Environmental conditions are subject to unprecedented changes due to recent progressive anthropogenic activities on our planet. Plants, as the frontline of food security, are susceptible to these changes, resulting in the generation of unavoidable byproducts of metabolism (ROS), which eventually affect their productivity. The response of plants to these unfavorable conditions is highly intricate and depends on several factors, among them are the species/genotype tolerance level, intensity, and duration of stress factors. Defensive mechanisms in plant systems, by nature, are concerned primarily with generating enzymatic and non-enzymatic antioxidants. In addition to this, plant-microbe interactions have been found to improve immune systems in plants suffering from drought and salinity stress.Plants are exposed to various environmental stresses in their lifespan that threaten their survival. Reactive oxygen species (ROS), the byproducts of aerobic metabolism, are essential signalling molecules in regulating multiple plant developmental processes as well as in reinforcing plant tolerance to biotic and abiotic stimuli. However, intensified environmental challenges such as salinity, drought, UV irradiation, and heavy metals usually interfere with natural ROS metabolism and homeostasis, thus aggravating ROS generation excessively and ultimately resulting in oxidative stress. Cellular damage is confined to the degradation of biomolecular structures, including carbohydrates, proteins, lipids, pigments, and DNA. The nature of the double-edged function of ROS as a secondary messenger or harmful oxidant has been attributed to the degree of existing balance between cellular ROS production and ROS removal machinery. The activities of enzyme-based antioxidants, catalase , monodehydroascorbate reductase , dehydroascorbate reductase , superoxide dismutase , ascorbate peroxidase , glutathione reductase , and guaiacol peroxidase ; and non-enzyme based antioxidant molecules, ascorbate (AA), glutathione (GSH), carotenoids, α-tocopherol, prolines, flavonoids, and phenolics, are indeed parts of the defensive strategies developed by plants to scavenge excess ROS and to maintain cellular redox homeostasis during oxidative stress. This review briefly summarises current knowledge on enzymatic and non-enzymatic antioxidant machinery in plants. Moreover, additional information about the beneficial impact of the microbiome on countering abiotic/biotic stresses in association with roots and plant tissues has also been provided. Constant changes in environmental conditions exacerbates unfavorable, stressful conditions such as salinity, drought, extreme temperatures, waterlogging, or heavy metal stress, severely affecting plant growth, development, and yield through inducing changes in plant physiological and biochemical characteristics ,2.2) consumption [2•−), hydroxyl radical (OH•), perhydroxyl radical (HO2•), singlet oxygen (1O2) and hydrogen peroxide (H2O2) [Plenty of studies have shown that plants are prone to generate both highly reactive oxygen free radicals and slightly reactive non-radicals of oxygen derivatives after subjection to various environmental biotic and/or abiotic stresses ,4,5. Thisumption , is highsumption ,8,9. Thee (H2O2) ,10. The e (H2O2) . Both ROe (H2O2) ,13, durie (H2O2) , or in ae (H2O2) ,16. Generally, ROS basic levels generated under optimal environmental conditions cannot cause cellular damage due to the expression of stress-responsive genes . This lePhaseolus vulgaris L.) plants affected by salinity stress, a subtle increase in the activity of CAT and APX antioxidants was observed in salt-tolerant GS57 and salt-sensitive 4F-89 genotypes, which was consistent with increased fresh and dry weight in these plants [Colobanthus quitensis (Kunth) Bartl by copper stress treatment [Plant adaptation to the oxidative stress caused by elevated ROS concentrations is often consistent with many key factors, among which are stress duration and severity, instantaneous cellular energy status, plant growth stages, ROS cellular level, and antioxidant capacity . Generale plants . Similare plants . The uprreatment . Bacillus subtilis SM21, Bacillus cereus AR156, and Serratia sp. XY21, have been shown to enhance the drought tolerance of cucumber (Cucumis sativus L.) plants by increasing the activity of SOD and mitigating the expression of genes encoding the cytosolic APX in cucumber leaf tissues [Plant growth-promoting rhizobacteria (PGPRs) are known to be beneficial microorganisms and play a contributing role in reinforcing plant response to stressful conditions . Typical tissues .This review aimed to further extend our understanding of antioxidants and their function in protecting plants against oxidative stress, as well as of plant–microbe interactions that confer abiotic stress tolerance in planta. Therefore, we provided a relatively comprehensive overview of major enzymatic and non-enzymatic antioxidants, pointed out the importance of PGPRs in alleviating plant stress, and summarised some current knowledge on them. Preventive mechanisms against ROS overaccumulation are the antioxidant capacity by which enzymatic activities confer plant tolerance to adverse environmental conditions ,46. Many•−2) into H2O2 and O2, such that it eventually wards off the formation of OH• by the metal-catalysed Haber–Weiss reaction [2 and H2O2, which are the Haber–Weiss reaction substrates, and reduces the risk of producing highly active OH− radicals. Based on the metal ion it binds, three main isozymes/isoforms (SODs) of this enzyme have been introduced thus far in the Arabidopsis thaliana genome: Cu/Zn-SOD gene localised to the chloroplast thylakoids (CSD2), peroxisomes (CSD3), and the cytosol (CSD1), Fe-SOD gene localised to the chloroplast stroma (FSD1), cytosol (FSD1), thylakoids , and the nucleus (FSD1), and Mn-SOD (MSD1) gene localised to the mitochondrial matrix [2O2 scavenger) and enhancement of the activity of chloroplastidial Fe-SOD usually leads to plant protection when CO2 assimilation is intensely reduced; or otherwise results in elevated cytotoxicity in the Haber–Weiss reaction [This omnipresent metalloenzyme, which can be found in almost all aerobic organisms including plants, in all intracellular organelles and apoplastic spaces , is actireaction ,49,51. Il matrix ,53,54. Tl matrix . Additiol matrix . The exireaction ,58. Suchreaction , tobaccoreaction , onion [reaction  and cottreaction  plants. reaction , anotherreaction ,65.2O2 [Regardless of SOD types, they are initially encrypted in the nucleus and then transferred to various organs ,66. In c2O2 . Most of2O2 ,67 or co2O2 ,69,70 wi2O2 , it was 2O2 . Apart f2O2 , oxidati2O2 , or phot2O2  stresses2O2 , germina2O2 , floweri2O2 , and not2O2 . 2O2 overproduced during light respiration or photorespiration in peroxisomes, H2O2 produced during β-oxidation of fatty acids in glyoxysomes, or H2O2 produced by SOD [2O2) into water and oxygen in an energy-efficient way [2O2 production as a result of oxidative stress, purine catabolism, photorespiration, and not least the β-oxidation of fatty acids [2O2, CAT showed a low affinity for organic peroxides [Gossypium hirsutum L.) [Cucumis sativus L.) [Oryza sativa L.) [ Heynh. [Cucurbita Linn.) [Zea mays L.) [Nicotiana plumbaginifolia Viviani) [Hordeum vulgare L.) [Lycopersicon esculentum Mill.) [Ipomoea batatas L.) Poir. [Ricinus communis L. cv. Hale) [As a Fe-containing homotetrameric enzyme, catalase  is responsible for the catalysation of Hd by SOD ,75. In aient way . Peroxisty acids ,76. Nevety acids . Despiteeroxides . One of eroxides . This enutum L.) , four inivus L.) , and in tiva L.) , three i) Heynh. , pumpkina Linn.) , maize (mays L.) , Tex-MexViviani) , two in gare L.) , and in m Mill.) , and one.) Poir. , and in v. Hale) , among o1 and CAT2) genes are predominantly present in glyoxysomes, peroxisomes and cytosol, and the latter (CAT3) is present in mitochondria and cytosol, with no presence reported in chloroplasts [1, CAT2, CAT3), pollens (CAT1), roots (CAT2), photosynthetic tissues (CAT2), and vascular tissues (CAT3) [In Arabidopsis, the first two  ,123,124.s (CAT3) , shoots,s (CAT3) ,123, ands (CAT3)  has been2O2, only high concentrations of H2O2 have the possibility of being eliminated by CATs [1 is responsible for scavenging H2O2 under abiotic stress conditions, and CAT2 and CAT3 for removing H2O2 to maintain ROS homeostasis both in light and darkness [2 rather than CAT1 in tobacco [Arabidopsis, all three CATs are engaged with photooxidation responses [1 is involved in the plant’s response to drought and salt stress [2 is implicated in toxic metal [3 is incorporated in drought stress responses [Because of low affinity of CATs to H by CATs . In partdarkness . Stimuladarkness . Leaf se tobacco . As for esponses ,124,128,t stress , CAT2 isic metal , heat [1ic metal , cold anic metal  stress responses .Arabidopsis genome revealed the presence of nearly nine putative APX genes, localised in the cytosol , peroxisomes (APX3 and APX5), chloroplast stroma (sAPX), chloroplast thylakoid (tAPX) [4 and APX7) being annotated as genes lacking H2O2 detoxifying activity and pseudogene, respectively [2), high light (APX2), heat and drought (APX1) [1) which tends to inactivate chloroplastic H2O2 detoxification [The ascorbate peroxidase  enzyme functions in the ascorbate-glutathione cycle and regularly exists in chloroplasts (stroma and thylakoid), mitochondria, peroxisomes, cytosols, vacuoles, and the apoplast ,11. Fived (tAPX) ,136, andd (tAPX) , with twectively . The criectively , salinitt (APX1) ,141 strefication ,143.2O2 signalling during developmental stages [2O2 toxicity in the chloroplast and cytosol [2O2, indicating that the ascorbate-glutathione (AA-GSH) cycle plays a vital role in controlling ROS levels in cellular organelles [2O2 into water and oxygen is to use ascorbate (ascorbic acid-AA) as a reducing factor [More importantly, APXs deal with Hl stages ,145 thro cytosol ,146. APXganelles . The wayg factor . In thisg factor .2O2; hence it is a better scavenger at times of stress [Pteris vittata in proportion to the HM-sensitive plant Pteris ensiformis suggests that the so-called enzymes play a central role in the detoxification of zinc oxide nanoparticles [Similar to the enzyme CAT, APX also presents either in the thylakoid membrane or as a solution in the stroma. The form attached to the thylakoid membrane immediately detoxifies the hydrogen peroxide at the production site, and the stroma-soluble type decomposes the hydrogen peroxide released into the stroma. In comparison to CAT, APX has a stronger affinity for Hf stress . The notarticles .2O2, whether by normal or stress-driven metabolism [2O2 utilisation in the process [2O2 removal [3) [With its 40–50 kDa monomers, this heme-containing enzyme, GPX (EC 1.11.1.7), is capable of eliminating excess Htabolism . GPX isotabolism . The two process . GPX pre process . As a co removal . Because removal . A numbe removal , potenti removal ,156, pol removal , and ozooval [3)  are invo2) and partially in the cytosol (GR1), mitochondria (GR2) [1) [1, GR2) [1) [2) [2+)-induced oxidative stress (GR2) [1) [2 has been proven to interfere with developmental processes involving embryo development [This enzyme , also known as flavoprotein oxidoreductase, is the last in the ascorbate-glutathione cycle that is found predominantly in chloroplasts (GRia (GR2) ,26,159 aGR2) [1) . The proGR2) [1) . The abo[1, GR2) ,162, salGR2) [1) ,164, chi [1) [2) ,166, metss (GR2) , and toxGR2) [1) ,169. Morelopment , seed geelopment , root grelopment . Thus, ielopment ,174 and elopment  stress f2-stabilizing chloroplast enzymes [+/NADPH ratio, thus elevating the amount of available NADP+ as the last electron receptor in photosynthetic light reactions, and eventually reducing the likelihood of electrons being transferred to O2 for superoxide radical production [GR mediated plant tolerance to stress conditions often refers to its role in ROS detoxification through GSH (reduced glutathione) regeneration . GR has  enzymes . Also, G enzymes , signify enzymes . Increasoduction ,178,179.Arabidopsis genes. MDHAR isozymes are laid out in different subcellular compartments, including mitochondria (MDHAR5), peroxisomes (MDHAR1) and its membrane glyoxysomes (MDHAR4), chloroplast (MDHAR6), and cytosol (MDHAR2 and MDHAR3) [4 in different growth stages of Arabidopsis, from germination to post-germination, through senescence. Based on studies conducted so far on rice, tobacco and Arabidopsis, it was revealed that overexpression of MDHAR genes resulted in an increased tolerance to salt, ozone, and osmotic stress as well as higher germination rate and grain weight [AA regeneration from transient MDHA is catalysed by the FAD (flavin adenine dinucleotide)-dependent MDHAR (E.C.1.6.5.4) enzyme using electrons donated by NADPH ,180,181. MDHAR3) ,184. Oth MDHAR3)  suggesten weight ,187.3) and cytosol  [1 is involved in plant responses to high light, high temperature, and MV2+-induced oxidative stresses [2 in plant protection against Polyethylene glycol (PEG), salt, drought, and ozone [3 in high light response conference [What is certain in the reduction process of DHA to AA is the DHAR utilisation and transformation of the reduced GSH as an electron donor, which eventually maintains the redox state of plant cells ,188. Thi, DHAR2) ,188. A pstresses ,191,192,nd ozone ,193, or nference .Non-enzymatic antioxidants are low molecular weight molecules with specific structures, chemical properties, and locations. Possessing a pivotal role in eliminating free radicals by virtue of donating electrons or hydrogen, these compounds are divided into two groups ,26: (1) As one of the most abundant antioxidants in plant systems, ascorbic acid ((AA),vitamin C) has a critical function in plant growth and development . The metjaponica rice genotypes with high DHAR expression had increased concentrations of AA and higher crop productivity than wild-type rice. Based on these results, one may speculate on the existence of a direct relationship between AA pools and the grain yield and decreased ROS in plants lacking sufficient environmental adaptation. DHAR down-regulating tobacco plants have shown lower ascorbate levels, signifying the importance of DHAR proteins in AA regeneration [Arabidopsis thaliana) mutant without all three DHAR proteins and a wild-type plant with respect to AA regeneration levels, as well as of growth and development. In addition to the analysis of DHAR mutants that show DHARs are required to tie the H2O2 metabolism with GSH oxidation, this finding has brought uncertainty in the efficiency of DAHRs in AA recycling [DHA reduction to AA is mediated by the coupled activity of DHAR and GSH (hydrogen donor) oxidation . There aneration . An opponeration . Despiteneration  demonstrecycling .Ddhar mutant identified that the DHAR proteins contributed to increased growth and ascorbate homeostasis under low-light stress [Ddhar mutant and wild-type plants elevate their ascorbate concentration, with a minor reduction seen in the ascorbate accumulation of Ddhar compared to the wild type that was ascribed to an ascorbate degradation product, the threonate [Ddhar pad2-1 quadruple mutants. This was attributed to lower photochemical activity, bleaching, and increased accumulation of threonate [Physiological studies demonstrate that DAHRs function properly in the course of ascorbate oxidation and increase in ascorbate pool size in a state of high-light stress conditions ,207. A st stress . Under hhreonate . In agrehreonate , a recenhreonate . Howeverhreonate . This sthreonate ,205, rechreonate .Even though AA is naturally synthesised in the inner membrane of mitochondria, it may also be found in the cytosol, cell walls, chloroplasts, vacuoles, and apoplasts . AA perftripeptide, α-glutamylcysteinyl glycine present in all cell parts, including cytosol, chloroplast, endoplasmic reticulum vacuole and mitochondria, with the highest amount reported in the chloroplast [2O2 through the ascorbate–glutathione cycle and the glutathione cycle [1 phase and cell differentiation and death. It is believed that this compound is one of the primary sources of non-protein thiols in most plant cells. Given the high reactivity domain of thiol (Sulfhydryl -SH) groups of the low molecular weight compound, GSH, these substances are tapped into many chemical reactions [Glutathione (GSH) is an intact oroplast ,212. Thene cycle . Furtherne cycle . The anteactions . The nuceactions . This reeactions ,214. Ano2O2 and OH• to generate stable free radicals by adducting to prolines and hydroxyproline derivatives such as as 4-hydroxyproline and 3-hydroxyproline [2O2 or O2•−. In Brassica juncea plants, proline involvement could dramatically suppress the production of 1O2 in the thylakoids [1O2 quenching feature of prolines has been suggested to help stabilise proteins, DNA, and membranes [Armeria maritime (Mill.) Willd [The amino acid proline has also been regarded as a non-enzymatic antioxidant in plant systems that can easily counteract the harmful effect of ROS ,215,216.yproline ,220. Furyproline  indicateylakoids . The 1O2embranes . One of embranes . Sharma embranes  reported.) Willd . Multipl.) Willd ,226, SOD.) Willd , or enzy.) Willd , and AA-.) Willd .α-tocopherol with its three methyl substituents is considered to be one of the most reactive antioxidants among the four well-known lipoperophilic isomers of tocopherol  [, and δ) . Regardl, and δ) ,231,232., and δ) . After t, and δ) ,234. Giv, and δ) . There a, and δ) ,238,239.1), and two precursors (homogentisic acid and phytyl diphosphate) compounds are required for tocopherol biosynthesis [α-tocopherol from γ-tocopherol [1 deficiency would result in a significant reduction in tocopherol synthesis [A combination of five catalysing enzymes  has identified them as protectors of biological membranes [α-tocopherol represses the chain propagation step in the lipid peroxidation (LPO) cycle [•) is often driven by coenzyme Q [1O2 neutralisation [α-tocopherols are capable of protecting seed storage lipids, activating plant defence responses, functioning in membrane stability, or involving in seedling germination, transcript regulation and intracellular signalling [The way tocopherols are utilised to indirectly protect the structure and function of photosystem II (PSII) is to establish a chemical reaction with Oroplasts . The preembranes ,244. On O) cycle ,152,245.enzyme Q  or AA anenzyme Q ,248. Morlisation ,250,251.lisation , α-tocopgnalling ,253,254.Carotenoids are tetraterpene antioxidants found in the plastids of photosynthetic and non-photosynthetic tissues of plants ,256 and Flavonoids, anthocyanins, tannins, hydroxycinnamic acid esters, and lignins are phenolic compounds that belong to the secondary metabolites arising from the phenylpropanoid pathway in plant tissues . The enz1O2 on the outer envelope of the chloroplastic membrane [Flavonoids are a large group of secondary metabolites that are widely distributed among plants and have multiple roles, including contributing to colouring of flowers, seeds and pollen grains, helping in pollination, germination and pollen tube growth, and auxin transport , as wellmembrane ,270. Themembrane . Aside f+ donor. Polyphenols have been shown to chelate intermediate metals such as iron, thus preventing the Fenton and/or Haber–Weiss reaction [Considering the fundamental role of phenolic compounds in reducing or inhibiting lipid auto-oxidation, eliminating oxygen free radicals, quenching singlet oxygen or decomposing peroxides, they have also been known as essential antioxidants responsible for protection against proliferation and advancement of the oxidation chain and defence against reactive oxygen species . The antreaction . Numeroureaction .2O2 perception and transduction by Wu et al. [2O2 generated in plastids at the expense of activating the defence gene expression [2+ [2), nitroxyl anion (NO−), nitrosonium cation (NO+), peroxynitrite (ONOO−), nitrate (NO•3), nitric oxide (NO•), and nitric dioxide (NO•2) [2O2 during plant hypertension responses. Usually, the crosstalk of ROS and RNS accompanies, by direct or indirect modulation, antioxidant enzymes [ROS signals are initially perceived and transduced in plants before being translated into sufficient responses . The oxiu et al. . Proteinu et al. . This pru et al. ,279. ROSu et al. . Also, Ru et al. . As suchpression . There asion [2+ ,282,283.e (NO•2) ,286,287.e (NO•2) . Delledoe (NO•2)  were the enzymes  and may  enzymes ,291.Increased ROS generation as a result of different environmental stresses has always been naturally responded to in biological systems in multiple ways under the disguise of antioxidative defence mechanisms ,292. In Cucumis sativus (cucumber) with a consortium of three PGPR strains could induce systemic tolerance in drought-imposed plants by maintaining root vigour, photosynthetic performance, and increased generation and activity of SOD, CAT and prolines in the leaves. In Lycopersicon esculentum (tomato) plants treated by a Bacillus cereus AR156 supernatant, an appreciable enhancement to drought stress was reported by Wang et al. [Panicum turgidum Forssk plants has been attributed to the Arbuscular mycorrhizal fungi contribution by modifying photosynthetic and antioxidant pathways [Solanum tuberosum L.) plants treated with Bacillus subtilis HAS31 by maintaining higher photosynthetic processes, total soluble sugars, proteins and prolines with elevated activity of POX, CAT, and SOD [Agrostis palustris by lowering MDA accumulation and developing osmotic adjustments associated with higher synthesis and accumulation of compatible solutes, including soluble sugars, free amino acids, proteins, and not least the non-enzymatic prolines. Plant inoculation with PGPRs adds up to the available quantity of proline in plant systems under stress conditions [Zea mays L. after inoculation with