CD147 also known as EMMPRIN, basigin, and HAb18G, is a single-chain type I transmembrane protein shown to be overexpressed in aggressive human cancers of CNS, head and neck, breasts, lungs, gastrointestinal, genitourinary, skin, hematological, and musculoskeletal. In these malignancies, the molecule is integral to the diverse but complimentary hallmarks of cancer: it is pivotal in cancerous proliferative signaling, growth propagation, cellular survival, replicative immortality, angiogenesis, metabolic reprogramming, immune evasion, invasion, and metastasis. CD147 also has regulatory functions in cancer-enabling characteristics such as DNA damage response (DDR) and immune evasion. These neoplastic functions of CD147 are executed through numerous and sometimes overlapping molecular pathways: it transduces signals from upstream molecules or ligands such as cyclophilin A (CyPA), CD98, and S100A9; activates a repertoire of downstream molecules and pathways including matrix metalloproteinases (MMPs)-2,3,9, hypoxia-inducible factors (HIF)-1/2α, PI3K/Akt/mTOR/HIF-1α, and ATM/ATR/p53; and also functions as an indispensable chaperone or regulator to monocarboxylate, fatty acid, and amino acid transporters. Interestingly, induced loss of functions to CD147 prevents and reverses the acquired hallmarks of cancer in neoplastic diseases. Silencing of Cd147 also alleviates known resistance to chemoradiotherapy exhibited by malignant tumors like carcinomas of the breast, lung, pancreas, liver, gastric, colon, ovary, cervix, prostate, urinary bladder, glioblastoma, and melanoma. Targeting CD147 antigen in chimeric and induced-chimeric antigen T cell or antibody therapies is also shown to be safer and more effective. Moreover, incorporating anti-CD147 monoclonal antibodies in chemoradiotherapy, oncolytic viral therapy, and oncolytic virus-based-gene therapies increases effectiveness and reduces on and off-target toxicity. This study advocates the expedition and expansion by further exploiting the evidence acquired from the experimental studies that modulate CD147 functions in hallmarks of cancer and cancer-enabling features and strive to translate them into clinical practice to alleviate the emergency and propagation of cancer, as well as the associated clinical and social consequences. These not only strikingly differentiate the cancer cells from normal cells of the same tissue but also bring about variations of characteristics between cancer types, the same cancer type in different patients , and cellular subpopulations within the same tumor  , 2. DespBehind the emanation of the hallmarks of cancer and cancer-enabling characteristics is a repertoire of genes and proteins that form the switches and pathways that drive the transcriptomic and proteomic machinery of cancer. Understanding and exploiting the cancer characteristics therefore demands the in-depth exploration and understanding of individual molecules and their roles in the web of cancer machinery. In this study, we review the role of CD147 in the emanation and propagation of the hallmarks of cancer and cancer-enabling characteristics. We found that CD147 has a regulatory or mediatory role in the upstream and downstream, single, or complex molecules and pathways that propagate most of the hallmarks of cancer and cancer-enabling characteristics. Inducing loss of functions to CD147 could prevent or reverse the acquisition of the hallmarks of cancers in the neoplastic cells and tissues.1.2BSG gene located on chromosome 19p13.3. It belongs to the immunoglobulin superfamily (IgSF) of receptors (Human Cluster of Differentiation 147 (CD147) also known as extracellular matrix metalloproteinase inducer (EMMPRIN), HAb18G, and basigin, is a single-chain type I transmembrane protein, coded by the eceptors .CD147 plays a critical and extensive role in both infectious and non-infectious disease development and/or propagation. In infectious pathologies for example, during malarial parasitic infection, CD147 interacts with Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) and rhoptry-associated protein 2 (RAP2). This is the essential interaction among the four modes of PfRhs-erythrocyte interactions that occurs during the reorientation stage (second stage) of parasite invasion into the erythrocyte , 12; agaCD147 is also integral in non-transmissible pathologies such as stroke, heart disease, and Alzheimer’s disease , and mor1.2.1CD147 is integral to the diverse but complimentary hallmarks of cancers. The molecule is pivotal in cancerous proliferative signaling, growth propagation, cellular survival, replicative immortality, angiogenesis, metabolic reprogramming, immune evasion, and invasion and metastasis –38. The 22.1One of the key pathological properties of cancer cells is their ability to switch from a quiescent state into a vicious replicative mode associated with the evasion of anti-growth signals . To commUnderlying cancerous cellular division and multiplication processes is the stemness characteristic found in subpopulations of cells existing in almost all types of cancer . Whetherin vitro, as well as tumor initiation and growth in vivo , a group of helix-loop-helix heterodimers which masters the cellular adaptation to mitigate the constant oxygen, nutrients, and excretory gap . The twoogenesis   and lactate even in the presence of oxygen (fermentation or aerobic glycolysis), later suggesting a dysfunctional mitochondrial metabolism as normal cells would generate ATP, water, and CO glucose . Follow- glucose , up to d glucose . In addi glucose ; rapid g glucose . Integrath among the 12 transmembrane domains of MCT1; the conformational CD147-MCT1 interaction enables the inward and outward opening of MCT1 with resulting shuttling of lactate , CD147 had a striking influence on several cancer metabolic activities such as lactate co-transportation with MCT1 and MCT4, regulated phenotypic expression of MCT1 and MCT4, and also the regulation of key metabolic enzymes including aldehyde dehydrogenase 1 A3, transglutaminase 2, pyruvate kinase isoenzyme 2, and glucose-6-phosphate dehydrogenase  which is crucial for lactic glycolysis by cancers in normoxia and hypoxia  expression through Akt/mTOR pathway; SREBP1c then activates FASN and ACC1 gene transcription with resultant de novo fatty acid synthesis. CD147 also inhibited fatty acid degradation through p38/MAPK activation which in turn downregulates PPARα and target genes ACOX1 and CPT1A, involved in β-oxidation of fatty acids  for the expression of CD147 and genes responsible for aberrant fatty acid metabolism in HCC, as well as ty acids   such as MMP-2, MMP-3, MMP-9, hence the name matrix metalloproteinase inducer, EMMPRIN , 80. CD133.1in vitro and in vivo studies, induction of DSBs through treatment with radiations of cervical cancer SiHa and ME-180 cell lines resulted in CD147-dependent evasion of DDR and cell cycle rescue; exposure to 10-Gy of CD147+ SiHa and ME-180 had a rapid decrease of γH2AX within 1 and 6 hours of exposure respectively meanwhile CD147- cells had a rapid increase of γH2AX, reaching maximal values within 30 minutes of exposure and remained at maximal for 24 hours post-exposure. On cell cycle regulation, CD147 was denoted as responsible for limiting the number of cells in the G2/M phase (DSBs repair point) whereby CD147- cells had a significantly increased percentage of cells in the G2/M phase as compared to CD147+ cells . Radiations, for example, inflict DNA damage as single-strand breaks (SSBs) or double-strand breaks (DSBs). Normally, SSBs are repaired through base excision repair or nucleotide excision repair. Homologous recombination and non-homologous end-joining (NHEJ) are methods of repair that cells deploy in DSB-damage; usually, homologous recombination is a conservative process aiming at restoring the original DNA sequence before damage and it occurs in the S-phase and G2-phase of the cell cycle under the mediation of cyclin and cyclin-dependent kinases (CDKs) . Therape7+ cells .3.2in vitro and in vivo mice study by Chen Y. et al. on B16-F10 melanoma and Lewis lung cancer cell lines, there was upregulation of CD147 on the cell membrane of TILs which also co-expressed with Tim-3 and PD-1, known immune checkpoint molecules. CD147 knockout was associated with an increase in TILs, CD8+ cytotoxicity, PD-1+ CD8+, cytotoxic molecules such as perforin, TNF-α, and INF-γ, and reversed PD-1+Tim-3+CD8+ TILs dysfunction  dysfunction following reduced glucose uptake and reduced GLUT1 expression . CD147-Mfunction .4Heuristic conceptualization of cancer disease into key features named hallmarks of cancer and cancer-enabling characteristics provided an additional roadmap for deciphering the complex and aberrant genotypic and phenotypic cancer manifestations. These features of cancer manifestation are known to be distinct and yet overlapping which in turn presents an avalanche of evasive and transformative custodianship of roles, this in turn presents uncertainty in identifying hotspot targets for developing effective and safe cancer treatments. In that light, determining a molecule common across heterotypic cancer types, cancer characteristics, and neoplastic signal transductions would provide a viable hotspot with an explosive anti-cancer effect. Apart from being involved in cancer development, progression, metastasis, and recurrence, CD147 has also been associated with resistance to multiple anti-cancer therapies such as Temozolomide , Cisplat4.1Therapeutics targeting CD147 are more likely to be successful because the molecule is involved in numerous pro-oncogenic and oncogenic molecules and pathways, such as MMPs , MCTs, G+ HCC cells, in vitro and in vivo  domain of CD147 increasingly expressed in HepG2 and SK-Hep1 cell lines were shown to have high cytotoxicity specific for CD147 in vivo . The the in vivo , 101. Crn tissue . Also, dIn antibody-antigen targeted therapies, CD147 could be an effective target molecule in both monoclonal antibodies, chimeric antibodies, antibody-drug conjugate (ADCs), and radioimmunotherapy therapeutic designs. Again, tumor-specific expression of CD147 renders its corresponding antibody potential for incorporation in both oncolytic viral and gene therapies.+ human HCC tissue showed a 99.55% binding rate against human antigen CD147/HAb18G with evident FAK-PI3K-Akt-Girdin pathway-mediated tumor-specific cytotoxicity (131mA HAb18 (I131-Metuximab), had a radiation absorption of 2.5 to 18.6 times that of nontumor tissue in an animal study, improvement of hematologic indices in 62.5% during phase I clinical trial, and during phase II trial had grade 3 hematologic toxicity in only 1 out of 14 (7.14%) and 4 out of 89 patients (4.49%) during preliminary and trial period respectively, with only <10% aggravated hepatic damage  of 43.4% versus 21.7% in the control group, with a hazard ratio of 0.49, p=0.0031, and only seven out of 78 treatment group patients had grade 3 or 4 adverse events  and was well tolerated without adverse events in animal studies (Metuzumab is another CD147-antibody dependent cellular cytotoxic (ADCC) agent, developed by recombination and glycoengineering of human and mouse chimeric immunoglobulin G1 monoclonal antibody against CD147 extracellular C2 domain; it is non-fucosylated and therefore, has optimized affinity to the Fc fragment subtype FcγRIIIa, which in turn increases the affinity to ADCC promoters such as NK cells , 111. Thin vitro and in vivo, was shown to be specifically internalized by CD147+ tumor cells, successfully delivering the payload (DM1) and releasing the active metabolites through lysosomal cleavage, leading to cell cycle arrest at G2/M (ADC designed using maytansinoid-based cytotoxic agent emtansine (DM1) linked by the non-cleavable chemical linker SMCC to CD147 antibody HcHAb18 (HcHAb18-DM1) in NSCLC cells both  at G2/M , 113. Th at G2/M .in vitro and in vivo, gallbladder cancer EH-GB1 cell line (131mA HAb18 through addition and replacement of the CArG element of early growth response-1 (Egr-1) and P53 respectively from AdSup-P53 to form AdeSurp-hSulf1- I131mA HAb18 combined therapy, resulted into an even more effective tumor-specific cytotoxic activity against HCC cells, a very potential CD147 based combined therapy   .in vitro and in vivo cytotoxic and anti-metastatic activities against HCC SMMC-7721 cell line  between the HN and F genes of the oncolytic Newcastle disease virus (NDV) by the reverse genetic system to form a recombinant NDV carrying CHAb18 (rNDV-18HL) maintained the same virulence as the wild NDV strain while additionally attained optimal active cHAb18 expression with increased ell line .In vitro and in vivo studies on