtain the 35S:: GsWRKY23::eGFP recombinant plasmid, and then the empty vector pCAMBIA1300-GFP (35S::eGFP) and the fusion vector 35S::GsWRKY23::eGFP were transferred into the young leaves of tobacco plants by A. tumefaciens GV3101-mediated transformation. As shown in GsWRKY23::eGFP. These results may indicate that the GsWRKY23 protein was mainly located in the nucleus and functioned as a transcription factor.To explore the subcellular localisation of the GsWRKY23 protein, the CDS of GsWRKY23-overexpressed vectors (GsWRKY23-OE) and RNA interference (GsWRKY23-RNAi) , GsWRKY23-OE, and GsWRKY23-RNAi soybean hairy-root composite plants were obtained . However, the salt damage to GsWRKY23-RNAi plants was seriously aggravated, with more stunted plant height, clearly decreased leaf chlorophyll content, and increased root and leaf REL values and MDA contents, especially in the aspect of leaf-related physiological indices, e.g., leaf chlorophyll content decreased by 22.22%, and leaf REL value and MDA content increased by 62.62% and 42.94%, respectively, which all reached significant levels in comparison with EV (p ≤ 0.05) (Using the 23-RNAi)  and a haobtained . Under nn plants . When ex ≤ 0.05) .+ and Cl− significantly increased in the roots, stems, and leaves of EV, GsWRKY23-OE, and GsWRKY23-RNAi wild soybean plants under NaCl stress, while the K+ content was significantly decreased and the NO3− content showed no visible change. When compared with GsWRKY23-RNAi plants, the Na+ contents in the roots, stems, and leaves of OE plants decreased by 28.44%, 13.53%, and 4.86%, respectively, while the K+ contents increased by 8.35%, 21.73%, and 3.12%, respectively; correspondingly, the Na+/K+ ratios of roots, stems, and leaves were markedly decreased, by 33.89%, 28.87%, and 7.68%, respectively (p ≤ 0.05)   A–C. Unde ≤ 0.05) D–F.GsWRKY23-OE and GsWRKY23-RNAi soybean composite seedlings were subjected to 120 mM NaCl solution for 6 h, compared with the control conditions, and except for the obvious downregulated expression of GsNRT2, the expression levels of GsHKT1-1, GsSOS1, GsNHX2, GsNRT2, GsCLC1, GsCLC-b2, and GsCLC-c2 in roots were significantly upregulated under salt treatment. Among them, the expression levels of GsHKT1-1 and GsSOS1 were significantly upregulated in GsWRKY23-OE plants in contrast to EV plants but were clearly downregulated in GsWRKY23-RNAi plants, and GsCLC-c2 and GsCLC-b2 exhibited the opposite trend. In addition, there were no remarkable differences in the expression levels of GsNHX2, GsNRT2, and GsCLC1 among the three soybean materials  were generated in this study  than WT and atwrky23  under 150 mM NaCl treatment on the 7th day , and the mitigation effect of salt damage on atwrky23-GsWRKY23 was more obvious   B,E.+/K+ and Cl−/NO3− in the roots and shoots of WT and atwrky23 plants under salt stress being significantly higher than the control levels, and the increases in atwrky23 plants were greater. The ratios of Na+/K+ and Cl−/NO3− in roots increased by 8.16 and 12.41 times, respectively, while those in shoots increased by 30.02 and 10.85 times, respectively. Under the transformation of the GsWRKY23 gene into Arabidopsis WT and the atwrky23 mutant, the ratios of Na+/K+ and Cl−/NO3− in the roots and shoots of WT-GsWRKY23 and atwrky23-GsWRKY23 plants decreased significantly (p ≤ 0.05), especially in atwrky23-GsWRKY23 plants, whose ratios of Na+/K+ and Cl−/NO3− in roots decreased by 69.42% and 72.88%, respectively, while those in the shoots decreased by 80.89% and 75.10%, respectively  and G. max cultivars (such as Jackson and N23674) [G. max cv. N23674, G. soja BB52 materials display stronger salt tolerance, which is mainly related to their superior ability to accumulate the absorbed Na+ and Cl− in the roots and reduce the transport and distribution to the stems and leaves under salt stress [3− levels in the stems and leaves of soybean plants to reduce the Cl−/NO3− ratio under salt treatment is also attributed to their stronger salt tolerance [Under saline environments, a great amount of Nahibition . The salsuch as N227 and G N23674) ,29. Compt stress ,25. Furtolerance ,29.GsWRKY23 was screened downstream of the MPK4 regulation pathway  in the roots of salt-stressed BB52 seedlings, and the significantly upregulated  pathway A. This ireatment A, and itaptation . The deeeedlings B may sugt system A, and trt system B, which WRKY23 gene and its homologues have been reported in plants such as Arabidopsis thaliana, Cucumis