P. fluorescens under drought stress [Lavandula dentate plants treated with PGPR B. thuringiensis was recorded under drought conditions, which was attributed to the increased shoot proline contents [Lycopersicon esculentum Mill cv. Anakha) plants after exposition to Bacillus polymyxa [The PGPRs function in inducing plant tolerance against abiotic stresses, such as salt, drought, and extreme temperatures, is of key importance in alleviating the adverse effect of climate change on sustainable crop production . As outlg et al. . The indpathways . Similar and SOD . In anot and SOD , it was nditions . As sucht stress . A noticcontents . Also, tpolymyxa . Similarpolymyxa .Trichoderma afroharzianum strain T22 has been shown to enhance tomato (Solanum lycopersicum L.) seed germination under biotic stress conditions by alleviating oxidative damage in salt-stressed seedlings [Trichoderma longibrachiatum strain T6 induced the tolerance of wheat seedlings to salt stress through upregulation of SOD, CAT and POX genes, improving the antioxidant defence machinery. Compared to uninoculated rice genotypes Swarna and Swarna sub-1 under severe drought conditions, the PGPFs  inoculated rice genotypes showed higher activity of SOD, POX, and CAT [The application of eedlings . Similareedlings  demonstr and CAT . Table 2ROS are natural byproducts of many metabolic pathways or their respective electron transport activities in different cellular compartments. Thanks to cellular auto adjustment mechanisms, there is always a homeostatic balance between ROS production and removal machinery which make ROS less harmful to plants in normal environments. However, prolonged environmental stresses such as salinity, chilling, drought, water deficit, and UV radiation can severely exacerbate the production level of ROS by disrupting the natural cellular homeostasis, changing the ROS role as a signalling molecule to a damaging oxidant capable of harming lipids, proteins and DNA. Plants have developed different enzymatic and non-enzymatic antioxidative pathways to alleviate the adverse effects of oxidative damages. The term Induced Systemic Tolerance (IST) has been coined in this review to capture the concept of microbial modulation of enhanced plant tolerance against abiotic stresses through inducing physical and chemical alterations. The beneficial microbiome related to roots and plant tissues suppresses plant stress by a variety of processes. As an example among them, PGPRs are capable of enhancing plant micronutrient uptake, regulating phytohormones homeostasis, and stimulating the plant immune system against biotic and abiotic stresses.Insofar as the results of ROS experiments under a variety of imposed stresses are concerned, it is not entirely clear how plants deal with stress-induced ROS homeostasis disruption and the consequent cellular degradation. There are still many unsolved questions regarding the simultaneous antioxidative activities of enzymatic antioxidants and their non-enzymatic counterparts. These uncertainties can be considered as hints for further studies in ROS formation and ROS removal machinery. In light of ROS studies, the use of state of art analytical and/or imaging techniques might provide a broader insight into understanding the complex antioxidant networks involved in plant responses to elevated ROS levels. Further, the combined application of advanced functional genomics, proteomics and metabolomics might be helpful in ROS network elaboration. Transgenic techniques can also be used for producing plants with high tolerance to multiple stresses.


Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are foodborne pathogens that cause hemolytic uremic syndrome and fatal infant diarrhea, respectively, but the characterization of these bacteria from imported food in China are unknown. A total of 1577 food samples from various countries during 2015–2021 were screened for STEC and EPEC, and the obtained isolates were tested for antimicrobial resistance and whole genome sequencing analysis was performed. The prevalence of STEC and EPEC was 1.01% (16/1577) and 0.51% (8/1577), respectively. Antimicrobial resistances to tetracycline (8%), chloramphenicol (8%), ampicillin (4%), ceftazidime (4%), cefotaxime (4%), and trimethoprim-sulfamethoxazole (4%) were observed. The antimicrobial resistance phenotypes corresponded with genotypes for most strains, and some resistance genes were related to mobile genetic elements. All 16 STEC isolates were eae negative, two solely contained stx1 (stx1a or stx1c), 12 merely carried stx2 , and two had both stx1 and stx2 . Although they were eae negative, several STEC isolates carried other adherence factors, such as iha (5/16), sab (1/16), and lpfA (8/16), and belonged to serotypes  or STs , which have caused human infections. All the eight EPEC isolates were atypical EPEC; six serotypes and seven STs were found, and clinically relevant EPEC serotypes O26:H11, O103:H2, and O145:H28 were identified. Two STEC/ETEC (enterotoxigenic E. coli) hybrids and one EPEC/ETEC hybrid were observed, since they harbored sta1 and/or stb. The results revealed that food can act as a reservoir of STEC/EPEC with pathogenic potential, and had the potential ability to transfer antibiotic resistance and virulence genes.Shiga toxin-producing  E. coli (STEC) are major foodborne pathogens, which can cause watery diarrhea, bloody diarrhea, or hemorrhagic colitis, and even life-threatening hemolytic uremic syndrome (HUS). It is estimated that STEC leads to 2,801,000 acute illnesses, 3890 HUS cases, and 230 deaths worldwide [E. coli bacteria have emerged and the human illnesses caused by these bacteria increased gradually each year from 2010 to 2019, and at present they rank 5th among all major foodborne pathogens. It is also worth noting that, among the emerging pathogenic E. coli, STEC infections increased most markedly [Shiga toxin-producing markedly ,5.stx1 and stx2 genes, respectively. Stx1 includes three subtypes , while Stx2 contains at least seven subtypes . Stx subtypes display dramatic differences in disease-causing potency. Stx2a (with or without Stx2c) and Stx2d are regarded to be more potent than other subtypes and highly associated with HUS [eae gene located on the locus of enterocyte and effacement (LEE), is described in highly virulent isolates; it produces the typical “attaching and effacing” lesion on intestinal mucosa [iha, saa, sab, paa, efa1, ompA, lpfA, and toxB [ehxA, espP, etpD, katP, and subA, usually located in a virulence plasmid were also reported [The main virulence factors of STEC are Shiga toxins, which can be divided into two subfamilies, Stx1 and Stx2, encoded by with HUS . Howeverwith HUS . The prol mucosa . This prand toxB . Other preported ,10. Althreported .E. coli (EPEC) can cause infant fatal diarrhea mainly in developing countries, and are isolated from various kinds of food and animals. Previous studies demonstrated that in China, EPEC is one of the most common diarrheagenic E. coli [E. coli strains that are positive for eae but negative for stx. In addition to eae, typical EPEC (tEPEC) strains carried the EPEC adherence factor (pEAF) plasmid that encodes the bundle forming pili (BFP), while atypical EPEC (aEPEC) do not possess this pEAF. A number of genes have been identified as being of particular importance for the pathogenicity of EPEC, including the eae gene; espA-B (encoding type III secretion system proteins); tir (encoding the translocated intimin receptor), katP ; etpD (associated with a type III secretion system); toxB (the adherence-associated gene); iha (encoding the IrgA homologue adhesin); and saa (encoding an auto agglutinating adhesin) [Enteropathogenic  E. coli ,12 and w E. coli ,14. EPECadhesin) ,16,17.The pathogenicity of STEC and EPEC is complex, thus the virulence potential should be assessed not only based on serotypes, but also other virulence associated markers, for instance, virulence genes, virulence gene subtypes, and phylogenetic markers. Whole genome sequencing (WGS) reveals the entire spectrum of pathogen information; moreover, the phylogenetic relationship between strains from different sources and geographic regions can be deduced by WGS.E. coli has become a major public health threat all over the world [The increase in antimicrobial-resistant he world . AntimicE. coli in foods.With the accelerating globalization of food trade, STEC and EEPC can be transmitted all over the world through contaminated food. Until recently, there has been no report on the characterization of STEC and EPEC from imported food in China. Therefore, we isolated these bacteria from food imported from different continents, and performed the antimicrobial resistance and whole genome sequencing analysis, so as to provide technical support for food safety investigations and monitor the emergence of stx and eae, respectively. A total of 16 (1.01%) food samples were confirmed to be contaminated with STEC, and 8 (0.51%) food samples contaminated with EPEC  and 41 (2.60%) were tested positive for ith EPEC . The STEThe antimicrobial resistance of 16 STEC and 8 EPEC strains is presented in AMP, ampicillin; CAZ, ceftazidime; AMS, ampicillin-sulbactam; IPM, imipenem; TET, tetracycline; NAL, nalidixic acid; ERY, erythromycin; FOX, cefoxitin; CHL, chloramphenicol; CTX, cefotaxime; CFZ, cefazolin; GEN, gentamicin; T/SUL, trimethoprim-sulfamethoxazole; AZI, azithromycin; and CIP, ciprofloxacin.tetA + tetB and tetA, respectively; two chloramphenicol-resistant strains  carried chloramphenicol-resistant genes, catA1 and cmlA1, respectively; one ampicillin-resistant isolate (1509-1) carried the blaTEM-1B gene, which encodes resistances to β-lactams and is linked to extended spectrum β-lactamases (ESBLs) production, thus this isolate also showed intermediate resistance to cefazolin; one trimethoprim-sulfamethoxazole-resistant isolate carried the respective sul3 and dfrA12 genes; and one gentamicin-resistant isolate carried multiple gentamicin-resistant genes, including aadA1, aph(4)-Ia, aac(3)-IV, aph(6)-Id, and aph(3″)-Ib.All the antimicrobial resistant strains carried the respective resistance genes, except for one strain (816b) that showed resistance to ceftazidime and cefotaxime, while no resistance genes, such as the extended spectrum β-lactamase genes, were found . Two tetsul1, but did not show resistance or intermediate resistance to trimethoprim-sulfamethoxazole. The strain 1509-1 carried the ciprofloxacin resistance gene qnrS1, thus the ciprofloxacin MIC reached 0.25, while for other strains it was 0.03 -Ib and aph(6)-Id were on contig 321; aac(3)-IV and aph(4)-Ia were on contig 279; the co-occurrence of aadA1, cmlA1, and sul3 on contig 102; aadA8b, aadA2, and dfrA12 on contig 103 were found for 1095a; and for strain 1509-1, the co-occurrence of qnrS1 and blaTEM-1B were observed on contig 54. Furthermore, the relations of these antimicrobial resistance genes to mobile genetic elements were evaluated. The aac(3)-IV and aph(4)-Ia