the combination of metuzumab with the nucleoside-derived chemotherapies, gemcitabine and cytarabine (Ara-C) have revealed promising results: in vitro and in vivo study under 18F-FDG PET/CT of combined gemcitabine/cHAb18 therapy in MIA PaCa-2 pancreatic cancer cell line showed an additively superior cytotoxic activity than either of the two therapies alone evidenced by relatively decreased Ki-67 expression (p=0.008)  ; while ip=0.008) . Cytosinp=0.008) ; Treatmep=0.008) , an evidp=0.008) .5CD147 is a molecule critically involved in the acquisition and propagation of hallmarks of cancer and cancer-enabling characteristics. It is therefore one of the key driver molecules in cancer development, microenvironment adaptation, progression, metastasis, and resistance to therapies witnessed in many human cancers.Because CD147 promotes cancer progression and metastasis through multiple signaling pathways, a single block of CD147 would therefore translate into fissional phenomena of arresting multiple and alternate cancer signaling cascades; this ensures minimal treatment-evasion of cancer cells that would result from epigenetic modulations, mutations of a single or multiple pro- and anti-oncogenes, and supplementary molecules that interplay within the hallmarks of cancer. Since CD147 is differentially expressed in cancer cells than in normal tissue cells, targeted therapies as well as immunotherapies against CD147 are therefore less likely to attack normal tissue and organs hence avoiding severe adverse events, this being one of the significant challenges with current immunotherapies. In that light, further experimental and clinical studies have a promising potential of yielding efficacious and safe treatment against aggressive human cancers that remain challenging to date.FL, AN, AL, and YX developed conceptualization for the study. AN, HL, TR, XS, and JZ performed literature curation and data analysis. FL was responsible for funding acquisition and project administration. FL, AN, and HL oversaw resource distribution and supervision throughout the study. AN, AL, XZ, TR, and JZ performed information synthesis and validation. FL, AN, and XS managed visualization designs. The original draft was written by FL, AN, AL, YX, TR, and XZ. All authors contributed to the article and approved the submitted version.


Vitamin D, adropin, proinflammatory cytokines, and oxidative stress closely related with metabolic homeostasis and endothelial dysfunction. The aim of the present study is to investigate how vitamin D levels affect serum adropin, IL-1ß, IL-6, and oxidative stress.A total of 77 female subjects were divided into 3 groups according to vitamin D levels. Biochemical parameters, adropin, IL-1ß, IL-6, oxidative stress markers were studied in these groups, and the results were compared statistically.Serum adropin, IL-1ß, IL-6, total oxidant status (TOS) and total antioxidant status (TAS) and oxidative stress index (OSI) levels differed significantly between the vitamin D groups (p < 0.05). A significant positive correlation was detected between vitamin D, and adropin and TAS . A significant negative correlation was detected between vitamin D, and IL-1ß, IL-6, TOS, OSI .Vitamin D could show its effects through vitamin D receptors on tissues or on the ENHO gene in adropin secreting tissues via direct or indirect mechanisms. Proinflammatory cytokines, oxidative stress, and adropin targeted studies could contribute to the prevention and treatment of diseases associated with vitamin D deficiency in future. While vitamin D is mainly synthesized in the skin under the influence of sunlight in humans, a small portion is obtained from foods. Vitamin D mainly regulates calcium, phosphorus and bone metabolism, and is also known as an immunomodulator hormone –3. VitamAdropin is a peptide hormone coded by the energy homeostasis-associated gene (ENHO) mainly in the liver and brain. It is closely related to glucose and lipid homeostasis and insulin resistance. It is produced in many tissues and organs such as the liver, brain, heart, kidney, pancreas, and gastrointestinal system –9. AdropOxidative stress (OS) is a result of impaired balance between the reactive oxygen species (ROS) formation and antioxidant defense mechanisms. Free radical production occurs continuously in all cells as a part of normal cellular functioning. High amounts of free radicals and ROS formed in tissues due to OS interact with intracellular molecules and cause cellular damage by injuring various biological molecules including proteins, lipids, and nucleic acids ,15. ExceInterleukin-1 beta (IL-1β) and interleukin-6 (IL-6) are proinflammatory cytokines especially produced by myelomonocytic cells such as monocytes, macrophages, and dendritic cells. They can be the pathogenic precursor to autoinflammatory, autoimmune, infectious, and degenerative diseases by affecting many cells and organs ,23. MoreMany chronic illnesses including endothelial dysfunction, diabetes mellitus, cardiovascular diseases, malignancy, autoimmune disorders which, as shown in the studies, were associated with vitamin D were also similarly associated with adropin, IL-1β, IL-6 and oxidative stress. In the literature search we performed, there were limited number of studies individually evaluating the relationship between vitamin D level, and oxidative status or proinflammatory cytokines  in some diseases, however, there was no study evaluating the relationship between vitamin D and adropin or evaluating these parameters together. We aimed to evaluate the relationship between the levels of vitamin D and adropin, IL-1β, IL-6 and oxidative status, which, we think might affect the pathological and metabolic conditions seen in vitamin D deficiency.This cross-sectional, case-controlled trial was initiated after approval of Bezmialem Foundation University, ethics committee. Our study included 77 female subjects aged 18 to 65 years who consulted to Bezmialem Foundation University, Internal Diseases Outpatient Clinic between July 2020 and December 2020, did not have a known history of chronic illness, were not pregnant or lactating, did not have a history of surgical operation within the last 6 months, did not use antioxidant medication, vitamin supplement, lipid-lowering agent, tobacco or alcohol, did not use vitamin D within the last 3 months, did not do heavy exercise recently and have normal C-reactive protein (CRP) levels. All study subjects gave written informed consent. The subjects were divided into three groups by their vitamin D level  .All volunteers underwent a thorough physical examination with their height and weight being recorded. Weight and height were rounded to the nearest kg and cm, respectively, and body mass index (BMI) was calculated [BMI = weight/(height)2O2 Equiv./L. Coefficients of variation values were less than 10%.Total oxidant status and total antioxidant status were determined by a recently developed method, colorimetric assay . The TAS2O2 Equiv./L/TAS, and mmol Trolox Equiv./L [OSI was determined as TOS/TAS ratio (resulting TAS unit was changed to μmol/L) and calculated as follows: OSI (arbitrary unit) = TOS, μmol HEquiv./L .Concentrations of adropin, IL-1β, and IL-6 in the serum were measured by a specific commercial ELISA kit according to the manufacturer’s instructions . Concentrations were determined with a spectrophotometric microtiter plate reader  at 450 nm optical density .IBM SPSS  statistics 22.0 software was used for the statistical analyses for the study. While evaluating the study data, descriptive statistical methods  were used. Skewness and kurtosis values were used together with the Shapiro-Wilk test to evaluate the normal distribution of the data. While the one-way ANOVA test was used to compare more than two normally distributed variables, the Kruskal Wallis test was used to evaluate more than two nonnormally distributed variables. Tukey and Games Howell tests were used for post-hoc pairwise comparison of the parameters that were significant after ANOVA and the Kruskal Wallis test. For the assessment of correlation between the data, Pearson’s correlation analysis was used for the normally distributed data and Spearman’s correlation analysis for the nonnormally distributed data. Results were evaluated within a 95% confidence interval with significance at a p level of <0.05.The mean age of the 77 women participating in the study was 33.79 ± 10.15 years and the mean BMI (body mass index) was 24.90 ± 5.20 kg/m2. The mean age of the groups that we divided into three groups according to their vitamin D levels  was G1:32.35 ± 10.67, G2:35.14 ± 10.85, and G3:35.41 ± 7.84 were determined. The mean BMI of the three groups was G1:24.41 ± 5.26, G2:26.70 ± 5.79, and G3:23.79 ± 3.81. There was no statistically significant difference between the groups in terms of age and BMI  .There was no statistically significant difference between three groups in terms of glucose, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), calcium, magnesium, phosphorus, thyroid stimulating hormone (TSH) and free tetraiodothyronine (fT4) (p > 0.05). As for adropin, IL-1β, IL-6, TOS, TAS, OSI, and parathyroid hormone (PTH) levels, a statistically significant difference was detected between the groups (p < 0.05) .In the correlation assessment between vitamin D, and age, BMI, adropin, IL-1β, IL-6, TOS, TAS, OSI and other biochemical parameters, significant positive correlation was detected between vitamin D, and adropin and TAS . Significant negative correlation was detected between vitamin D, and IL-1β, IL-6, TOS, OSI and PTH  . EvaluatFigures 1In the correlation assessment between adropin, and vitamin D, IL-1β, IL-6, TOS, TAS, OSI and PTH, statistically significant positive correlation was detected between adropin and TAS , and statistically significant negative correlation was detected between adropin, and IL-1β, IL-6, TOS, OSI and PTH  . AdditioWhile vitamin D is mainly synthesized in the skin under the influence of sunlight in humans, a small portion is obtained from foods. Vitamin D mainly regulates calcium, phosphorus and bone metabolism, and is also known as an immunomodulator hormone –3. In thOur study detected significant difference between the vitamin D groups  in terms of adropin level, and also, a significant positive correlation between vitamin D level and adropin level (p < 0.001). Adropin has been reported to have lipid and glucose homeostasis-regulating, angiogenesis and antiinflammatory effects in addition to preventive effects against insulin resistance and endothelial dysfunction ,10,28,29In our study, significant difference was detected between vitamin D groups in terms of IL-1β and IL-6 levels, and also a significant negative correlation between vitamin D level, and IL-1β and IL-6 levels (p < 0.001). Proinflammatory cytokines  have multiple effects on many events including the initiation and maintenance of inflammation, endothelial dysfunction, metabolic syndrome, insulin resistance, diabetes mellitus, oxidative stress and cardiovascular events ,24–26. IThere was a significant negative correlation between vitamin D, and adropin level and oxidative stress marker, TOS, and a significant positive correlation with TAS (p < 0.05). Antioxidants prevent or remove excessive endogenous or exogenous free radical production leading to prevention of free radical-induced cellular damage. The increased oxidative stress as a result of the shift in this balance towards the oxidative stress was detected to play a role in the pathogenesis of various conditions including metabolic syndrome, endothelial dysfunction, diabetes mellitus, hypertension, cardiovascular diseases, malignancy, and kidney and neurological diseases ,15,18–20This study has several limitations. First, the number of patients included is relatively low and it is a single-center study, and second, it is a female-only study. We think that the strength of our study is the combined evaluation of several parameters related with each other.Significantly decreased adropin levels and significantly increased levels of proinflammatory cytokines  were detected in vitamin D deficiency with the oxidative/antioxidative balance being changed in favor of oxidative status. Vitamin D could show its effects through vitamin D receptors on tissues via direct or indirect mechanisms. In addition, it may affect adropin release with positive or negative effects on the ENHO gene in adropin secreting tissues. Proinflammatory cytokines, oxidative stress, and adropin targeted studies could contribute to the prevention and treatment of diseases associated with vitamin D deficiency in future. Larger studies are needed to confirm these results.