sativus, Populus tremula, Oryza sativa, and G. max, and this suggests biological functions involved in the responses of plants to stressful environments. For example, overexpression of AtWRKY30 enhanced the seed germination rate of transgenic Arabidopsis thaliana under salt and oxidative stresses [OsWRKY42, as a negative regulator of jasmonic acid (JA)-mediated plant stress responses, conferred enhanced salt tolerance to OsWRKY42-transgenic Arabidopsis, which was mainly related to inducing anthocyanin production and reducing ROS levels [G. max, Li et al. found that GmWRKY45-transgenic Arabidopsis plants displayed a significantly higher survival rate and soluble sugar content and, thus, improved salt tolerance compared to WT plants [GmWRKY21-transgenic Arabidopsis plants were tolerant to cold stress, whereas GmWRKY54 conferred salt and drought tolerance, and GmWRKY13-transgenic Arabidopsis seedlings were more sensitive to NaCl and mannitol stress [PcWRKY33 from Polygonum cuspidatum increased salt sensitivity in transgenic Arabidopsis seedlings by inhibiting the increased expression of AtSOS1, AtSOS2, AtSOS3, and AtNHX1 under salt treatment, accumulating excessive Na+ and thereafter increasing the Na+/K+ ratio [Arabidopsis by binding to the W-box in the upstream promoter of AtSOS1 and, accordingly, significantly increased the Na+ content and Na+/K+ ratio in roots [G. soja transcription factor WRKY gene family, such as GsWRKY15a, which is involved in the control of seed size in wild soybeans and was significantly more weakly expressed than its orthologous GmWRKY15a in G. max during pod development [GsWRKY20-transgenic Arabidopsis displayed enhanced drought tolerance, which was associated with increased susceptibility to ABA and facilitation of ABA-induced stomatal closure [GsWRKY20-overexpressing alfalfa showed increased salt tolerance as a result of accumulating less Na+ and more K+ in both leaves and roots [GsWRKY20-overexpressing soybeans displayed strongly increased drought tolerance, with lower stomatal density and faster stomatal closure [To date, the stresses . OsWRKY4S levels . In G. mT plants . Zhou etl stress . Bao et K+ ratio . Song etin roots . Currentelopment . Compare closure . Furthernd roots , and GsW closure .GsWRKY23 gene of wild soybeans and its related functions under salt stress or other stressful conditions. Therefore, the physiological functions of GsWRKY23 in the responses of overexpressed hairy-root composite soybean plants and transgenic Arabidopsis to NaCl treatment were investigated for the first time in this study, to further decipher the molecular mechanisms of the G. soja BB52 accession with strong salt tolerance in terms of transcription factors regulating functional gene expression. In this work, the Na+/K+ ratios in the roots, stems, and leaves of GsWRKY23-OE soybean hairy-root composite plants, as well as Cl−/NO3− in roots and stems, were significantly lower than those of GsWRKY23-RNAi plants under NaCl treatment  was d GsSOS1 D–H. WhenGsWRKY23 C. Corresatwrky23 C,F. Overreatment . These r quality . Howeverequences B,C, joinGsWRKY23 gene, which was significantly upregulated under NaCl stress, was screened for the first time from the genome of the salt-tolerant G. soja BB52 accession. Both the GsWRKY23 gene and its promoter displayed an enhanced response to NaCl stress. GsWRKY23 is a nucleus-localised protein that functions as a transcription factor. Analysis of phenotypic and physiological indices of EV, GsWRKY23-OE, and GsWRKY23-RNAi hairy-root composite soybean plants under salt stress indicated that GsWRKY23 played a positive role in the process of salt stress response and adaptation, which could reduce the contents of Na+ and Cl− in plants and increase their K+ content to maintain lower Na+/K+ and Cl−/NO3− ratios, along with ionic homeostasis for contributing to stronger salt tolerance. These similar effects were further verified in GsWRKY23-transgenic Arabidopsis. These findings not only provide an important research basis for further revealing the molecular functions of the GsWRKY23 gene and the mechanisms underlying the strong salt tolerance of G. soja BB52 materials, but also provide a valuable theoretical basis and gene resources for molecular genetic improvement and germplasm innovation of salt-tolerant soybean cultivars and other crops.In conclusion, the transcription factor Glycine soja BB52 accessions, tobacco (Nicotiana benthamiana LAB), Arabidopsis thaliana , and atwrky23 mutants (SALK_006855C). The bacterial strains included Escherichia coli DH5α, A. tumefaciens strain GV3101, and A. rhizogenes strain K599. The yeast strain Y2HGold, yeast expression vector pGBKT7, and plant binary vectors pCAMBIA1300-GFP, PBI101, and pFGC5941 were used in this study.Plant seeds included http://www.wildsoydb.org/Gsoja_W05/ (accessed on 5 June 2023)), and amino acid sequences of the WRKY23 family members with high homology to GsWRKY23 were retrieved and downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/ (accessed on 6 June 2023)). The phylogenetic tree of the WRKY family was constructed using the neighbour-jointing (NJ) method with MEGA X software, and the amino acid sequence alignment of WRKY family members was performed using DNAMAN 8 software.The amino acid sequence of GsWRKY23 (Glysoja.02G002639) was obtained from the wild soybean database . The reverse transcription was performed using Hifair II 1st Strand cDNA Synthesis SuperMix . Quantitative real-time PCR (qRT–PCR) was performed on an ABI 7500 fluorescence quantitative PCR instrument  using the 2 × Hieff qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen). Three independent biological replicates and three independent technical replicates were included for each sample. The soybean UBI3 gene (LOC114401261) was used as an internal control, and the relative expression was calculated using the 2−ΔΔCt method [Seeds of t method . All of GsWRKY23 was inserted upstream of the GUS (uidA) gene of the PBI101 vector to obtain the recombinant vector GsWRKY23pro::GUS. The CDS of GsWRKY23 was amplified by PCR using the specific primers , and 35S::GsWRKY23 overexpression (OE) vector. A 200 bp fragment was amplified from the CDS of GsWRKY23 as the RNAi target and inserted upstream and downstream of the CHSA intron of the pFGC5941 vector in forward and reverse orientations, respectively, to obtain the GsWRKY23-RNAi vector. The CDS of OsMADS (encoding a nuclear locator protein as a label) without a stop codon was inserted into the pCX-DR-RFP vector to obtaine the RFP fusion construct 35S::OsMADS::RFP.The promoter (1632 bp upstream of ATG) of  primers  and inseGsWRKY23pro::GUS was transformed into Arabidopsis Col-0 plants using the A. tumefaciens strain GV3101-mediated floral dip method [Arabidopsis seedlings planted on ½ MS agar medium were stressed with 120 mM NaCl solution for 24 h, and the untreated seedlings were used as a control. Then, these seedlings were sampled for the GUS staining assay as described by Jefferson et al. [The fusion construct p method . Ten-dayn et al. .GsWRKY23 was introduced into the yeast strain Y2HGold, and the empty vector pGBKT7 was used as a negative control. The strains carrying pGBKT7 or pGBKT7-GsWRKY23 and the Y2H strain were inoculated on YPDA, SD/-Trp/-His, and SD/-Trp/-His/X-α-Gal media to observe the growth of the strains and identify the transcriptional activation of GsWRKY23.A transcriptional activation assay of GsWRKY23 was conducted as described by Liu et al. . BrieflyGsWRKY23::GFP and 35S::OsMADS::RFP were transformed into the A. tumefaciens strain GV3101. Leaves of four-week-old tobacco plants were co-transformed with the fusion expression vector 35S::GsWRKY23::eGFP or 35S::OsMADS::RFP as described by He et al. [The fusion constructs 35S::e et al. , and theGsWRKY23-OE vector, -RNAi vector, and empty vector (pFGC5941) were transformed into A. rhizogenes K599, and soybean hairy-root transformation was mediated according to the methods described in our previous work [GsWRKY23 and the ion-transporter-related genes  in EV, GsWRKY23-OE, and GsWRKY23-RNAi plants. Three independent biological replicates and three independent technical replicates were included for each sample. The specific primers used for qRT–PCR are also listed in GsWRKY23-OE, and GsWRKY23-RNAi plants was performed. Physiological parameters such as FW per plant, plant height, leaf chlorophyll content and RWC, REL values, and MDA contents in the roots and leaves were measured as described in our previous work [+, K+, Cl−, and NO3− according to the method described by Zhou and Yu [The 35S::ous work . Positivous work . The roou and Yu .Arabidopsis atwrky23 mutant was obtained from the Arashare platform, and homozygous mutants were screened using the three-primer method. The vector GsWRKY23-OE was transformed into Arabidopsis WT and mutant plants using the floral dip method [3 lines of WT-GsWRKY23  and atwrky23-GsWRKY23  were