were associated with insertion sequence ISEc59. The gene blaTEM-1B was related to insertion sequence ISKpn19 while qnrS1 was related to unit transposon Tn2.The multiple antimicrobial resistant strains also had multiple resistance genes. The location of these resistance genes was evaluated. For 1053l-2, the stx1 (stx1a or stx1c), 12 merely carried stx2 , and two had both stx1 and stx2  , sab (1/16), and lpfA (8/16), were found. Six isolates carried the hemolytic gene ehxA , and all belonged to phylogroup B1. The most widespread genes that were detected in nearly all the STEC isolates were terC (tellurium ion resistance protein), traT (outer membrane protein complement resistance), iss , and ompT (outer membrane protease). Some isolates carried toxins, for instance, the sublitase toxin subA (4/16), EAEC heat-stable enterotoxin I astA , colicin ia (cia) (7/16), colicin B (cba) (3/16), colicin M (cma) (4/16), colicin ib (cib) (4/16), colicin E1 (cea) (4/16), and colicin H (mchB) (1/16). Some isolates had the enteroaggregative immunoglobulin repeat protein encoding genes, air (4/16) and epeA (4/16), and the plasmid-encoded extracellular serine protease gene espP (4/16). One STEC isolate had ETEC specific genes sta1 (heat-stabile enterotoxin ST-Ia) and stb (heat-stabile enterotoxin II), and the other one carried sta1.Among the 16 STEC strains, two solely contained + stx2c) . All theterC, tir, espF, espA, and nleB. More than half the EPEC isolates harbored ehxA (5/8), ompT (5/8), iss (6/8), traT (7/8), gad (6/8), espB (6/8), nleA (5/8), tccP (5/8), espJ (4/8), etpD (4/8), and nleC (4/8). Apart from eae, some isolates also had other adherence associated genes, including afaD (2/8), efa1 (1/8), and iha (1/8). One isolate also had the ETEC-specific gene sta1.None of the eight EPEC isolates had EAF, and thus were aEPEC. All the EPEC isolates had the following genes, including Among the 16 STEC isolates, 10 serotypes were found: O142: UN, O8:H19, O79:H40, O15:H45, O163:H19, O130:H11, O100:H30, O43:H2, O175:H21, and O128:H2. Ten STs were identified: ST1112, ST360, ST10, ST1011, ST679, ST297, ST993, ST937, ST223, and ST25. The serotypes corresponded with the STs, and five, two, and two and one serotypes (STs) belonged to phylogroups B1, A, E, and C, respectively .Regarding the 8 EPEC isolates, 6 serotypes were observed: O26:H11, O145:H28, O103:H2, O56:H6, O107:H7, and O153:H19, and 7 STs were found, including ST137, ST17, ST48, ST6323, ST7583, ST517, and one newly identified ST12360 named by EnteroBase. Serotype was highly correlated with ST, except for O26:H11, which belonged to different STs. Three, one, one, one, and one STs belonged to phylogroups B1, B2, E, D, and A, respectively .lpfA, ehxA, iha, subA, espP, and epeA. Certain stx subtypes were found to be associated with food type or serotype. For instance, stx2e was mainly found in pork, stx2a and stx2d were more commonly found in beef. Genotype stx1c + stx2b was observed in O128:H2 from mutton. There was no apparent correlation between country, food type, serotype, and ST; however, five isolates from beef in country A showed highly similar characteristics, regarding serotype, ST, and virulence genes.The phylogenetic tree based on cgMLST showed that the strains in this study were diverse. Regardless of the pathotype (STEC/EPEC), serotype, or ST, all the strains were grouped according to their phylogroups, as isolates belonging to different phylogroups  clustered together . Some viThe characterization of STEC and EPEC from imported food in China has not been reported previously. Therefore, in this study, we isolated these bacteria from various kinds of food, mainly animal source frozen meat, imported from different countries, and studied their antimicrobial resistance, genetic diversity, and virulence profile.stx, the low levels of STEC in the samples, or the presence of free Stx phages can lead to the failure of STEC isolation [TM STEC media, which has inhibitory effects on uncommon non-O157 serotypes [E. coli differentiating agar, e.g., TBX or MacConkey, to facilitate the isolation.The overall PCR screening rates for STEC (2.16%) and EPEC (2.60%) and the isolation rates for STEC (1.01%) and EPEC (0.51%) in this study were both lower than in the previous reports ,19,20,21solation . Currenterotypes ,24. TherE. coli has become a worldwide issue. Overall, the antimicrobial resistance is not severe in the studied isolates. Resistance to β-lactam, tetracycline, chloramphenicol, and sulfonamide are most frequently detected in this study, quite different from the situation in China, for which streptomycin (46.94%) and ciprofloxacin (20.41%) resistances were most common for the STEC from retail meats [Drug resistance in il meats . It is wil meats ,26. The stx subtype, virulence gene, phylogroup, and ST. We detected several STEC serotypes that have been related to human infections, such as O8:H19, O128:H2, O100:H30, and O130:H11, based on previous reports [stx2 subtypes  were reported to be linked with serious human diseases [stx2e in this study should not be neglected. Specifically, the two stx2e-positive O8:H19-phylogroup C isolates belonged to ST360, which is principally found in human diseases based on the EnteroBase database, and possessed adhesion and colonization factor lpfA; thus, they should also be attached importance. Strains belonging to E. coli phylogroups B1, C, and E2(O157) are often pathogenic and of interest to medical research [lpfA, ehxA, iha, subA, espP, and epeA. Strains positive for these virulence genes may have a pathogenic potential. Iha is an adherence conferring protein and a siderophore receptor distributed among STEC, and it may be involved as an alternative mechanism of adhesin in eae-negative STEC strains [subA and subB genes, is harbored by the O113:H21 outbreak strain and other eae-negative strains associated with human diseases [stx2a and eae/aggR were assessed to be on the highest level for their estimated potential to cause diarrhea, bloody diarrhea, and HUS [stx2a + iha + subA + ehxA + lpfA may have a high pathogenic potential.The virulence potential of STEC should be assessed based on various factors, including serotype,  reports  and the diseases . Nine is strains . Subtilastx genes are carried by lambdoid phages, which are highly mobile genetic elements, and the horizontal transfer and the dissemination, as well as the loss of the stx genes, are facilitated [stx genes and bfp genes during passage through a host or the environment or after culture in the laboratory; thus, these bacteria should not be underestimated. Clinically relevant serotypes O26:H11 and O103:H2 were found for two EPEC isolates in phylogroup B1, thus posing a great health threat. Although sharing the same serotype, O26:H11, the virulome was quite different for those belonging to B1 (12360) and A (ST48), indicating that their virulence potential may be distinct.None of the isolates obtained in this study were typical EPEC, which is in line with the previous results on various kinds of food ,31. The ilitated . The aEPsta1 and/or stb, which were the virulence markers of enterotoxigenic Escherichia coli (ETEC), and thus were STEC/ETEC and EPEC/ETEC hybrids, respectively. The E. coli genome is dramatically plastic and this accelerates the adaptation of this species into various environments, which provides numerous opportunities for new variants to emerge via the gains and losses of genes. Recently, hybrid E. coli pathotypes are representing emerging public health threats with enhanced virulence from different pathotypes. The most notorious hybrid was the STEC/EAEC strain O104:H4, which caused a large outbreak with numerous HUS cases and deaths in Germany in 2011 [stx and/or sta/stb genes by the independent acquisition of the Stx-phages and/or plasmids carrying these genes lead to the emergence of STEC/ETEC hybrids. The STEC/ETEC hybrid (826a) identified in this study had the same virulence characteristics as the STEC/ETEC strain causing diarrhea in an 82-year-old patient in Sweden. For instance, both strains belonged to O100:H30, ST993, and phylogroup A and carried stx2e + sta1; therefore, it also had a high potential to cause human diseases [st variants, sta1 and stb, had a distinctly mucoid and thread-drawing morphology on the nutrient agar. The genetic mechanism should be further explored.Notably, we found two STEC and one EPEC carrying  in 2011 . STEC/ET in 2011 ,36, and  in 2011 . The hordiseases . It is aE. coli are usually located on plasmids or prophages, the emerging E. coli hybrids in foods may have enhanced virulence and thus should be attached with great importance.The contamination frequency of STEC and EPEC from imported foods during the period study was relatively low, and the antimicrobial resistance was not severe. However, multidrug-resistant isolates harboring the respective multiple antimicrobial resistance genes, which are related to mobile genetic elements, were identified, and thus have the potential to transfer antibiotic resistance. Some isolates carried the virulence factors described in pathogenic strains and thus have a high pathogenic potential. STEC/ETEC and EPEC/ETEC hybrid strains were identified. Since the virulence genes of n = 1066), frozen pork (n = 172), fresh and frozen aquatic products (n = 198), frozen mutton (n = 102), milk products (n = 27) and frozen chicken (n = 12). The samples were collected according to the National Sampling Plan by authorities and sent directly to the laboratory for testing.A total of 1577 samples from different kinds of foods imported from different continents were collected from the containers or airplanes at Shanghai port from 2015 to 2021, including frozen beef (stx and eae according to the method of USDA FSIS MLG-5 [stx (stx1 and stx2) primers, 1 μM of eae primers, 0.16 μM of 16 s primers, 0.25 μM of stx1 probe, 0.25 μM of stx2 probe, 0.2 μM of eae probe, 0.1 μM of 16 s probe, and 5 μL of DNA template. The PCR reaction conditions were as follows: 95 °C pre-denaturation for 2 min, followed by 40 cycles of 95 °C denaturation (5 s) and 60 °C (34 s) annealing. The EHEC O157: H7 strain EDL933 and nucleotide-free water were included as the positive and blank controls, respectively. For the stx positive samples, the enrichment was streaked onto three solid media: CHROMagar™ STEC agar , MacConkey agar , and TBX agar ; for the eae positive samples, the enrichment was streaked onto two solid media: MacConkey agar  and TBX agar . Approximately 40 colonies with E. coli morphology were picked from the above agars for further stx or/and eae detection. Each stx/eae-positive isolate was confirmed to be E. coli by the API 20 E system .The enrichment method was based on the ISO/TS 13136:2012 . BrieflyIS MLG-5 . The 25 The antimicrobial resistance patterns of the 24 isolates were determined by the micro-dilution method, following the standards of the Clinical and Laboratory Standards Institute . The folGenomic DNA from each strain was extracted from overnight cultures using the bacterial genomic DNA extraction kit , following the manufacturer’s instructions. The DNA concentration was determined using the Qubit™ dsDNA HS Assay Kit . The DNA integrity was determined by 1% gel electrophoresis. The qualified DNA was stored in −20 °C until use.