K/Z RIL population of 198 lines was screened in the field at Fayetteville (AR) for three consecutive years under controlled drought stress (DS) and well-watered (WW) treatment during the reproductive stage. The effects of DS were quantified by measuring morphological traits, grain yield components, and root architectural traits. A QTL analysis using a set of 4133 single nucleotide polymorphism (SNP) markers and the QTL IciMapping identified 41 QTLs and 184 candidate genes for drought-related traits within the DR-QTL regions. RT-qPCR in parental lines was used to confirm the putative candidate genes. The comparison between the drought-resistant parent (Kaybonnet) and the drought-sensitive parent (ZHE733) under DS conditions revealed that the gene expression of 15 candidate DR genes with known annotations and two candidate DR genes with unknown annotations within the DR-QTL regions was up-regulated in the drought-resistant parent (Kaybonnet). The outcomes of this research provide essential information that can be utilized in developing drought-resistant rice cultivars that have higher productivity when DS conditions are prevalent.Rice is the most important staple crop for the sustenance of the world’s population, and drought is a major factor limiting rice production. Quantitative trait locus (QTL) analysis of drought-resistance-related traits was conducted on a recombinant inbred line (RIL) population derived from the self-fed progeny of a cross between the drought-resistant  Oryza sativa L.) has a compact diploid genome size of about 500 Mb and 12 chromosomes, making it one of the rich sources of carbohydrates and commercially productive cereal crops that serves as the primary food source for about 2.5 billion people worldwide [A model species for monocot and cereal plants, rice  are regions of DNA that encode genes for quantitative traits, the effects of which can be measured quantitatively. Plant height, grain yield, and susceptibility to abiotic and biotic stress are all examples of quantitative traits that are controlled by one or more genes and affected by environmental variation. Molecular markers are used to define and map QTLs. Recent advances in genomic technology and statistical analysis methodologies have made this mapping approach particularly cost-effective for plant research projects ,19. SNPsIn addition, single nucleotide polymorphisms are both productive and economical ,21. As aOryza sativa L. [In the past few decades, QTL mapping for agronomic and physiological traits under abiotic stress conditions has involved in the improvement of rice production. A number of QTLs associated with drought-resistant (DR) traits have been identified in ativa L. . Furtherativa L. . HoweverOryza sativa [temperate japonica and aus. In 2010, Gomez et al. [A number of QTLs associated with drought-resistant (DR) traits have been identified in a sativa . In 1996a sativa . The QTLa sativa . Price ea sativa ,28, mappa sativa  in the da sativa  in the bz et al.  also detSeveral techniques, such as expressed sequence tag (EST) profiling, transcript profiling via massively parallel signature sequencing (MPSS), microarrays and quantitative real-time PCR, RNA gel blot analysis , and comBy controlling stomatal closure and thereby reducing water loss through transpiration, the plant hormone abscisic acid (ABA) plays a crucial role in adaptive responses to drought stress ,37. DrouThe identification of genomic regions related to the expression of complex drought traits using recombinant inbred line (RIL) populations, which contain many gene recombinations, and subsequent validations of the associated loci with gene expression using additional studies of genomes from a diverse panel representing six distinct subpopulations for the role of SNPs regulating the expression of the QTL genes are among the few recent studies on drought tolerance. This study provides extensive phenotypic data associated with the RIL population to find drought-resistant QTLs and candidate genes in the K/Z RIL population under both drought stress and well-watered conditions by examining changes in the morphological traits, grain yield components, imposed drought conditions, and root architectural traits linked to the ABA response. Gene expression analysis was used to further validate the QTLs that showed high logarithm of odd (LOD) and phenotypic variation explanation (PVE) in both the tolerant and sensitive parental lines. The identification of QTLs for drought-related traits in the two contrasting cultivars will contribute to our understanding of the genetic control of rice productivity at the sensitive reproductive stage under drought-stress conditions, which in turn will accelerate the development of drought-resistant rice varieties with improved grain yields under drought stress conditions.2, which is larger than that of ZHE733 (0.29 mm2). According to Zhu et al.’s [The variations in the root anatomy and root morphology of drought-resistant Kaybonnet and drought-sensitive ZHE733 are presented in et al.’s  findingsBased on the screening of grain yield in DS conditions at the reproductive stage (R3), Kaybonnet is the more drought-resistant of the two parental genotypes, while ZHE733 exhibits a phenotype indicative of sensitivity to drought. Under DS conditions, Kaybonnet showed a 20% reduction in filled grains per panicle, while ZHE733 showed a 50% decrease in grain filling . The proA normal frequency distribution  in the RThe total plant biomass (TPB), spikelet per panicle number (SP), filled grain per panicle number (FG), panicle length (PL), and primary panicle branch number (PPB) mean values of the grain yield components revealed a significant difference in drought-tolerance for both parents (KB and ZHE 733) and K/Z RIL population under DS conditions compared to WW . Under WUnder WW conditions, KB-WW (66) and ZHE733-WW (43.2) did not show a significant difference in the filled grain per panicle number; while under drought stress, the KB-DS (50) showed a significant decrease compared to ZHE733-DS (16), suggesting a more severe reduction in ZHE733 (62.96%) than KB (24.24%) under DS. Under WW conditions, KB-WW (21.7 cm) and ZHE733-WW (18.6 cm) did not show a significant difference in panicle length; while under drought stress, the KB-DS (19.3 cm) showed a significant decrease compared to ZHE733-DS (15.6 cm), suggesting a more severe reduction in ZHE733 (16.13%) than KB (11.06%) under DS. The primary panicle branch number showed significant differences under WW conditions between KB-WW (19) and ZHE733-WW (8); while under drought stress, the KB-DS (13) showed significant decreases compared to ZHE733-DS (4), suggesting a more severe reduction in ZHE733 (50%) than KB (31.58%) under DS.The phenotypic trait TPB under WW showed normal distribution; while under DS, the overall RIL population was better performing than parent KB. The SPP showed normal distribution under WW, but under DS, the trait showed transgressive segregation. The RIL population was more weighted towards ZHE733 parent for trait FG under both WW and DS. The trait PL has a normal for RIL population under both WW and DS. Based on PPB, the RIL population was distributed between both parents under both WW and DS. When comparing the average values of traits in RIL populations, there is a reduction under DS compared to WW for following traits: TPB (12.5%), SP (38.75%), FG (47.62%), PL (15.82%), and PPB (16.92%).The responses of the parents, Kaybonnet and ZHE733, to ABA varied; Kaybonnet, a parent that is drought-resistant, displayed greater sensitivity to ABA than ZHE733, a parent that is drought-sensitive. Under ABA conditions compared to control conditions, both parents showed reduced root length (RL), total root number (TRN), shallow root number (SRN), deep root number (DRN), and root fresh weight (RFW). In contrast to ZHE733, Kaybonnet showed a greater reduction in RL, TRN, SRN, DRN, and RFW . Under AThe K/Z RIL population showed variations in their root architectural traits in response to ABA , such as reduction in RL , increasIn addition, the average root architectural traits of the RILs lie between the parents under ABA conditions. The average RL, TRN, SRN, and RFW in the RIL population under ABA conditions 3 µM all showed a reduction, with 18.69%, 16.46%, 33.78%, 9.26%, and 2.94%, respectively. In addition, when the concentration of ABA was increased to 5 µM, the average levels of RL, TRN, SRN, and RFW in the RIL population all decreased by 21.16%, 21.07%, 36.51%, and 12.61%, respectively. Despite the fact that the RL, TRN, SRN, DRN, and RFW of both parents and the RIL population decreased in response to ABA conditions, the reduction in Kaybonnet was significantly greater. The root–shoot ratio (RSR) increased by 38% and 16% for Kaybonnet under ABA conditions 3 µM and 5 µM; for the RIL population under ABA conditions, 3 µM and 5 µM increased 22.92% and 22.69%, respectively.According to Gibert et al. , correlaThree RIL lines  showed drought resistance based on morphological traits (PH and TN), grain yield components , and ABA sensitivity response characteristics similar to Kaybonnet as the drought-resistant parent. Under DS conditions, these three groups showed a decrease of 30% or less in PH, TN, TPB, SP, FG, PL, and PPB. Meanwhile, these three lines showed greater than a 50% reduction in root architectural traits . In addition, these three lines showed short PH and high numbers in TN, TPB, SP, PL, FG, and PPB based on the morphological features and grain yield components under WW conditions, making them a valuable genetic resource for the development of drought-resistant varieties. In WW conditions, values for PH, TN, TPB, SP, PL, FG, and PPB ranged from 42 to 51 cm, 5 to 9, 17 to 24 g, 104 to 130, 17.5 to 23.7 cm, 100 to 103, and 10 to 12, respectively.2, while drought-sensitive lines have an average metaxylem size of 0.0008 mm2. In drought-resistant lines, aerenchyma typically covers 60% of the total root cross section, compared to only 40% in drought-sensitive lines. In drought-sensitive lines, there are generally 11 cortical cell files, which is more than the average of 5 in drought-resistant lines.In comparison to ZHE733 and drought-sensitive lines, parental Kaybonnet and drought-resistant lines displayed more metaxylem vessels . AccordiUnderstanding the plant genome and learning about allele introgression through plant breeding are both aided by genetic linkage maps . More prOn average, there were 344.42 SNP markers mapped to each chromosome. This ranged from a low of 182 SNP markers on chromosome 12 to a high of 562 SNP markers on chromosome 1. With an average of 505.26 cM per chromosome, the total length of the genetic linkage map was 6063.12 cM (ranging from 343.72 cM on chromosome 10 to 676.52 cM on chromosome 2). From 0.92 cM on chromosome 6 to 2.89 cM on chromosome 12, the average genetic distance between pairs of SNP markers was calculated to be 1.58 cM. One single nucleotide polymorphism (SNP) marker was located approximately every 1.5 centimorgans (cM). This high-density genetic linkage map covered 373 Mb of the rice genome, allowing for more precise and reliable identification of QTLs for use in rice breeding environments experiencing DS. As a result of the high-density genetic linkage map, candidate genes inside the QTL areas that contribute to enhanced drought resistance in rice plants were identified and selected, and new drought-resistant rice cultivars can be developed. High-density linkage maps have been utilized in numerous earlier studies for QTL mapping ,52,53,54qPHC1.1, qPHC2.1, and qPHC2.2, were discovered. Moreover, 23 QTLs were detected for grain yield component traits, such as