A plant’s C2H2-type zinc finger proteins (C2H2-ZFPs) play crucial roles in the process of plant growth and development, as well as various stress responses. The Q-type ZFP family, which contains a conserved “QALGGH”, has been reported in many plants. Sugar beet is an important crop for sugar production. Salt stress and viral infection significantly reduce both sugar yield and processing quality of sugar beet. So far, the genome-wide analysis of Q-type C2H2 ZPFs and their expression pattern in sugar beet have not been analyzed yet. This study analyzed 35 Q-type ZFPs in sugar beet and their expression patterns under salt stress and virus. These results will provide theoretical evidence for understanding the functions of Q-type ZFPs.Beta vulgaris L.) is an important crop for sugar production. Salt stress and viral infection significantly reduce the root yield and sugar content of sugar beet. However, there is a lack of comprehensive genome-wide analyses of Q-type C2H2 ZFPs and their expression patterns in sugar beet under stress. In this study, 35 sugar beet Q-type C2H2 ZFPs (BvZFPs) containing at least one conserved “QALGGH” motif were identified via bioinformatics techniques using TBtools software. According to their evolutionary relationship, the BvZFPs were classified into five subclasses. Within each subclass, the physicochemical properties and motif compositions showed strong similarities. A Ka/Ks analysis indicated that the BvZFPs were conserved during evolution. Promoter cis-element analysis revealed that most BvZFPs are associated with elements related to phytohormone, biotic or abiotic stress, and plant development. The expression data showed that the BvZFPs in sugar beet are predominantly expressed in the root. In addition, BvZFPs are involved in the response to abiotic and biotic stresses, including salt stress and viral infection. Overall, these results will extend our understanding of the Q-type C2H2 gene family and provide valuable information for the biological breeding of sugar beet against abiotic and biotic stresses in the future.A plant’s Q-type C2H2-type ZFP plays key roles in plant growth and development and responses to biotic and abiotic stresses. Sugar beet ( Transcription factors (TFs) function as crucial molecular regulators for gene expression in all organisms . In planGlycine max [Brassica rapa [Medicago truncatula [Triticum aestivum [Oryza sativa [Arabidopsis thaliana [Zea mays [Sorghum bicolor [Cucumis sativus [Populus trichocarpa [Solanum lycopersicum [A. thaliana are categorized into three sets , with each set further divided into several different subsets, such as C1, C2, and C3 [2–4CX3FX3QALGGHX3–5H), identifying them as the plant-specific Q-type subfamily of C2H2 ZFP.C2H2-type ZFPs, also known as TFIIIA-type zinc fingers, are among the most extensively studied and abundant ZFPs in eukaryotes . Recentlcine max , 301 in ica rapa , 218 in uncatula , 204 in aestivum , 189 in a sativa , 176 in thaliana , 150 in Zea mays , 145 in  sativus , 109 in , and C3 . To datepetunia, and a total of 21 Q-type ZFPs were identified based on their specific structures [The first Q-type C2H2 ZFP was discovered in ructures . The conructures . In addiructures .AZF1, AZF2, AZF3, STZ/ZAT10, ZAT11, and ZAT18 is strongly induced by drought, salt, cold stresses, or abscisic acid treatment [ZAT7 and ZAT12 are upregulated by oxidative stress, heat shock, or wounding in A. thaliana [ZAT7 has been shown to be involved in plant growth suppression and increased tolerance to salinity stress depending on its EAR motif [Zat7 was more tolerant to salinity stress than seedlings of wild-type plants in Arabidopsis [Zat12 could tolerate oxidative stress in Arabidopsis [ZAT18 was initially identified as a positive regulator of drought stress tolerance in Arabidopsis [Pseudomonas syringae induces ZAT18 expression to repress the transcription of EDS1 for bacterial infection [ZAT18 overexpressing plants were more susceptible to Pst DC3000 compared to Col-0, while ZAT18-KO plants displayed enhanced resistance in Arabidopsis [T. aestivum, 47 Q-type C2H2-ZFPs were identified, and the expression of the majority of TaZFP genes was responsive to drought stress in either leaf or root [O. sativa, OsZFP179 was characterized as a salt-responsive gene, and it was found to enhance salt tolerance in transgenic rice plants [A. thaliana, T. aestivum, Brassica oleracea, S. lycopersicum, and Medicago. sativa via genome-wide identification [The plant Q-type C2H2 ZFP is involved in plant development and various abiotic stress responses, such as drought, salt, osmotic, low temperature, and oxidative stresses . For exareatment , while tthaliana . ZAT7 haAR motif . Overexpbidopsis . Transgebidopsis . More inbidopsis . A recennfection . ZAT18 o or root . In O. se plants . Until nfication ,31,33,34Beta vulgaris), a member of the Amaranthaceae family [BvbZIP and BvWRKY family, as well as their expression pattern under salt stress, have been genome-wide analyzed for sugar beet [Sugar beet  in sugar beet comprising different numbers of zinc finger domains. The phylogenetic relationships, genomic location, gene structure, chromosome distribution, gene duplication, and cis-regulatory elements are also explored. In addition, we determined their mRNA expression profiles in both leaf and root tissues under salt stress and BNYVV infection. This study aims to provide a comprehensive understanding of the Q-type C2H2 gene family and shed light on the roles of the BvZFP family in sugar beet, especially the functional characterization of plant development processes and stress responses. This research study can help improve plant quality, stress resistance, and enhanced crop production via genetic modifications in Q-type C2H2 gene family members in sugar beet.In this study, we identified 35 Q-type http://pfam.xfam.org/ (accessed on 15 March 2023)). Subsequently, C2H2 zinc finger protein candidates were identified based on the HMM profiles using Simple HMM Search in TBtools [http://smart.embl.de/smart/set_mode.cgi?NORMAL=1 (accessed on 22 March 2023)), InterPro (https://www.ebi.ac.uk/interpro/ (accessed on 22 March 2023)), and NCBI-CDD search (https://www.ncbi.nlm.nih.gov/cdd/ (accessed on 22 March 2023)). The physicochemical properties of the family members were analyzed using the online tool ExPASy Proteomics Server (https://web.expasy.org/protparam/ (accessed on 27 March 2023)). The subcellular localization analysis of each gene family member was carried out using the Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 27 March 2023)) online tool.The protein, cDNA, and genome annotation files of sugar beet were retrieved from the NCBI database (sugar beet EL10_1.0) . The HMM TBtools . To furtBvZFP genes on each chromosome were retrieved from the sugar beet gene annotation database, and the results were visualized using the Gene Location Visualize program of Tbtools. Out of 35 genes, 34 BvZFP genes were located on the nine chromosomes, while the remaining 1 may be located on unannotated intergenic regions of the genome. The members of sugar beet BvZFP were renamed according to the location on the chromosome. Protein properties, including the length of the amino acid (aa), molecular masses (MW), theoretical isoelectric point (pI), instability index, and subcellular localization, were predicted using ExPASy Server (https://web.expasy.org/protparam/ (accessed on 27 March 2023)).The starting and ending positions of all http://www.arabidopsis.org/ (accessed on 16 March 2023)). The multiple sequence alignment of Q-type zinc finger protein members in sugar beet and Arabidopsis was performed using MEGA software with the default parameters. Full-length amino acid sequences were aligned using ClustalX, and the phylogenetic tree was constructed using the neighbor-joining method with the following parameters: bootstrap method with 1000 replicates and partial deletion.The full-length sequences of Arabidopsis C2H2 C1-2i zinc finger proteins (Q-type ZFPs) were downloaded from the TAIR database (https://meme-suite.org/meme/ (accessed on 22 March 2023)) [The full-length protein sequences of each subfamily were analyzed for conserved motifs using Multiple Expectation Maximization for Motif Elicitation ) . We usedBvZFP start codons were extracted from the sugar beet EL10_1.0 genome. The predicted cis-acting elements on promoters with their positional information were identified using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 23 March 2023)). The obtained results were used to predict putative stress- and hormone-responsive cis-acting elements and visualized by Simple BioSequence Viewer in TBtools.The 2000 bp promoter sequences upstream of the 35 Arabidopsis was selected for collinearity analysis with sugar beet. The genome sequences and annotation files of Arabidopsis were downloaded from NCBI databases, and the chromosome length and location information for the ZFP genes on the genome of the two species were extracted. Using the One Step MCScanX tool of TBtools, we investigated gene replication events and collinearity relationships for gene pairs between two species. All data were visualized via the use of the Advanced Circos program of TBtools software. Ka and Ks substitution between gene pairs were also calculated using the Simple Ka/Ks Calculator tool.