® Ultra™ DNA Library Prep Kit for Illumina . The genomes of the strains sequenced used an Illumina HiSeq sequencer , with the 2 × 150 bp pair-end chemistry according to the manufacturer’s instructions, at approximately 350X average coverage. The sequencing reads were quality-control processed and quality evaluated with FastQC . The processed reads were assembled de novo with SPAdes (version: 3.12.0) in “careful mode”.The library was constructed using NEB Nexthttp://www.genomicepidemiology.org, accessed on 21 October 2021) for E. coli as part of their web-based serotyping tool , with a similarity of 85% and minimum length of 60%; the ST of each strain was in silico analyzed using the MLST E. coli#1 approach  provided by the Center for Genomic Epidemiology ; and the phylogroup was determined by the using the E.coli phylotype analysis available in the EnteroBase website . The virulence genes present in each strain were determined using the genes deposited in the Center for Genomic Epidemiology for E. coli as part of their web-based VirulenceFinder 2.0 tool , with the similarity of 90% and minimum length of 60%; the resistance genes present in each strain were identified using the genes deposited in the Center for Genomic Epidemiology for E. coli as part of their ResFinder 4.1 tool , with a similarity of 90% and minimum length of 60%. The relations of the antimicrobial resistance genes to the mobile genetic elements were evaluated using the mobile element finder v1.0.3 tool provided by the Center for Genomic Epidemiology .The serotype of each strain was determined using the genes deposited in the Center for Genomic Epidemiology  analysis. The core genome of all the PHY 1.12 . Specifioutgroup . The phyoutgroup ,45.E. coli strains used in this study are available in GenBank under the following accession numbers: JAIPMO000000000; JAJIYZ000000000; JAIPMP000000000; JAIPMQ000000000; JAJIZA000000000; JAJIZB000000000; JAJIZC000000000; JAIPMR000000000; JAIPMT000000000; JAJIZD000000000; JAIPMU000000000; JAJIZE000000000; JAJIZF000000000; JAIPMV000000000; JAIPMW000000000; JAIPMX000000000; JAIPMY000000000; JAIPMZ000000000; JAIPNA000000000; JAJIZG000000000; JAIPNB000000000; JAJIZH000000000; JAJTCC000000000; and JAJTCB000000000.The draft genome sequences of the


Inorganic phosphate (Pi) starvation is an important abiotic constraint that affects plant cellular homeostasis, especially in tropical regions with high acidic soil and less solubilizable Pi. In the current work, oil palm seedlings were hydroponically maintained under optimal Pi-supply and no Pi-supply conditions for 14 days, and metabolites were measured by gas chromatography–mass spectrometry (GC–MS), from leaves and roots, after seven and 14 days of treatment, to investigate biochemical pathways in relation to P-utilizing strategy. After seven days of limited Pi, plant leaves showed increased levels of most soluble sugars, and after 14 days, the sugars’ level decrease, except for erythritol, mannose, fructose, and glucose, which showed the highest levels. Rather in root samples, there were different but overlapping alterations, mainly on sugars, amino acids, and organic acids. The leaf sample was shown to have the highest response of sugars with myo-inositol playing a vital role in the redistribution of sugars, while maltose levels increased, indicating active degradation of starch in the root. High levels of glycerol and stearate in both roots and leaves suggest the metabolism of storage lipids for cellular energy during Pi-deficient conditions. Orthophosphate (Pi) is an important macronutrient that plays a central role in plant metabolic processes predominantly in photosynthesis and respiration. In spite of its importance, most of the Pi in the soil is unavailable for plant uptake because of adsorption, precipitation, or conversion to organic forms . TropicaPlant Pi-starvation response is a dynamic process and has a complex regulon that involves several genes responding to Pi-loss stimulus ,10. Pi iHigh-throughput omics approaches, including genomics, transcriptomics, proteomics, and metabolomics have been widely used for a comprehensive characterizing of molecular mechanisms involving multiple plant physiological and biochemical processes. This provides an understanding of the various layers of the cellular responses to environmental perturbations delineating the function of biological networks . Plant mRecent studies on plant P-deficiency reported the abundance of di and trisaccharides in Stylosanthes roots and a significant reduction in the levels of phosphorylated metabolites like P-containing sugars, suggesting the enhancement of internal P utilization in low-Pi-stress stylo roots , in LupiOil palm is a monoecious oil-producing tree belonging to the family of flowering plants, Arecaceae. It has a ubiquitous application in food ingredients, industrial produce , and, recently, in biofuel prospecting. Oil palm has the highest oil output per hectare among all oil-producing plants ; hence, In this study, we profiled metabolites from the leaves and roots of oil palm after two weeks of phosphate withdrawal using GC-MS analysis. To reduce and visualize the complex metabolomics datasets, multivariate statistical tools, including supervised partial least-squares-discriminate analysis (PLS-DA), and unsupervised principal component analysis (PCA), were employed. From the GC-MS profile analysis, a broad range of phosphate-deficiency responsive metabolites were quantified, and the variations in several metabolic pathways were highlighted. Moreover, to unravel the integrative biochemical networks of the oil palm in response to Pi deficiency, this work provides a better framework for understanding the mechanisms that govern cellular response to phosphate stress in oil palm leaves and roots at the molecular level.2PO4 (P) as control or without (NP). Leaves and roots were harvested at two different time points, after one week (seven days) and two weeks (14 days) of treatments. We observed the establishment of P-starvation in the seedlings with higher root mass in the P-deficient treatment (-Pi), as compared to the control (+Pi)  A. Figureol (+Pi) A. Conseqol (+Pi) .A GC-MS metabolomic method was employed to compare the metabolite profile of leaves and roots of seedlings subjected to P-deficient and P-sufficient treatments at the two different time points (NP1 and NP2). Metabolites were extracted from leaf and root samples, in three replicates, for each experimental group, i.e., P-deficient and P-sufficient seedlings. A total of about 90 metabolites were detected in both leaves and roots of oil palm seedlings. After the filtering of repetitive metabolites and some noises, 38 and 39 metabolites were finally quantified from the different treatments in leaves and roots, respectively. To display a visual pattern of metabolites concentration, a heatmap was generated to show metabolite changes under P-deficiency and P-sufficient conditions at different time points. From the heatmap, leaf and root metabolic profiles were compared from seedlings subjected to either seven days (NP1) or 14 days of no P supply (NP2) to control. Increased levels of metabolite under a given treatment condition are represented by red and the decreased level is indicated by green color. Our result showed up-accumulation of 26 metabolites in the leaves of seedlings subjected to seven days (one week) of Pi-deficiency (LNP1) and 10 up-accumulation in the leaves of seedlings after 14 days (two weeks) of no P supply (LNP2). Under the Pi sufficient condition, eight metabolites showed increased levels in both the first week (LP1) and second week (LP2). Out of the 26 metabolites that showed increased levels in LNP1, 18 , glucopyranose, erythritol, mannose, isoleucine, glyceric acid, malic acid, mannonic acid, glycerol, proline, xylose, succinate, and galactopyranoside) are significantly higher than in LP1. Interestingly, only 10 metabolites from LNP2 treatment  showed higher levels than in LP2. It is obvious that LNP1 seedlings resulted in more metabolites being elevated in production, compared to the controls. Some metabolites were commonly being detected in both LNP1 and LNP2 treatments but at different levels. We discovered that seven metabolites  showed higher levels in LNP1 than in LNP2. The increased levels of two metabolites (fructose and glucose) were only detected in LNP2 while five metabolites  were only detected in LNP1 AIn root samples, we detected 39 metabolites from the P-deficient (RNP) and Pi-sufficient seedlings (RP). Fifteen metabolites each from seven days (RNP1) and 14 days (RNP2) days of no phosphate treatment showed up-accumulation in the root. On the other hand, six and 14 metabolites were up-accumulated in the root sample after seven days (RP1) and 14 days (RP2) of P-sufficient conditions, respectively. At the first week of no phosphate supply (RNP1), 10 metabolites’  levels were up-accumulated, compared to P-sufficient root (RP1) samples. In the second week of P-deficient roots (RNP2), eight metabolites  were significantly higher in RNP2 than in P-sufficient roots (RP2). Two of the eight metabolites  were specifically up-accumulated in the RNP2 sample. In contrast, six metabolites  were distinctively present only in RNP1 treatment B.p < 0.05). The PCA for the first and second components generated do not show a clear pattern between different treatments  was performed on the dataset with MINITAB 14 statistical software, using correlation  database because of the depth of rice metabolic data; it is also a monocotyledonous plant, like oil palm. The leaf (38) and root (39) metabolites were mapped to 40 , six metabolites  from leaf samples were significantly assigned to the first 4 metabolic pathways: Inositolphosphate metabolism, Phosphatidylinositol signaling system, Ascorbate and aldarate metabolism, and Galactose metabolism . Meanwhile, in the ascorbate and aldarate metabolism, it is also a substrate for uridine diphosphate glucuronic acid (UDP-glucuronate) . D-glucuFour metabolic pathways were mapped in the root samples: amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, galactose metabolism, and fructose and mannose metabolism .These pathways were related to eleven metabolites  from the root sample data. Like in the leaf sample, all the metabolites that were mapped in the root are carbohydrate metabolism related. In the amino sugar and nucleotide sugar metabolism, D-fructose, which was highly accumulated in NP2, serves as a precursor for the production of β-D-fructose-6P, while mannose with higher levels in RP1 was mapped as a precursor to GDP-L-galactose-1P and GDP-L-glucose through GDP-mannose