qTPBC4.2, qTPBC6.2, qTPBC10.1, qTPBD3.1, qTPBD9.1, qTPBD10.2, qTPBD10.3, qTPBD12.2, qPPBC4.1, qPPBC7.1, qPPBC8.1, qPLC7.1, qPLD4.2, qSPC7.1, qSPC7.2, qSPC8.1, qSPD7.1, qSPD10.1, qFGC5.1, qFGC6.2, qFGC7.1, qFGD5.1, and qFGD8.1. For root architectural traits, 15 QTLs were identified, including qRLA3-2.3, qRLA3-3.1, qRLA3-4.2, qRLA3-5.1, qRLA3-6.1, qRLA3-8.1, qRLA3-10.1, qRLA3-10.3, qRLA5-2.2, qRLA5-3.1, qRLA5-4.2, qRLA5-5.1, qRLA5-6.1, qRLA5-8.1, and qRLA59.1. For both grain yield components and root architecture features, 2–4 QTLs were found to be co-localized in a total of 12 QTL clusters or hot spots, suggesting that these QTLs have the pleiotropic effect of a single gene or multiple connected genes [qRLA3-2.3 and qRLA5-2.2 are located on chromosome 2; in cluster 2, qRLA3-3.1, qRLA5-3.1, and qTPBD3.1 are on chromosome 3; in cluster 3, qRLA3-4.2, qRLA5-4.2, qPLD4.2, and qTPBC4.2 are on chromosome 4; in cluster 4, on chromosome 5. The significant positive association between grain yield components under DS and root architectural features under ABA conditions  with the highest LOD and PVE scores among the 41 QTL regions were examined for changes in gene expression levels under control and drought conditions in the parents (KB and ZHE733).As a result of our study, we were able to identify candidate genes that are involved in a wide variety of biological processes, molecular functions, cell components, and the response to drought . Out of qPHC2.1 region has a LOD score of 24.42 and a PVE of 13.81% for the trait PHC. This qPHC2.1 window region of 25 Kb upstream and downstream was chosen based on the location of the polymorphic SNP on chromosome 2. This region contains six genes with unknown annotations: LOC_Os02g44590, LOC_Os02g44599, LOC_Os02g44610, LOC_Os02g44620, LOC_Os02g44630, and LOC_Os02g44642  has nine genes spanning the 25 kb region and one of gene LOC_Os01g66270  is associated with abiotic stress [qRLA3-2.3 and qRLA5-2.2 had common overlapping regions comprising five genes; one was the gene LOC_Os02g54160, with a APETALA2/ERF transcription factor, which has been studied for abiotic stress [qTPBD3.1, qRLA3-3.1, and qRLA5-3.1, has six common genes, with one gene LOC_Os03g56280 annotated as a malate dehydrogenase [qTPBC4.2, qRLA3-4.2, and qRLA5-4.2 were identified and shared the same region that had seven genes with one gene, LOC_Os04g44190 annotated for light reaction photosystem II [qFGC5.1, qFGD5.1, qRLA3-5.1, qTPBC6.2, qFGC6.2, qRLA3-6.1, and qRLA5-6.1 had a majority of 12 genes, 3 of which are linked to the response to drought-WRKY transcription factor (LOC_Os05g49210) [qSPD7.1 contains nine genes and one potential candidate gene, LOC_Os07g02520, which regulates sugar partitioning in carbon-demanding young leaves and developing leaf sheaths [qFGD8.1, qRLA3-8.1, and qRLA5-8.1 contain six genes, one of which is involved in stress, LOC_Os08g06344 [qTPBD9.1 and qRLA59.1 contains 7 genes, 1 of which is involved in carbohydrate metabolism (LOC_Os09g08120) [qTPBD12.2 has 10 genes and LOC_Os12g02700 is annoted as a late embryogenesis abundant/LEA protein [The 02g44642 . The regc stress ,57,58,595g49210) ,65, MYB 5g49210) ,68 and B5g49210) ,70,71,72 sheaths . The reg08g06344 ,74,75. T9g08120)  and qTPB protein ,79,80,81qRLA3-2.3, qRLA5-2.2), LOC_Os03g56280 , LOC_Os08g06344 , LOC_Os10g41460 (qRFWA3-10.1), LOC_Os11g30760 (qRFWA3-11.1), LOC_Os12g29330 (qTRN12.1), LOC_Os02g44610 (qPHC2.1), LOC_Os10g07040 (qTPBD10.2), and LOC_Os10g07080 (qTPBD10.2) suggesting that these genes are constitutively highly expressed in control conditions and are also maintained with higher expression in drought conditions in parent KB compared to ZHE733.The 26 potential candidate genes identified from multiple QTL regions that had high LOD and PVE scores were selected for gene expression variations in parents KB and ZHE733 under control and drought conditions. Amongst them, 15 candidate genes have been previously studied for abiotic stress and 11 are novel candidate genes. The QTL regions that were up-regulated under control and drought for KB parent compared to ZHE733 were LOC_Os02g54160 (qPHC1.1 (LOC_Os01g66270) was down-regulated in KB under control conditions compared to ZHE733, but under drought conditions, this gene shows almost 3.5 log2FC up-regulation in KB compared to ZHE733. The following genes in the QTL region, LOC_Os07g02520 (qSPD7.1), LOC_Os09g08120 , LOC_Os12g02700 (qTPBD12.2), LOC_Os02g44620 (qPHC2.1), LOC_Os02g44630 (qPHC2.1), and LOC_Os02g44642 (qPHC2.1) are up-regulated under control conditions but down-regulated in KB compared to ZHE733 under drought.The gene in the QTL region qTPBD10.3 with high LOD and PVE scores, the two identified novel genes, LOC_Os10g07040 and LOC_Os10g07080, could be potential candidates for total plant biomass under drought based on gene expression levels in control and drought conditions. Among the candidate drought-resistance genes not annotated to drought stress functions, LOC_Os10g07040 showed high up-regulation in Kaybonnet compared to ZHE733 under DS and WW conditions  and chromosome 10 (5 loci). The up- and down-stream region of the 26 drought loci was used for dissecting the genetic diversity in and surrounding these loci in the diversity panel. Depending upon the proximity to the nearest neighbor gene, the region of interest was defined between 400 bp to 22 Kbp for the upstream and 800 bp to 18 Kbp for the downstream region.indica; Aus, aus; TeJ, temperate japonica; TrJ, tropical japonica; Adm, admixture; and Aro, aromatic). Each individual SNP has multiple effects on individual loci depending upon the ‘impact’, ‘functional class’, ‘type’, and ‘region’ where it is present as shown  from the indica and aus subpopulations, and explains 84% of the variation in the data in doing so  on the vcf files containing the SNP datasets. We performed three separate runs of PCA for (i) all 26 loci, (ii) 6-loci cluster on chromosome 2, and (iii) 5-loci cluster on chromosome 10 containing 7475 SNPs, 565 SNPs, and 4375 SNPs, respectively . In the enotypes . This isthe data . Howeverdoing so . This intropical japonica) and ZHE733 (indica), using the SSD method. This was performed in order to create segregating progenies with high genetic variability for selection-desirable genes. The many genotypes of rice investigated here exhibit a wide variety of patterns of root growth and development. Greub [O. glaberrima, IR64, Nagina-22 (N22), Vandana, and the parental line ZHE733. There is a correlation between the number of metaxylem vessels and their size and the water conductivity of the tissue. Among these lines, Bengal and Nagina-22 (N22) are examples of drought-resistant rice genotypes. On the other hand, IR64 and Nippobare serve as references for drought-sensitive rice genotypes.The K/Z RIL population was developed at the USDA Dale Bumpers National Rice Research Center in Stuttgart, AR, USA. This was accomplished by crossing diverse parental genotypes from different subspecies, namely Kaybonnet . Most of the QTLs connected to grain yields were found on chromosomes 5 and 6  [qRLA3-10.1 and qRLA3-10.3) for root architectural traits.The majority of the QTL found in this study were mapped to regions that were similar to those found in earlier studies ,133,134.qFGC6.2) ,141,142.qFGC6.2) , Kitomi qFGC6.2) , and GimqFGC6.2) , chromosIdentification of candidate genes within a QTL region is essential for developing transgenic rice with enhanced drought resistance . Within sd1 and QTL for drought-related traits. The sd1 locus is also linked to above- and below-ground traits in rice, such as plant height and root architectural traits [A transcription factor family well-known for drought response such as APETALA2 (AP2)/ethylene response factor (ERF) ,57,58,59l traits . The adal traits ,61,62. Al traits  observedLOC_Os03g56280, a significant candidate gene known to modulate malate dehydrogenase in response to drought stress, was identified in the QTL regions for TPB-DS, RL-ABA3, and RL-ABA5, on chromosome 3. In the QTL regions for TPB-DS and RL-ABA5, a candidate gene for carbohydrate metabolism was identified on chromosome 9 (LOC_Os09g08120). Malate dehydrogenase is an enzyme that uses NAD(H)/NADP(H) as a cofactor to catalyze the oxidation of malate to oxaloacetate. In addition, this enzyme can be expressed in various rice plant tissues, including the root, leaf, panicle, and stem, and was induced by water deficit . TransgeA photosynthesis-encoding gene (LOC_Os04g44190) is present in the TPB-WW, RL-ABA3, and RL-ABA5 QTL regions on chromosome 4. This gene regulates stomatal closure and protects plants from dehydration . It is iOn chromosome 5 (LOC_Os05g49210) of the QTL regions for FGC and FGD, a WRKY transcription factor involved in drought stress response and plant development was identified. Shen et al.  reportedOn chromosome 5 (LOC_Os05g49240) in the QTL regions for SP-DS and PPB-DS; and also on chromosome 10 (LOC_Os10g41460) in the QTL regions for TPB-DS and RL-ABA3, were identified MYB transcription factor, a well-known transcription factor in drought response ,67,68. RZinc finger (ZFN) protein (LOC_Os06g49080), a stress-responsive transcription factor, is located on chromosome 6 in the QTL regions for FG-WW, BY-WW, RL-ABA3, and RL-ABA5. It has been reported that the ZFN protein enhances drought resistance in plants, suggesting that the ZFN protein contributes to the higher yield under DS conditions by regulating stomatal closure ,70,71,72The genomic region on chromosome 8 (LOC_Os08g06344) within the QTLs for FG-DS, RL-ABA3, and RL-ABA5 encodes a DEAD-box RNA helicase that has been reported to enhance drought resistance in rice ,67,68. OA gene regulating late embryogenesis abundant (LEA) protein was identified on chromosome 12 (LOC_Os12g02700) between the TPB-DS QTL regions. The LEA protein is essential for drought resistance in plants ,79,80,81Within the QTL region for SP-DS on chromosome 12, LOC_Os12g29330 (OsNAC139) was identified. OsNAC139 is a member of the NAC transcription factor family that is known to control plant response to drought ,159,160 Among all the candidate genes identified within the QTL regions, various transcriptomes correlated with drought stress resistance were detected. Drought resistance of the rice plants can be either associated with metabolic regulation or osmoregulation. This study has revealed a number of potential candidate genes for developing drought-resistant rice varieties. Information about the differences in the expression of drought-resistance genes between drought-resistant and sensitive genotypes and their relationship to morphological characteristics, grain yield components, and root architectural traits under DS conditions has been limited. However, the study of the differences in gene expression between resistance and sensitive genotypes could improve the efficiency and opportunities of developing drought-resistant varieties.A large number of genes were up-regulated in Kaybonnet (drought-resistant parent), indicating that the drought-resistant cultivar had a higher capability to modulate drought-resistant genes when exposed to DS conditions, thereby enhancing its resistance level compared to drought-sensitive parent (ZHE733). Modulation of a higher number of up-regulated expressed genes with different transcription factor gene families is a crucial characteristic of drought-resistant genotypes. Similar results were also obtained by Hayano-Kanashiro et al.  who showCandidate genes within QTL regions involved in regulatory response to drought include a large family of genes expressed under DS conditions. Proteins expressed by known and candidate drought-resistance genes played important roles in (1) cellular protection, including structural adaptation and osmotic adjustment, and (2) drought responses by interaction with other proteins and transcription factors, such as MYB, NAP, NAC, bZIP, and