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA666117/ (accessed on 27 March 2023)), which could be used to analyze the expression pattern of BvZFP genes under 300 mM salt stress. In addition, the database of E-MTAB-8187 from NCBI was used to analyze the expression pattern of BvZFP genes under BNYVV infection. GraphPad Prism was used to map gene expressions.The transcriptome data of sugar beet under salt stress and BNYVV were downloaded from the NCBI SRA database . RSEM (http://deweylab.biostat.wisc.edu/rsem/ (accessed on 1 April 2023) was used to determine gene abundance. The R statistical package software edgeR ) was used for differential expression analysis.To identify B. vulgaris genome, we utilized hidden Markov model (HMM) files  to conduct a genome-wide HMM search. This search yielded 104 putative non-redundant sugar beet C2H2 ZFP proteins (BvZFPs). This number was greater than that present in PlantTFDB, where 64 BvZFPs have been deposited for sugar beet (http://planttfdb.gao-lab.org/family.php?sp=Bvu&fam=C2H2 (accessed on 17 March 2023)). Subsequently, 35 Q-type ZFPs, characterized by the specific CX2CX3FX3QALGGHX3–5HX domain, were manually selected . All BvZFP genes were distributed widely and unevenly on nine Chrs, except for BvZFP35, which was located on unmapped scaffolds , 6 members in the C1-2i group, 6 members in the C1-3i group, and 1 member in the C1-4i group , we constructed a phylogenetic tree using MEGA10 based on the alignment of 93 Q-type ZFPs amino acid sequences at the whole protein level, which included 35 from sugar beet and 58 from Arabidopsis. The resulting tree classified the members into six major clades, including C1-1Q-A, C1-1Q-B, C1-2Q, C1-QM-A, C1-QM-B, and C1-QM-C  is widely used for evaluating the selective pressure of duplication events [BvZAT7 and BvZAT32 is 0.1419  , stress response elements (low temperature and drought), and growth- and development-related response elements. These results spotlight potential candidate genes for anti-abiotic stress, although their specific functions need to be confirmed by further investigation. Previous studies have shown that genes with fewer introns are more prone to be activated in response to stress [BvZFPs have no introns, except for five BvZFPs, which indicates that BvZFPs could rapidly react to external stimuli. Collinearity analysis, an essential analytical strategy in comparative genomics, illuminates both large-scale and small-scale molecular evolutionary events across species. In this study, about 70% of the 34 mapped BvZFP genes are collinear with respect to four C2H2-ZFP genes on Arabidopsis chromosomes. The Ka/Ks ratios showed the conservation of BvZFPs throughout evolution, aligning with the previous views that regarded the C2H2 family as evolutionarily stable [A total of 104 C2H2 . annuum . Among t genomes . These Q4893654) . Based o genomes ,20. Inte C1-QM-B . Proteinactivity . Cis-actactivity . A previaestivum . In planaestivum ,62. Howeo stress . In thisy stable ,65. All PR1 defense gene in Arabidopsis [AtZP1, which contains an EAR motif, negatively regulates Arabidopsis root hair initiation and elongation [Besides the conserved zinc finger domains, many previously identified plant ZFP proteins have EAR motifs. These motifs, characterized by conserved amino acid signatures “LxLxL” or “DLNxxP” in their respective C-terminal regions , play a bidopsis . AtZP1, ongation . Most stongation .ZFP genes exhibited different expression patterns across various tissues [CsZFP genes in group 1 showed high expression levels, while CsZFPs in group 4 exhibited little to no expression in all tissues [StZFP genes displayed a tissue-specific expression pattern. Specifically, PG0005486 and PG0030311 were predominantly expressed in leaves and roots, respectively [FaZAT10 was highly expressed in roots, followed by leaves and stems [TaZFPs in different wheat tissues showed that 75% of TaZFPs were predominantly expressed in roots, implying their potential role in regulating wheat root development [BvZFPs displayed a specific expression pattern between leaves and roots (BvZFPs (16 out of 35) were not observed to be expressed in either leaf or root tissue, suggesting that these genes might not influence development at this growth stage. In contrast, 23% BvZFPs (8 out of 35 genes) were expressed preferentially in roots while 29% BvZFPs (10 out of 35) were expressed in both leaf and root tissues to variable degrees. Among the genes expressed in both tissues, root expression levels for BvZFPs were relatively higher than in leaf tissue except for one gene. In addition, BvZFP6, BvZFP27, and BvZFP30, each containing two “QALGGH” domains, were significantly expressed in roots. Therefore, the results suggested that many Q-type BvZFP genes are involved in sugar beet development under normal growth conditions, especially root development.Previous studies demonstrated that many plant Q-type  tissues ,74,75,76 tissues . In potaectively . In strand roots . With reNHX1 and ABI1 to actively regulate anthocyanin synthesis and increases sensitivity to salt stress in apple calli and Arabidopsis [Csa6G303740 was significantly up-regulated in response to drought, heat, and salt stresses, indicating it could be involved in the regulation of abiotic stresses in cucumbers [StZFP1, which responds to salt, dehydration, and infection by Phytophthora infestans, has the potential to improve salt tolerance in transgenic tobacco [Plants are frequently affected by various abiotic and biotic. To adapt to these stresses, plants have evolved sophisticated mechanisms. Numerous Q-type ZFP genes have been found in response to diverse stresses, such as drought, salt, osmotic, low temperature, oxidative stress, and pathogen infection in many plant species ,30,32. Sbidopsis . The ZFPucumbers . Potato  tobacco . BvZFP genes, BvZFP2, BvZFP6, BvZFP17, BvZFP30, and BvZFP34, were up-regulated in leaves or/and roots under salt treatment, indicating that these genes may play roles in salt response in sugar beet. These genes belong to the C1-2i and C1-3i groups, and all contain two Q-type zinc finger domains. In addition, BvZFP2, BvZFP17, and BvZFP 30 are grouped into the same C1-QM clade, while BvZFP6 and BvZFP34 are clustered into the C1-2Q clade. For biotic stress, twelve BvZFP genes were differentially expressed by BNYVV infection. Interestingly, BvZFP2, BvZFP6, BvZFP17, BvZFP30, and BvZFP34 were induced either by salt treatment or viral infection. On the other hand, some BvZFP genes only responded to viral infection; for example, BvZFP8 and BvZFP28 were significantly up-regulated in the BNYVV-infected samples, and BvZFP13, BvZFP25, BvZFP26, and BvZFP33 were down-regulated exclusively due to viral infection. These four down-regulated genes all belong to the C1-1i group and are clustered into the C1-1Q-A clade. BvZFP8 and BvZFP28 are in the C1-1Q-B and C1-2Q clades, respectively.In this work, we analyzed publicly available transcriptome data and found that five Arabidopsis ZAT6 is strongly induced by various stresses, including salt, cold, dehydration, and pathogen infection [AtZAT6 improved resistance to salt, drought, freezing stresses, and pathogen infection. In our study, the BvZFP30 clustered with AtZAT6 in C1-QM was differentially expressed under salt in sugar beet, indicating that BvZFP30 may have functions that are similar to AtZAT6 under abiotic stress. ZAT18 was initially shown to be a positive regulator of drought stress tolerance in Arabidopsis [Pseudomonas syringae induces ZAT18 expression to repress EDS1 transcription during bacterial infection [BvZFP6, BvZFP34, and BvZFP28, which are clustered with ZAT18, are induced by BNYVV infection. Given that both BvZFPs and ZAT18 are highly expressed under the attack of pathogens, it is plausible that these genes may have similar functions with respect to responding to biotic stress. Taken together, these results indicate that BvZFP genes are involved in plant development and stress resistance in sugar beet, particularly in response to both abiotic and biotic stresses.Generally, members in the same cluster may have conserved functions. Previous studies have shown that nfection . Plants bidopsis . A recenBvZFP genes that contain at least one conserved “QALGGH” motif were identified. We analyzed the physiochemical properties, genomic location, gene duplication event, gene structure, conserved motif compositions, and cis-regulatory elements of these BvZFP genes. Phylogenetic analysis revealed that 35 Q-type BvZFP genes were classified into five subclades. Moreover, the BvZFPs clustered within the same clade generally shared a similar motif composition. In addition, the expression profiles of BvZFPs in leaf and root tissues, as well as their responses to salt stress and viral infection, were analyzed. Q-type BvZFPs are predominantly expressed in roots and are enriched with members that are responsive to abiotic and biotic stresses. These results will enhance our understanding of the BvZFP gene family and provide valuable information for the further functional analysis of Q-type BvZFPs relative to the abiotic and biotic stress tolerance of sugar beet. Meanwhile, this study provides a theoretical basis for the biological breeding of sugar beet against salt stress and viral infection in the future.In summary, we performed a genome-wide exploration of Q-type ZFPs in sugar beet, and a total of 35 Q-type


Increased PMN-FOXO3389 presence predicted colon cancer invasion  and poor survival. Validated DEGs from PMN-FOXO3389  are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs’ coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes  involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3 Inflammatory bowel disease (IBD), a chronic inflammation of the intestinal tract, includes two distinct clinical entities, Crohn’s disease (CD) and ulcerative colitis (UC) . Both CDPMNs, accounting for ~60% of circulating immune cells, are the first and most rapidly migrating cells to the sites of tissue damage and microbial invasion, where they neutralize microorganisms, recruit other immune cells, and remodel damaged tissues to resolve injury ,11. In tFOXO3 (leading to its reduced levels) to the severity of inflammation in IBD [In mouse models of colonic inflammation and inflammatory tumors, LDs are elevated in colonic cells and infiltrated immune cells ,28,29,30n in IBD . Addition in IBD ,36. GiveWe have previously demonstrated in the inflamed mouse colon elevated levels of LDs in colonic and infiltrating immune cells . To detep < 0.05). Further, FOXO3 KO PMNs, relative to WT, had significantly increased migration in response to fMLP, as shown by the MPO activity (p < 0.05). These findings demonstrate that PMNs migration is increased with LDs accumulation and FOXO3 deficiency, suggesting that LDs and FOXO3 regulatory networks in PMNs may play a role in driving inflammation and tumorigenesis.We have demonstrated the critical role of elevated LDs and loss of FOXO3, an established regulatory network in colonic cells, in driving inflammation and tumorigenesis in the colon, the role of which in immune cells is not well understood. As PMNs are critical players in inflammatory and tumorigenic processes in the colon ,9,38,39,Next, we determined in FOXO3-deficient PMNs systemic transcriptional changes and associated molecular pathways driving their activity in the colon. Transcriptional assessment of intraperitoneal PMNs from FOXO3 KO mice showed 212 increased and 242 decreased differentially expressed genes (DEGs) relative to WT . The diseases and functions associated with these DEGs include gastrointestinal disorders, metabolic diseases, lipid metabolism, immune responses, immune cell trafficking, inflammatory pathways, and cancer A. To detBased on our above findings, we sought to determine the significance of the loss of FOXO3 in PMNs in IBD and human colon cancer. The transcriptomes from PMNs of FOXO3 KO mouse have shared regulators with those representing publicly available transcriptomes of affected tissue of different patient cohorts with UC  and CD  A,B. Simi389), with