Moreover, lithium is synergistic with standard cancer therapies, enhancing their anti-tumor effects. In addition, lithium has a neuroprotective role in cancer patients, by improving their quality of life. Interestingly, nano-sized lithium enhances its anti-tumor activities and protects vital organs from the damage caused by lipid peroxidation during tumor development. However, these potential therapeutic activities of lithium depend on various factors, such as the nature and aggressiveness of the tumor, the type of lithium salt, and its form of administration and dosage. Since lithium has been used to treat bipolar disorder, the current study provides an overview of its role in medicine and how this has changed. This review also highlights the importance of this repurposed drug, which appears to have therapeutic cancer potential, and underlines its molecular mechanisms.Lithium is a therapeutic cation used to treat bipolar disorders but also has some important features as an anti-cancer agent. In this review, we provide a general overview of lithium, from its transport into cells, to its innovative administration forms, and based on genomic, transcriptomic, and proteomic data. Lithium formulations such as lithium acetoacetate (LiAcAc), lithium chloride (LiCl), lithium citrate (Li Cancer is a disease characterized by the uncontrolled growth of certain cells. The phenotypic and genotypic complexity of cancer cells translates into the acquisition of functional capabilities, to continue to grow and spread. Capabilities for sustaining proliferative signaling, evading growth suppressors, non-mutational epigenetic reprogramming, avoiding immune destruction, enabling replicative immortality, tumor-promoting inflammation, polymorphic microbiomes, activating invasion and metastasis, inducing or accessing vasculature, senescent cells, genome instability and mutation, resisting cell death, deregulating cellular metabolism, and unlocking phenotypic plasticity are all hallmarks of cancer . Metabol+ in seawater and in minerals [+ cation is therapeutically relevant, given that it has been used to treat bipolar disorders [2+, resulting in the bioactive form of lithium that modulates purine receptor activity in neuronal cells [3C6H5O7), and lithium carbonate (Li2CO3) on cell proliferation, tumor growth, and cancer metabolism. According to this evidence, LiAcAc inhibited cell proliferation in colon, breast, pancreatic, and neuroblastoma cancers [Lithium is a highly reactive alkali metal that exists as Liminerals . The Li+isorders ,8,9, Alzisorders ,11, and isorders ,13. For isorders , whereasisorders . Lithiumal cells . Lithiumal cells . Some moal cells . Interes cancers . Table 1+ ion shares transport and permeation pathways with other ions, such as sodium [+ ion depend on its ingestion [+ ion exposure. Differential expression of MCTs in healthy and cancer tissues has been reported. In this way, MCT1 is expressed by healthy or cancer tissues [+ ion. The transport of lithium is in both directions across the cellular membrane through a Na+-dependent counter-transport system. Furthermore, Li+ ion enters the voltage-dependent Na+ channel, but the Na+-K+ pump mediates the lithium uptake but not its release in cells [+ ion below 1 mM in patients exposed to 1–2 mM of lithium [The Lis sodium . Therefongestion . Howeverngestion . MCT2 hangestion , but thengestion  and MCT4ngestion . LiAcAc ngestion ,43,44. T tissues . Further tissues ,53,54,55 tissues ,56, in a tissues ,58 and i tissues . Moreove tissues ,62,63,64 tissues ,66. Whil tissues ,68,69,70in cells . Therefo lithium .Regarding MCTs, evidence indicates that MCT1 and MCT4 are expressed in cancer cells, whereas scant information is available regarding MCT2 and MCT3 expression in cancer. Expression of MCT1 was reported for lung , stomach, colon, bladder, prostate, breast, ovarian cervical, head and neck cancers, and gliomas ,76,77,78Furthermore, chaperone proteins of MCTs are important mediators in cancer. CD147 and CD44 function as chaperones of MCT1. In this way, the expression of MCT1 and CD147 correlated in bladder and ovarian carcinomas , whereasV600E mutation found in malignant melanomas, hairy cell leukemia, colorectal cancer, and multiple myeloma [V600E binding and activates the protein kinase MEK1  [V600E mutation cancers [Cancer cells use several metabolic substrates such as glucose, glutamine, lipids, and lactate to grow and survive. Therefore, MCTs maintain a high-rate lactate transport, allowing glycolysis to preferentially fuel lactic fermentation . On the  myeloma ,85,86,87 myeloma , resulti kinase) . Further kinase) . Therefo cancers . Interes cancers ,90. In c cancers ,92,93. TThe chlorinated form of lithium inhibits Glycogen Synthase Kinase 3β (GSK3β), which is a serine/threonine protein kinase. GSK3β phosphorylates β-catenin in the Wnt metabolic pathway, leading to growth arrest ,95. MoreThe expression of GSK3β is higher in colon cancer cells and in colorectal cancer patients in comparison to normal counterparts. Furthermore, the inhibition of GSK3β activity resulted in apoptosis induction and reduced proliferation in cell culture . InteresFurthermore, when lithium blocks GSK3β activity, ovarian cancer cell proliferation was reduced and tumor suppressor activity was observed in nude mice inoculated with human ovarian cancer cells . MoreoveBmi1 is increased in various cancers such as medulloblastoma and glioblastoma multiforme [Bmi1 is downregulated, it triggers differentiation and reduces the expression of the stem cell markers Sox2 and Nestin [Bmi1, inhibition of GSK3β activity by lithium triggered tumor cell differentiation, increased apoptosis, the disintegration of tumor spheroids, and decreased clonogenicity [The polycomb group gene ltiforme . When Bmd Nestin . Since Ggenicity .The role of lithium as a proapoptotic or antiapoptotic compound is contradictory. In HepG2 cells, lithium confers resistance to the apoptosis induced by etoposide and camptothecin, by preventing the activation of caspase-8 and caspase-3 and by inhibiting the Bax translocation to mitochondria . TherefoOn the other hand, in human SH-SY5Y neuroblastoma cells, lithium inhibits GSK3β, contributing to antiapoptotic signaling mechanisms . FurtherIn glioblastoma cells, low and higher LiCl doses had no effect on the expression of Bcl-2, procaspase-3, and PARP . In cont3) levels, resulting in a novel form of autophagy induction that is independent of the mammalian target rapamycin (mTOR) [+ ion downregulated autophagy  via activation of mTOR [Sc) in prion-infected neuronal and non-neuronal cells [Lithium induces autophagy and enhances the clearance of aggregated proteins, such as mutant huntingtin and α-synucleins neuronal precursor cell lines and non-neuronal cells. Interestingly, this effect is not mediated by GSK3β, but it was mediated by inhibition of IMPase, leading to free inositol depletion and reducing myo-inositol-1,4,5-triphosphate (IPn (mTOR) ,128. In  of mTOR ,130. Fural cells . Lithiumal cells .P62, Tmem74, Tm9sf1, and Tmem166, indicating that lithium increased autophagy that may have contributed to increased endothelial cell survival in a mouse model of Fuchs endothelial corneal dystrophy [2CO3 induces apoptosis and arrests the cell cycle in the G2/M phase in hepatocellular carcinoma-29 cells [Additionally, lithium carbonate promotes autophagic vacuole formation in hepatocellular carcinoma (HCC) cells in vivo, suggesting that lithium-mediated autophagy might be a novel approach for treating hepatocellular carcinoma . Interesystrophy . Moreoveystrophy . Blockinystrophy . Finallyystrophy . In addiystrophy . Additio29 cells .Tumor cell growth is controlled by various signaling pathways, such as PI3K/Akt and the raf-1/mitogen-regulated extracellular kinase (MEK)/extracellular regulated kinase (ERK) pathway (raf-1/MEK/ERK). LiCl decreases hormonal secretion and suppresses the growth of MTC in vitro and in vivo due to GSK3β inhibition . The rafSix2 (sine oculis homeobox homolog 2 gene). In contrast, higher concentrations of LiCl (50 mM) inhibit proliferation and downregulate the expression of Six2 [Six2 expression, while high LiCl concentration decreases both proliferation and Six2 expression, which is connected to the Wnt/β-catenin signaling pathway. Furthermore, in hippocampal neural stem/progenitor cells, low concentrations of LiCl increase cell proliferation [Interestingly, low concentrations of LiCl  promote the proliferation of mk3 and mk4 cells (metanephric mesenchyme cells), which play an essential role in nephron generation, by increasing the expression of the transcription factor  of Six2 . Therefoferation . In addiferation . In addiferation .Lithium causes nuclear localization of the claudin-1 and β-catenin that form the apical junctional complex, which is associated with colorectal cancer progression. In addition, lithium reduces colony formation, inhibiting cell migration and cell proliferation through G2/M cell cycle arrest. In colorectal cancer, the aberrant activation of epidermal growth factor (EGF) disrupts the association between E-cadherin and β-catenin, increasing cell proliferation and migration ,146. SinCip) in a p53-dependent pathway, suggesting that lithium stabilizes β-catenin and activates p53 [In colon cancer, lithium reduces the expression of transforming growth factor-β-induced protein (TGFBIp) by inhibiting Smad3 phosphorylation through GSK3β inhibition . In addiates p53 . In the ates p53 ,155,156.cdc6), cyclin A, cyclin E, and cdc25C, which are regulated by E2F [Genome-wide screening has been used to determine that LiCl inhibits cell proliferation, reduces replication, and arrests the cell cycle in multiple prostate cancer cells . In prosd by E2F . Furtherd by E2F . In addid by E2F . In thisd by E2F .The anti-cancer properties of lithium salts are summarized in In some types of cancer, before the cell becomes malignant, inflammatory conditions occur. Therefore, inflammation is associated with the proliferation and survival of cancer cells. In addition, this promotes angiogenesis and metastasis . Since iThe anti-inflammatory activity of lithium results from its strong inhibitory effect on GSK3β, leading to a reduction in TNF-α production via attenuated activation of NF-κB and JNK signaling cascades  and indu+ ion have toxic effects in patients [+ ion levels for therapy were 0.8 to 1.2 mEq/L (0.8–1.2 mM) and the cytotoxicity effect began at 1.5 to 2 mEq/L (1.5–2 mM) [High concentrations of lithium (4–5 mM) inhibit inositol 1-monophosphatase, resulting in a reduction of phosphatidylinositol turnover. However, these high concentrations of Lipatients . Reporte.5–2 mM) . Interes.5–2 mM) . These r.5–2 mM) , suggest50 dose of etoposide enhances apoptosis and reduces the percentage of cells in the G2/M phase in prostate cancer LNCap cells [Bax signaling, but LiCl also prevented MMC-induced necrosis [Conventional cancer therapies such as chemotherapy and radiotherapy inhibit cell division, causing neurocognitive deterioration. Preliminary studies have shown that lithium has a neuroprotective role in cancer patients undergoing radiotherapy or chemotherapy treatment, improving their quality of life . Combinaap cells . In pancap cells . Additioap cells . Furtherap cells . In addiap cells . Neuriteap cells . Pre-trenecrosis .