APETALA2/ERF. Under DS conditions, in the drought-resistant genotype Kaybonnet, exogenous ABA significantly improved the expression of ABA biosynthetic genes suggesting the Kaybonnet genotype must be maintaining the water potential and cellular activity of the cell by closing the stomata.Based on the RT-qPCR results, it may also be suggested that there is a correlation between gene expression, transcriptional regulation, and resistance to drought across resistance and sensitive genotypes. Therefore, the up-regulation of the drought genes and novel candidate genes in Kaybonnet compared to ZHE733 provided important information to characterize the function of candidate drought-resistance genes. All in all, these results enhance our understanding of the role of candidate drought-resistance genes in the regulation of drought stress response, and this research has also revealed a number of potential candidate drought-resistance genes that could be used to develop rice cultivars with greater drought resistance. Additional study is needed to alter the gene sequences of locally grown rice cultivars to improve their drought tolerance when exposed to abiotic stress.Oryza sativa, an upland japonica type) and ZHE733 , K/Z RILs  after selfing for F10 generations. An extensive study was performed for various morphological traits, such as grain yield components under well-watered (WW) and drought stress (DS) conditions, and also the response of ABA on root architectural traits.A total of 198 RILs from an F2 population was generated by single seed descent (SSD) from the cross between varieties Kaybonnet  were germinated, grown in the greenhouse, and uniform plants were transplanted to the field separately in 6 batches . This RIL population was grown in the field in Fayetteville, Arkansas, during the growth seasons (May to November) in 2016, 2017, and 2018, with an average yearly rainfall of 849.63 mm. A randomized full-block design was implemented to grow the population, with five replications and two treatments—well-watered (WW) and drought stress (DS) conditions. The DS treatment was imposed at reproductive stage (R3). Using a tensiometer, the DS conditions were maintained continuously below −70 kPa (severe stress), and the WW plot was continuously watered.A number of morphological traits and grain yield indicators, including heading day (HD), plant height (PH), productive tiller number (TN), flag leaf width (FLW), leaf rolling score (LR), spikelet per panicle number (SP), panicle length (PL), primary panicle branch number (PPB), filled grain per panicle number (FG), hundred grain weight (HGW), and total plant biomass (TPB) with five replications per line, were measured to determine the impact of drought stress.Heading date was recorded as the period from the germinating date to the time when 50% of the panicles have exerted. Plant height was measured from the ground surface to the tallest panicle tip before harvesting with a ruler. The total of the productive tiller number per plant was recorded after 10 days of DS treatment. Productive tiller number means number of tillers bearing panicle at the reproductive stage. Flag leaf width was measured in the widest area of the leaf on the tallest culm of each plant by using a ruler after 10 days of the DS treatment. The leaf rolling score on the first five leaves on the tallest tiller of each plant was identified after 10 days of DS treatment based on the standard evaluation system for rice . The ranThe spikelet per panicle number was counted manually for each panicle. Panicle length, the length per panicle, was measured from the panicle neck to the panicle tip. Primary panicle branch number was determined by counting the branches that directly come out from the peduncle. Manual counting was used to determine filled grain per panicle number. The hundred grain weight was calculated based on the weight of one hundred filled grains of each plant with 14% grain moisture content. Total plant biomass was determined using the total above-ground parts of the rice plant  at the maturity stage, which were oven-dried at 80 °C for 72 h and then weighed.Using JMP version 12.0, the data for morphological features and grain yield components under WW and DS conditions were evaluated using an analysis of variance and the Tukey’s HSD was used to compare the means of the two treatments (WW and DS) . Using the Shapiro–Wilk test, a normal distribution for each trait was found by using SAS 9.4.2/s, relative humidity: 60%) for uniform seedling growth. The Kaybonnet, ZHE733, and 198 lines’ seedlings at the S3 stage were transplanted into ABA media at various concentrations—0 M (control), 3 M, and 5 M—and then cultivated in a growth chamber until the V3 stage. The root architectural traits of maximum root length (RL), root–shoot ratio (RSR), total root number (TRN), number of roots with a shallow angle (0–45°) (SRN), number of roots with a deep angle (45–90°) (DRN), and root fresh weight (RFW) were measured with five replications per line/treatment in order to quantify the effect of ABA sensitivity.The seeds from 198 recombinant inbred lines and a total of 2 parental genotypes were sterilized using 70% ethanol for 60 s and 30% bleach solution for 45 min, followed by four rinses with sterilized water. To ensure consistent seedling growth, sterilized seeds (S0 stage) were germinated in 2 mL tubes containing germination media  until S3 stage in a growth chamber ) was used to measure the metaxylem, aerenchyma, and cortical cell properties of roots [The root anatomical phenes of the genotypes (Kaybonnet and ZHE733), 18 drought-resistant lines, and 18 drought-sensitive lines were measured with five biological replications using root samples from the V3 growth stage. A section of approximately 10 cm from the midsection of the primary root was used for fixing. For fixation, root segments were immersed in an FAA  solution for at least 24 h at 4 °C, followed by dehydration in a series of ethanol concentrations of 70%, 80%, 85%, 95%, and 100% for one hour each. The dehydrated samples were then treated twice for one hour with toluene and then embedded in paraffin. The embedded samples were sectioned 80–250 m with a Microtome . The root sections were mounted on microscope slides and dried overnight at 37 °C in a forced air dryer. The root sections were stained for 3 min with 0.05% toluidine blue, de-stained with distilled water, air dried for 2 min, and then embedded in xylene. Images of root sections were captured using a digital camera (Nikon Optihot 2 and Nikon D1) mounted on a microscope with a 40× magnification. RootScan2 software (of roots .Pstl and Mspl were used to generate a single-end library. One million base pairs (bp) of reads were produced by NextSeq for each sample. The average quality ratings of the selected reads were higher than Q30.Genomic DNA was extracted from two-week-old seedlings of the 198 selected lines and parents (Kaybonnet and ZHE733) using the cetyl tri-methyl ammonium bromide (CTAB) approach. The GBS libraries and analysis were handled by the Genomics Center at the University of Minnesota. The enzymes Oryza sativa spp. Japonica version MSU7. Aligned and mismatched nucleotides were used for SNP calling. All samples were concurrently collectively called for variations using Freebayes. All samples were collectively called variations using Freebayes. Freebayes’ raw variant call format (VCF) file was filtered using VCF tools to remove variants with minor allele frequency of less than 1%, variants with genotype rates less than 95%, samples with genotype rates less than 50%, variants with 100% missing data, variants with monomorphic markers between parents, and variants with more than 50% heterozygosity. The filtered data file containing the filtered SNPs in nucleotide-based hap map format was changed to an ABH-based format, where “A” represents donor allele, “B” represents recipient allele, and “H” represents heterozygous allele.Illumina BCL2FASTQ created de-multiplexed FASTQ sequencing readings to identify SNPs. FASTQ files with more than 2,000,000 readings were subsampled. Trimmomatic deleted adapter sequences at the 3′ ends of reads by removing the first 12 bases from each read. Burrows–Wheeler Alignment (BWA) linked the FASTQ data to the reference genome of Nipponbare, The QTL IciMapping software version 4.2.53  linkage Based on the position of the SNP markers bordering the QTL areas and the nearest predicted/annotated gene in the region, the MSU rice japonica reference genome annotation release 7.0 was used to identify potential genes. To identify the key functional genes regulating drought-related traits and ABA sensitivity, all genes within the sliding window of 25 Kb upstream and downstream of the identified QTLs were classified into the following three major functional categories: biological process, molecular function, and cellular component. RNA from the parental genotypes was extracted and used to analyze gene expression under DS conditions.® Plant Mini Kit  with a 260/280 ratio of 1.8–2.1, or 260/230 ratio 2.0, was used to extract the RNA. The GoScript® reverse transcription system  was used to reverse-transcribe a one microgram sample of RNA. The 20 L total volume of the RT-qPCR reaction samples was made up of 10 L of SYBR green master mix, 1 L of cDNA template, 8 L of ddH2O, and 1 L of each primer. A BIO-RAD CFX-96 device  was used to conduct RT-qPCR. The threshold cycle (Ct) value for each gene was normalized against the Ct value of ubiquitin and calculated relative to the relevant control samples as a calibrator using the equation 2-Ct. Each expression value was calculated as the average of three biological replicates and three technical replicates for each sample [The two parental genotypes’ leaves were collected for RNA extraction and gene expression measurement . The RNeh sample ,178,179.The raw FASTQ reads from 200 genotypes comprising 2 parental genotypes and 198 lines of the K/Z RIL population were mapped to the reference rice genome cv. Nipponbare (IRGSP 1.0) using the Burrows–Wheeler Aligner (BWA) . The bamAll morphological variables, components of grain yield, and root architecture traits revealed normal frequency distributions in the RIL population, indicating quantitative inheritance. A total of 11 lines from K/Z RIL population were identified as highly drought resistance based on the morphological traits and grain yield components under WW and DS conditions with root architectural traits under ABA conditions, including 100005, 100032, 100034, 100133, 100135, 100162, 100169, 100242, 100295, 100334, and 100337. Meanwhile, 10 highly drought sensitive were found, such as 100092, 100164, 100170, 100222, 100237, 100239, 100259, 100263, 100324, and 100345. In addition, ABA-mediated cell signaling aids drought-tolerant rice plants maintain grain yields under DS conditions by fostering cell elongation, preserving cellular membrane integrity, and regulating osmotic stress tolerance. All of these traits show a positive correlation with FG-DS, and this is also true for the major root architectural traits. Using QTL IciMapping, we analyzed QTL with a total of 4133 SNPs as markers. For traits affected by drought, a total of 41 QTLs and 184 candidate genes were found inside the QTL areas. The results of the RT-qPCR analysis showed that a large number of genes were up-regulated in the drought-resistant parent, Kaybonnet. Of these genes, 15 were candidates for drought resistance within QTL regions with known annotations, showing higher intrinsic values in Kaybonnet. Important for the development of drought-resistant rice varieties is a thorough understanding of the control of gene expression in response to drought stress, which can be gained by identifying candidate genes within the QTL areas.