stringent differential expression and statistical thresholds relative to WT . Unsupervised hierarchical clustering demonstrated that the PMN-FOXO3389 signature separated transcriptomes  of affected IBD tissue (UC and CD) and colon cancer samples (adenomas and adenocarcinomas) from the control (unaffected) group  and p = 3.7 × 10−3 for colon cancer   group A. Additie normal B,C. The  normal) D,E.389 in a large number of colon cancer patients using publicly available transcriptomes from the TCGA database. In unsupervised hierarchal clustering, PMN-FOXO3389 signature separated transcriptomes of colon cancer samples from normal, which was further quantified by PCA index score between two groups (p = 2 × 10−16) , lymphovasculature , and perineural space   when compared to 65% (55–74%) for lower presence (p = 8.2 × 10−4) (TCGA)  A,B. In a = 0.03) C–E. More) (TCGA) F. TogethP2RX1), which belongs to G-protein-coupled receptors, RalA-binding protein 1 (RALBP1), which plays a role in receptor-mediated endocytosis, and melanoma cell adhesion molecule (MCAM), as well as cell cycle progression gene 1 (CCPG1), which is involved in the immune response to endoplasmic reticulum stress and cyclin-dependent kinase inhibitor 1A (CDKN1A). Further, Phospholipase A2 Group VII (PLA2G7) is responsible for phospholipid metabolism, and Monoacylglycerol lipase (MGLL) catalyzes the conversion of monoacylglycerides to free fatty acids and glycerol. In addition to MGLL, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were lowered in DEGs from FOXO3 KO PMNs (NS). These lipases are responsible for the breakdown of triacylglycerols from LDs [We identified the top FOXO3 KO-dependent DEGs in PMNs, as shown in the heatmap A and Tabfrom LDs , suggest389 transcriptional signature, which is increased in IBD and human colon cancer and is highly significant in their pathobiology. Ultimately, we identified FOXO3-dependent differentially expressed genes in PMNs with roles in metabolism, inflammation, and tumorigenesis. Together, these findings establish a novel mechanism in PMNs involving LDs and FOXO3, driving inflammatory and tumorigenesis processes in the colon.Chronic inflammation in IBD, characterized by massive infiltration of PMNs, is associated with increased risk and progression of colon cancer ,5,6,7. HTLR9, suggesting their sensitivity to viral components. Moreover, this LDs and FOXO3-mediated immune sensitivity to infection is shown in FOXO3 KO colon with increased bacterial LPS sensing TLR4 [Aberrant PMN function exacerbates inflammation and tumorigenesis in the colon ,9,14,38.ing TLR4 . Additioing TLR4 . Furthering TLR4 . For insing TLR4 . Moreoveing TLR4 . Hence, NR1H4), a sensor of intracellular bile acid levels. FXR protects against bile acid toxicity, and it is reduced in CD patients [Moreover, we demonstrated that in PMNs, a negative regulatory network of LDs and FOXO3 might be one of the mechanisms of colonic inflammation and tumorigenesis within IBD and colon cancer. We have previously demonstrated that this regulatory network drives pathobiological processes in human colonic cells ,29,30,31patients .MGLL is involved in fatty acid metabolism and plays roles in tumorigenesis and metastasis [P2RX1 is linked to the modulation of microbiota and the alleviation of inflammation in colitis [PLA2G7, which is shown to be reduced in FOXO3 KO PMNs, is associated with inflammation [MCAM and CDKN1A, in FOXO3 KO PMNs, are involved in tumorigenesis [RALBP1 has a key effector function in cancer cell survival [MGLL, ATGL, and HSL lipase, critical for the breakdown of triacylglycerols stored in LDs [Further, we identified several FOXO3-dependent differentially expressed genes in PMNs involved in metabolism, inflammation, and tumorigenesis. Specifically, tastasis . P2RX1 i colitis . PLA2G7,ammation . Two othigenesis ,57. RALBsurvival . Moreoved in LDs , are decd in LDs . These fd in LDs ,30,31,35One of the hallmarks of IBD is excessive infiltration of PMNs that intensify the inflammatory pathobiology, leading to tumorigenesis ,12,13,15n = 10, n = 2 of each for normal, UC, CD, tumor-adjacent mucosa, and adenocarcinoma).Tissue microarray samples included human intestinal tissue representing normal, Inflammatory Bowel Diseases (Ulcerative Colitis and Crohn’s Disease), and colon adenocarcinoma with tumor-adjacent colonic mucosa (tissuearray.com LLC). These tissues were obtained from different individuals, males and females, aged 20 to 66  and CD . Publicly available transcriptomic data from tumor tissue obtained from three colon cancer patient cohorts  were used. Moreover, publicly available transcriptomic data of colon cancer patients, including normal controls, were also utilized . These data were acquired by using NCBI’s GEO2R.Publicly available transcriptomes were obtained for control and affected tissue from three different patient cohorts of UC  [Mice, strain C57BL/6, male and female, were housed under pathogen-free conditions at Tulane University School of Medicine. Both wild type (WT) and FOXO3 knock-out (FOXO3 KO) mice had free access to a standard chow diet and water. All littermates were genotyped to identify homozygous WT and FOXO3 KO, according to the guidelines of Tulane Institutional Animal Care and Use Committee . Transcrr group) .n = 3) and dysplasia (n = 3) were utilized (GSE31106) [Moreover, publicly available transcriptomes obtained from mice with colonic inflammation (SE31106) .Immunohistostaining against antibody PLIN2  of tissue microarray with samples from IBD and human colon cancer patients was performed by The Pathology Core Laboratory at Tulane University Health Sciences Center as described previously . Images g for 3 times). Next, PMNs were isolated from the peritoneal fluid using histopaque separation media using a density gradient centrifugation method [Experimental mice (six to eight weeks old) were injected intraperitoneally with 1 mL of sterile casein  solution, followed by a second injection the next day, which caused a response of peritoneal PMNs . Three hn method .6 cells in assay buffer, HEPES containing 10 mM glucose, 0.1% BSA, pH 7.4, and 1% penicillin/streptomycin, was incubated with oleic acid (OA) (50 µM), for 2 h, and were placed on the top of transwells, an 8 mM pore size polycarbonate filter. In the lower compartments of transwells, 1 mL of the assay media with N-Formylmethionyl-leucyl-phenylalanine (fMLP) (1 mM) or chemokine KC (29 ng/mL) was added. After 30 min, at 37 °C, assay media from the lower compartment was collected and centrifuged at 400× g for 5 min to assess for migrated PMNs.Mice peritoneal PMNs, 10 × 10n = 6) and FOXO3 KO (n = 6) mice (n = 3 wells per mouse) using MPO Colorimetric Activity Assay Kit according to the manufacturer’s protocol . Briefly, collected PMN samples were rapidly homogenized in MPO assay buffer and centrifuged at 13,000× g for 10 min at 4 °C to remove insoluble material. These samples were then plated in a 96-well plate, further assessed, and used for colorimetric detection of MPO activity at 412 nm.Enzymatic activity of the activated PMN’s marker, known as myeloperoxidase (MPO), was quantified in PMNs from WT (n = 5–6 mice per group) was isolated using the miRNeasy kit , following the manufacturer’s instructions. First, RNA was evaluated for quality using Agilent Bioanalyzer . Samples having RNA integrity numbers (RIN) of more than 8 were utilized. RNA was then reverse transcribed to cDNA with qScript cDNA SuperMix  and used for qPCR as described previously [Total RNA from harvested PMNs (eviously . The priRNA sequencing (RNAseq) was performed as described previously . SequencHierarchical clustering of transcriptomes among experimental groups was performed using an uncentered correlation as a symmetric matrix, as described before .Principal component analysis (PCA) of FOXO3-deficient PMNs’ signatures with IBD (GSE4183) and human colon cancer (TCGA) transcriptomes was performed with the FactoMineR R package with the PCA function, as described before .t-test or through ANOVA for one-way analysis of variance in Graph Pad Software. A p-value of <0.05 was considered significant.All results are represented as mean ± SD. The statistical analysis of experiments was carried out by Student’s unpaired


Prader-Willi syndrome (PWS) is a rare genetic disorder characterized by loss of expression of paternal chromosome 15q11.2-q13 genes. Individuals with PWS exhibit unique physical, endocrine, and metabolic traits associated with severe obesity. Identifying liver steatosis in PWS is challenging, despite its lower prevalence compared to non-syndromic obesity. Reliable biomarkers are crucial for the early detection and management of this condition associated with the complex metabolic profile and cardiovascular risks in PWS.Circulating proteome profiling was conducted in 29 individuals with PWS  using the Olink Target 96 metabolism and cardiometabolic panels. Correlation analysis was performed to identify the association between protein biomarkes and clinical variables, while the gene enrichment analysis was conducted to identify pathways linked to deregulated proteins. Receiver operating characteristic (ROC) curves assessed the discriminatory power of circulating protein while a logistic regression model evaluated the potential of a combination of protein biomarkers.CDH2, CTSO, QDPR, CANT1, ALDH1A1, TYMP, ADGRE, KYAT1, MCFD, SEMA3F, THOP1, TXND5, SSC4D, FBP1, and CES1 exhibited a significant differential expression in liver steatosis, with a progressive increase from grade 1 to grade 3. FBP1, CES1, and QDPR showed predominant liver expression. The logistic regression model, -34.19 + 0.85 * QDPR*QDPR + 0.75 * CANT1*TYMP - 0.46 * THOP1*ALDH1A, achieved an AUC of 0.93 (95% CI: 0.63-0.99), with a sensitivity of 93% and specificity of 80% for detecting steatosis in individuals with PWS. These biomarkers showed strong correlations among themselves and were involved in an interconnected network of 62 nodes, related to seven metabolic pathways. They were also significantly associated with cholesterol, LDL, triglycerides, transaminases, HbA1c, FLI, APRI, and HOMA, and showed a negative correlation with HDL levels.The biomarkers identified in this study offer the potential for improved patient stratification and personalized therapeutic protocols. It is caused by the lack of expression of genes located on the paternal chromosome 15q11.2-q13, resulting in various metabolic abnormalities and hormonal dysregulation , 5.While obesity is a prominent feature of PWS, the metabolic profile and cardiovascular complications associated with the syndrome differ from those observed in individuals with non-syndromic obesity , 7. Indi22.1Thirty-one individuals with PWS , hospitalized for a three-week multidisciplinary body weight reduction program at the Division of Auxology, Istituto Auxologico Italiano, IRCCS, Piancavallo-Verbania, Italy, were recruited for the present study. All subjects with PWS showed the typical clinical phenotype , 26, as 2.2® serum separating tubes . The tubes were then centrifuged at 1900 x g for 10 minutes at 4°C. Following the initial centrifugation, the resulting supernatants were carefully transferred into new tubes. Subsequently, samples were centrifug ed at 16000 x g for 10 minutes at 4°C. Finally, the supernatants were aliquoted into new tubes and promptly frozen at -80°C to ensure long-term storage until further analysis.After an overnight fast, blood samples were obtained through standard venipuncture using BD Vacutainer2.3Blood samples were analyzed using the proximity extension assay (PEA) on Olink Target 96 metabolism and cardiometabolic panels . In this process, the target proteins specifically bind to double oligonucleotide-labeled antibody probes. Subsequently, microfluidic real-time PCR amplifies the oligonucleotide sequence for quantitative DNA sequence detection . Thresho2.4http://geneontology.