+ prevents the debilitating cognitive effects of cranial radiotherapy [Radiotherapy has various consequences in children, such as debilitating cognitive decline, partly linked to impaired neurogenesis. In this context, LiCl has a protective role in the juvenile brain against radiotherapy and improves the quality of life of these children . In addiotherapy ,173. Sinotherapy . LiCl haotherapy . In colootherapy . Therefootherapy .Chemotherapy-induced peripheral neuropathy (CIPN) is an adverse effect of cancer treatment using taxanes, such as paclitaxel. Adding lithium to taxane treatment prevents calcium overloading in mitochondria, resulting in the prevention of calpain activation and preventing the loss of cell functions related to the progression of peripheral neuropathy , suggestIn a systems biology analysis, the interactomes of lithium-sensitive enzymes and the pathways associated with several types of cancer suggested that Li affects the incidence and progression of cancer . Despitehttp://www.clinicaltrials.gov (accessed on 20 January 2023)), thirteen clinical trials have been conducted  nanoparticles. The TiO2 nanomaterial exerted cytotoxicity and induced apoptosis through lipid peroxidation in breast cancer cells. The anti-tumor efficacy and safety of TiO2 nanomaterial were evaluated in vivo using a subcutaneous 4T1 breast BALB/c mouse tumor model, displaying significantly less cardiotoxicity and body weight loss compared to doxorubicin [+ ion administration for bipolar disorders, but its pharmacodynamics and pharmacokinetic properties need to be addressed [+ ion at therapeutic levels [In addition, leaf extract of orubicin . These fddressed . Howeverc levels . Cellulac levels , but apoc levels . A mesopc levels .Lithium formate monohydrate was irradiated by X-rays, electrons, and protons for use as a dosimeter in radiotherapy . In addiThe biocompatibility and multifunctionality of Li-doped bioceramics with polymers provided new biomaterials used in 3D printing for clinical applications of tissue engineering . Further3 and WO3 treated with lithium transformed white-colored micrometer-long MoO3 nanobelts into blue-colored short, thin, defective, interlayer gap-expanded MoO3−x nanobelts with strong NIR-II absorption. With this surface modification, the MoO3−x nanobelts could be a promising nanomaterial for photoacoustic imaging-guided photothermal therapy, resulting in an efficient cancer cell ablation and tumor eradication under irradiation from a 1064 nm laser [Furthermore, lithium has been used for the synthesis of new nanomaterials with potential applications in cancer therapy. Layered metal oxides such as MoOnm laser . Lithiumnm laser . To overnm laser . In addinm laser .2CO3 induced a cutaneous adverse reaction [Prolonged treatment with lithium causes renal, endocrine, and dermatological side effects. Four weeks of therapy with lithium caused renal tubular concentration defects, polyuria, polydipsia, and nephrogenic diabetes insipidus . Adminisreaction  and parareaction . Furtherreaction . In addireaction .The inhibition of GSK3β using lithium has contradictory effects. On one hand, chemoresistance to apoptosis is observed, but on the other hand, it induces tumor suppressor activity and reduces proliferation. Those effects depend on the type of cancer, the origin of cells, and doses. The anti-cancer effect of lithium has been reported for colorectal cancer, neuroblastoma cells, ovarian cancer cells, medullary thyroid TT cancer cells, esophageal cancer, medulloblastoma, and glioblastoma multiforme, hematological tumors (multiple myeloma), breast cancer cells, MTC, prostate cancer cells, pancreatic ductal adenocarcinoma cells, corneal endothelial cells, and colon cancer cells. However, opposite effects of lithium have been reported for neuroblastoma, hepatoblastoma cells, and malignant glioma cell lines.bax/bcl-2 ratio (bax: pro-apoptotic gene and bcl-2: anti-apoptotic gene). Lithium resistance then results when this ratio is reversed, leading to lithium resistance accompanied by DNA fragmentation, and decreasing cell viability [Lithium induces apoptosis in promyelocytic leukemia cells, but a small fraction of these cells remain viable and survive the cytotoxic effect of this cation, resulting in altered gene expression patterns , suggestiability .+ ion has potential for cancer treatment, the selection of the delivery form of lithium, such as nanosized or original forms, plays a key role in triggering the antitumor response, which depends on its transport into the cell, the cancer type, doses, and administration. Li+ ion selectivity for tumor cells, however, needs to be further investigated, as well as doses and administration.The expression of transporters of lithium is linked to various types of cancer, such as NSCLC, colon, stomach, bladder, prostate, breast, ovarian, cervical, head and neck cancer, and gliomas. In both in vivo and in vitro models of cancer, lithium is capable of activating the Wnt/β-catenin pathway. The GSK3β kinase is activated when a secreted glycoprotein (Wnt) binds to its receptor. GSK3β is inhibited by lithium through Ser-9 phosphorylation, stabilizing free β-catenin in the cytoplasm. Therefore, the GSK3β might have anti- or pro-apoptotic activity depending upon the cell type. According to genomics and proteomics data, lithium salts such as citrate, carbonate, chloride, and acetoacetate may stimulate antitumor effects, such as apoptosis, autophagy, cell death, tumor growth, tumor proliferation, invasion, and metastasis and arrest the cell cycle in in vivo and in vitro models . Since L


Avena sativa L.) have been conducted to date. The recently published complete genome sequence of oat enables the systematic analysis of the AsWRKYs. Based on a genome-wide study of oat, we identified 162 AsWRKYs that were unevenly distributed across 21 chromosomes; a phylogenetic tree of WRKY domains divided these genes into three groups . We also analyzed the gene duplication events and identified a total of 111 gene pairs that showed strong purifying selection during the evolutionary process. Surprisingly, almost all genes evolved after the completion of subgenomic differentiation of hexaploid oat. Further studies on the functional analysis indicated that AsWRKYs were widely involved in various biological processes. Notably, expression patterns of 16 AsWRKY genes revealed that the response of AsWRKYs were affected by stress level and time. In conclusion, this study provides a reference for further analysis of the role of WRKY transcription factors in species evolution and functional differentiation.The WRKY family is widely involved in the regulation of plant growth and stress response and is one of the largest gene families related to plant environmental adaptation. However, no systematic studies on the WRKY family in oat ( Plants and animals are subjected to different evolutionary stresses in nature because plants are incapable of escaping predation and environmental changes , and in SPF1) [ABF1,2) [ZAP1) [stp mutants led to the derepression of NST2 and C3H zinc-finger transcription factors, activating downstream gene expression and thereby increasing stem biomass [atwrky62 negatively regulated the plant growth [AtWRKY8 and AtWRKY28 positively enhanced resistance to Botrytis cinerea, and AtWRKY8 and AtWRKY48 did not positively enhance resistance to Pseudomonas syringae [OsWRKY08 [OsWRKY11 [OsWRKY23 [OsWRKY45 [OsWRKY72 [OsWRKY89 [TaWRKY2 and TaWRKY19 combined with RD29B, RD29A, RD29B, or COR6.6, increased the expression of downstream genes under drought stress [TaWRKY51 promoted lateral roots by negatively regulating ethylene biosynthesis [TaWRKY146/2/19 both enhanced the resistance of salt or drought [AtWRKY18, AtWRKY40, and AtWRKY60 were relatively high, which not only formed three different homologous complexes through their own interaction, but also formed heterologous complexes through their interactions with each other. Hwang et al. [OsWRKY51 and OsWRKY71 inhibited the signal of gibberellic acid (GA) in aleurone cells of rice seeds.Since the first WRKY sequence discovery in sweet potato (SPF1) , wild oa[ABF1,2) , and Ara) [ZAP1) , accumul biomass ; and thet growth . In addisyringae ,12,13. IOsWRKY08 , OsWRKY1OsWRKY11 , OsWRKY2OsWRKY23 , OsWRKY4OsWRKY45 , OsWRKY7OsWRKY72 , and OsWOsWRKY89  positiveOsWRKY89 . In wheat stress ; TaWRKY5ynthesis ; overexp drought  showed tg et al.  found th4–5-C-X22–23-HXH (C2H2) or C-X7-C-X23-HXC (C2HC) [2H2 motif; group II and III proteins only have a single WRKY domain and have a distinct zinc finger motif. The former has the C2H2 motif and is further divided into five subgroups (IIa-e) [2HC motif. Interestingly, group I proteins are not restricted to the plant lineage and were also found in the unicellular eukaryote Giardia lamblia and slime mold Dictyostelium discoideum [Some WRKY proteins may contain multiple domains, whereas others contain only highly conserved WRKY DNA-binding domains, which are a common feature of WRKY TFs ,27,28. T (IIa-e) , whereasscoideum ,30,31, bscoideum . Among tscoideum . Once a scoideum .Arabidopsis thaliana [Triticum aestivum [Oryza sativa [Glycine max [Zea mays [The WRKY family already evolved during the moss and fern era . Althougthaliana , 171 in aestivum , 31 in Oa sativa , 182 in cine max , and 119Zea mays . TherefoA. sativa) is an allohexaploid (2n = 6× = 42) plant with a large and complex genome and is one of the least explored in genome and transcriptome among cereal grain crops [https://wheat.pw.usda.gov/GG3/graingenes-downloads/pepsico-oat-ot3098-v2-files-2021) (accessed on 3 March 2022). In this study, we identify WRKY TFs from oat and analyze features of AsWRKYs and expression profiles. The results will lay the foundation for further studies of WRKY TFs in other crops, including their evolutionary relationships and phylogeny.Oat (in crops . Oats cain crops . Given thttps://wheat.pw.usda.gov/) (accessed on 3 March 2022) [https://pfam.xfam.org/) (accessed on 3 March 2022) [https://www.ncbi.nlm.nih.gov/cdd/) (accessed on 3 March 2022) [http://smart.embl.de/smart/batch) (accessed on 3 March 2022) [https://web.expasy.org/compute_pi/) (accessed on 3 March 2022) [http://cello.life.nctu.edu.tw/) (accessed on 3 March 2022) [https://www.genscript.com/wolf-psort.html) (accessed on 3 March 2022) [In this study, the oat proteins sequence and HMM file of the WRKY domain (PF03106) were downloaded from the grain genes database (ch 2022)  and Pfamch 2022) , respectch 2022) , and SMAch 2022)  databasech 2022) . Furtherch 2022)  and WoLFch 2022) . The SSRch 2022) .https://meme-suite.org/meme/tools/meme) (accessed on 3 March 2022) [cis-elements of the promoter, the upstream 1500 bp sequences of all AsWRKY genes were screened using PlantCare (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 3 March 2022) [The WRKY domain of AsWRKY proteins were subjected to multiple sequence alignments using MEGA 7. The amino acid sequences subjected to BLAST were manually adjusted for group classification using GeneDoc tools . Then, phylogenetic reconstruction based on the WRKY domain of Arabidopsis and oat was performed using the neighbor-joining method, and the program parameters were as follows: p-distance, 80% cutoff of partial deletion, and 1000 bootstrap repeats. The conserved motifs of all WRKY proteins were analyzed using MEME (ch 2022) , and thech 2022) . To idench 2022) .https://github.com/kullrich/kakscalculator2, accessed on 3 March 2022)was used to calculate non-synonymous (Ka) and synonymous (Ks) substitutions for each duplicated gene pair [−9 substitution/synonymous site in oat [−6.Based on the genome annotation file of oat, TBtools  was usedene pair . Based oe in oat , the apphttp://eggnog-mapper.embl.de/) (accessed on 10 March 2022) [The Gene Ontology of oat WRKY proteins were obtained from eggNOG databases (ch 2022) . The fulch 2022) . Based on the previously obtained miRNA data, 623 miRNAs were identified by sRNAminer . The AsWhttps://www.ncbi.nlm.nih.gov/sra/?term=oat) (accessed on 15 March 2022) were analyzed with Kallisto [In this study, data SRP294982, SRP235083, SRP094911, SRP094513, and SRP237902 from NCBI , and the PCR program was conducted as follows: 10 s at 95 °C, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Each reaction had three biological replicates, and data were analyzed by the 2−ΔΔCT method.The total RNA of all samples was isolated using the plant RNA Kit , and then cDNA was synthesized by a PrimeScript™ RT reagent Kit . The 16 AsWRKYs were selected to validate the expression level under drought and salt stress and primers, as listed in AsWRKY1 to AsWRKY162  in the oat genome, and these were further checked for the conserved domain in the Pfam and CDD databases. Finally, a total of 162 candidate genes were identified; these encode 162 WRKY proteins. According to their order of chromosomal distributions, these genes were designated as sWRKY162 . Among tAsWRKY132 has the greatest protein length (4494 aa) and MW (168.78 kDa), while the smallest one was AsWRKY95 (399 aa and 14.46 kDa). The pI of proteins ranged from 4.61 to 10.32, which were identified in AsWRKY158 and AsWRKY156, respectively. Further analysis using protein subcellular localization predictions revealed that all proteins, except for AsWRKY2/7/19, were located in the nuclear region. In addition, we also predicted the SSR site of AsWRKYs. In total, 92 putative new SSRs were detected from 62 sequences (Gene signatures such as coding sequence (CDS) length, protein sequence length, protein molecular weight (MW), isoelectric point (pI), and subcellular localization were analyzed . There wequences , which wAtWRKY58, 40, 61, 28, 7, 14, and 54) and all oat WRKY domains for multiple sequence alignment. Alignment of the amino acid sequences found two significant regions, the first of which is conserved heptapeptide (WRKYGQK) in the WRKY domain that is the binding site of the W box (3–5-C-X22–24-H-X-H (C2H2) and C-X4–7-C-X23–29-H-X-C (C2HC). Among these, 112 AsWRKYs contained C2H2 zinc finger motifs, 46 AsWRKYs included C2HC, and the others (AsWRKY28/95/100/103) lost their zinc finger motifs.To investigate the evolutionary relationship among members of the oat WRKY family, we selected Arabidopsis  contains a single WRKY domain. Thirteen members were found in subgroup IIa, 11 in IIb, 27 in IIc, 14 in IId, and 18 in IIe.A phylogenetic tree containing about 60 amino acids of the WRKY domains was constructed. According to previous studies on WRKY genes, their phylogenetic relationships, and WRKY domain characteristics of  and III . Group IAsWRKY73/131/132 contain 9 introns and have the highest number of detected introns. It is worth noting that groups I and IIb have higher transcript abundance–the average number of introns is greater than 3.5 and was higher than that of other groups   . NotablyKY12/13) .To further deduce the evolutionary mechanism of the oat AsWRKY gene family, we constructed a collinearity relationship with four representative model plants, such as Arabidopsis, maize, rice, and wheat . The numcis-acting elements were identified to be involved in abiotic and biotic stress, growth and development, and hormone signal transduction . Two members of the miR664 family target four genes , and the miRN7 family also targets four genes . Another known and novel miRNA targets one or multiple genes. The regulation function of the miRNAs in the WRKY family needs additional research to explain.miRNA can directly bind to TFs and regulate the expression of genes . At the AsWRKY4), 9 (AsWRKY116/117/140/4/78/79/80/106/108), 11 (AsWRKY32/43/44/67/86/105/137/143/145/146/147), 7 (AsWRKY67/86/32/136/145/44/146), 2 (AsWRKY143 and AsWRKY67), and 1 (AsWRKY95) in lemma development, seed vigor, salt, drought with silicon, phosphorus deficit, and drought stress, respectively. Other WRKY genes may be upregulated or downregulated depending on the treatment, time, and developmental stage. For example, water-sensitive cultivars had more WRKY genes that exhibited upregulated expression under drought stress , qRT-PCR analysis was performed on 16 genes from AsWRKY members. Overall, some AsWRKY genes were significantly inhibited by PEG or salt treatment . For exaAsWRKY1 to AsWRKY162  is significantly different from group II. Surprisingly, AsWRKY131 contains two common WRKY motifs, but the zinc finger structure (C2HC) has undergone a great change, which is different from IN and IC, but closer to group III. Brand et al. [AsWRKY131 may be a novel subgroup of Group I or that it is intending to evolve into Group III. There is evidence that both groups IIc and I evolved from a IIc-like ancestral WRKY, and the members of IIc in this study are more similar to group IC. Therefore, we could speculate that group IIc may have differentiated from Group I, or they may have come from the same ancestor.The loss of a domain does not always result in negative consequences. Although gene function is lost or changed temporarily, it may cause the expansion of gene families once again. Of course, this process can be extremely lengthy. For example, group II (except II c) and III may be the WRKY domain deletion of the N-terminal in I. Interestingly, we also have a similar event in the WRKY family of oat. IIa and IIb are divided into the same subgroup, and the same is true for IId and IIe. Both have a high similarity with the IC WRKY domain, but the former is closer to IC. Therefore, both clades may be derived from IC. Interestingly, phylogenetic analysis of WRKY sequences from Arabidopsis and rice merged the four clades of group II into two new sister groups IIa + b and IId + e; this similar model hints that they could also have an evolutionary relationship. Group III is considered to be the youngest branch in evolution, and its zinc finger structure . After analyzing gene duplication events, we found 111 duplication WRKY gene pairs, including 108 segmental duplications and three tandem duplications, indicating that segmental duplication is the main means of oat WRK family expansion. In addition, all duplicated gene pairs were subjected to strong purifying selection during evolution, indicating that the functional limitation of oat WRKY mainly comes from purifying selection. Hexaploid cultivated oats had completed the differentiation of each subgenome as early as 16 Mya, much earlier than the subgenome differentiation time of common wheat about 7 Mya. In this study, only five duplication gene pairs occurred before the doubling of the whole genome, and all of these occurred in the A and D subgenomes; the rest of the duplicate gene pairs occurred after subgenome differentiation, and the evolution time was between 0.18 and 15.3 Mya. Gene collinearity analysis among different species showed that oat and wheat had the highest conservation, followed by rice and maize, and the lowest was Arabidopsis. This difference may be related to the genomic characteristics and evolution of oat, which has many similarities in evolutionary origin with wheat because they both are allohexaploid. Meanwhile, Arabidopsis is dicotyledonous, suggesting that the WRKY gene pair for oat may have arisen after the differentiation of dicotyledonous and monocotyledonous plants.Most plants have undergone an ancient whole-genome duplication event or polyploidy, which is a large-scale chromosome doubling . Every dcis-acting elements to regulate the expression of related target genes [cis-acting sites determine the diversity of regulatory functions of TFs [Moringa oleifera [D. candidum [TFs specifically bind to et genes ,75, and s of TFs . In thisoleifera , and thecandidum . This inMd-miR156ab and Md-miR395 could regulate MdWRKYN1 and MdWRKY26 in the response to fungal pathogens in M. domestica [tae-mir159a was observed to target wheat WRKY gene under heat stress [Ha-miR396 targeted WRKY6 to regulate the early responses to high temperature in H. annuus [miRNA169 and miRN83, which specifically targeted IIa and III. However, it remains to be considered whether a miRNA targeting genes in multiple WRKY subgroups, in addition to indicating that they share the same target, can indicate that they have a certain relationship in evolution.miRNAs play an important role in repressing and cleaving genes at the post-transcriptional level. omestica ; tae-mirt stress ; Ha-miR3. annuus ; moreove. annuus . In the AtWRKY25/26/33/40/57/63 in Arabidopsis, OsWRKY1/2/5/7/11/43/45 in rice, GmWRKY13/21/54 in soybean, and TaWRKY2/10/19 in wheat [TFs often regulate plant development and stress in the form of families, so that they can quickly respond to environmental changes ,82. Consin wheat ,89,90,91In this study, we identified 162 AsWRKY genes from the whole oat genome. Phylogenetic relationships, gene structure, conserved motifs, chromosomal locations, promoter regions, and transcriptome expression were systematically analyzed. Amplification of the WRKY gene family in oat mainly occurred by segmental duplications, and most genes evolved after chromosome hexaploidy. The analysis of AsWRKYs function and expression pattern showed that most AsWRKY genes are actively involved in various processes of oat growth, development, and stress response, which provides extensive information for the continued study of AsWRKY genes.