Cuproptosis is a novel type of copper-induced cell death that primarily occurs in cells that utilize oxidative phosphorylation as the main metabolic pathway to produce energy. Copper directly associates with the lipoylated proteins of the tricarboxylic acid cycle, leading to the disulfide-bond-dependent aggregation of these lipoylated proteins, destabilization of the iron-sulfur cluster proteins, and consequent proteotoxic stress. Cancer cells prefer glycolysis (Warburg effect) to oxidative phosphorylation for producing intermediate metabolites and energy, thereby achieving resistance to cuproptosis. Interestingly, the tumor suppressor p53 is a crucial metabolic regulator that inhibits glycolysis and drives a metabolic switch towards oxidative phosphorylation in cancer cells. Additionally, p53 regulates the biogenesis of iron-sulfur clusters and the copper chelator glutathione, which are two critical components of the cuproptotic pathway, suggesting that this tumor suppressor might play a role in cuproptosis. Furthermore, the possible roles of mutant p53 in regulating cuproptosis are discussed. In this essay, we review the recent progress in the understanding of the mechanism underlying cuproptosis, revisit the roles of p53 in metabolic regulation and iron-sulfur cluster and glutathione biosynthesis, and propose several potential mechanisms for wild-type and mutant p53-mediated cuproptosis regulation. Copper is an essential cofactor for enzymes and it is required for biological processes—including oxidative phosphorylation, reactive oxygen species (ROS) detoxification, iron homeostasis, connective tissue cross-linking, and signal transmission—in almost all organisms , 2. CoppTsvetkov et al.  investigIn addition, it was found that cells addicted to mitochondrial metabolism are more sensitive to copper ionophores than those using glycolysis as the main source of energy . ConsistFDX1 encodes a reductase that converts Cu2+ to Cu+ which is a more cytotoxic form of copper; LIPT1, LIAS, and DLD encode the components of the lipoic acid pathway; and DLAT, PDHA1, and PDHB encode the lipoylated components of the pyruvate dehydrogenase (PDH) complex, suggesting that the lipoic acid pathway and protein lipoylation may be important for cuproptosis. Interestingly, copper was found to directly bind to lipoylated TCA cycle proteins, leading to disulfide-bond-dependent aggregation of these proteins and degradation of iron-sulfur (Fe-S) cluster proteins. More importantly, FDX1 was considered a central regulator of cuproptosis, because FDX1 depletion led to complete loss of protein lipoylation, marked decrease in cellular respiration, accumulation of pyruvate and α-ketoglutarate, reduction of succinate, and stabilization of Fe-S cluster proteins. Altogether, excessive copper promotes aggregation of lipoylated TCA cycle proteins and destabilization of Fe-S cluster proteins, both of which are mediated by FDX1, consequently resulting in cuproptosis.To elucidate the mechanistic basis underlying cuproptosis, genome-wide CRISPR-Cas9 screening was performed . Seven g2-induced aggregation of lipoylated proteins and degradation of Fe-S cluster proteins. Consistently, SLC31A1-sensitized, copper-induced cell death was partially rescued by copper chelators and depletion of FDX1 or LIAS. Remarkably, these in vitro findings were further validated in a Wilson’s disease mouse model with Atp7b depletion (Atp7b−/−). Compared with the livers of Atp7b+/− and wild-type mice, those of Atp7b−/− mice showed significant loss of lipoylated and Fe-S cluster proteins, demonstrating that accumulation of intracellular copper leads to cuproptosis in vivo.Finally, the authors investigated the effects of intracellular copper fluctuation on cuproptosis by experimentally manipulating the genes encoding the copper importer SLC31A1 and exporter ATP7B in vitro and in vivo [Collectively, the study reported by Tsvetkov et al.  providesThe tumor suppressor p53 inhibits cancer progression mainly by promoting different types of RCD, such as apoptosis, ferroptosis, parthanatos, programmed necrosis, and autophagic cell death, in response to various cellular stresses –10. The PFKFB3/4 and TIGAR [Cancer cells tend to utilize glycolysis, but not the TCA cycle, to produce intermediate metabolites and energy, even under oxygen-rich conditions, which is known as aerobic glycolysis or the Warburg effect. Inhibition of glucose uptake or glycolysis may induce a metabolic shift from glycolytic metabolism to mitochondrial metabolism by using alternate energy sources, such as galactose or glutamine, which sensitizes cells to cuproptosis , 21. p53nd TIGAR –29, 3-phnd TIGAR , and 2-pnd TIGAR . In the nd TIGAR  or promond TIGAR . This mand TIGAR . AMPK acnd TIGAR , thus fond TIGAR , 37. ThuAIF, which encodes a mitochondrial membrane protein, is required for efficient oxidative phosphorylation as it is involved in the proper assembly of the mitochondrial respiratory complex I [p53 activity has been found to maintain mitochondrial integrity and performance, as mutation or deficiency of p53 leads to a significant reduction in mitochondrial content, cytochrome c oxidase (COX) activity, and respiratory metabolism –40, whicomplex I . Interesomplex I  Fig. 1)AIF, whicomplex I . Under tFDXR, which encodes a ferredoxin reductase responsible for electron transport from NADPH to FDX1/2 and then to cytochrome P450 for Fe-S cluster biogenesis [FDXR was originally identified as a p53-target gene required for p53-induced oxidative stress and apoptosis by two independent groups [Fe-S clusters are ubiquitous proteins that act as cofactors involved in a wide range of biological processes, including enzymatic catalysis, electron transport, and metabolic stress sensing . Tsvetkoogenesis . FDXR wat groups , 59. It t groups . In addit groups  and FXN t groups , 63, bott groups . Inhibitt groups . These fTIGAR [GLS2 [SESN1/2 [CDKN1A, also known as p21, triggers the NRF2 antioxidant pathway, resulting in increased GSH and NADPH synthesis [GSH not only acts as a central antioxidant mediator but also as a copper chelator, as GSH deficiency results in increased labile copper levels . ConsistTIGAR , GLS2 [4AR [GLS2 , 48, and[SESN1/2 . Additioynthesis . These synthesis , 21. ForTP53 is the most frequently mutated gene in human cancers. The majority of cancer-associated p53 mutations are missense mutations that often occur in the DNA-binding domain. Many of these mutant p53 (mtp53) proteins acquire “gain-of-function” (GOF) activities to promote cancer development [elopment , 72 indeelopment , 20. Forelopment  and promelopment  and PLA2elopment . Mtp53 aelopment , 77. In elopment , or downelopment . It is aelopment . Moreoveelopment , 82, traelopment , and supelopment , 84, 85.Cuproptosis is a novel form of copper-induced cell death. Cells that utilize glycolysis as the main metabolic pathway to produce energy are resistant to cuproptosis, whereas those that are dependent on the TCA cycle and oxidative phosphorylation are sensitive to cuproptosis. Excessive labile copper can directly associate with lipoylated TCA cycle proteins, leading to aggregation of these lipoylated proteins, destabilization of Fe-S cluster proteins, and consequent cell death. p53 acts as an important metabolic regulator that mediates the transition from glycolysis to oxidative phosphorylation, enhances Fe-S cluster biogenesis and orchestrates the level of the copper chelator GSH. The p53- or mtp53-regulated genes, which may be associated with cuproptosis as discussed above, are illustrated in Tables reproducibility checklist


BIRC5/HIF1A/FLT4 pro-angiogenic genes. Furthermore, we used in silico molecular docking analysis to determine whether HN-N07 could potentially inhibit BIRC5/HIF1A/FLT4. Interestingly, the results of docking HN-N07 with the BIRC5, FLT4, and HIF1A oncogenes revealed unique binding affinities, which were significantly higher than those of standard inhibitors. In summary, these results indicate that HN-N07 shows promise as a potential inhibitor of oncogenic signaling pathways in NSCLC. Ongoing studies that involve in vitro experiments and in vivo investigations using tumor-bearing mice are in progress, aiming to evaluate the therapeutic effectiveness of the HN-N07 small molecule.Tumor angiogenesis and lymphangiogenesis pathways have been identified as important therapeutic targets in non-small cell lung cancer (NSCLC). Bevacizumab, which is a monoclonal antibody, was the initial inhibitor of angiogenesis and lymphangiogenesis that received approval for use in the treatment of advanced non-small cell lung cancer (NSCLC) in combination with chemotherapy. Despite its usage, patients may still develop resistance to the treatment, which can be attributed to various histological subtypes and the initiation of treatment at advanced stages of cancer. Due to their better specificity, selectivity, and safety compared to chemotherapy, small molecules have been approved for treating advanced NSCLC. Based on the development of multiple small-molecule antiangiogenic drugs either in house and abroad or in other laboratories to treat NSCLC, we used a quinoline-derived small molecule—HN-N07—as a potential target drug for NSCLC. Accordingly, we used computational simulation tools and evaluated the drug-likeness properties of HN-N07. Moreover, we identified target genes, resulting in the discovery of the target  BIRC5 overexpression is associated with mitosis, proliferation, migration, and immune infiltration in different cancers; however, its clinical impacts and associations with the tumor microenvironment (TME) are still not well understood [Globally, non-small cell lung cancer (NSCLC) is responsible for a significant number of cancer-related deaths and is the second most prevalent cause of cancer-related morbidity. It accounts for approximately 85% of newly diagnosed cases each year  and has derstood ,16,17,18BIRC5 is reported to be an immune-associated gene that promotes tumor progression, many studies related to survivin in NSCLC focus on the sensitization to chemotherapy, radiotherapy, and targeted therapy, with little success achieved due the heterogeneity of this disease [BIRC5 overexpression is associated with angiogenesis and inhibition of cell apoptosis in lung cancer, thus making it a potential anti-NSCLC therapeutic biomarker [VEGF) and secreting it to nearby tissues [VEGFs, including VEGF  and placental growth factor. These factors