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/pathway.html), Reactome (https://reactome.org/), and GeneCards (https://www.genecards.org/). During the enrichment analyses, we associated all the significantly expressed proteins with their respective terms or pathways in the GO or KEGG database. The analysis was carried out using the hypergeometric test or Fisher’s exact test with default settings. This process allowed us to gain insights into the functional implications of the identified proteins in the context of liver steatosis. In addition, the expression data of the proteins of interest were investigated in the GTEx data portal . By querying the selected tissue expression database, a heatmap with pseudocolor representation was generated. This is to visualize the expression patterns of these proteins across different tissues. STRING Version 11.5 (https://string-db.org) was employed to construct a protein-protein interaction network. The network was generated by using a query of five proteins, as described in the results section.To identify relevant pathways and perform enrichment analyses, we utilized various databases including Gene Ontology (GO) (2.5Correlations between variables were investigated using the Spearman rank correlation test (considering the low sample size). Correlation matrices were generated with R 4.2.2  by using2.6We assessed the robustness of the logistic regression model using a non-parametric bootstrap simulation with out-of-bag prediction over 10,000 iterations. The corresponding R script can be found in 2.7T-test was used to determine significant differences among normally distributed continuous variables, while the Mann-Whitney was used for non-normally distributed variables. The Chi-Squares test was used for categorical variables. The Kruskal-Wallis test in the One-Way ANOVA procedure with Benjamini Hochberg correction was used for multiple comparisons. The receiver operating characteristic (ROC) curves were plotted to estimate the discriminatory potential of the circulating protein biomarkers. A hierarchical forward selection with switching one-way logistic analysis was used to estimate the discriminatory potential of the protein biomarker combination. Analyses were performed using NCSS 11 Software (2016) .33.1Olink proteomic analysis of individuals with PWS was performed to identify circulating biomarkers associated with the presence of steatosis. The studied population was grouped according to absence (S0) or presence (S1-S2-S3) of steatosis. 3.2The circulating proteome was investigated using the Olink metabolism and cardiometabolic panels, each consisting of 92 different human protein biomarkers Table S13.3https://gtexportal.org/home/), we found that the liver exhibited the highest mRNA expression levels of FBP1, CES1, and QDPR among the analyzed organs  and cut-off values based on the Youden index, which maximizes sensitivity and specificity for each candidate. The results, including the AUC values and cut-off values, are presented in in silico simulation provided additional validation for our model. We evaluated the logistic regression model using observations from the bootstrap sample, resulting in an AUC of 0.83, a sensitivity of 0.81 and aspecificity of 0.87 at a determined cut-off of 0.554  and KYAT1 , also known as CCBL1, which in turn correlated with THOP1  Figure 6The role of those candidates was confirmed by the corrplot matrix, which showed a significant positive correlation of QDPR, ALDH1A1, TYMP, and THOP1 with several clinical variables associated with liver disease, such as cholesterol level, LDL, triglycerides, transaminases levels, and HbA1c, as well as with disease-associated scores such as FLI, APRI, and HOMA Figure 84The use of biomarkers for detecting and monitoring liver steatosis holds significant potential in individuals with PWS. Liver steatosis, characterized by fat accumulation in the liver cells, is a common complication in PWS and is associated with various metabolic abnormalities and an increased risk of liver-related complications , 13, 34.Circulating biomarkers provide a minimally invasive approach easily obtained from individuals with PWS. This minimally invasive nature of biomarkers not only improves patient comfort and compliance but also allows for repeated measurements, facilitating longitudinal monitoring of liver health and response to therapy. For these reasons, the present study aimed to identify circulating protein biomarkers associated with liver steatosis in individuals with PWS.Patients with liver steatosis showed a significant increase in BMI, diastolic pressure, insulin, HOMA index, glycated hemoglobin (HbA1c), C-reactive protein, triglycerides, fatty liver index (FLI), and the presence of metabolic syndrome. As for non-syndromic obesity, these findings are consistent with previous studies linking these factors to the development and progression of liver steatosis –37.To further characterize the disease, a circulating proteome analysis was performed.To the best of our knowledge, our study represents the first attempt to comprehensively investigate the circulating proteome profile in individuals with PWS and its association with steatosis. We identified 15 circulating proteins that exhibited higher expression levels in individuals with PWS and steatosis as compared with those without. These proteins include CDH2, CTSO, QDPR, CANT1, ALDH1A1, TYMP, ADGRE, KYAT1, MCFD2, SEMA3F, THOP1, TXND5, SSC4D, FBP1, and CES1. These proteins have been implicated in various biological processes and metabolic pathways, including those related to liver function.Upon examining mRNA expression profiles in different organs within the GTEx database, it became evident that the liver exhibited the highest mRNA expression levels of FBP1, CES1, and QDPR, suggesting that these proteins play crucial roles in liver functions. They may be specifically involved in the angiopoietin-like protein 8 regulatory pathway, disorders of fructose metabolism, glycolysis and gluconeogenesis, cholesterol, and sphingolipid transport. Interestingly, the circulating angiopoietin Like 8 (ANGPTL8) has already been associated with liver steatosis in PWS .in vivo, have been shown to prevent high-fat diet-induced lipid accumulation in the liver and reduce plasma triacylglycerol levels . It is noteworthy that SIGLEC7 expression in subjects with steatosis grades 2 and 3 was higher compared to subjects without steatosis or with steatosis grade 1. In parallel, DDP7 was higher in the group with grade 3 steatosis suggesting that these candidates perform better in discriminating subjects with more severe steatosis. Both proteins participate in immune system-related pathways. SIGLEC 7 has a sialic acid binding activity involved in cell adhesion and it is expressed in immune cells, while DPP7 is involved in the maintenance of quiescent lymphocytes , 43. A rIn this study, we evaluated the discriminatory potential of the 15 protein candidates in the detection of liver steatosis in individuals with PWS. When a logistic regression model including QDPR, CANT1, TYMP, THOP1, and ALDH1A1 was constructed the model exhibited an AUC of 0.93 (95% CI: 0.63-0.99), with a sensitivity of 93% and specificity of 80%, highlighting the potential of these proteins as biomarkers for the detection of liver steatosis in PWS. Interestingly, these biomarkers well correlated in the serum of individuals with PWS and they are part of a cellular network connecting several metabolic pathways, including 5-Hydroxytryptamine biosynthesis, Salvage pyrimidine deoxyribonucleotides, Tetrahydrofolate biosynthesis, Pyrimidine Metabolism, Salvage pyrimidine ribonucleotides, Beta2 adrenergic receptor signaling pathway, and Beta1 adrenergic receptor signaling pathway.Consistently, 5-Hydroxytryptamine (5-HT) has been demonstrated to regulate lipid metabolism in the liver through the activation of the mTOR pathway , 49. In By using an imprinting center deletion mouse model for PWS (PWSICdel), Davies and colleagues (2019) demonstrated the abnormal serotonin 2C receptor (5-HT2CR) function in this genetic syndrome , 52. SinDisruption of pyrimidine metabolism was associated with lipid accumulation in the liver  and the Our study has some limitations. Firstly, histological confirmation of hepatic steatosis was not performed, as a liver biopsy is invasive, scarcely acceptable by these individuals, and has potential complications. Instead, we relied on non-invasive imaging and blood biomarker assessments as screening tools . SecondlThe original contributions presented in the study are included in the article/The studies involving humans were approved by ethics Committee of Istituto Auxologico Italiano Milan, Italy (ethical committee code: CE: 2022_03_15_07; research code: 01C216; acronym: PROTEOMARKER. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.DP: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. PG: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. CB: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. SG: Conceptualization, Investigation, Methodology, Writing – review & editing. CT: Conceptualization, Supervision, Writing – original draft, Writing – review & editing. AB: Data curation, Investigation, Methodology, Writing – review & editing. DC: Data curation, Investigation, Methodology, Writing – review & editing. AM: Investigation, Methodology, Writing – review & editing. GG: Data curation, Investigation, Methodology, Writing – review & editing. AS: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.