intermediate.Additionally, D-xylose, a pentose sugar mostly found in lignocellulose, had a high accumulation in RNP2, where it can be channeled to power the alternative pathway to glycolysis, like PPP, to keep producing energy for the plant, despite the decrease in Pi A.Plants react to Pi stress through defined molecular , biochemSoluble sugars show inconsistent levels in both leaf and root samples across treatments. Eleven sugars showed an early increase at NP1 in leaves, compared to the control, and then started to diminish at NP2, while only five sugars started to respond with increased levels in leaves at NP2, compared to the control. In general, most sugars were detected in the leaves of P-deficient seedlings earlier. Furthermore, in root samples, six sugars showed an early increase in response NP1, five sugars showed increased levels at NP2, and only one sugar showed an increased level in both NP1 and NP2, compared the to control . Our finThe levels of glycerol and stearate increased under P-deficiency in the leaves . This maIn conclusion, metabolomic responses by GC-MS analysis for oil palm roots and leaves during P-stress showed different but overlapping alterations, mainly on sugars, amino acids, and organic acids. The leaf sample was shown to have the highest response of sugars, with myo-inositol playing a vital role in the remobilization of sugars and maltose showing high degradation of starch in the root. There was a general decrease in protein anabolism products throughout Pii withdrawal treatment. These findings have shed more light on oil palm’s response to P-deficiency.In this study, we used three-month-old GH500 genotype (DxP) oil palm seedlings, which were procured from Sime Derby Seeds & Agricultural Services Sdn Bhd . Seedlings were maintained at Transgenic Greenhouse (TGH) Universiti Putra Malaysia, under controlled climatic conditions of temperature 30 °C /22 °C day/night and a photoperiod of 12 h throughout the treatment period.3, 4.25 mM Ca(NO3)2, 2.1 mM MgSO4, 0.2 mM Fe-Na2EDTA, 36 µM MnSO4, 27 µM H3BO3, 1.56 µM CuSO4, (NH4)6Mo7O24.4H2O, and 1.5 µM ZnSO4.7H2O. After acclimation, the seedlings were equally shared into phosphate (+Pi) sufficient (12 seedlings) and Pi-limited (−Pi) treatment solution (12 seedlings) for two weeks that is when the seedlings were five months old. In the Pi withdrawn treatment solution, the Pi was replaced with 0.97 mM K2SO4. Both leaves and roots were harvested weekly from four seedlings for phosphate analysis, while only three seedlings from the four were used for GC-MS analysis.Twenty-four oil palm seedlings were acclimatized for two months, at TGH, in a hydroponic Cooper 1976 solution. The Cooper 1976 medium solution contains: 1.93 mM KH2PO4, 5.77 mM KNOv/v) and 10 mL of 20% HNO3 (v/v) were added to digest the sample, and the sample solution was heated on a hotplate for 1 h. After cooling, the solution was transferred into a 100 mL flask via a filter funnel, and the volume was topped up to 100 mL with deionized water. Finally, the solution was filtered for the second time, using filter paper No. 2. The final solution was sent for atomic absorption spectroscopic (AAS) for Pi measurement.To estimate phosphate from oil palm leave and root, samples were prepared according to Murphy and Riley’s (1962)  protocolg, at RT, for 20 min. About 400 µL of the supernatant from each tube was transferred into a 1.5 mL Eppendorf tube and evaporated, using a vacuum, for more than 5 h, until the tubes were completely dry. To derivatize the samples, a two-step procedure was followed; 100 µL of methoxylamine hydrochloride in pyridine (20 mg/mL) was added to each tube, incubated for 90 min, at 37 °C, for oximation; and, subsequently, 100 µL of MSTFA was added to each tube incubated at 37 °C, for 30 min, for silylation. GC-MS analysis was carried out in three biological replicates.Fresh samples of leaves and roots were immediately flashed-frozen in liquid nitrogen, for storage, before analysis. Metabolite extraction was carried out according to a method by Lisec et al. (2006) , with mim/z were recorded. Metabolites were detected by blasting of each metabolite’s mass-charge ration against the mass chromatogram database of the National Institute of Standards and Technology (NIST). Each spectral area was normalized sample fresh weight and internal standard concentration; the data were filtered and sorted in an Excel sheet, before statistical analyses.After metabolite derivatization, 0.4 µL of derivatized sample was injected in splitless mode in a Shimadzu-QP2010 Plus GC-MS system  containing ZB-5MS 30 m × 0.25 mm ID × 0.25 µm film thickness , at a constant helium flow rate of 1 mL/min. The sample was evaporated at 250 °C, while interphase and ion source temperatures were set at 250 and 220 °C, respectively. Oven temperature was kept constant at 80 °C, for 3 min, and gradually raised to 325 °C, at a flow rate of 6 mL/min. Mass spectra of 1 scan/0.5 s with ion scanning range of 50 to 750 Before GC-MS statistical analysis, missing values were assumed to be below the limits of detection, and these values were assigned with a minimum compound value. The relative abundance of each metabolite was log-transformed before analysis, to meet normality.p-value (p ≤ 0.05). Multivariate analysis (MVA) for principal component analysis (PCA) was carried out by using MINITAB 14  and partial-least-squares-discriminant analysis PLS-DA by using online software Metaboanalyst  [The phosphate levels’ data were analyzed in SAS software , for mean comparison, using least significant difference (LSD), with er 2020) . Heatmap


NFE2L2, is the master regulator of phase II antioxidant enzymes that protect against oxidative stress and inflammation. NRF2/ARE signaling has been considered as a promising target against oxidative stress-mediated diseases like diabetes, fibrosis, neurotoxicity, and cancer. The consumption of dietary phytochemicals acts as an effective modulator of NRF2/ARE in various acute and chronic diseases. In the present review, we discussed the role of NRF2 in diabetes, Alzheimer’s disease (AD), Parkinson’s disease (PD), cancer, and atherosclerosis. Additionally, we discussed the phytochemicals like curcumin, quercetin, resveratrol, epigallocatechin gallate, apigenin, sulforaphane, and ursolic acid that have effectively modified NRF2 signaling and prevented various diseases in both in vitro and in vivo models. Based on the literature, it is clear that dietary phytochemicals can prevent diseases by (1) blocking oxidative stress-inhibiting inflammatory mediators through inhibiting Keap1 or activating Nrf2 expression and its downstream targets in the nucleus, including HO-1, SOD, and CAT; (2) regulating NRF2 signaling by various kinases like GSK3beta, PI3/AKT, and MAPK; and (3) modifying epigenetic modulation, such as methylation, at the NRF2 promoter region; however, further investigation into other upstream signaling molecules like NRF2 and the effect of phytochemicals on them still need to be investigated in the near future. Oxidative stress is a pathological condition occurring due to an imbalance between the oxidants and antioxidant defense systems in the body. Nuclear factor E2-related factor 2 (NRF2), encoded by the gene  Reactive oxygen species (ROS) and reactive nitrogen species (RNS) production are essential for maintaining the body homeostasis and play a key role as regulatory mediators necessary for cell–cell communication or triggering certain signaling pathways. However, at an increased concentration of ROS/RNS in the cellular system, they react with protein, lipids, and DNA molecules and cause detrimental effects in living organisms. In normal conditions, the antioxidant systems, such as catalase, superoxide dismutase (SOD), and glutathione, react with deleterious ROS/RNS into less toxic molecules. During pathological conditions, ROS/RNS overproduction exceeds the antioxidant defense system or dysfunction of antioxidants that are essential to neutralize oxidants, resulting in stress conditions known as oxidative stress. Oxidative stress (OS) will develop into chronic inflammation, leading to various diseases, including diabetes, cardiovascular disease, arthritis, cancer, etc. ,2.NFE2L2 that is also the master regulator of phase II antioxidant enzymes that protect against oxidative stress and inflammation. Under normal conditions, NRF2 is sequestered by Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (KEAP1) as a complex in the cytoplasm where its level is regulated by ubiquitination and proteasomal degradation. Under stress conditions, the interaction between the NRF2 and KEAP1 complexes is broken down, and NRF2 is accumulated in the cytoplasm and then translocated into the nucleus, where it binds to phase 2 of the antioxidant response element (ARE) and initiates the transcription of antioxidative enzymes, such as SOD, catalase, etc. . . 14]. ApOxidative stress is a pathological condition occurring due to an imbalance between oxidants and antioxidant defense systems in the body. Oxidative stress is linked with the induction of various diseases, both chronic and acute pathologies, the aging process, and neurodegeneration. In this section, the role of the NRF2 signaling pathway in various diseases, including diabetes, Alzheimer’s disease, kidney injury, and cancer, is discussed .In type 2 diabetes mellitus (T2DM), an increased glycemic overload affects the electron transfer with the mitochondrial membrane, causing an accumulation of free radicals and a depletion of antioxidants, leading to oxidative stress . Oxidati−/−ApoE−/−) fed with an atherogenic diet, the data revealed that there was decreased hepatic cholesterol and small atherosclerotic plaques developed in the NRF2−/−, ApoE−/− when compared to NRF2+/+, ApoE−/− control mice [Atherosclerosis is a chronic inflammatory disease of large- or medium-sized muscle arteries, while oxidative stress acts mainly as a trigger of atherosclerosis. Due to macrophages’ increased production of ROS, circulating LDL is oxidized, resulting in the formation of foam cells and lipid deposition in the arteries, finally resulting in atherosclerotic plaque . To underol mice . In low-rol mice . In a strol mice .Oxidative stress has been associated with neurological disorders like multiple sclerosis, Alzheimer’s (AD), Parkinson’s disease (PD), etc. AD is mainly due to the abnormal deposition of amyloid β-peptides (Aβ) and accumulation of neurofibrillary tangles containing hyperphosphorylated tau protein and dementia. Studies have shown that oxidative stress increases the aggregation and production of Aβ and, also, stimulates phosphorylation of the tau protein, leading to neurotoxicity. Further, accumulated Aβ and tau protein causes redox imbalance by stimulating dysfunction of the mitochondria, thereby increasing ROS production. Thus, oxidative stress is necessary for the initiation and, also, the progression of AD . SimilarDamage to macromolecules such as protein, DNA, RNA, and membrane lipids