obtained. The seeds of WT, atwrky23, WT-GsWRKY23, and atwrky23-GsWRKY23 were sterilised with 75% ethanol and sown on 1/2 MS containing 0 (control), 75, and 150 mM NaCl, kept at 4 °C for 2 d and cultured horizontally in a greenhouse. Germination rates were calculated daily and photographed on the 7th day. Furthermore, seeds of the WT, atwrky23, and transgenic lines were sown in pots filled with peat moss and vermiculite (1:1). Three-week-old Arabidopsis plants were stressed with 120 mM NaCl for 7 d for phenotypic analysis, and the untreated plants were used as controls. Measurements of FW per plant, leaf chlorophyll content and RWC, REL values, and MDA contents in the shoots were also conducted. Finally, the shoots and roots were sampled, dried, and ground into powder for the assays of the contents of Na+, K+, Cl−, and NO3−.The p method . Homozygn = 3, except for FW per plant and plant height, where n = 6, and seed germination of Arabidopsis where n = 32) using SPSS software (version 20.0). Significant differences among means were determined by Duncan’s test at the p ≤ 0.05 significance level.Data were analysed and presented as the mean ± SD for each treatment (


Thematic narratives were used to report data into six themes: attitudes and perceptions about COVID-19 vaccines, intention to uptake COVID-19 vaccines; factors and barriers associated with COVID-19 vaccine uptake; socio-demographic determinants affecting the intention and uptake; and information sources for COVID-19 vaccines. The intention for uptake ranged from 25% to 80.9%, resulting in a suboptimal uptake intention rate (54.2%) on the African continent. Factors that promoted vaccine acceptance included confidence in the COVID-19 vaccines and the desire to protect people. Age, education, and gender were the most common factors significantly associated with vaccine acceptance. Most studies revealed that considerable barriers to vaccine uptake exist in Africa. Concerns about potential side effects, vaccine ineffectiveness, a perceived lack of information, and inaccessibility were among the individual, interpersonal, and structural barriers to COVID-19 vaccine uptake. The unwillingness to receive the COVID-19 vaccine was strongly correlated with being female. Mass and social media were the main sources of information regarding COVID-19 vaccines. To encourage vaccine uptake, governments should pay attention to refuting misinformation through integrated community-based approaches, such as creating messages that convey more than just information.The COVID-19 pandemic is a severe concern worldwide, particularly in Africa. Vaccines are crucial in the fight against the COVID-19 pandemic. This scoping review examined existing literature from 2020 to 2022 on individual, interpersonal, and structural barriers and facilitators to COVID-19 vaccination within Africa to facilitate more informed health promotion interventions to improve vaccine uptake. This review was conducted using Arksey and O’Malley’s five-stage methodological framework. A comprehensive search was undertaken from 2021 to 2022 using six electronic databases: EBSCOhost, PubMed, Web of Science, ProQuest, WorldCat Discovery, and Google Scholar. Data was collected, charted into themes, and summarized using a standard data extraction sheet in Microsoft Excel. A total of forty ( The novel coronavirus 2019, termed COVID-19, is a highly transmissible and pathogenic viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , and COVSARS-CoV-2 has spread to most African countries , and theVaccine uptake is dependent on various psychological states, behaviors, and contextual factors. More specifically, vaccine hesitancy (VH) refers to indecisiveness or uncertainty among individuals about vaccine uptake, and those vaccine-hesitant individuals can exhibit a spectrum of attitudes , dependeDespite recent systematic reviews in low- and middle-income countries (LMICs) ,19,20, tAccordingly, this review attempts to explore factors influencing the intention and uptake of COVID-19 vaccines on the African continent. The present study will shed light on the existing literature by understanding the determinants of uptake and factors influencing people’s decisions to vaccinate against COVID-19, which is necessary to inform health promotion interventions to improve the uptake of COVID-19 vaccination in this context. The review addressed the following questions: (i) What are the perceptions and attitudes regarding COVID-19 vaccines