Showing noteworthy potencies to neutralize all of the radicals at the ng/mL level, the active EOs were also characterized as protectors of DNA double strands from damage induced by peroxyl radicals (ROO•), emerging from 2,2′-azobis-2-methyl-propanimidamide (AAPH) as a source, and OH•, indicating some genome protectivity and antigenotoxicity effectiveness in vitro. The chemical compositions of the EOs associated with the obtained activities were then analyzed by means of machine learning (ML) classification algorithms to generate quantitative composition–activity relationships (QCARs) models (models published in the AI4EssOil database available online). The QCARs models enabled us to highlight the key features  for exerting the redox potencies and to define the partial dependencies of the features, viz. percentages in the mixture required to exert a given potency. The ML-based models explained either the positive or negative contribution of the most important chemical components: limonene, linalool, carvacrol, eucalyptol, α-pinene, thymol, caryophyllene, p-cymene, eugenol, and chrysanthone. Finally, the most potent EOs in vitro, Ylang-ylang (Cananga odorata (Lam.)) and Ceylon cinnamon peel (Cinnamomum verum J. Presl), were promptly administered in vivo to evaluate the rescue ability against redox damage caused by CCl4, thereby verifying their antioxidant and antigenotoxic properties either in the liver or in the kidney. The antioxidant activity of essential oils (EOs) is an important and frequently studied property, yet it is not sufficiently understood in terms of the contribution of EOs mixtures’ constituents and biological properties. In this study, a series of 61 commercial EOs were first evaluated as antioxidants in vitro, following as closely as possible the cellular pathways of reactive oxygen species (ROS) generation. Hence, EOs were assessed for the ability either to chelate metal ions, thus interfering with ROS generation within the respiratory chain, or to neutralize 2,2-diphenyl-1-picrylhydrazyl (DPPH O- and O,S-heterocycles, as well as N- and N,S-heterocycles, that contribute to free radical scavenging activity , hydroperoxyl radical [HOO•], and hydroxyl radical [OH•]), reactive lipid species , reactive nitrogen species (RNS), such as nitric oxide radical [NO•], and non-free radicals, like hydrogen peroxide (H2O2) and peroxynitrite (ONOO), are in vivo oxidative stress inducers by interacting with cellular biomolecules  , s, s111], pred values. For details, see Refined models were investigated with 10,000 and 100,000 random combination runs from all possible considered combinations , while aCV obtained from 50 random CV iterations ,,2•− has cid (11) ,135, on DA) (14) .n+ metal ions play an important role at the very beginning of redox signaling or later in the redox signaling cascade  showed chelating properties in the range of EC50 = 0.63–1.72 μg/mL. Nevertheless, no significant linear regression correlation was obtained between TAC and Mn+ potencies. Interestingly, the YY, CCP, Rosemary, and Savory EOs were active in all antioxidant assays and characterized by medium to high EC50 values of 0.84, 095, 1.39, and 1.46 μg/mL, respectively  during the rate-limiting propagation step of lipid peroxidation (13)  LOO• = 1.0511 × [EC50] DPPH• + 0.0599), indicating some predictivity ability for LPI from a DPPH assay. Among the 17 EOs found active in the DPPH• assay above, Red thyme, Oregano, YY, Lemongrass, Rosemary, Natural nutmeg, Thymus vulgaris, Coridothymus capitatus, and White thyme were more potent against LOO• and Cade and Birch EO were equally potent against both radicals, whereas Cedar leaf, CCP, Clove, and Origanum hirtum EO were more potent against DPPH• with respect to LOO•. Interestingly, CCP, Savory, Rosemary, and YY EOs, potent against any given metal ion/radical species, showed EC50s against LOO• of 0.032, 0.180, 0.230, and 0.230 µg/mL, respectively   OH• = 0.6389 × [EC50] ABTS•+ + 0.0501, implying that the ABTS assay should be taken with caution as a predictor of potency against OH•.Based on the results of the ABTS assay, EOs were further tested for their ability to neutralize the hydroxyl radicals in vitro  by means radical  and maloDA) (14) . The neu50 concentration of active EOs against OH• ranged from 0.01 (Red thyme EO) to 0.72 µg/mL . In summary, eight EOs  were about 10% to 600% more sensitive to ABTS•+, whereas Origanum hirtum, YY, Savory, White thyme, CCP, Thymus vulgaris, Cedar leaf, and Lemongrass EOs were from 1.12- to 3.25-folds more sensitive to OH•. Moreover, Savory-, CCP-, YY-, and Rosemary-derived EOs were also active against OH•, with EC50s of 0.032, 0.078, 0.35, and 0.42 µg/mL, respectively , released from AAPH by the homolytic cleavage of the two nitrogen–carbon bonds to form two isobutyrimidamide radicals (R•) and subsequent oxidation with O2 [• (and its homolog LOO•) is associated with the modification of DNA, such as 8-oxoG (17), which could lead to inflammation and degenerative pathologies, including cardiovascular and neurological diseases, as well as the development of some cancers [•, here formed in vitro in the Fenton-type oxidation of H2O2 catalyzed by FeSO4, also attacked the salmon DNA double-strand [17), thymine glycol (18), and 6-hydroxy-5,6-dihydrocytosine (19) [The damage within the DNA double-strand was induced by the 2-hydroperoxy-2-methylpropanimidamide radical  ,142.1 indicate better protection , were calculated  of DNA  knowing otection . Each EOormation ; (2) wasormation . An anallculated  to diffeEucalyptus globulus, Artemisia, White thyme, Marjoram, Cypress, Peppermint, Palmarosa, Natural anise, Frankincense, Mentha suaveolens, and Coridothymus capitatus) protected salmon DNA from damage induced by either ROO• or OH• in a dose-dependent manner, with the level of protection increasing with increasing concentrations , Normalized MCC (nMCC) was considered, defined as nMCC = MCC + 1/2, and linearly projecting the original range into the 0-1 interval. A model was generated for each of the evaluated in vitro assays .As mentioned above, the correlations of the in vitro antioxidant potencies of EOs with their chemical compositions were analyzed by means of ML algorithms to generate QCAR models . The preo assays . In genePred and nMCCCV values , eugenol , and chrysanthone  as the most important chemical components, which were characterized by the WFI  where an alternating line trend can be seen when the concentration of this component is between 0 and 20%. The descending curve was in part a reflection of 10.36% of linalool in the composition of YY EO   . A very  (RBD50) A, despitD50)   . To counteract the hazardous behavior of CCl4, EOs were i.p.administered in a separate protocol simultaneously with CCl4 [4 is likely to be oxidized by rat CYP2E1 for the production of trichloromethyl radical , an intermediate metabolite that is further oxidized to trichloromethylperoxy radical  [rCCl3OO• could initiate the peroxidation of polyunsaturated fatty acids in the membrane of either hepatocytes or kidneys (rMDA formation (14), whereas its excess may generate cellular rO2•− (5) and H2O2 (5) [YY and CCP EOs, the EOs exerting high potencies against each redox species in in vitro assays, , were pr4  to adult4   ,157,158.H2O2 (5) ,157,158.rTBARS) [rGSH) [rSOD) [rCAT) [rTBARS [rGSH [rCAT [rTP) [rAST) [rALT) [rALP) [rγ-GT) [4-induced intoxication and its prevention by EOs were investigated by monitoring the catalytic activities of renal toxicity markers, such as xanthine oxidase (rXO) [rNOX) [rNO) [rGPx) [Therefore, the efficacies of EOs as lipid peroxidation inhibitors and consequent redox-stress-reducing mediators in vivo within either the liver or kidneys were assessed by monitoring the concentrations of the thiobarbituric-acid-reactive substance (rTBARS)  and redu) [rGSH) , as well) [rSOD)  and cata) [rCAT)   . On the ) [rAST) ,160 and ) [rALT) , while d) [rALP)  and γ-gl [rγ-GT)  Table 2rTBARS [r [rγ-GT)  after CCse (rXO)  and NADP) [rNOX) , as wellX) [rNO)  and glut) [rGPx) .In general, within all of the observed results, dose-dependent effects were observed upon administration of either YY EO or CCP EO.rCCl3OO• radical can induce the formation of malondialdehyde (rMDA), a toxic metabolite major side-product of lipid peroxidation (14) [rMDA with thiobarbituric acid, leading to the formation of rTBARS  . Therefof rTBARS , whose irSOD for the rCCl3OO• to rCCl3• and rO2•− conversion  [rO2•- likely acts as a secondary source of lipid peroxidation, and rSOD catalyzes the dismutation of rO2•− into oxygen (O2) and hydrogen peroxide (H2O2)  . Moreovee stress . This scrH2O2 is promptly decomposed to rOH• (9) and this could lead to 8-oxo-dG, thymineglycol, and 6-hydroxy-5,6-dihydrocytosine  [rCAT induces oxidative stress in tissue [rCAT, so that an excess of rH2O2 is promptly decomposed to water and molecular oxygen (4 caused a moderate decrease (58%) in the catalytic activity of rCAT  . Considen tissue , either rGSH concentration (rCCl3OO• to form rCCl3OOH (23) and rCCl3OH (24) [rH2O2 to rH2O  was observed than in the negative control   or by co4-induced damage was further monitored through the catalytic activities of rAST and rALT, whereas the condition of the bile duct was determined by means of the catalytic activities of rALP and rγ-GT  and 114-fold (vs. 1.07-fold of YY EO) above the CCl4 and negative control groups , ending in an additional generation of rO2•− [rXO’s catalytic activity  within kidneys’ endoplasmic reticulum and may lead to the progression of oxidative stress. Therefore, a significant upregulation of NOX  represents an indicator of renal insufficiency [rNOX did respond intensively to the oxidative damage caused by CCl4, as the positive control sample was characterized by a 3.67-fold higher enzyme catalytic activity than the negative control (compare group II with I in NADPH oxidase (NOX), usually considered as a marker for cardiovascular, hemorrhagic-shock-induced organ injury ,163, hepficiency . In factrNOX response agrees with the EOs’ rescued catalytic activity of rCAT, which can decompose rH2O2 generated by rSOD from the rO2•− decomposition. In this scenario, rNOX then captures rO2 and converts it into rO2•−  cells using micronucleus assay [Bacillus subtilis DNA-repair test (Rec-assay), Escherichia coli WP2uvrA reversion test [Drosophila melanogaster Somatic Mutation and Recombination Test [rCCl3OO• that could cause the formation of rMDA and transform the DNA into the aberrant adducts, like M1G, M1A, and M1C , which root the transversions and transitions of bases and, upon the quantitative ring-opening, ultimately form N2-oxopropenyl-dG, N2-oxopropenyl-dA, and