MoADE12 in Magnaporthe oryzae by screening for growth-defective T-DNA insertional mutants. Gene replacement was performed to investigate the biological role of MoADE12. Δmoade12 mutants were adenine auxotrophs that failed to produce conidia, and showed reduced perithecia formation and pathogenicity. Moreover, the Δmoade12 mutant was hypersensitive to Congo red and oxidants, indicating that MoADE12 was required for cell wall integrity and oxidative stress resistance. Transcriptomic analysis identified the underlying mechanisms and indicated that several pathogenicity-related genes were regulated in the Δmoade12 mutant. Therefore, our data suggest that the adenylosuccinate synthase MoADE12 is involved in the de novo AMP biosynthesis pathway and is important for conidiation and pathogenicity in the rice blast fungus.Purines are basic components of nucleotides in living organisms. In this study, we identified the ortholog of adenylosuccinate synthase  Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice blast disease, one of the most devastating rice diseases worldwide [M. oryzae infection is initiated by the attachment of a three-celled conidium on the host surface. After germination and appressorial maturation on the surface of the plant, the penetration peg forms under the appressorium and penetrates the plant cuticle under high turgor pressure [M. oryzae and the plant host, the fungus derives its nutrients from living plant cells by conserved sensitive global genetic regulatory mechanisms of carbon and nitrogen metabolism before switching to the necrotrophic stage [M. oryzae, the metabolic regulation of carbon and nitrogen, as well as their roles in growth, development, and pathogenicity have been well described [M. oryzae, several amino acid synthesis pathways, including those of arginine, lysine, leucine, and methionine, reportedly play important roles in development and pathogenicity [orldwide ,2. M. orpressure ,4. Endogpressure . Once thic stage ,7. Theseic stage ,8. In M.escribed ,10,11. Iescribed . In M. ogenicity ,14,15,16M. oryzae, two enzymes involved in purine synthesis were identified [MoADE1 encodes an N-succinyl-5-aminoimidazole-4-carboxamide ribotide synthetase, which ligates aspartate with 5’-phosphoribosyl-4-carboxy-5-aminoimidazole (CAIR) to form 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR), a key step in the conversion of PRPP into IMP. Disrupting MoADE1 blocks the de novo synthesis of IMP. During plant development, the Δmoade1 mutant is aborted, which may be attributed to the prevalence of purine nutritional deficiency [MoGUK2, which encodes guanylate kinase that catalyzes the conversion of GMP to guanosine diphosphate (GDP), is involved in conidial germination, appressorial formation, and infectious hyphae growth, resulting in attenuated virulence [Δmoguk2 mutant formed normal conidiophores with decreased conidia, and its sexual development ability was abolished. The activities of MoADE1 and MoGUK2 indicate differences in the biological functions of related genes in purine metabolism. Therefore, to better understand the biological function of de novo purine synthesis in M. oryzae, further analysis of the synthesis of different purine types is required.Purines are the basic components of nucleotides in living organisms. In yeast, purines are synthesized via the de novo biosynthetic and salvage pathways . De novoentified ,19. MoADirulence . HoweverADE12) catalyzes the first committed step in the conversion of IMP to AMP, which is the precursor of adenosine triphosphate (ATP) synthesis [S. cerevisiae, ADE12 inactivation resulted in adenine auxotrophy [ADE12 orthologous gene MoADE12 in M. oryzae. MoADE12 is involved in the de novo adenosine biosynthesis pathway and is important for development and virulence in the rice blast fungus.Adenylsuccinate synthetase (ynthesis . In S. cxotrophy . In thisM. oryzae strain Guy11 and its derivatives were grown at 28 °C on complete medium (CM) plates, as described by Talbot et al. [t et al. . Myceliahttp://broadinstitute.github.io/picard/, accessed on 5 December 2019, Version 4.1.0.0) was used to mark duplicate reads, and SAMtools (version 1.4) was used to convert the alignment result format. GATK (version 4.1.0.0) was used to identify the InDel sites.Sequencing and analysis were performed by OE Biotech Co., Ltd. . Libraries were constructed using the TruSeq Nano DNA LT Sample Preparation Kit . Briefly, genomic DNA was sheared into fragments of ~350 bp using S220 focused ultrasonicators . The adapters were ligated to the 3’-end of the sheared fragments. After PCR amplification and purification, the final libraries were sequenced on the Illumina HiSeq X Ten platform  and 150 bp paired-end reads were generated. Raw reads were subjected to a quality check and filtered using Fastp (Version 0.19.5). Clean reads were aligned to the reference genome (GCF_000002495.2) using the Burrows Wheeler Aligner . After alignment, Picard (MoADE12 (~1 kb upstream and 1 kb downstream) were amplified from M. oryzae genomic DNA and inserted into the p1300-KO plasmid [M. oryzae Guy11 by Agrobacterium tumefaciens-mediated transformation (AtMT). In order to complement the gene deletion mutant, the full-length MoADE12 gene and its native promoter region (~1500 bp upstream from the start codon) were amplified by PCR and cloned into the p1300BAR (with glufosinate ammonium resistance) plasmid [MoADE12 deletion mutant in order to obtain a full-length complemented transformant. The primers used in this study are listed in A dual plasmid for gene knockout was generated using homologous recombination as previously described . Briefly plasmid , which f plasmid . The finOryza sativa cv. CO-39) were infected with six milliliters of conidial suspensions prepared in 0.25% gelatin at a concentration of 5 × 104 conidia mL−1 using an artist’s airbrush with high-pressure air. The inoculated plants were placed in a moist chamber at 25 °C for 24 h in the dark. One day after inoculation, rice seedlings were maintained in a moist chamber with a photoperiod of twelve hours under fluorescent light for an additional five days to enable the full development of disease symptoms. Disease severity was scaled to evaluate the virulence of the tested strains, as described previously [For the pathogenicity assays, about 100 four-week-old rice seedlings , and mating partner strain TH3 (MAT1-1) were point-inoculated 3 cm apart on oatmeal medium (OMA) plates and cultured at 20 °C with continuous light for 3–4 weeks. Mature perithecia were crushed and photographed, in order to examine the asci and ascospores.For conidiation observation, mycelial plugs of the indicated strains were placed on glass slides in order to induce conidiation under continuous light for 24 h and observed under a light microscope. For sexual reproduction, plugs of the Oryza sativa cv. CO-39), as described previously [In order to analyze infection-related morphogenesis, conidia suspensions were inoculated onto plastic coverslips  to examine conidial germination and appressorium formation. The appressorium turgor pressure was determined using a cytorrhysis assay . Infectieviously .2O2 (30% (w/w) in H2O) at the different concentrations described in the Results at 28 °C. All reagents were purchased from Sinopharm Chemical Reagent Co. Ltd , except for CFW. Growth rates of all tested strains were determined by measuring the colony diameter of 6-day-old cultures. All experiments were repeated at least three times. The mean ±SD of growth rate was determined using SPSS Statistics 22 . Asterisks indicate a statistically significant difference at p < 0.05.In order to test the sensitivity against osmotic regulators, cell-wall-disrupting agents, or oxidants, vegetative growth was assayed on CM plates with sodium chloride, sorbitol, calcofluor white , Congo red (CR), rose Bengal, or Hq-value < 0.05 and a fold-change > 2 or <1/2 were set as the threshold for significantly differential expression.Mycelia of the tested strains from three-day-old cultures in liquid CM were harvested. Total RNA was extracted using the TRIzol reagent according to the manufacturer’s protocol . The libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit , according to the manufacturer’s instructions. Transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. The libraries were sequenced on an Illumina HiSeq X Ten platform, and 150 bp paired-end reads were generated. Differential expression analysis was performed using the DESeq2 R package (2012). A M. oryzae, we screened for T-DNA insertion mutants. The mutant M76.8 exhibited impaired aerial mycelial growth. In order to determine the insertion site of T-DNA in M76.8, whole-genome resequencing was performed. Sequence analyses showed that the T-DNA insertion occurred 573 bp before the start codon of the MGG_17000 open reading frame  containing GMM. The growth of the mutants was recovered by supplementation with adenine, but not by hypoxanthine, in the GMM medium .In order to determine whether formants A. HoweveΔmoade12 mutant produced perithecia, and the ability of the Δmoade12 mutant to produce perithecia was visibly reduced compared that of the wild type and complemented transformant , indicating that loss of MoADE12 does not impair the normal function of appressoria derived from the conidia formed on adenine-supplemented media. Next, infectious hyphae of the Δmoade12 mutant were observed in the epidermal cells of the rice leaf sheath. At 40 h post-inoculation, wild-type Guy11 had elaborated bulbous infectious hyphae within host cells, whereas the infectious development of the Δmoade12 mutant was severely impaired, as it had formed primary hyphae alone and had not branched  and CFW (50 μg/mL). For the same molar concentration, the osmotic pressure of the NaCl solution was higher than that of sorbitol. CFW and CR interfere with cell wall assembly by binding to chitin and β-1,3 glucan, respectively [MoADE12 is required for cell wall integrity and resistance to high osmotic stress.In order to examine whether deletion of of Guy11 . Howeverectively . These fΔmoade12 mutant was significantly inhibited in the presence of the tested oxidants, compared with that of Guy11  are shown in p-value < 0.05) included tyrosine (19); beta-alanine (12); glycine, serine, and threonine metabolism (19); isoquinoline alkaloid biosynthesis (13); phenylalanine metabolism (11); vitamin b6 metabolism (6); tropane, piperidine, and