occurs as a result of oxidative stress, initiating carcinogenesis. Cancer is a genetic disease in which cells grow out of control and is a multistage process, including initiation, promotion, and progression. Cancer is initiated by ROS-mediated DNA damage, causing gene mutations, genome instability, and altering the DNA structure. In the promotion stage, there will be increased cancer cell proliferation and decreased apoptosis due to abnormal gene expression, improper cell-to-cell communication, and alterations in the second messenger system. In the final stage, oxidative stress involves further DNA alterations and leads to the progression stage of cancer . AdditioOxidative stress, along with inflammation, is an important factor necessary for the initiation and progression of acute and chronic kidney diseases like diabetic nephropathy, hypertension-associated kidney disease, polycystic disease, and glomerulonephritis. C-reactive protein, IL-6, TNF-α, and fibrinogen, in combination with oxidative stress, induce apoptosis, necrosis, and fibrosis in kidney tissues, eventually leading to chronic kidney disease. Additionally, dysregulated metabolic products like uric acid are also involved in the stimulation of oxidative stress, which would worsen chronic kidney disease . NormallNrf2 gene through epigenetic modifications, including histone modifications, miRNA alterations, DNA methylation, etc. In this section, this review provides an update on the role of phytochemicals in regulating NRF2 in various diseases . It has been known to possess a wide range of biological activities, such as anti-inflammatory, antioxidant, and ameliorating oxidative stress in C2C12 myoblast cells [2O2-treated RAW264.7 macrophage cells [HO-1 [Keap1 and, also, decreasing CpG demethylation at the NRF2 promoter by inhibiting DNMT enzymes and HDAC 4 [Curcumin is a yellow-colored polyphenol and an active component of the plant st cells . Curcumige cells . In a hils [HO-1 . Due to ls [HO-1 . Interesls [HO-1 . Curcumils [HO-1 . In anotd HDAC 4 . In rhabd HDAC 4 . Curcumid HDAC 4 . Thus, cmiR-27a and increase Nrf2 and its responsive genes, including SOD, GPX, and CAT in streptozotocin–diabetic rats [Quercetin is the most abundant flavonol and is found in onions, berries, apples, cereals, black and green tea, red grapes, broccoli, cherries, etc. Quercetin has beneficial effects in preventing various diseases, such as lung and cardiovascular diseases and cancer, and also acts neuroprotective, antioxidative, antidiabetic, and hepatoprotective . Quercettic rats . In the tic rats . In breatic rats .Nrf2 expression is enhanced to counteract oxidative stress. However, NRF2 is not translocated into the nucleus, showing a functional impairment of NRF2. Surprisingly, EGCG acts as a booster in translocating NRF2 into the nucleus and activates the NRF2/ARE pathway, which downregulates KEAP1 and its interaction with NRF2, thereby mitigating DN [Nrf2 expression via KEAP1-dependent and -independent mechanisms  in two adenocarcinoma cells [Epigallocatechin-3-gallate (EGCG) is the main catechin of green tea and is reported to possess antioxidant, anti-inflammatory effects . In diabating DN . In anotating DN . The neuating DN . EGCG exma cells . EGCG efma cells . SOD, heme oxygenase-1 (HO-1), and metallothionein (MT) [HO-1 expression in amyloid-beta Aβ1-42 cytotoxicity-induced PC12 cells [Resveratrol is a natural polyphenolic compound present in berries, grapes, peanuts, and food products such as red wine, and it possesses various biological properties, including antioxidant, anti-inflammatory, and antiplatelet aggregation potential. Resveratrol inhibits diabetes-related cardiac dysfunction and hypertrophy in multiple low doses of streptozotocin-induced diabetic mice by restoring NRF2 and its downstream antioxidative genes, such as ein (MT) . Resvera12 cells . In rote12 cells . Resvera12 cells . In a mo12 cells . In high12 cells .HO-1, and ameliorating cognitive deficits and preventing accumulation in AD-induced mice models [Sulforaphane is an isothiocyanate that is found abundantly in cruciferous vegetables such as cabbage, broccoli, and radishes. It showed many biological activities, such as antioxidant, anti-inflammatory, and antitumor activities . In vivoe models . In an ie models . Sulforae models .HO-1 and NQO1, and ultimately prevents metabolic syndrome [Apigenin is a naturally occurring plant flavone found richly in fruits and vegetables, including oranges, parsley, celery, etc. It has a variety of biological properties, including the ability to induce apoptosis; prevent DNA damage; and exert immunomodulatory, antioxidant, and anti-inflammatory effects . In highsyndrome . In stresyndrome . In LPS-syndrome . Apigenisyndrome . PI3K/Aksyndrome . Apigenisyndrome . A highlsyndrome .(−/−) group, ursolic acid inhibited its neuroprotective effect in a traumatic brain injury mice model [Nrf2 and its target gene HO-1 is reduced in skin tumors. Upon ursolic acid treatment, Nrf2 expression is restored by decreasing hypermethylated CpG islands of the Nrf2 gene promoter region in mouse epidermal cells by reducing the expression of epigenetic-modifying enzymes, including the DNA methyltransferases [Ursolic acid is a natural pentacyclic triterpenoid acid and is one of the major components present in apples, basil, berries, and fruit peels and is also found in certain medicinal plants. It shows a wide range of biological properties, including antioxidant, anticancer, and anti-inflammatory activities . In middce model . In anotce model . The ursce model . In CCL4ce model . In a sksferases .HO1, SOD, and CAT and decreased the inflammation and inhibited apoptosis via Bax and caspase-3 in the liver tissue of perfluorooctane sulfonate-exposed mice [HO-1 and NQO1 in hypoxia-induced neurons of Sprague–Dawley rats [Nrf2 and its target genes HO-1 and NQO1, together with a decrease in the Keap-1 level, while naringenin effectively modulates the NRF2-mediated-signaling pathway and decreases the invasion of endometrial cells and induces apoptosis [Nrf-2 and mRNA expression are decreased while naringenin targets the NRF2/HO-1 pathway by its antioxidant and anti-inflammatory potential and thereby decreases myocardial injury [Naringenin is a bioactive flavonoid found widely in citrus fruits like oranges, grapes, lemons, and tomatoes and is reported to be a well-known antioxidant, anti-inflammatory, and anticancer agent. In a study, naringenin has been reported to possess antidiabetic properties by inhibiting gluconeogenesis, stimulating glycolysis, restoring the insulin levels, and inhibiting apoptosis in both STZ-induced mouse insulinoma cell lines and, also, albino Swiss mice. One of the important mechanisms for the antidiabetic effect of naringenin is by promoting the dissociation of KEAP1 and NRF2 and elevating the antioxidant enzymes like GST in a dose-dependent manner, thereby protecting pancreatic beta cells . Upon exsed mice . Naringeley rats . Controvpoptosis . In arsel injury .Nrf2 [GPx, GR, CAT, and NQO1 [Pterostilbene is a natural compound and a demethylated analog of resveratrol found in blueberries and grapes. Pterostilbene prevents several diseases, including aging, cardiovascular disease, diabetes, and cancer . ArsenicNrf2 . Pterostand NQO1 . In anotand NQO1 .Ginkgo biloba extracts (GBE), rutin and procyanidin B2 were reported to suppress t-butyl hydroperoxide (t-BHP)-induced oxidative stress in retinal pigment epithelial cells by stimulating Nrf2 expression together with activating Erk1/2 signaling in injured retinal epithelial cells, thus suggesting that rutin can act as a protective agent in inhibiting oxidative stress-mediated retinal diseases [Nrf2 expression, and it was suggested that the PI3K/AKT/GSK-3β/NRF-2 pathway is involved in the neuroprotective effect of rutin against acrylamide and g-radiation (5Gy)-induced brain damage [Rutin is a flavonol predominantly found in buckwheat, mulberry, grapefruit, orange, lemon, and cranberries. Rutin prevents angiogenesis, decreases oxidative stress, decreases cholesterol accumulation, and inhibits inflammation . Rutin pdiseases . In anotn damage .Nrf2 and its target genes HO-1 and NQO-1 in diabetic mice. However, in Nrf2-downregulated mice, the protective effects of cinnamaldehyde decreased, which suggested the crucial role of NRF2 in cinnamaldehyde-treated mice [HO-1, NQO-1, and SOD in cognitively impaired mice induced by d-galactose and aluminum chloride [Cinnamomum cassia extract and its active compound cinnamaldehyde activated the NRF2/HO1 pathway and inhibited aryl hydrocarbon receptor signaling, thereby suggested to exhibit a beneficial effect on oxidative stress-mediated diseases [Nrf2 and its target genes HO-1, GPx-1, and NQO-1 and inhibiting cardiomyocyte hypertrophy in diabetic mice [Nrf-2/HO-1 and downregulation of the NF-κB pathway and reduced IL-6 and TNF-α levels in neurons suggesting the neuroprotective effect of three phytochemicals [Nrf2 expression and promoted NRF2 nuclear translocation in HUVECs and ultimately suggested that cinnamaldehyde acts as an effective NRF2 activator in high glucose-treated HUVECs [Cinnamaldehyde, an active component derived from cinnamon, has been described to show diverse biological activities like anticancer, antimicrobial, antidiabetic, and anticoagulant effects . In a stted mice . In LPS-ted mice . In anotchloride . In benzdiseases . In humadiseases . Cinnamatic mice . Upon thhemicals . Cinnamad HUVECs .Humulus lupulus L.) and has been shown to have antioxidant and anti-inflammatory properties [Nrf2 expression and its downstream targets GST, NQO1, and SOD and ultimately led to a decreased proliferation of PANC-1 cells, which suggested the anticancer effects of xanthohumol and phenethyl isothiocyanate [−/− (knockout) C57BL/6 mice, xanthohumol was reported to suppress acute lung injury via inducing the expression of Nrf2 through activation of the AMPK/GSK3β pathway and inhibiting the LPS-induced NF-κB-signaling pathway and Txnip/NLRP3 inflammasome [Nrf2 and its associated gene expression, including phase II enzymes, in normal cells, while, in cancerous cells, xanthohumol exhibits cytotoxic effects. Additionally, xanthohumol also elevated the expression of GST, HO-1, NQO1, and P53 in normal hepatocytes [GSH, HO-1, and NQO1 in LPS-induced BV2 cells, thereby suggesting the protective role of xanthohumol against brain injury induced by LPS [Nrf2 expression, effectively protects neuronal cells and is suggested as a potential candidate against neurodegenerative diseases [Xanthohumol is a significant prenylflavonoid found primarily in hop plants  [Nrf2 gene, leading to the re-expression of Nrf2 and its target gene, NQO-1, and thereby exerting a chemopreventive effect against prostate cancer [Sulforaphane promoted demethylation at the NRF2 promoter region, which leads to the activation of n cancer . Using ienzymes) . In prose cancer .