N2-oxopropenyl-dC adducts , leading to the DNA–DNA inter-strand cross-links or DNA–protein inter-strand crosslinks [In parallel with the in vivo antioxidant evaluation, the Wistar rats’ liver and kidney samples were used for determining the selected YY and CCP EOs’ antigenotoxic activities by means of the comet assay  128,165165. Whilus assay , Ames Saion test , and Droion Test . Herein,osslinks  to which0) and a small number of comets associated with very low damage (T1)  of 89.8% and an absence of comet types T3 and T4. Although the CCP EO delivered 1.8-fold higher TCS than in negative control group  compared to that observed for CCP EO could be, in part, associated with the higher percentage of caryophyllene  , I, or Col group , 31.24% in vitro . Eugenolin vitro , was a l4-induced DNA damage were also assessed in the kidney cells of albino Wistar rats (0) and a few of them indicated very low damage (type T1) . Differently from what was observed in the case of the liver, CCP EO was more potent than YY EO at the highest tested concentration. Both YY and CCP EO showed lower DNA protection  , I, or C•, LOO•, ABTS•+, and OH•. The titled EOs also protected the DNA from damage induced by ROO• or OH•. Among the tested EOs, those obtained from YY and CCP showed the best antioxidant profile and were selected for in vivo investigations.Extensive in vitro assays on a list of 61 EOs were performed, leading to interesting and unique results. Many EO samples were shown to be able to modulate the TAC, to chelate the transition metal ions, and to neutralize the DPPHn+, DPPH•, LOO•, ABTS•+, OH• (either on the level of nucleotides or a DNA double-strand), or ROO• (protecting the DNA) by means of the machine learning (ML) classification algorithms, like SVM, RF, GB, DT, and KNN, various QCAR models were generated, yielding the most important features to be limonene, linalool, carvacrol, eucalyptol, α-pinene, thymol, caryophyllene, p-cymene, eugenol, and chrysanthone, and characterizing their either positive or negative contributions through features importance plots as well as indicating the percentages of features within the EO mixtures required to either positively or negatively contribute to the listed antioxidant activities through the Partial Dependencies plots. The features’ predicted contributions were in good agreement with their previously reported experimentally defined biological profiles while exerting antioxidant potential, thus validating the models’ accuracies. As an added value of this research, all of the generated models can be freely used for the prediction of any untested EO for its antioxidant potencies, as a part of the AI4EssOil project .By correlating the chemical compositions of 61 commercial EOs with their in vitro experimentally determined abilities against M4. Experimental in vivo data strongly suggested that both YY and CCP EO represent potential new and effective antioxidant agents able to protect the cell from injuries caused by toxicants, such as CCl4. Further studies are in due course to investigate the isolated components indicated by the ML models as possible new antioxidant compounds.The features’ potential contributions in vivo were thereafter anticipated upon the administration of the most potent EOs (from YY and CCP) in adult Wistar rats against the redox and genotoxic damage caused by CCl


Salmonella Isangi is an infrequent serovar that has recently been reported in several countries due to nosocomial infections. A considerable number of reports indicate Salmonella Isangi multidrug resistance, especially to cephalosporins, which could potentially pose a risk to public health worldwide. Genomic analysis is an excellent tool for monitoring the emergence of microorganisms and related factors. In this context, the aim of this study was to carry out a genomic analysis of Salmonella Isangi isolated from poultry in Brazil, and to compare it with the available genomes from the Pathogen Detection database and Sequence Read Archive. A total of 142 genomes isolated from 11 different countries were investigated. A broad distribution of extended-spectrum beta-lactamase (ESBL) genes was identified in the Salmonella Isangi genomes examined , primarily in South Africa. Resistome analysis revealed predicted resistance to aminoglycoside, sulfonamide, macrolide, tetracycline, trimethoprim, phenicol, chloramphenicol, and quaternary ammonium. Additionally, PMQR (plasmid-mediated quinolone resistance) genes qnr19, qnrB1, and qnrS1 were identified, along with point mutations in the genes gyrAD87N, gyrAS83F, and gyrBS464F, which confer resistance to ciprofloxacin and nalidixic acid. With regard to plasmids, we identified 17 different incompatibility groups, including IncC, Col(pHAD28), IncHI2, IncHI2A, IncM2, ColpVC, Col(Ye4449), Col156, IncR, IncI1(Alpha), IncFIB (pTU3), Col(B5512), IncQ1, IncL, IncN, IncFIB(pHCM2), and IncFIB (pN55391). Phylogenetic analysis revealed five clusters grouped by sequence type and antimicrobial gene distribution. The study highlights the need for monitoring rare serovars that may become emergent due to multidrug resistance.  Salmonella (NTS) remains one of the most critical enterobacteria, causing foodborne gastroenteritis worldwide . Salmonella Typhimurium and Salmonella Enteritidis continue to be the most reported NTS serovars due to their high endemicity in numerous countries , sulfonamide , macrolide , tetracycline [tet(A)], trimethoprim , phenicol [floR], beta-lactams , chloramphenicol , and quaternary ammonium [qacE]  and the United States (blaCTX-M-2). Furthermore, we detected 83 genomes exhibiting plasmid-mediated quinolone resistance (PMQR) genes (qnrB1 (n = 47) was the most common quinolone resistance gene observed, followed by qnrB19 (n = 36) and qnrS1 (n = 2). All 47 genomes carrying the qnrB1 gene co-produced at least one ESBL gene , with many of them co-producing more than one. Ten of the genomes carrying the qnrB19 gene co-produced ESBL gene (bla2CTX-M-) and were isolated from the United States, whereas the others were positive only for qnrB19. All genomes carrying qnrS1 were positive for qnrB19 and co-produced ESBL genes . Our sequenced genome  presented only the plasmid-mediated quinolone resistance gene qnrB19, a characteristic shared with the other Salmonella Isangi isolated from Brazil , followed by Col(pHAD28) [n = 39], IncHI2 [n = 37], IncHI2A [n = 28], IncM2 [n = 12], ColpVC [n = 6], Col(Ye4449) [n = 5], Col156 [n = 5], IncR [n = 5], IncI1(Alpha) [n = 4], IncFIB (pTU3) [n = 3], Col(B5512) [n = 2], IncQ1 [n = 2], IncL [n = 2], IncN [n = 2], IncFIB(pHCM2) [n = 1], and IncFIB (pN55391) [n = 1]. The most frequent plasmid incompatibility group identified among the analyzed qnrB19 mapped to the Col(pHAD28) incompatibility group, with different distributions of other incompatibility groups  , IncL, and IncFIB (pHCM2)]. The genomes carrying the qnrS1 displayed the same distribution of incompatibility groups as the genomes carrying the qnrB19 gene  displayed the ctamases ,31. The H4-ST131 ,33, and la genes . We evalce genes , and wasce genes , we obseqnrB1 that confers low-level quinolone resistance [qnrB1 has raised concerns among health authorities in recent years precisely because of their potential to increase resistance among bacteria. Because they are plasmodial genes, their spread is facilitated by horizontal gene transfer [Salmonella Isangi, which are not commonly associated with severe infection episodes. The high incidence of ST335 in South Africa, with multiple ESBL genes alongside the PMQR, may justify its endemic status across Africa. We also identified Salmonella Isangi ST335 in Uganda and the United Kingdom, which were positive for ESBL genes and more closely related to the South African genomes (Genomes belonging to ST335 presented transfer . Therefo genomes . These rqnrB19. The presence of qnrB19 dated from 2012 is relatively recent compared to genomes isolated before (2001–2008)  . These r in 2008 . Quinolo in 2008 . Therefod Europe ,41,42. Sreported ,44,45. ASalmonella Isangi isolated from poultry [gyrA) and the topoisomerase C (parC)  [gyrAD87N, gyrAS83F, and gyrBS464F), all of which were isolated from South Africa in 2001. In contrast, seven genomes from the United Kingdom and India presented point mutation (parCT57S) and were isolated in recent years, indicating that this specific point mutation is more widespread within recent Salmonella Isangi. No relation between QRDR and PMQR was observed.Previous studies have identified an increased phenotypic and genotypic resistance to quinolones in  poultry ,46. Mostistance) , and PMQSalmonella Isangi genomes from the United States belonging to ST216 displayed blaCTX-M-2. In the United States, blaCTX-M-producing Salmonella Isangi were associated with hospital outbreaks [blaCTX-M-2 in Salmonella Isangi isolated from the United States. The genomes described in this study were available in 2015, but, so far, we have not identified studies concerning the CTX-M-2 in Salmonella spp. in the United States. However, several recent studies in Brazil, Colombia, and South Africa have identified CTX-M-2 in various Salmonella serovars associated with the poultry industry [E. coli [Eight utbreaks . Howeverindustry ,49,50,51industry , CTX-M-2industry . This in[E. coli ,54,55. CSalmonella Isangi genomes were grouped based on the country and ST profile  that increases iron uptake. These genes are part of the Yersinia High Pathogenicity Island (HPI) [Yersinia, Salmonella spp., and E. coli [Salmonella Isangi dissemination and persistence across these countries and the established population.Regarding virulence genes, three genomes from South Africa (2020) and the United States (2010) presented the complete yersiniabactin siderophore (nd (HPI) , which i E. coli ,58. The  E. coli . This stSalmonella Isangi in South Africa and how it could be related to the acquisition of multiple ESBL genes associated with PMQR genes, mainly qnrB1. These results reaffirm the need to constantly monitor Salmonella Isangi and other rare serovar dissemination across continents, as MDR Salmonella spp. pose a significant risk to public health. Moreover, the presence of antimicrobial-resistant Salmonella Isangi in the Brazilian poultry industry could significantly impact One Health, given the international importance of Brazil in the production and export of poultry products and their potential risk of transmission to humans. Lastly, our results highlight the importance of large-scale genomic analysis as an epidemiological and monitoring tool for the surveillance of Salmonella and other microorganisms. In summary, our study highlights the endemic success of