pyridine alkaloid biosynthesis (6); tryptophan metabolism (13); sulfur metabolism (8); cysteine and methionine metabolism (14); taurine and hypotaurine metabolism (5); starch and sucrose metabolism (16); glycosphingolipid biosynthesis-globo and isoglobo series (4); valine, leucine, and isoleucine biosynthesis (7); histidine metabolism (7); alanine, aspartate, and glutamate metabolism (9); glycerolipid metabolism (10); non-homologous end-joining (4); nitrogen metabolism (6); and betalain biosynthesis (6).In order to identify the biological processes regulated by MoADE12, gene ontology (GO) term analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations were performed. Based on sequence homology, 673 genes were categorized into different functional groups, of which 35  and mitogen-activated protein kinase (MAPK) signaling pathways, in order to regulate a variety of cellular functions such as conidiation, mating, and pathogenicity [The growth rate of the  mutants ,19. In t mutants . PhenotyGG_02246 . COS1 is protein  were dowgenicity ,54. MoADΔmoade12 mutant harvested from a CM plate supplemented with adenine could germinate normally and form functional appressoria on an inductive surface, suggesting that adenines stored in conidia are sufficient to complete the invasion of host cells. Additionally, the initial colonization process does not appear to require de novo AMP biosynthesis during the early infection stage. However, the infectious hyphae of the Δmoade12 mutant were inhibited in host cells compared with those of the wild type, suggesting that de novo AMP biosynthesis is required for the growth of infectious hyphae. Although adenine is abundant in rice leaf cells [Δmoade1 mutant is limited to the acquisition of adenine from rice cells, despite up-regulated expressions of two putative purine transporters (putative uric acid-xanthine permease gene UAP1 and putative purine-cytosine permease gene FCY2) [M. oryzae genome based on homology search with the purine transporters of Aspergillus nidulans [UAP1 and FCY2, including MGG_06740, MGG_08279, and MGG_00158. UAP1 is homologous to UapA and UapC of A. nidulans, MGG_06740, and MGG_08279 to AzgA, FCY2 and MGG_00158 to FcyB. The purine transporters have the highly specific ability to transport purines [A. nidulans, AzgA and FcyB are identified to transport adenine, but UapA and UapC transport xanthine and uric acid, respectively. In the present study, only the expression of UAP1 were found to be down-regulated in the Δmoade12 mutant, and the expressions of AzgA and FcyB homologous genes were not perturbed. Our results showed that the growth rate of the Δmoade12 mutant was comparable to that of the wild type on CM media, even though the mutant is not able to synthesize AMP de novo. In fact, AMP or adenine and their analogues should be present in CM media due to the presence of yeast extract and peptone in the medium formulation. These facts imply that it may involve diverse salvage pathways. In human erythrocytes, four different pathways of adenine salvage and twelve different pathways of adenosine salvage have been found [Δmoade12 mutant to synthesize the necessary AMP in CM medium for growth.Our results showed that the conidia of the af cells , Fernandne FCY2) . Five punidulans  besides  purines . In A. nen found . AccordiMoADE12 deletion mutant was hypersensitive to oxidative stress. Reactive oxygen species (ROS) are scavenged by antioxidant systems, including catalase and various peroxidases. In the genome of M. oryzae, a total of 27 peroxidase genes had been revealed [MoADE12. Among those, 10 genes were down-regulated, including MoLIP2, CPXB, CATB, MoHPX3, MoHPX2, NOX1, MoLIP3, MoPRX1, NOX2, and MoAPX1. According to Mir et al. [CPXB, CATB, NOX1, MoLIP3, NOX2, and MoAPX1 were up-regulated under H2O2 stress, and nine of those genes, except for MoHPX3, were induced during the early phase of host infection. Furthermore, CATB [NOX1 [NOX2 [MoPRX1 [M. oryzae. Although deletion of CXPB does not affect pathogenicity, CXPB is involved in fungal defense against H2O2 accumulated in epidermal cells of rice during the early stage of infection [Δmoade12 mutant.Furthermore, our results showed that the revealed . In the r et al. , six of re, CATB , NOX1 [4TB [NOX1 , NOX2 [4X1 [NOX2 , and MoP [MoPRX1  were reqnfection . Such evM. oryzae that exhibited impaired aerial mycelial growth and attenuated virulence. Genome resequencing of the mutant led to the identification of the adenylosuccinate synthase encoding gene MoADE12, which catalyzes the first step in the de novo synthesis of AMP from IMP. Loss of MoADE12 leads to the abolition of asexual development as well as hypersensitivity to oxidative stress. MoADE12 is involved in many biological processes, including carbohydrate metabolism, amino acid metabolism, signal transduction, and stress responses. The associated regulatory networks should be evaluated in future studies.In conclusion, we identified a T-DNA mutant of


Participants were followed up after receivingacid-suppression agents or on the corresponding date until the occurrence of end-stage renal disease (ESRD) in the presence of competing mortality, death, or through the end of 2016. Compared toneither users, H2RAand PPI users demonstrated adjusted hazard ratios of 0.40  for ESRDand 0.64 (0.57–0.72) for death and 1.15 (0.91–1.45) for ESRD and 1.83 (1.65–2.03) for death, respectively. A dose-response relationship betweenH2RA use with ESRD and overall, cardiovascular, and non-cardiovascular mortality was detected. H2RA consistently provided renal and survival benefits on multivariable stratified analyses and multiple sensitivity analyses. In conclusion, dose-dependent H2RA use was associated with a reduced risk of ESRD and overall mortality in CKD patients, whereas PPI use was associated with an increased risk of overall mortality, not in a dose-dependent manner.Histamine-2-receptor antagonist (H2RA) has shown beneficial effects on the kidney, heart, and sepsis in animal models and on the heart and COVID-19 infection in clinical studies. However, H2RAshave been used as a reference in most epidemiological studies examining the association of proton pump inhibitors (PPI) with outcomes. Therefore, we aimed to evaluate the effect of H2RA on renal and survival outcomes in chronic kidney disease (CKD) patients. We used a Taiwanese nationalhealth insurance database from 2001 to 2016 to screen 45,767 CKD patients for eligibility. We identified new users of PPI ( Histamine-2-receptor (H2R) antagonist (H2RA) and proton pump inhibitor (PPI) are the main options for acid suppression therapy available at present, with PPI being more expensive, high-profile, and over-prescribed worldwide  than H2RAnimal interventional models of ischemic acute renal failure, pressure-induced heart failure, and sepsis and clinical studies of patients with heart failure and COVID-19 infection have provided evidence that H2RA could improve heart and renal functions, such as reduced levels of serum creatinine and blood urea nitrogen, and lower mortality ,16,17,18CKD is associated with an increased incidence of acid peptic disease , which cThis was a retrospective cohort study using claims data from the 2005 Longitudinal Generation Tracking Database (LGTD2005), a de-identified database of 2 million individuals who were randomly sampled from all beneficiaries of Taiwan’s single-payer compulsory NHI program between 2000 and 2016. This study did not require informed consent and was exempted from full review by the Institutional Review Board of the Dalin Tzu Chi Hospital . Further details about the LGTD2005 have been described in our previous research . In brien = 7121) and those on first-time H2RA prescriptions containingranitidine, cimetidine, and famotidine without PPIsprescribedduring follow-up were counted as new H2RA users . Patients without the use of PPIsor H2RAs during follow-up were counted as nonusers  , which was also used in another study [n = 34,229). No ESRD occurrence was ascertained again between CKD inception date and first-time acid suppression therapy prescription. We considered the following covariates: preexisting comorbidities within one year before CKD inception date including hypertension, diabetes, coronary heart disease, chronic liver disease, and acid peptic diseases such asupper gastrointestinal bleeding, reflux esophagitis, and peptic ulcer disease [We included 405,767 patients who had a primary diagnosis of CKD between 1 January 2001 and 31 December 2016 from the outpatient and inpatient claims . The fol disease ,24 becau disease , and two disease ,26 with  disease . We did  disease ,22,26. Tp-value less than 0.05 was considered statistically significant.The three cohorts were followed from their index date to the occurrence of ESRD, death, or the end of 2016, whichever ensued first; the latter two were considered as censored observations. Nevertheless, death before reaching ESRD, which could lead to informative censoring, was considered a competing risk event in estimating the cumulative incidence and risk of ESRD ,26. ESRDBefore propensity matching, PPI, H2RA, and control cohorts accounted for 7%, 47%, and 46%, respectively, of the overall 102,802 CKD patients . Comparep < 0.0001) and 1.15  and that for overall mortality were 0.64  and 1.83  in the H2RA and PPI cohorts, respectively  (p < 0.0001). When analyzing the Kaplan–Meier curve for ESRD-free survival in three study cohorts by log-rank test (p < 0.0001).During follow-up, 490 (3.7%) developed ESRD, 2572 (19.7%) died, and 2264 (17.3%) died before developing ESRD. Furthermore, 26.5% of the PPI cohort died before developing ESRD; this percentage was higher than the H2RA (13.2%) and control (12.2%) cohorts ( 0.0001) . The 15- 0.0001)  and overank test , survivaThe association between the median cDDD and prescription frequency of H2RA and PPI and study outcomes was explored. Considering neither users as the reference, the H2RA cohort receiving ≥ 10 cDDD, compared with those receiving < 10 cDDD, showed significantly lowered risks across all the outcomes . This doThe association between H2RA and lower risks of ESRD and overall mortality, as well as between PPI and higher overall mortality, seemed consistent in several sensitivity tests, including the exclusion of CKD patients who died or developed ESRD within 30, 60, and 90 days after the index date , multivaAfter propensity score matching, the