among people in Africa? (ii) What are the facilitating factors associated with COVID-19 vaccine uptake in this geographical setting? (iii) What barriers are associated with COVID-19 vaccine uptake?This study utilized a scoping review approach because of its ability to identify trends and gaps in an existing knowledge base to inform research, policy, and practice . This scThe following five-stage framework proposed was as follows: (1) identifying the research questions; (2) searching for relevant studies; (3) selecting studies; (4) charting the data; and (5) collating, summarizing, and reporting the results  (p. 22).A systematic search was conducted utilizing the relevant electronic databases such as EBSCOhost, PubMed, Web of Science, ProQuest, WorldCat Discovery, and Google Scholar. The search followed a process to identify studies that addressed the research questions outlined above. These four electronic databases  were searched from 1 October 2021 to 13 October 2021. Another search was conducted via the remaining two electronic databases (ProQuest and WorldCat Discovery) from 25 October 2022 to 8 November 2022. The following restrictions were placed on all four databases to produce the relevant studies needed for this scoping review. Studies were searched from 2020 to 2022 and published in English and in peer-reviewed journals.The COVID-19 pandemic was the motivating factor behind this timeline. Additional limiters were placed to only search for full-text studies conducted on the African continent. The following search terms were included: “COVID-19 vaccines”, “COVID-19”, “SARS-CoV-2 vaccines”, “perceptions”, “attitudes”, “barriers”, “drivers”, “acceptance”, “hesitancy”, “Africa”, “vaccine uptake”, “vaccine refusal”, “COVID-19 vaccination uptake”, “COVID-19 vaccination intention”, and “COVID-19 vaccine willingness”. The final search strategies for EBSCO host can be found in the After conducting a complete title and abstract screening in the databases mentioned above, studies were screened using the Population–Concept–Context (PCC) framework to establish their eligibility for this review. The researcher excluded all studies that did not answer the review’s research query: published research on psychosocial and contextual factors influencing intention and uptake of COVID-19 vaccines in Africa.Furthermore, the inclusion and exclusion criteria guided the selection of full-text studies to determine which studies were most appropriate to include in this review. The researcher focused on quantitative, qualitative, and mixed-methods academic/published journals  published in English between 2020 and 2022 on attitudes, beliefs, barriers, factors, facilitators, perceptions, acceptance, intentions, concerns, uptake, and hesitancy toward COVID-19 vaccines on the African continent among the general population. The researcher excluded gray literature , summaries, and other reviews. Additional exclusions included studies that focused solely on populations other than the general population .A standardized data extraction sheet in Microsoft Excel was used to collate and chart the data into themes and summarize studies and reports. The following headings were used to extract detailed information for the included studies: authors and year of publication; study setting, i.e., country and data collection period; and methodology. The methodology section consisted of study characteristics, i.e., study design, population target, and sample size.Due to the heterogeneity of studies, a narrative synthesis approach was used to collect, synthesize, and map the literature . The foln = 247), PubMed (n = 53), Web of Science (n = 43), ProQuest (n = 26), WorldCat Discovery (n = 13), and Google Scholar (n = 13). After removing duplicates with EndNote (V.X9), 274 studies were screened by title and abstract to find those that met the initial screening criteria. One hundred and sixty (n = 160) studies were excluded because they were irrelevant to the research question, leaving 114 studies for full-text review. Following the inclusion and exclusion assessment criteria, studies were further excluded because they did not address research questions (n = 11), a summary of studies (n = 1), focusing only on VH (n = 1), non-peer-reviewed articles (n = 7), not applicable population targets (n = 38), and research reports and documents (n = 16), resulting in 40 published articles for the final analysis. The PRISMA flow diagram below illustrates the selection