activate their specific receptors, including VEGF receptor-1, receptor-2, and receptor-3, as well as co-receptors, and play distinct roles in vascular development [Since  disease ,20. Numeiomarker ,22,23,24iomarker , and, heiomarker . The groiomarker . In addi tissues ,29,30. Telopment ,31. Prevelopment ,33.VEGF-C) and its receptor VEGFR-3, which is also known as Fms-like tyrosine kinase 4 (FLT4), are expressed in tumor cells and associated with growth of blood vessels within tumors, as well as the progression of cancer cells to other parts of the body and the overall prognosis of the disease. FLT4 is mainly expressed in the lymphatic endothelium and plays a crucial role in lymphangiogenesis and metastasis in malignant tumors [FLT4 expression is developmentally regulated and mainly restricted to lymphatic endothelial tissues [FLT4 expression was identified in various cancers; however, its involvement in NSCLC remains elusive, hence it is necessary to investigate its molecular mechanism in this disease [VEGFR/FLT4 pathway may offer a promising strategy for preventing tumor lymphangiogenesis and metastasis in NSCLC [HIF)-1α, which is a transcription factor, may have a significant impact on tumor growth and metastasis through its regulation of angiogenesis and lymphangiogenesis, which enables cellular survival in hypoxic conditions [HIF-1α in the NSCLC TME, leading to VEGF expression [BIRC5/HIF1α/FLT4, thus driving lymphangiogenesis and metastasis in NSCLC.Specifically, vascular endothelial growth factor C . Accordingly, the database contained a total of 56,938 samples, which consisted of both RNA-seq and gene array samples. After undergoing pre-processing, there were 34,350 distinct gene array samples available, which covered 40 different tissue types. Among these samples, there were 3781 normal samples, 30,276 tumorous samples, and 462 metastatic samples. Data analyzed from selected oncogenes were based on rapid RNA-Sequencing (RNA-Seq) profiling, and the Kruskal–Wallis test was used to compare results [http://gepia2.cancer-pku.cn/, 28 November 2022) to analyze correlations between BIRC5/HIF1A/FLT4 oncogenic signatures in NSCLC. A statistically significant association was determined based on positive Pearson’s correlation coefficients and a p-value of less than 0.05.Expression profiles of BIRC5/HIF1A/FLT4 in NSCLC were analyzed using GEPIA2 online software, with the major stage option used for plotting, and all data were selected from lung adenocarcinoma (LUAD) datasets, with log2 and transcripts per million +1 (TPM+1) used for the log-scale. Furthermore, we explored The Cancer Genome Atlas (TCGA) database sourced from the UALCAN online platform  to analyze BIRC5/HIF1A/FLT4 expression in LUAD based on histological subtypes from TCGA samples, and these included LUAD-not otherwise specified (NOS), lung clear cell adenocarcinoma (clear cell), predominant adenocarcinoma with a solid pattern in the lungs, mucinous lung bronchoalveolar carcinoma (LBC-mucinous), lung papillary adenocarcinoma (papillary), lung micro-papillary adenocarcinoma (micro-papillary), lung adenocarcinoma with mixed subtypes (mixed), non-mucinous lung bronchoalveolar carcinoma (LBC), lung acinar adenocarcinoma (acinar), mucinous (colloid), and lung signet ring adenocarcinoma (signet ring). In addition, we determined overall survival (OS) in relation to expression of BIRC5/HIF1A/FLT4 oncogenes using GEPIA2 software, with a significance level of p < 0.05 considered to be statistically significant.Pathological stage plots of https://string-db.org/ 21 December 2022) [https://david.ncifcrf.gov/.jsp, 22 December 2022), Funrich, and Network Analyst software to construct the graphs. The minimum level of significance was set at p < 0.05.Protein interactions were constructed using STRING (er 2022) . For furBIRC5/HIF1A/FLT4 oncogenes and their altered expression in LUAD were analyzed using the online mutation target (muTarget) bioinformatics tool  [p < 0.05, helping us to determine the differences in expression levels between the mutant and wild-type (WT) groups. Moreover, we utilized the oncoprint webtool, which is embedded in cBioPortal software , to further analyze genetic alterations and copy number variations (CNVs) in the BIRC5/HIF1A/FLT4 genes in LUAD, which were based on mutation spectra and alteration frequencies.Associations between genetic mutations of the ry 2023) . Herein,scRNA-Seq is widely used to study communication between cells and their TME, and Single-cell RNA sequencing (scRNA-Seq) offered a comprehensive approach that we used to gain deeper insights into the diverse populations within the tumor microenvironment (TME). It enabled the identification of novel cell types and the exploration of previously unknown associations within the TME ,66.Herein, we explored the Tumor Immune Single-cell Hub (TISCH), which is a powerful web-tool designed to comprehensively dissect the intricate components of the tumor microenvironment (TME) at a single-cell resolution. It provided a comprehensive and user-friendly platform through which to analyze and explore the complex characteristics of the TME at the cellular level .http://timer.cistrome.org/1, 4 January 2023) to examine the associations between the expression levels of BIRC5, HIF1A, and FLT4 and the extent of tumor infiltration. This tool allowed us to analyze and assess the correlations between these examples of gene expression and the immune cell infiltration within the tumor microenvironment [BIRC5/HIF1A/FLT4 and infiltration of cancer-associated fibroblasts (CAFs) in LUAD. In addition, we analyzed mutations of BIRC5/HIF1A/FLT4 in CAFs using the mutation module from TCGA clinical outcomes in the TIMER 2.0 algorithm. For further analysis, we determined distributions of BIRC5/HIF1A/FLT4 expression in LUAD across different molecular subtypes, including wound healing, interferon (IFN)-γ dominant, inflammation, lymphocyte, and transforming growth factor (TGF)-β subtypes using TISIDB  [We utilized the Tumor Immune Estimation Resource  .https://cibersortx.stanford.edu/, 25 February 2023) that allowed us to extract a signature from single-cell RNA-Seq data and quantify cell fractions sourced from differential bulk tumor gene expression [To profile the infiltration of bulk RNA-Seq expression tumors, we utilized CIBERSORTx, which is a versatile online analytical tool , which applies similar prediction principles to predict drug targets based on a “probability” target score [To determine the physicochemical properties, drug likeness, pharmacokinetics (PKs), physicochemical properties, and medicinal chemistry of HN-N07, we used SwissADME software, and to create target predictions, we used DTP-COMPARE algorithms . These tet score .BIRC5/HIF1A/FLT4, as previously predicted from DTP-COMPARE and Swisstarget software, we performed in silico molecular docking analysis of HN-N07 using the BIRC5, FLT4, and HIF1A genes. To enable further analysis, we used standard inhibitors of BIRC5 , FLT4 , and HIF1A , which were retrieved from PubChem as SDF files. The obtained SDF files were converted to PDB format using PyMol software. We obtained the crystal structures of BIRC5 (1xox), FLT4 (4bsk), and HIF1A (1l3e) from the Protein Data Bank in PDB format. The PDB files were then converted to PDBQT file format using autodock software [A docking analysis was conducted by examining interactions of three-dimensional (3D) structures of receptors and ligands at the lowest possible binding energy . Accordisoftware , which esoftware .p-value as p < 0.05 and ∣log 2FC∣ > 1.5 as the statistical standards. To perform the analysis, from GSE2088, we sourced 48 tumor samples and 30 normal samples; from GSE6044, we sourced 9 tumor samples and 5 normal samples; from GSE19188, we sourced 91 tumor samples and 65 normal samples; and from GSE68465, we sourced 89 tumor samples and 67 normal samples. The expression data, which included upregulated and downregulated genes, were presented in volcano plots  were analyzed using muTarget software. The top two highly expressed genes linked to rognoses A–F. Morerognoses G. We furr graphs H,I, withBIRC5, HIF1A, and FLT4 were overexpressed in malignant tissues [BIRC5 and HIF1A, occurred in epithelial cells, CD8 T cells, T proliferative cells, mono/macro cells, basal cells, fibroblasts, and malignant cells ; accordingly, we found abundances of major linear cell types within the NSCLC TME, and these cell types included malignant cells, fibroblasts, epithelial cells, plasma, CD8 T cells, T proliferation cells, endothelial cells, basal cells, alveolar cells, and mono cells A. The an tissues . By condnt cells B,C, whilal cells D.BIRC5, HIF1A, and TLF4 and infiltrating immune cells. In order to understand the connections between BIRC5, HIF1A, and FLT4 expression and specific immune cells, we conducted a correlation analysis between these oncogenes and markers of cancer-associated fibroblasts (CAFs), considering the influence of sample purity. As expected, results showed correlations between BIRC5/HIF1A/FLT4 and CAFs in NSCLC  to explore how our target gene correlates with the TME in NSCLC. Accordingly, we used a TIMER database analysis to determine correlations between in NSCLC A–C. In ain NSCLC D–F. Morein NSCLC G–I.