associations between H2RA and lower ESRD and mortality risks and between PPI and higher mortality risk remained in stages 1–4 CKD populations. The assTo our knowledge, this is the first large nationwide cohort study of the CKD population using the new-user design, competing for mortality, and propensity score matching of PPI, H2RA, and neither substance to demonstrate a significantly lower risk for ESRD by 60% and overall mortality by 37% with H2RA use. These findings were reinforced by results from animal models suggesting plausible mechanisms for benefits ,5. NotabIn disagreement with our results that H2RA exerts potential renal and survival benefits, a clinic-based non-matching cohort study of 25,455 CKD patients by Cholin et al. , which mExperimental evidence has accumulated over time, suggesting the pathophysiological roles of histamine and H2R that belong to theG-protein-coupled-receptor family ,4. In reIt is note worthy that this is the first human report on the dose-dependent effect of H2RA on kidney, heart, and survival outcomes, which builds on prior experimental research, after adjusting for the number of medical visits. In an experimental study, the effect of histamine on renal vascular resistance via H2R was dose-dependent; a higher dose of histamine caused a drop in creatinine and urea clearance and stimulated renal sympathetic nerve activity ,4. This We found the statistically non-significant relationship of PPI with ESRD risk in fully multivariable regression, indicating that the adverse renal effect of PPI was dwarfed by the complex covariate influences that might lead to renal injury. We also found the excess risk of death associated with PPI use in CKD patients was not in a dose-dependent manner. Some discordant associations of PPI with renal and survival outcomes existed in prior research onrenal transplants, CKD patients, and normal renal function populations. When considering H2RA users as the reference in the normal renal function population, one study reported no significantly increased risks for AKI and ESRD in PPI users ; three sBy analyzing the NHI claims data with a highly representative sample, the present study demonstrated five strengths. First, recall bias of acid-suppression agents was avoided, and cumulative dose and prescription frequency could be identified. Second, the new-use design minimized the immortal bias and the potential residual effect of using acid-suppression agents before CKD diagnosis. Third, follow-up of ESRD and death outcomes was completed, and theuse of competing mortality minimized the risk of overestimating the ESRD. Fourth, the use of propensity score matching and stratified analyses minimized confounding effects. Fifth, consideration of the use of medical services minimized detection bias ,24,25. HThis national cohort study on CKD patients indicated that H2RA, especially in a dose-response relationship, was associated with areduced risk of ESRD, overall, cardiovascular, and non-cardiovascular mortality, while PPI, not in a dose-response relationship, was associated with anincreased risk of overall mortality, but not ESRD. Further prospective research is warranted to improve our understanding of the causal and dose-response relationships underlying this association.


In recent years, porcine reproductive and respiratory syndrome virus (PRRSV) strains have been experiencing extensive recombination in Chinese swine farms. This recombination usually happens in NADC30/34 strains and highly pathogenic (HP) PRRSV strains. This study identified a new PRRSV isolate that shared 99% and 99.1% nucleotide identity with CH-1a and CH-1R at the genomic level, respectively. After purification by viral plaque assay, this isolate was named PRRSV CSR1801. The isolate did not experience any recombination with other PRRSV strains common in swine herd epidemics in China, which means it still maintains the stable features of the classical PRRSV strain and did not easily recombine with other PRRSV strains. Further analysis of the pathogenicity of the PRRSV isolate CSR1801 was performed in piglets. The results indicated that none of the inoculated piglets showed the typical clinical manifestations of PRRS, which presented with runny noses, rough back hair, rectal temperatures always below 40.5 °C, and no deaths. Additionally, no obvious histopathological lesions such as severe interstitial pneumonia could be observed in the lungs of the piglets. Hence, the PRRSV isolate CSR1801 should be classified as a classical-like PRRSV strain. This classical PRRSV strain showed genetic stability and maintained low pathogenicity. This study may provide new clues for further understanding the genetic evolution and pathogenicity of PRRSV and may also be an important reference for the prevention and control of PRRS in swine farms. Arteriviridae family. The PRRSV genome comprises a positive-sense single-stranded RNA of approximately 15 kb in length and encodes 10 open reading frames (ORFs) [Porcine reproductive and respiratory syndrome (PRRS) has become one of the most economically significant pig diseases all over the world since it was first reported in 1986 and 1990 in North America and Europe, respectively [s (ORFs) . The ORFs (ORFs) .PRRSV was recognized as two distinct virus species (PRRSV-1 and PRRSV-2) instead of two genotypes (genotype I and genotype II) by the International Committee on Taxonomy of Viruses in 2016, although many researchers usually evaluate the effect of cross-protection of one PRRSV MLV against both PRRSV-1 and PRRSV-2 . In ChinAlthough numerous variant PRRSVs have been reported, most recombination happens among NADC30 and HP-PRRSV or mutations/deletions in HP-PRRSV strains. The impact of these strains varies among swine herds, but the consensus is that the pathogenicity trends to be intermediate between the parental strains ,10,11. P2 atmosphere. When approximately 80% of the cells exhibited a cytopathic effect (CPE) in the fourth generation, the supernatant of the virus culture was collected after two freeze-thaw repeats and stored at −80 °C for further use. PRRSV-positive lung samples were collected from a PRRS suspected pig farm uninoculated with PRRSV vaccine from Shandong Province, China, in 2018. These samples were homogenized with Dulbecco’s modified Eagle medium . The treated samples were inoculated after passing through 0.22 µm filters to MARC-145 cells. The inoculated cells were maintained in DMEM supplemented with 2% fetal bovine serum (FBS) and 100 U/mL of penicillin plus 100 μg/mL streptomycin at 37 °C in a 5% COg for 5 min, the supernatant was subjected to two additional rounds of plaque purification.For purification of the isolated virus, a plaque assay was performed using MARC-145 cells as previously described [MARC-145 cells were infected with PRRSV strain CSR1801 at a multiplicity of infection (MOI) of 0.1 when the cells reached approximately 90% confluence. At 24 h post-infection (hpi), the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.3% Triton X 100. After three washes with PBS, they were blocked with 5% fetal bovine serum albumin at 37 °C for 1 h. The cells were incubated overnight at 4 °C with the monoclonal antibody (mAb) against PRRSV N protein at a dilution of 1:5000. Then, the cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG  at 37 °C for 1 h. After a final washing, the cells were observed using a fluorescent microscope .Total RNA was extracted from the CSR1801-infected MARC-145 cells’ supernatants using TRIzol reagent . The full-length genome of the CSR1801 virus was obtained by amplifying each gene fragment using RT-PCR based on the specific primers  accordinThe evolutionary relationship of the CSR1801 isolate with other represented Chinese PRRSV isolates was analyzed using MegAlign in the Lasergene software (DNASTAR). Phylogenetic trees were constructed using the MEGA software (version X) using the neighbor-joining method, and the reliability of the tree was assessed by bootstrap analysis with 1000 replications. The nucleotide and deduced amino acid (AA) sequences were aligned using the MegAlign program of the DNASTAR software (DNASTAR) to determine the sequence homology. A phylogenetic tree was constructed using the MEGA5 software with the neighbor-joining method; bootstrap values were calculated for 1000 replicates for alignment with multiple sequences of representative PRRSV sequences available in GenBank.Furthermore, the software MegAlign was used to compare the full-length gene sequences of VR2332, CH-1R, JXA1, NADC30, and HLHDZD32-1901 (NADC34-like) strains. The above file was saved and homologous recombination analysis was performed using Simplot software (version 3.5.1). The CSR1801 strain was selected as the recombined parent sequence and Do BootScan was used to carry out a homologous recombination analysis.The experimental protocol for the pig studies was approved by the Shandong Agri-cultural University Animal Care and Use Committee . The animal experiments were conducted in strict accordance with the Chinese Regulations on Laboratory Animals and the Guidelines for the Care of Laboratory Animals (the Ministry of Science and Technology of the People’s Republic of China).n = 5) were all nasally inoculated with 2 mL/pig of the PRRSV CSR1801 strain at a dose of 1 × 105 TCID50. The piglets in group 2 (n = 5) were inoculated with the cell culture medium as the negative control. The piglets were monitored daily for clinical signs of PRRS, including fever , respiratory symptoms , were randomly divided into two groups. The piglets in group 1 (eviously ), and abThe viral loads of PRRSV in the sera collected at serial timepoints  were determined by conducting absolute RT-qPCR as reported previously . First, 450 value based on the corresponding established standard curve.The concentrations of IFN-α, IFN-γ, IL-10, and TGF-β1 in pig sera collected at serial timepoints  were detected using commercial kits for IFN-α, IFN-γ, IL-10, and TGF-β1  detection in accordance with the manufacturers’ instructions. The concentrations of the cytokines were calculated according to the corresponding ODHistopathological examinations of the collected lungs were performed using the H&E method. Briefly, the dried tissue sections were passed through the following alcohol gradient: xylene I (15 min), xylene II (15 min), alcohol xylene (1:1) (5 min), 100% alcohol (3 min), 90% alcohol (3 min), 80% alcohol (3 min), 70% alcohol (3 min), and