process in In the initial search, 395 studies were identified from database searches: EBSCOhost (n = 37) that were conducted used a quantitative research approach. Of those studies, (n = 33) adopted a cross-sectional design. Two studies (n = 2) adopted a qualitative research approach, and one (n = 1) applied a mixed-methods research approach.Many studies (See n = 30) made up most of the study sample, while the general population comprised (n = 10). Three studies by Elhadi et al. [n = 2) and pregnant women (n = 1). However, these sub-populations were not considered due to the abovementioned exclusion criteria. Only the general population sample was used. The sample sizes for the current review ranged from 14 to 11,971 respondents. Male respondents ranged from 15% to 91.7%, while female respondents ranged from 8.3% to 100%. Additional baseline characteristics of the selected studies are listed in The general adult population (aged ≥ 18 years)  reported on attitudes or perceptions regarding COVID-19 vaccines in Africa. Six of those studies (n = 6) found that the participants were hesitant toward the vaccine [In this review, a total of eighteen studies  studies assessed the willingness, refusal, hesitancy, vaccinated, and unvaccinated rates regarding COVID-19 vaccination among the general African population. (see Thirty-five (ion. see .n = 30) studies [Thirty  studies, the intention for uptake ranged from 25% to 80.9%. Only nine studies (n = 9) reported a lower-than-average rate . In comparison, twenty-one studies (n = 21) illustrate an intention rate of 50% or higher . Across the 13 studies these were grouped as confidence in the COVID-19 vaccine [Various factors that promoted the intention for vaccine uptake were reported in thirteen studies  highlighted the barriers associated with COVID-19 vaccine uptake. Of those twenty-nine studies (n = 29) cited fears over potential side effects and the newly developed vaccine being unsafe for the African population [n = 11) reported concerns regarding the vaccine’s ineffectiveness in protecting against COVID-19 [n = 8)  [n = 5) attributed barriers to a perceived lack of information to make informed decisions about its uptake [n = 3) reported on vaccine inaccessibility [n = 2) [n = 1) reported on participants being afraid of needles [Thirty-three studies  ,41,42,56dies n =  cited fes uptake ,50,54,64s uptake ,55,58,62sibility ,35,50—desibility ,38,45 en =  highlighsibility ,64; partsibility ,49,63. A [n = 2) ,60, and dies n =  cited fe needles , having  needles  and nega needles .Individual, interpersonal, and structural barriers to COVID-19 vaccine uptake were identified  among Ghn = 27) reported socio-demographic determinants influencing COVID-19 vaccine intention and uptake. Twenty studies (n = 20) found a statistically positive relationship between socio-demographic characteristics and vaccine uptake. Age [n = 5) marital status [n = 4) suggested an income level [n = 3) occupation [Twenty-seven studies  included in this review. Having a low perception of the use of vaccination; being unemployed, having a low level of acceptance of COVID-19 vaccines, being unwilling to test for COVID, and having an extremely low (illiterate) education level were significant predictors for uptake.According to Jabessa and Bekele , Ethiopin = 9) revealed a statistically negative relationship between socio-demographic characteristics and vaccine uptake, resulting in the rejection of the COVID-19 vaccine. Gender [Further analysis indicated that nine studies  reported on information sources [n = 17) [n = 13) [n = 10) [n = 5) reported respondents receiving information from HCWs [Seventeen studies  ,62,63,64[n = 13) ,62,63,64[n = 10) ,41,57,63rom HCWs ,34,57,63rom HCWs ,29,32,34n = 4) reported on participants who received their information from religious and traditional leaders [Four studies  and Health Promotion Practitioners (HPPs) to engage with the public through information, education, and communication platforms to improve vaccine literacy in general and for COVID-19 vaccines, as well as cultivate positive beliefs and attitudes towards COVID-19 vaccines. Greater awareness of social responsibility in protecting oneself, loved ones, and the vulnerable in communities may augur well for increased uptake of vaccines. However, COVID-19 vaccination uptake in the African context will hinge greatly on building trust between the general population, scientists, the health care system, and governments.