+), epithelial/cancer (EpCAM), and stromal (CD10+ and CD34+) subsets. Genes that were either not predicted to be expressed or were eliminated through adaptive noise filtration were indicated by items that were navy blue in color. To profile bulk RNA-Seq expression of tumor-infiltrating cells, we used CIBERSORTx, which is an online and versatile analytical tool that allows a signature to be extracted from single-cell RNA-Seq data, as well as quantification of cell fractions from differential gene expression of bulk tumors. For each sample size, a subset of tumors was randomly selected from a larger cohort (n = 302) in 10 iterations. The results were displayed with and without adaptive noise filtration. The data were presented using boxplots, where the center line represents the median, the box limits indicate the upper and lower quartiles, the whiskers extend to 1.5 times the interquartile range, and any points beyond the whiskers represent outliers A. HeatmaPharmacophore hybridization and the exploration of different bioactive compound scaffolds are valuable strategies in the design and development of new drugs . In the BIRC5, FLT4, HIF1A, dopamine receptor D2 (DRD2), mammalian target of rapamycin (mTOR), dipeptidyl peptidase-4 inhibitor (DPP-4), proto-oncogene B-Raf (BRAF), and others, as shown in We used the Swisstarget prediction tool to investigate druggable target genes for HN-N07. Interestingly, we identified several targets which included kinases, family A–G protein-coupled receptors, proteases, and transcription factors. The HN-N07 small molecule was also shown to target oncogenes, including BIRC5, FLT4, and HIFA oncogenes. Accordingly, after docking our compound with these individual genes, we obtained the following the Gibbs free binding energy (ΔG) results: BIRC5 ; FLT4 ; and HIF1A  (BIRC5 (flavokawain A), FLT4 (sorafenib), and HIF1A (belzutifan), and compared those results to our HN-N07 small molecule. Interestingly, our compound had higher binding affinities than flavokawain A  and belzutifan . However, sorafenib exhibited a much higher binding energy of −7.9 kcal/mol  A,B. The kcal/mol C,D, whicin NSCLC .Despite the advanced optional treatment modalities for NSCLC, including surgery, radiation therapy, and chemotherapy, which depend on various factors, such as cancer types and histological subtypes, that make treatments less effective, OS is still approximately <5 years ,80,81,82BIRC5, which are also known as survivin, is an immune-associated gene, which has also been shown to regulate metastasis and angiogenesis in tumors and is highly expressed in NSCLC [BIRC5 and survivin was shown in NSCLC, thus illustrating its great potential as a therapeutic target for treatment development [Angiogenic pathways are essential targets in the molecular regulation of the NSCLC tumor microenvironment (TME), influencing tumor progression and metastasis . Vasculain NSCLC . The co-elopment . Moreoveelopment . Additioelopment ,91,92,93elopment . Additioelopment ,95.BIRC5, FLT, and HIF1A were more clearly associated with primary NSCLC tumor progression and metastasis than normal samples. These findings are in line with the study conducted by Aldo et al. in 2019. Interestingly, based on results of the correlation analysis, all of the oncogenic signatures were also coexpressed in NSCLC. Since current treatment is influenced by factors such as cancer types and histological subtypes, we determined the prognostic relevance of BIRC5/FLT4/HIF1A at different stages of LUAD, and we found that BIRC5, FLT4, and HIF1A were higher in stages 2, 3, and 4 than in stage 1, where they were significantly lower. This result suggests that BIRC5/HIF1A/FLT4 signaling may promote tumor progression in NSCLC. The expression of this signature in LUAD based on histological subtypes from TCGA samples exhibited a high presence of a solid pattern-predominant adenocarcinoma, which is an aggressive large tumor associated with poor clinical outcomes.In the present study, we have analyzed a sample of patients affected by metastatic disease and compared them to normal samples using bioinformatics analysis of non-small cell lung cancer patients. We used the TNM plot tool, and samples were obtained using RNA-Seq data. Our results showed that high expression levels of BIRC5/HIF1A/FLT4 and CAFs in NSCLC. Moreover, we exploited a CIBERSORTx digital flow cytometric analysis of NSCLC RNA-Seq bulk tumors, and we identified that immune (CD45+), epithelial/cancer (EpCAM), and stromal (CD10+ and CD34+) subsets were expressed in NSCLC and associated with poor prognoses. Due to their broad efficacy and safety compared to traditional chemotherapeutic regimens, small molecules, which are kinase inhibitors, have become the most recognized cancer treatments [BIRC5, FLT4, and HIF1A oncogenes revealed unique binding energies. These energies were significantly higher than the standard inhibitors of flavokawain-A and belzutifan. However, sorafenib exhibited a binding energy that was slightly higher than that of HN-N07. Collectively, these findings suggest that HN-N07 may be a potentially inhibitor of the oncogenic signaling pathway in NSCLC.The TME plays crucial roles in cancer initiation and progression. However, the association between the TME and tumor prognosis remains elusive. Herein, we utilized a web-based program with a tumor-infiltrating immune cell algorithm (TIMER 2.0) to explore correlations between our target genes and the TME in NSCLC. In order to determine the associations between BIRC5, HIF1A, and FLT4 expression and specific immune cells, we conducted a correlation analysis that accounted for the purity-adjusted CAF markers. As anticipated, results showed correlations between eatments ,96. HereBIRC5/HIF1A/FLT4 signature as a targetable signature correlated with angiogenic pathways in NSCLC. We used a computational analysis and identified the BIRC5/HIF1A/FLT4 oncogenes as being highly upregulated in NSCLC and associated with cancer progression and poor prognoses. Docking results of HN-N07 with the BIRC5, FLT4, and HIF1A oncogenes revealed unique binding energies of −8.2, −7.7, and −8.2 kcal/mol. These results were significantly higher than those of two standard inhibitors. Collectively, these findings suggest that HN-N07 may be a potential inhibitor of an oncogenic signaling pathway in NSCLC. Currently, further in vitro and in vivo investigations in tumor-bearing mice are in progress to study the potential treatment efficacies of the novel HN-N07 small molecule.In conclusion, we identified the


Acute bacterial sinusitis accounts for 5 million pediatric antibiotic prescriptions in the United States each year, with 65% amoxicillin or amoxicillin-clavulanate. More than 90% of pediatric patients are prescribed 10+ days of antibiotics. No study has compared shorter to longer treatment. This study compares 5-7 days versus 10 days of treatment in children and adolescents.This cohort study included patients < 18 years with an outpatient encounter for acute sinusitis and a same-day dispensation of either a 5–7 day supply or a 10 day supply of amoxicillin or amoxicillin-clavulanate between January 1, 2017 and December 31, 2020 in the MarketScan Commercial Claims and Encounters database. Treatment failure, defined as a new antibiotic dispensation, an ED or inpatient encounter for acute sinusitis, or an inpatient complication of acute sinusitis, was assessed in the first 5, 10, and 30 days after cohort entry. Adverse and control outcomes were captured. We used Cox proportional hazards regression with propensity score (PS) overlap weights for confounding control.The initial cohort included 254,629 patients, among which 19,627 patients were prescribed a 5-7-day treatment (exposure) and 235,002 patients were prescribed a 10-day treatment (referent). After PS overlap weighting, groups were balanced on all measured covariates (Table 1). The rate of 30-day treatment failure was 167.77 per 1,000 person-years  among patients dispensed 5-7 days of treatment and 150.19 per 1,000 person-years  among patients dispensed 10 days of treatment  (Table 2). Adverse events were rare, and there was no difference between groups in a negative control outcome (Table 3).The absolute risk of treatment failure was very low. Children and adolescents dispensed 5-7 days of antibiotic treatment had a slightly higher rate of treatment failure compared to 10 days of antibiotic treatment. Adverse effects were rare, with a potentially lower rate of yeast infections following shorter treatment. The marginal benefit and increased risk of side effects with longer treatment indicates that shorter treatment of acute bacterial sinusitis, 5-7 days, may be a reasonable initial approach in some populations.Timothy J. Savage, MD, MPH, MSc, UCB: Contract to Brigham and Women's Hospital Krista F. Huybrechts, PhD, MS, Takeda: Grant/Research Support|UCB: Grant/Research Support


Acute respiratory distress syndrome (ARDS) is a life-threatening pulmonary condition characterized by the sudden onset of respiratory failure, pulmonary edema, dysfunction of endothelial and epithelial barriers, and the activation of inflammatory cascades. Despite the increasing number of deaths attributed to ARDS, a comprehensive therapeutic approach for managing patients with ARDS remains elusive. To elucidate the pathological mechanisms underlying ARDS, numerous studies have employed various preclinical models, often utilizing lipopolysaccharide as the ARDS inducer. Accumulating evidence emphasizes the pivotal role of reactive oxygen species (ROS) in the pathophysiology of ARDS. Both preclinical and clinical investigations have asserted the potential of antioxidants in ameliorating ARDS. This review focuses on various sources of ROS, including NADPH oxidase, uncoupled endothelial nitric oxide synthase, cytochrome P450, and xanthine oxidase, and provides a comprehensive overview of their roles in ARDS. Additionally, we discuss the potential of using antioxidants as a strategy for treating ARDS. Historically, acute lung injury (ALI) referred to a milder form of lung injury, while ARDS signified a more severe lung condition [Acute respiratory distress syndrome (ARDS) imposes a huge burden in the context of critical care medicine, demanding immediate attention and a comprehensive approach due to its life-threatening nature. Initially described in the 1960s by Petty and Ashbaugh . In respondition . HoweverARDS can be attributed to various conditions, with trauma, aspiration, pneumonia, and sepsis serving as primary triggers for its development ,6,7. AddThe incidence of ARDS varies globally, with data from population-based studies revealing significant diversity ,10. A stUnder normal physiological conditions, lung fluid balance is maintained through fluid clearance. However, dysfunction of the alveolar barrier can impair alveolar fluid clearance, a prominent hallmark of patients with ARDS, leading to hypoxemia and pulmonary bilateral edema ,17. AddiThe acute phase, referred to as the exudative stage, typically spans from hours to the first week after the injury onset. During this stage, a cascading inflammatory response is activated, leading to an influx of neutrophils , a phenoTo gain a deeper understanding of the intricate pathophysiology of ARDS and to develop novel therapeutic strategies, researchers have extensively utilized controlled experimental models to mimic clinical syndromes. These models serve as invaluable tools for exploring the mechanisms underlying ARDS and evaluating potential interventions. The workshop report published in 2011 by the American Thoracic Society (ATS) proposed the following four features of ARDS for