+ cells showed that an apigenin dose dependently prevents skin cancer by effectively reversing the hypermethylated status at the promoter region of the NRF2 promoter, thereby enhancing Nrf2 expression and the nuclear translocation of Nrf2 in skin epidermal JB6 P+ cells [Radix Angelica Sinensis prevent DNA hypermethylation at the NRF2 gene promoter region via suppressing DNA methyltransferase activity, which leads to the re-expression of Nrf2 and its downstream gene targets in TRAMP mice, which underlies the anticancer potential of phytochemicals against prostate cancer [In high glucose-treated HepG2 cells, there is an increased methylation status in the promoter region of NRF2, which could be effectively prevented by resveratrol by inhibiting HG-induced ROS production through demethylation of the NRF2/ARE-signaling pathway . DNA metP+ cells . ResveraP+ cells . γ-tocopP+ cells . LuteoliP+ cells . Naturale cancer . In thisNrf2 expression and its downstream targets in the nucleus, including HO-1, SOD, and CAT; (2) regulating NRF2 signaling by various kinases-like Gsk3beta, PI3/AKT, and MAPK; and (3) altering epigenetic modulation like methylation at the promoter region of NRF2 is also one of the important targets of phytochemicals in preventing disease. Thus, the above three mechanisms can be targeted for oxidative stress-mediated diseases. We have also discussed the miRNA and HDACs that can modify NRF2 in various disease models. However, further investigation into another upstream signaling, histone methylation enzymes targeting NRF2 and the effect of phytochemicals on them still need to be investigated, which might provide additional targets for the prevention of oxidative stress-mediated diseases. This review clearly demonstrates that NRF2/KEAP1 signaling could be an effective target for various pathological conditions, including cancer, Alzheimer’s, etc. Phytochemicals are receiving increased attention due to their role as NRF2 activators and inhibitors; however, much research still needs to investigate the understanding that their pharmacokinetic and dynamic profiles and, also, clinical studies need to be conducted using various phytochemicals and its synthetic moieties against various diseases targeting NRF2 signaling.Oxidative stress and inflammation are the two important factors necessary for the pathogenesis of various diseases, including diabetes, atherosclerosis, kidney injury, AD, PD, cancer, etc. The important mechanism that is necessary for the cellular defense against oxidative stress is the NRF2-signaling pathway. With these concepts, researchers are paying more attention to identifying the agents that could target oxidative stress and inflammation via the NRF2 intracellular-signaling pathway. Among them, phytochemicals are widely used as important therapeutic agents targeting oxidative stress and inflammation. Several lines of evidence support the use of phytochemicals in modulating dysregulated signaling pathways in a variety of disease models. In the present review, we discussed the role of NRF2 during diabetes, AD, PD, cancer, and atherosclerosis. Additionally, we discussed the phytochemicals that have effectively modified NRF2 signaling and prevented various diseases in both in vitro and in vivo models. In this review, we discussed important phytochemicals like curcumin, quercetin, resveratrol, EGCG, apigenin, sulforaphane, and ursolic acid that act as effective NRF2 inducers. Throughout this review, we learned that dietary phytochemicals can prevent diseases by (1) blocking oxidative stress-inhibiting inflammatory mediators through inhibiting Keap1 or activating


Urothelial bladder cancer (BC) is one of the most prevalent malignancies with high mortality and high recurrence rate. Angiogenesis, tumor growth and metastasis of multiple cancers are partly modulated by CC chemokines. However, we know little about the function of distinct CC chemokines in BC.ONCOMINE, Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan–Meier plotter, cBioPortal, GeneMANIA, and TIMER were used for analyzing differential expression, prognostic value, protein–protein interaction, genetic alteration and immune cell infiltration of CC chemokines in BC patients based on bioinformatics.The results showed that transcriptional levels of CCL2/3/4/5/14/19/21/23 in BC patients were significantly reduced. A significant relation was observed between the expression of CCL2/11/14/18/19/21/23/24/26 and the pathological stage of BC patients. BC patients with high expression levels of CCL1, CCL2, CCL3, CCL4, CCL5, CCL8, CCL13, CCL15, CCL17, CCL18, CCL19, CCL22, CCL25, CCL27 were associated with a significantly better prognosis. Moreover, we found that differentially expressed CC chemokines are primarily correlated with cytokine activity, chemokines receptor binding, chemotaxis, immune cell migration. Further, there were significant correlations among the expression of CC chemokines and the infiltration of several types of immune cells .This study is an analysis to the potential role of CC chemokines in the therapeutic targets and prognostic biomarkers of BC, which gives a novel insight into the relationship between CC chemokines and BC. Bladder cancer is one of the most prevalent cancers, with an estimated 549,000 new cases and 200,000 deaths reported in 2018 –6 and wiChemokines, constituting the largest family of cytokines, are chemotactic cytokines that mediate immune cell migration and lymphoid tissue growth . SequencIn this study, we performed a comprehensive analysis of CC chemokines to evaluate their potential value as therapeutic targets and prognostic biomarkers based on several large public databases, thus supplying informative assistance to help clinicians select appropriate therapeutic drugs and more accurately prognosis in BC patients.www.oncomine.org), an online database providing powerful, genome-wide expression analysis with cancer microarray information [p-value < 0.05, a fold change of 2, and a gene rank in the top 10% were set as the significance thresholds. The mRNA expression of CC chemokines in clinical cancer specimens were compared with those in normal controls. Student’s t-test was used to analyze the difference in the expression of CC chemokines in BC.The mRNA levels of distinct CC chemokines in diverse cancer types were analysed in ONCOMINE (ormation . In thishttp://gepia.cancer-pku.cn/index.html) is a new analytical tool using a standard processing pipeline and consist of thousands of tumors and normal tissue samples data [GEPIA  [p-value was < 0.05. Patient samples were split into two groups by median expression (high versus low expression) and assessed by a Kaplan–Meier survival plot.The prognostic analysis of CC chemokines patients was also performed by using Kaplan–Meier plotter  , which iwww.cbioportal.org) is a comprehensive web resource, can visualize and analyze multidimensional cancer genomics data [cBioPortal (ics data . Based ohttps://string-db.org/) is a website that provides a comprehensive and objective global network of protein–protein interaction (PPI) [STRING (on (PPI) . A PPI nhttp://www.genemania.org) is a website about gene information, analyzing gene lists and prioritizing genes for functional assays [GeneMANIA (l assays . The pothttps://cistrome.shinyapps.io/timer/) is web interface that provides systematic evaluations of the infiltration of different immune cells and their clinical impact [TIMER  were also observed in BC compared with normal tissue. Similar results were found when we assessed the transcriptional levels of CC chemokines using the GEPIA database, CCL2, CCL14, CCL21 and CCL23 were lower in BC patients than normal patients   , patients with higher levels of CCL14 , neutrophils , and dendritic cells ; CCL2 with CD4+ T cells , CD8+ T cells , macrophages , neutrophils , and dendritic cells ; CCL3 with neutrophils , and dendritic cells ; CCL4 with CD8+ T cells , neutrophils , and dendritic cells ; CCL5 with CD8+ T cells , neutrophils , and dendritic cells ; CCL7 with B cell , CD8+ T cells , neutrophils , macrophages ; CCL8 with CD8+ T cells , neutrophils , and dendritic cells ; CC11 with CD4+ T cells , CD8+ T cells , macrophages , neutrophils , and dendritic cells ; CCL13 with CD8+ T cells , neutrophils , and dendritic cells ; CCL14 with CD4+ T cells , CD8+ T cells , macrophages , neutrophils , and dendritic cells ; CCL15 with CD8+ T cells , neutrophils , and dendritic cells ; CCL16 with CD8+ T cells  and dendritic cells ; CCL17 with B cell , CD4+ T cells , CD8+ T cells , and macrophages ; CCL18 with CD8+ T cells , neutrophils , and dendritic cells ; CCL19 with CD4+ T cells  and neutrophils ; CCL20 with CD8+ T cells , macrophages , neutrophils , and dendritic cells ; CCL21 with CD4+ T cells , CD8+ T cells , neutrophils , and dendritic cells ; CCL22 with CD4+ T cells , macrophages , neutrophils , and dendritic cells ; CCL23 with CD4+ T cells , CD8+ T cells , neutrophils , and dendritic cells ; CCL24 with CD8+ T cells , macrophages , neutrophils , and dendritic cells ; CCL25 with B cell , CD4+ T cells , neutrophils , and dendritic cells ; CCL26 with B cell , CD8+ T cells , macrophages , neutrophils , and dendritic cells ; CCL27 with macrophages  and neutrophils ; CCL28 with B cell , CD8+ T cells , macrophages , neutrophils , and dendritic cells .To explore the relation between immune cell level and cancer cell, the TIMER database are used to perform an analysis. The results Fig.  indicateBladder cancer is one of the most common causes of cancer-related deaths worldwide . CC chemAmong the CC chemokines, CCL2 is the most studied in BC. Expression of CCL2 was higher in BC tissues and in human BC cell lines . And thiFor the other CC chemokines, a previous study demonstrated that CCL1 can be up-regulated by estrogen receptors alpha and then enhance bladder cancer cell invasion . CCL1/CCOne of the limitations of our study was that a detailed description or stratified analysis was missed about characteristics of the patients and clinical subtypes of BC. The data in our study are extracted from several online databases and different studies. Particularly, part of the clinical course of bladder tumors, characteristics of the patients baseline data is not complete in these studies. Among these patients, stage Ois bladder urothelial carcinoma, superficial bladder cancer, infiltrating bladder urothelial carcinoma are included. Therefore, a detailed description or stratified analysis was missed about these items related to diagnosis or therapeutic. Moreover, in the Figs. In this research, we analyzed the prognostic and therapeutic value of CC chemokines in BC. Our results provided the information that CC chemokines might play an important role in BC oncogenesis which indicates a potential target of BC. We hope our results provide novel insights on the therapeutic targets of BC and help clinicians make a better personal treatment plan. However, further studies are needed to elucidate the difference between subgroup of BC classified by Infiltration degree or grade of these genes in BC.