deionized water (4 min). The sections were then dyed with hematoxylin for 5 min and rinsed for 1 s. The sections were subsequently covered with 1% hydrochloric acid alcohol for 2–3 s and flushed for 16 min. Next, the slices were placed in 85% alcohol for 2 s, 1% eosin for 3 s, dipped in 95% alcohol, 95% alcohol for 3 min, 100% alcohol for 2 min, and xylene for 10–30 min. Finally, the slices were sealed with neutral resin and the pathological changes in the lungs were observed under a light microscope .p < 0.05 was considered statistically significant.The data are expressed as the mean ± standard deviation (X ± SD), Duncan’s multi-sample test was used to analyze the differences between the groups, and SPSS 19.0 was used for statistical analysis. MARC-145 cells were inoculated with the grinding fluid of PRRSV-positive lung samples, and typical PRRSV-induced CPE appeared at 48 h after inoculation from the fourth-generation virus A. One ofThe full-length genome of the PRRSV CSR1801 strain was determined by segmental gene sequencing and splicing, which is 15,219 bp excluding the poly (A) tail (GenBank accession number: OM743305). To investigate the genetic relationships between the PRRSV CSR1801 strain and other PRRSV strains, the whole genome sequence of the CSR1801 strain and the representative PRRSV reference strains or vaccine strain registered in GenBank were used for phylogenetic tree analysis. Based on the phylogenetic tree result of full-length genome sequence analysis, the PRRSV isolate CSR1801 was classified into the CLASSIC-like PRRSV group A, which Finally, we analyzed the amino acid mutation of the CSR1801 strain compared with CH-1R and CH-1a strains, which showed that the ORF1a (2503 aa) amino acid mutation of the CSR1801 strain has 23 different loci—155, 235, 402, 509, 510, 582, 851, 860, 867, 997, 1023, 1259, 1307, 1445, 1508, 1549, 1640, 1643, 1755, 2166, 2273, 2341, and 2410; 12 mutations are identical to CH-1a but not to CH-1R—227, 560, 764, 964, 969, 1245, 1630, 2067, 2109, 2295, 2441, and 2448; 18 mutations are identical to CH-1R but not to CH-1a—573, 780, 865, 1015, 1038, 1193, 1254, 1429, 1522, 1523, 1543, 1705, 1742, 1797, 1994, 2063, 2105, and 2241. A detailed analysis of the corresponding amino acid changes is shown in The viral titers of PRRSV in the sera collected from piglets at regular intervals were detected by RT-qPCR. The results showed that the PRRSV CSR1801 isolate infection induced a low level of viremia. The viral titer peaked at 3 dpi, remained constant at about 4.2 log copies per microliter until 14 dpi, and declined rapidly to near zero after 14 dpi A, indicaIn general, the piglets inoculated with the PRRSV CSR1801 isolate showed mild clinical signs. In the animal challenge experiment, the rectal temperature of piglets inoculated with PRRSV CSR1801 strain increased to 39.7 °C at two dpi and it always fluctuated around 39.5 °C the rest of the time. The rectal temperature of the mock-inoculated pigs was relatively stable and almost always about 39.0–39.2 °C B. Mild cLymphocytic interstitial pneumonia characterized by a diffuse interstitial proliferation could be observed in the lungs in the PRRSV CSR1801 strain-inoculated piglets , which wIn the animal experiment, the concentrations of the corresponding swine cytokines, including IFN-α, IFN-γ, IL-10, and TGF-β1 in pig sera were determined by ELISA. Ten days after the challenge, the concentration of IL-10 did not change significantly. At 10 dpi and 14 dpi, the concentration of IL-10 in the CSR1801-inoculated piglets was significantly higher than that of the mock-inoculated piglets and then decreased rapidly A. The chThe PRRSV epidemic strains in China have experienced recombination since 2015 due to the introduction of the PRRSV NADC30 strain from North America that has undergone genetic exchange with the HP-PRRSVs in China ,20,21, pIn our study, the PRRSV CSR1801 strain still shows the classical PRRSV features, and no recombination or deletion was found in the amino acids (AAs) of Nsp2, the most variable genes in the whole genome of PRRSV and NADC30/NADC34/HP-PRRSV ,26,27,28The pathogenicity of the PRRSV CSR1801 strain for the piglets was mild and mainly manifested in clinical signs, rectal temperature, viral load, and lung lesions, which is consistent with the previous report [In China, PRRSV MLVs are used extensively in swine farms, and the MLV strains include VR-2332, R98, CH-1R, PC, JXA1-P80, HuN4-F112, GDr180, and TJM-F92, which can offer effective protection in homologous PRRSV strain challenges ,31,32,33In summary, this study looked at a new classical PRRSV strain from a swine farm in Shandong province, China, which maintained stable features and did not recombine with other PRRSV strains  as is common in swine farms. This study may provide clues for further understanding the genetic evolution and pathogenicity of PRRSV and may also have important reference value for the efficient prevention and control of PRRS in swine farms.


Zinc finger E-box binding homeobox 1 (ZEB1) is a master modulator of the epithelial–mesenchymal transition (EMT), a process whereby epithelial cells undergo a series of molecular changes and express certain characteristics of mesenchymal cells. ZEB1, in association with other EMT transcription factors, promotes neuroinflammation through changes in the production of inflammatory mediators, the morphology and function of immune cells, and multiple signaling pathways that mediate the inflammatory response. The ZEB1–neuroinflammation axis plays a pivotal role in the pathogenesis of different CNS disorders, such as brain tumors, multiple sclerosis, cerebrovascular diseases, and neuropathic pain, by promoting tumor cell proliferation and invasiveness, formation of the hostile inflammatory micromilieu surrounding neuronal tissues, dysfunction of microglia and astrocytes, impairment of angiogenesis, and dysfunction of the blood–brain barrier. Future studies are needed to elucidate whether the ZEB1–neuroinflammation axis could serve as a diagnostic, prognostic, and/or therapeutic target for CNS disorders. Central nervous system (CNS) disorders, such as multiple sclerosis (MS), Alzheimer’s disease (AD), neuropathic pain, and glioblastoma (GBM), are of high scientific interest worldwide due to their increasing prevalence and a lack of effective therapies , 2. FocuZeb1 gene could have also resulted in a cleft palate as well as other craniofacial and skeletal anomalies [Zeb1 gene mutation has been suggested [The expression of ZEB1 differs markedly among various adult human tissues, showing low expression in the prostate, pancreas, and liver, moderate expression in the heart, mammary gland, and ovary, and high expression in the thymus, aorta, uterus, and bladder , 17. ZEBnomalies . A link uggested . Moreoveuggested .Alterations of ZEB1 expression regulate neural stem cell renewal and cell fate in the brain . The expZEB1 gene on chromosome 10p11.2 [ZEB1, also known as TCF8 and δEF-1, belongs to the ZEB family encoded by the  10p11.2 , 42. ZEB 10p11.2 . Its DNA 10p11.2 , 44, 45. 10p11.2 . The HD  10p11.2 . ZEB1 pr 10p11.2 .E-cadherin gene (CDH1) and preventing its expression. It is downstream of many cascades linked to EMT, such as TGF-β, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), Ras-Raf-MEK-ERK (RAS/ERK), Wnt/β-catenin, and tyrosine kinase receptors. Recent studies have focused on ZEB1 and its influence on EMT function causing both malignancy and chemotherapy resistance in tumors. In addition, this important transcription factor has influenced many CNS diseases through various pathways  and the EMT process has been suggested . EvidencZeb1 expression, and inhibits cytokine expression [Zeb1 expression and regulate neuroinflammation and neuropathic pain [ZEB1-related neuroinflammatory responses could contribute to neuropathic pain. The miR-28-5p/ZEB1 pathway has been suggested as a potential therapeutic target for neuropathic pain. Overexpression of miR-28-5p reduces neuropathic pain behaviors in a chronic sciatic nerve injury model in rats through the inhibition of neuroinflammation induced by the release of Cox-2, IL-6, and IL-1β. MiR-28-5p binds to the 3-untranslated area of Zeb1, downregulates pression . Moreovehic pain , 185. Inhic pain . lncRNA hic pain . Circulahic pain , 138. Fuhic pain . Oxaliplhic pain .The induction of ZEB1 is part of a neuroprotective response by neurons after brain ischemic insults. High values of ZEB1 expression in neocortical tissues are observed in neocortical specimens of patients with stroke . ZEB1 reZeb2f in astrocytes lessened astrogliosis, induced greater lesions, and delayed functional motor recovery [The astrocytic response to CNS injury is implicated in EMT and upregulation in ZEB expression. CNS injury-related astrogliosis enhances EMT and its related gene expression. In experimental models of spinal cord injury or transient ischemic stroke, the knockdown of the recovery . Followirecovery . Expressrecovery . Thus, Zrecovery , 154.Considering the critical role of neuroinflammation in various diseases, EMT is known to be an important process of pathogenicity in various neurological disorders. ZEB1 as a focal transcription factor of EMT has been considered a potential target for the prognosis and treatment of various neurological diseases. Although recognition of the exact role of ZEB1 in CNS dysfunction requires additional analysis and evaluation, it was concluded that ZEB1, in partnership with other EMT transcription factors, has dominant functions in EMT and neuroinflammation. The complex interaction between ZEB1, immune cells, and various cytokines is tightly connected to neuroinflammation through the regulation of different signaling pathways. The ZEB1–inflammation axis plays a crucial role in the pathogenesis of various CNS disorders through promoting tumor cell proliferation and invasiveness, formation of the hostile inflammatory micromilieu surrounding neuronal tissues, dysfunction of BBB, dysfunction of microglia and astrocytes, and disturbances of angiogenesis. The molecular mechanisms and signaling pathways orchestrating the association between ZEB1 and inflammatory processes are linked with the initiation, progression, and outcomes of various CNS disorders. Future studies are required to demonstrate whether the modulation of ZEB1 could play a beneficial role as a diagnostic, prognostic, and/or therapeutic approach for CNS disorders.