preclinical models: histological evidence of tissue injury, alterations in the alveolar-capillary barrier, inflammatory response, and physiological dysfunction . HistoloARDS models have been generated through both direct and indirect methods to recapitulate human ARDS. The direct approach involves the stimulation of conditions such as pneumonia and acid aspiration, while the indirect approach involves the stimulation of pancreatitis and non-pulmonary sepsis. In direct models, ARDS is induced through the intranasal or intratracheal administration of agents such as lipopolysaccharides (LPS), HCl, bleomycin, bacteria, and viruses. Hyperoxia is also a direct cause of ARDS. In contrast, indirect lung injury can be induced via the intravenous injection of LPS, cecal ligation and puncture (CLP) to induce sepsis, hemorrhagic shock, mesenteric ischemia, and reperfusion.2/FiO2 ratio, increased total protein concentration in the BALF, infiltration of inflammatory cells, disruption of alveolar barrier function, and excessive generation of ROS and oxidative stress [2 is not sustained for an extended period [2 is sustained for more than 72 h and induces more pronounced hyaline membrane formation and edema. Additionally, several studies have reported that the intraperitoneal injection of LPS can lead to lung injury [The most frequently utilized ARDS model involves the instillation of LPS via either the intratracheal or intranasal routes. Research has also explored variations based on species, age, sex, and LPS concentrations. In the LPS-induced ARDS model, several key observations include a reduced PaOe stress ,26,27. Te stress . Howeverd period . These ld period . This twg injury ,31.In the clinical setting, the aspiration of gastric contents is a major contributor to ARDS. Given the role of pH in lung injury, hydrochloric acid (HCl) has been employed to mimic human ARDS. The orotracheal and intratracheal instillation of HCl leads to reduced oxygenation, increased respiratory elastance, and pulmonary inflammation . FurtherThe intravenous injection of oleic acid induces ARDS characterized by intra-alveolar edema and an increased infiltration of inflammatory cells ,35. An aCLP is a method commonly utilized to induce sepsis in models . In brieThe primary goal of managing ARDS is to improve blood oxygen levels. Despite administering high levels of inspired oxygen, oxygen saturation often remains inadequately low, underscoring the severity of impaired gas exchange or the failure to respond to conventional respiratory therapy in patients with ARDS. Furthermore, due to the diversity of ARDS causes and the complexity of the syndrome, there is currently no single therapy tailored specifically to ARDS. Additionally, a decline in the quality of life is frequently observed even after recovering from ARDS.Various professional societies, including the Faculty of Intensive Care Medicine and Intensive Care Society , The FreMechanical lung ventilation is a standard therapeutic approach used to enhance oxygenation in patients with ARDS. However, it can also lead to ventilation-induced lung injury (VILI) ,50. VILIVILI is characterized by four primary mechanisms: volutrauma, barotrauma, atelectrauma, and biotrauma . VolutraPatients with ARDS often exhibit reduced lung volume due to edema and inflammation, colloquially referred to as the “baby lung”. Consequently, traditional tidal volume ventilation (10 mL/kg or more) can lead to lung overdistension in patients with ARDS. LTVV is a technique that involves reducing the tidal volume, a strategy commonly used in individuals with healthy lungs to prevent lung tissue overdistension . In 1998PEEP involves maintaining a pressure higher than atmospheric pressure at the end of expiration, which helps prevent atelectrauma and corrects hypoxemia resulting from alveolar hypoventilation . Physiol2O for 20 to 40 s, with continuous monitoring for any adverse effects [LRMs are strategies designed to transiently increase the driving pressure to facilitate the recruitment of collapsed alveoli. This recruitment process improves oxygenation, promotes uniform volume distribution in the lungs, and mitigates the risk of overdistension . Sustain effects ,62. The  effects . However effects . Further2/FiO2 ratio was significantly higher in the prone position group on day 4. A recent meta-analysis also indicated that prone positioning during venovenous extracorporeal membrane oxygenation improved survival [The prone position is commonly employed to facilitate breathing in ventilated patients. It involves turning the patient from the supine (back) position to the prone (face down) position to improve oxygenation. ARDS patients in the supine position are affected by the weight of the heart and abdominal organs, leading to increased pleural pressure in the dorsal lung regions, resulting in lung collapse. In such cases, the leveraging of the prone position can help redistribute blood and airflow more evenly ,66. The survival ,70. Nevesurvival .Corticosteroids are potent anti-inflammatory and immunomodulatory drugs commonly employed to treat various inflammatory diseases, including asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), and pneumonia . The useThe overproduction and accumulation of ROS in ARDS can be attributed to several key factors, including the accumulation of leukocytes, elevated levels of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) and xanthine oxidase (XO), and the use of high-concentration oxygen during mechanical ventilation. A defining characteristic of ARDS is the excessive infiltration of neutrophils and macrophages into the lung tissue ,77. LeukLPS treatment increases the uncoupling of eNOS and mitochondrial ROS production, resulting in the activation of NLR family pyrin domain containing 3 protein (NLRP3) inflammasome in human lung microvascular endothelial cells . The inh2, generating ROS, including O2·− and H2O2 [2·− reacts with NO, contributing to the formation of reactive nitrogen species such as peroxynitrite [Xanthine oxidoreductase (XOR) directly transfers electrons to Oand H2O2 . Notablyynitrite . Grum etynitrite  showed tynitrite . Febuxosynitrite . The intynitrite . In addiynitrite  showed tynitrite .2 > 65%) can lead to an increase in ROS, inducing oxidative stress, worsening respiratory symptoms, and potentially causing lung injury [Supplementation with oxygen is a potential treatment option for patients with ARDS . Howeverg injury . Consistg injury  showed tg injury . Howeverg injury . In addi2 gains electrons. Under normal conditions, a stable equilibrium exists between ROS production and elimination. ROS include H2O2, •OH, 1O2, and O2•−. O2•− is generated through a single electron transfer to O2 and subsequently dismutates into H2O2, which is either spontaneously generated in the presence of water or catalyzed by SOD [2− and H2O2 are the two most abundant ROS subspecies, differing significantly in chemical properties and, consequently, behavior and function. O2•− is highly reactive and contributes to oxidative stress and cellular damage characterized by protein and amino acid oxidation, DNA damage, and lipid peroxidation. In contrast, H2O2, a relatively stable molecule, serves as a crucial cellular signaling factor. However, excessive production can lead to cytotoxicity.ROS are generated when Od by SOD . NotablyROS are primarily generated during tightly regulated enzymatic processes involving endothelial nitric oxide (NO) synthase, the mitochondrial respiratory chain, various NOX isoforms, XOs, and CYP enzymes ,101. Amo+ or NADP+. They catalyze the reduction of oxygen to produce O2•− and are known to activate other ROS sources, such as endothelial NO synthase (eNOS). Initially, NOX was discovered in phagocytic immune cells; however, it was later found in non-phagocytic cells. Activated NOX generates ROS, leading to endothelial cell dysfunction. The NOX family comprises seven isoforms: NOX1, NOX2, NOX3, NOX4, NOX5, dual oxidase 1 (Duox1), and Duox2. NOX2 and NOX4 play crucial roles in ROS production. In a spinal cord injury model, ROS had a harmful impact, contributing to oxidative stress, which is reversed by the deletion of NOX2 or inhibition of NOX4 to reduce ROS production and oxidative stress markers [NOXs are enzymes that oxidize NADH or NADPH to form NAD markers .phox, p67phox, p40phox, and Rac family small GTPase 2, along with transmembrane proteins p22phox and gp91phox [phox and gp91phox to the phagosomal membrane and induces association with the trimeric complex composed of p40phox, p47phox, and p67phox [phox and p67phox, a structural alteration and stabilization mediated by GTP-Rac2, p22phox, and gp91phox take place, inducing the overproduction of O2•− and MPO, which exists in the form of a ferric (Fe III) heme enzyme in lysosomes of phagocytic cells and chlorinates H2O2 to highly reactive hypochlorous acid (HOCl−) [2O2, •OH, and HOCl− [NOX2, in particular, comprises four cytoplasmic components, including p47gp91phox ,104. Upogp91phox . Simulta p67phox . Through (HOCl−) ,106. Thind HOCl− .l-arginine into NO. However, under conditions of oxidative stress, it shifts its production toward O2•− instead of NO; this phenomenon is known as eNOS uncoupling. eNOS uncoupling can occur under several conditions, including cofactor tetrahydrobiopterin (BH4) deficiency, a shortage of the eNOS substrate l-arginine, the accumulation of the NOS inhibitor asymmetric dimethylarginine (ADMA), and increased eNOS S-glutathionylation [2 and NO can oxidize BH4. As a result, BH4 depletion destabilizes NOS and induces its uncoupling, which, in turn, affects eNOS activity. Under conditions of reduced l-arginine availability, eNOS becomes less effective at converting it into NO, and this shift in activity can lead to eNOS uncoupling. ADMA is an endogenous inhibitor of eNOS. When ADMA levels are elevated, it competes with l-arginine to bind to eNOS, thereby reducing the availability of l-arginine for NO production.Under normal circumstances, eNOS generates NO. eNOS transfers the electrons donated by NADPH to the amino-terminal oxygenase domain of heme via a series of cofactors located in the carboxy-terminal reductase domain. These cofactors include flavin adenine dinucleotide and flavin mononucleotide, which facilitate the electron transfer process necessary for the enzymatic conversion of nylation . BH4 is CYP enzymes, a diverse group of heme monooxygenases, have been the focus of extensive research because of their central roles in the metabolism of drugs and xenobiotic compounds. Furthermore, they play crucial roles in the synthesis of sterols, fatty acids, eicosanoids, vitamins, and various other biochemical compounds, including ROS. Notably, upon modification by CYP enzymes, certain substrates give rise to reactive intermediates or products that can be implicated in the onset and progression of various diseases.