JAK1, JAK2, JAK3, SOCS1, IL7R, MCL1, BCL2, MTOR, and IL6ST genes. Interestingly, six mutations which were likely to be novel deleterious were identified: two in the SCH1 gene, two in the IL2 gene, and one in each of the IL7R and JUN genes. According to IVA analysis, interleukin 2, interleukin 6, and interleukin 7 signaling pathways were the most altered in 34.5%, 29%, and 23.6% of our BC group, respectively. Our multigene panel sequencing analysis reveals that our BC patients have altered interleukin signaling pathways. So, these results highlight the prominent role of interleukins in the carcinogenesis process and suggest its potential role as promising candidates for personalized therapy in Egyptian patients.This study aimed to investigate the impact of somatic mutations on various interleukin signaling pathways associated with grade II invasive breast cancer (BC) in Egyptian patients to broaden our understanding of their role in promoting carcinogenesis. Fifty-five grade II invasive BC patients were included in this study. Data for somatic mutations in 45 BC patients were already available from a previous study. Data for somatic mutations of 10 new BC patients were included in the current study. Somatic mutations were identified using targeted next-generation sequencing (NGS) to study their involvement in interleukin signaling pathways. For pathway analysis, we used ingenuity variant analysis (IVA) to identify the most significantly altered pathways. We identified somatic mutations in components of the interleukin-2, interleukin-6, and inter-leukin-7 signaling pathways, including mutations in  Breast cancer (BC) is the most common and first cause of cancer death among women . In 2020Fresh tissue samples were recruited from the National Cancer Institute (NCI) of Egypt from November 2019 to November 2020. Fifty-five grade II invasive BC patients were included in this study. Data for somatic mutations in 45 BC patients were already available from a previous study . In addi® 3.0 Fluorometer  with Qubit™ dsDNA HS assay kit . Then, the amplicon libraries were prepared using Ion Ampliseq Complete Cancer Panel (CCP) and Ion AmpliSeq™ Library Kit Plus . The CCP has 16,000 primer pairs for 409 genes . The isolated DNA was measured using Qubit09 genes , coverinFirstly, we filtered the somatic mutations according to our normal Egyptian population database. Then, we used the Catalogue of Somatic Mutations in Cancer (COSMIC) in the identification and classification of the somatic variants. Then, we used ANNOVAR package to annotate the somatic variants. All genetic annotations and nomenclature were based on GRCh37/hg19 build. Frameshift deletions/ insertions, and in-frame deletions/insertions were considered to be pathogenic. For missense mutations, we used amino acid prediction software, SIFT , PolyPheFor pathway analysis, we used ingenuity variant analysis  to identify the significantly altered interleukin signaling pathways. The Ingenuity Knowledge Base was used to link variants in an analyzed dataset to biologically relevant information. Based on the applied filter, the pathogenic variants and the most affected pathways could be identified.The clinical characteristics of the studied patients are summarized in JAK1, JAK2, SOCS1, IL7R, MCL1, BCL2, MTOR, and IL6ST genes. Interestingly we identified 18 out of 21 variants that were not previously reported in BC tissue. We identified four loss-of-function mutations in the JAK1 gene in 13 (23.6%), 8 (14.5%), 5 (9%), and 3 (5.4%) patients, respectively. This gene harbored four different mutations in 29 patients, representing 52.7%. The BCL2 gene harbored two different deletions in 8 patients (16.4%). The MCL1 gene harbored three different mutations in 11 (20%), one (1.8%), and one (1.8%) patients, respectively. In addition, two different mutations were identified in the SOCS gene and IL7R gene in 15 (27.3%) and 10 (18.2%) patients, respectively. One loss function mutation was identified in each of the JAK2 gene, IL6ST gene, and MTOR gene.Without departing from the scope of this study, we reported the somatic mutations involved in the significantly altered interleukin signaling pathways. SCH1 gene (p.K11fs*18 and p.F17fs*12); each has been identified in two samples. Two more likely novel mutations were identified in the IL-2 gene (p.C145fs*8 in 10 patients and p.L45* in 3 patients). In addition, one likely novel mutation was identified in each of the IL-7R gene (p.K157fs*4) and JUN gene (p.P220fs*6), as listed in Interestingly, two likely novel mutations predicted to be deleterious were identified in the p = 0.0021)   . Pathway Analysis reported the altered interleukin signaling pathways with its involved mutated genes. The most altered interleukin signaling pathways were IL-2 ( 0.0021) , IL-6 (p 0.0025) , and IL-=0.0012) , signaliCancers, including BC, are multifactorial diseases in which genetic and epigenetic changes significantly impact tumorigenesis, progression, and treatment response . These aJAK1 and gain of function mutations in JAK3 genes and hypothesized that it resulted in constitutive activation of their kinase activity. From here comes the importance of using next-generation sequencing techniques to identify the mutations likely to lead to constitutive activation of kinases. Such mutations are known to be a driving event in cancer, and their identification is of great importance as it might help identify their selective kinase inhibitors. Consequently, we claim that JAK1 and/or JAK3 selective kinase inhibitors can be used to block IL-2-mediated proliferative responses [IL-2 is a key regulator of normal immune functions and is critical for the activation and subsequent amplification of the immune response following antigenic stimulation . IL-2 biesponses ,31.SHC gene, the key pathway for IL-2 control of Ras/Raf/MEK/REK pathway. Tyrosine-phosphorylated SHC forms a complex with the SH2 domain of the adapter GRB2. This complex then binds to Ras guanine nucleotide exchange protein SOS (Son of Sevenless) and activates the Raf-ERK MAP kinase cascade [Moreover, we identified loss of function mutations in the  cascade . The refJAK1 gene might explain why the PI3K-AKT signaling pathway is upregulated in our studied cases.IL-2 can also regulate the activation of the PI3K-AKT signaling pathway ,34 throuSeveral studies suggested the relationship between inflammation and various cancer types and showed that anti-inflammatory agents could attenuate tumor growth. Interleukin-6 (IL-6) is a pro-inflammatory cytokine released by cancerous cells in the tumor microenvironment and plays a vital role in the differentiation and expansion of tumor cells ,38. The JAK1, gain of function mutation in JAK3, and loss of function mutation in SOCS1. Thus, we hypothesized that JAK1 mutations lead to a partial loss of its kinase activity. On the other hand, all the previously mentioned mutations promote the constitutive activation of the PI3K/AKT pathway and Ras/Raf/MEK/ERK pathway. We also identified mutations in the SOCS1 gene, the negative feedback gene of these signaling pathways.IL-6 leads to the activation of three major downstream pathways due to the dimerization of gp130/IL6ST: the JAK/STAT pathway, the MAPK/MERK/ERK signaling cascade, and the PI3K/AKT pathway. The tyrosine kinases in the JAK family that are associated with the cytoplasmic region of gp130/IL6ST by a non-covalent bond, initiate the signaling cascade. The dimerization of gp130/IL6ST causes auto-phosphorylation and activation of JAK. In turn, JAK activates the SH2 domain-containing cytoplasmic protein tyrosine phosphatase (SHP2), which activates the Ras/Raf/MEK/ERK pathway, responsible for different transcription factors linked to cell growth ,41. MoreIL-6 is mainly secreted in the tumor cells and tumor associated fibroblast and many studies have reported its immunopathogenicity and its signaling in tumor growth, metastasis, and therapeutic resistance in BC. However, herein, we shed light on IL-6 function in grade II invasive BC patients. Therefore, targeting IL-6 and/or its receptor in combination with other potent anticancer therapies may be a potent therapeutic approach for BC therapy ,45,46.IL7R, JAK1, and JAK3 would result in constitutive activation of the IL7R-JAK-STAT signaling pathway. In addition, we hypothesized that loss-of-function mutations in CCND1 and SOCS1 genes cause deregulation of expression of antiapoptotic (Bcl-2 and Mcl-1) and proapoptotic (Bad and Bax) proteins.IL-7/IL-7R signaling activates two main signal transduction pathways (the JAK/STAT and PI3K/Akt pathway). The first mandatory step in IL-7/IL-7R signaling is the phosphorylation of JAK1 and JAK3 kinases, which will result in constitutive activation of proteins regulating cell cycle progression, survival, and protein translation . AdditioThese findings shed light on the alteration of IL-signaling pathways in our BC patients. These results were in agreement with our previous published study in Egyptian colorectal cancer patients that reported the alteration of the interleukin pathways in those patients , suggestIn summary, our findings support the role of somatic mutations in IL-signaling pathways in breast oncogenesis in Egyptian patients with grade II BC.JAK1, JAK2, SOCS1, IL7R, MCL1, BCL2, MTOR, and IL6ST genes. Six mutations are likely to be novel and deleterious. In addition to the diagnostic benefit of detecting such IL-pathways alterations, this work would also assist in the development of personalized treatment regimens suited to Egyptian breast cancer patients. Further research is recommended to validate these identified somatic mutations in larger series of patients and to confirm their impact at the protein level.Using multigene panel sequencing analysis, we were able to identify somatic mutations in IL-signaling pathways including We recommend following up the response to treatment of those patients to investigate the better described therapy regimens that are suitable for the identified mutations in this study.