•− and H2O2, while (2) xanthine dehydrogenase is an NAD+-mediated type that utilizes NAD+ as a cofactor and does not generate ROS [2O2 or O2•− is generated [2•− and H2O2 levels in the XO inhibitor allopurinol treatment, preventing an increase in mitochondrial ROS levels in a model of cocaine-induced diastolic dysfunction [XOR plays a pivotal role in the final stages of purine catabolism, facilitating the oxidation of hypoxanthine to xanthine or subsequently xanthine to uric acid . It exisrate ROS . Importaenerated . In one function .In ARDS, ROS can be induced and activated through the activation of various signaling pathways, such as mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), nuclear factor kappa B (NFκB), hypoxia-inducible factor 1 (HIF1), and activator protein-1 (AP-1), as shown in MAPKs are serine-threonine protein kinases involved in various processes, such as cell growth, proliferation, and differentiation. They are categorized into subgroups, including extracellular signal-related kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAPKs ,113. The2O2 or other oxidants, it dissociates from ASK1, leading to the activation of JNK MAPKs [JNK, also known as stress-activated protein kinase, is activated by physical or chemical stresses, cytokines, and ROS via the phosphorylation of upstream components such as MEK4, MEK7, and apoptosis signal-regulated kinase 1 (ASK1) ,116. ASKNK MAPKs . ConsistNK MAPKs  showed t2O2 exposure in bovine lung microvascular endothelial cells [phox in ARDS models.The p38 MAPKs encompass isoforms such as p38α, p38β, p38γ, and p38δ, and they are activated by a variety of stress responses, including inflammation, oxidative stress, heat, and physical shock ,119. Intal cells . Additioal cells  demonstr2O2, thereby becoming activated [phox-mediated ROS production in rat alveolar macrophages (AMs) stimulated with IgG-opsonized Klebsiella pneumonia. Similarly, the inhibition of PKC attenuated hypochlorous acid-induced pulmonary artery pressure and vascular permeability in a rabbit lung injury model [phox was shown to be associated with LPS-induced ROS production (mediated by PKC activation) in murine AMs [PKC comprises at least 12 isoforms, including PKCα, PKCβI, and PKCδ, and it possesses structural features sensitive to oxidative modification ,124. It ctivated . Bourdonctivated  showed try model . Additiorine AMs .2O2 typically regulate the activation of NFκB signaling pathways through the phosphorylation of tyrosine residues, particularly Tyr42, on IκBα [NFκB is a redox-sensitive transcription factor that regulates the expression of various genes associated with inflammation and immune responses . It is c on IκBα . Ghobadi on IκBα  showed t on IκBα . Similar on IκBα . Additio on IκBα  revealedHIF1 is a conserved family member of the heterodimeric basic helix-loop-helix transcription factors; it is composed of oxygen-sensitive α subunits, including HIF1α, HIF2α, and HIF3α, and three constitutively expressed β subunits, including HIF1β, HIF2β, and HIF3β . HIF1 acAP-1 is a dimer comprising members of the JUN , FOS , ATF , and MAF  families, which contain a basic leucine zipper (bZIP) domain necessary for DNA interaction and the formation of homodimers and heterodimers . FurtherIn ARDS, the permeability of the alveolar-capillary barrier increases, causing the edema fluid containing a large amount of protein to flow into the lung parenchyma, leading to epithelial cell dysfunction and reduced lung compliance and gas exchange ,152. RecNRF2 is a transcriptional regulator of genes with antioxidant and anti-inflammatory effects that has emerged as a major target in the treatment of ARDS. As a master regulator, NRF2 crosstalks with multiple signaling pathways, controlling the activity of various antioxidant enzymes and oxidizing enzymes such as NOX, NO synthase, XOR, and CYP ,160. UpoThe phosphorylation of Akt induces the nuclear translocation of NRF2, leading to the up-regulation of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 through the PI3K/Akt pathway. This process reduces oxidative stress and inflammation mediated by forkhead box protein O1-NLRP3, reducing NLRP3 inflammasome activity in the lungs . The phoKelch-like ECH-associated protein 1 (Keap1) features a broad-complex, tramtrack and bric-a-brac domain (BTB) that engages with the Cul3-Rbx1-E3 ligase complex, governing Keap1 homodimerization. Additionally, it possesses a double glycine repeat domain (DGR) that binds to the ETGE and DLG motifs within the NRF2-erythroid-derived CNC homology domain (Neh) 2 of NRF2, orchestrating the ubiquitination and degradation of NRF2 ,166,167.Recent studies have shown that single-nucleotide polymorphisms (SNPs) in NRF2 correlate with the severity of ARDS. In one specific study, the hierarchical clustering of 72 inbred strains of mice identified a significant association between NRF2 haplotypes and hyperoxic lung injury phenotypes . AdditioMaintaining redox homeostasis and curbing excessive ROS production throughout the development, progression, and exacerbation of ARDS presents a promising avenue for alleviating and treating clinical symptoms. This can be achieved by regulating multiple signaling pathways implicated in ARDS pathogenesis. From this standpoint, antioxidant enzymes like SOD, glutathione (GSH), and various pharmacological and nonenzymatic antioxidants have been explored for their therapeutic potential in ARDS.2•− into H2O2 and is categorized into cytosolic copper-zinc SOD (CuZnSOD), mitochondrial manganese SOD (MnSOD), and extracellular SOD (ECSOD), found in fibroblasts and endothelial cells [2•− and inflammatory cytokine production in murine AMs. Additionally, commercialized SOD mimetics, such as manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin, demonstrated the alleviation of oxidative and nitrative stress-mediated MPO activity and vascular permeability in a murine lung inflammation model following LPS stimulation [2•− production, lung tissue damage, and necrosis in a murine lung inflammation model, as demonstrated by Suresh et al. [SOD catalyzes the conversion of Oal cells . While ial cells ,180,181.al cells . Researcal cells ,184. A sal cells , suppresmulation . Similarmulation . Anothermulation . Furtherh et al. .2O2, with GPx being the major enzyme responsible for this reducing mechanism in the redox cycle [2O2 or lipid hydroperoxides (LOOH), with reduced GSH serving as an electron donor [2•− scavenging induces a radical propagation reaction involving several steps, leading to the formation of thiyl radicals (GS·) and H2O2 [GSH plays a pivotal role in scavenging intracellular Hox cycle ,191. GPxon donor . Glutathon donor ,193. GPxon donor . Additioon donor . The GSHand H2O2 ,195. In and H2O2 . Kim et and H2O2  demonstrand H2O2 . In a stand H2O2 , pharmacand H2O2  demonstrphox expression and O2•− production in porcine lung arteries, thereby mitigating the progression of ARDS [phox expression and O2•− formation in porcine pulmonary artery endothelial cells. The NOX1/4 dual inhibitor GKT137831 ameliorated lung injuries and suppressed the expression of inflammation- and autophagy-related genes in a murine ischemic lung injury model [K. pneumonia-induced murine lung injury [Synthetic and pharmacological antioxidants play a crucial role in preventing ARDS development by inhibiting intracellular ROS production and associated oxidases. Notable antioxidants, such as apocynin, GKT137831, tempol, sivelestat, and NAC, have been developed for this purpose. Apocynin, a NOX inhibitor, demonstrated the attenuation of acute hypoxia-induced gp91 of ARDS . Muzaffa of ARDS  showed try model . In recery model . Sivelesg injury . Additiog injury .2+, Hg2+, and Pb2+, as well as Cu2+ and Fe3+, through the free thiols that bind with redox metal ions [2O2 levels in rats following intratracheal IL1α administration [NAC, in its acetylated form of cysteine, possesses antioxidant capabilities by virtue of its sulfhydryl functional group (-SH) . It actstal ions . Clinicatal ions  demonstrstration . Kolomazstration  observedstration  showed tl-ascorbic acid and l-dehydroascorbic acid [2O2. Studies have demonstrated the beneficial effects of vitamin C supplementation in various ARDS models [Nonenzymatic antioxidants such as vitamins and flavonoids have been explored as treatments for ARDS by inhibiting intracellular ROS production and associated oxidases. Vitamins, serving as coenzymes, metabolic activators, cellular components, and ROS scavengers, play essential roles in combating oxidative stress . Notablybic acid . It effeS models . DioxygeS models . Fisher S models  showed tS models . Kearns S models  revealedS models  demonstr2/FiO2 ratio in a lung inflammation model [The most abundant and potent form of vitamin E, a liposoluble antioxidant consisting of eight isoforms, is α-tocopherol . α-tocopon model .α-Lipoic acid (α-LA), a naturally synthesized short-chain fatty acid with sulfhydryl groups, and its reduced form, i.e., dihydrolipoic acid, are known as universal antioxidants that function in both hydrophobic membranes and hydrophilic cytosol . α-LA isFlavonoids, a class of polyphenols, exhibit anti-inflammatory, antioxidant, metal ion chelation, and free radical scavenging activities, countering oxidative stress by mitigating lipid oxidation and intracellular ROS . ComprisARDS results in sudden and severe lung failure; despite elevated mortality rates, the syndrome is currently managed primarily through supportive care due to the absence of specific targeted treatments. The identification of increased levels of ROS in ARDS models and the clinical correlation between mortality and oxidative stress highlights the role of ROS as a potential target for ARDS treatment. Inhibiting ROS signaling through antioxidants has emerged as a prospective therapeutic approach for patients with ARDS. Nevertheless, conducting additional research, particularly through clinical trials, is imperative for comprehensively assessing the therapeutic efficacy of antioxidants.