ETV6::NTRK3 fusion. The highly cellular tumors were composed of solid and dense cribriform nests, often with comedo-like necroses in the center, and minor areas with papillary, microcystic, and trabecular formations without secretions, mostly located at the periphery of the lesion. The cells displayed high-grade features, with enlarged, crowded, and often vesicular nuclei with conspicuous nucleoli and brisk mitotic activity. The tumor cells were immunonegative for mammaglobin while showing immunopositivity for p40/p63, S100, SOX10, and GATA3, as well as for cytokeratins 7, 18, and 19. For the first time, we describe 2 cases of primary high-grade non-intestinal type adenocarcinomas of the nasal cavity, distinct from secretory carcinoma by morphology and immunoprofile, harboring the ETV6::NTRK3 fusion.We report 2 cases of high-grade sinonasal adenocarcinoma with a distinct morphological and immunohistochemical phenotype. Albeit histologically different from secretory carcinoma of the salivary glands, both tumors presented here share an  Primary sinonasal adenocarcinomas are rare tumors encompassing a wide morphological spectrum, divided into two groups: intestinal-type sinonasal adenocarcinoma (ITAC) and non-intestinal-type sinonasal adenocarcinoma (non-ITAC) subtypes . While INUTM gene, most commonly NUTM::BRD4 [IDH2 gene [IDH1 gene mutations [With excessive solid areas, the differentiation between HG sinonasal non-ITAC and other poorly differentiated epithelial neoplasms of the salivary gland and non-salivary gland origin might pose a problem. Recent molecular-genetic findings had aided in the subclassification of primary sinonasal carcinomas, prompting the inclusion of a number of new entities in the 5th edition of the WHO Classification of Head and Neck Tumours . The aggTM::BRD4 . The diaMYB::NFIB and MYBL1::NFIB in adenoid cystic carcinoma [MAML2 fusions in mucoepidermoid carcinoma [NR4A3 upregulation by enhancer hijacking in acinic cell carcinoma [ETV6::NTRK3 in secretory carcinoma [Salivary gland tumors might rarely arise from the minor salivary glands located in the sinonasal tract, representing 5–10% of all sinonasal adenocarcinoma cases . Multiplarcinoma , 8, MAMLarcinoma , NR4A3 uarcinoma , 12.ETV6::NTRK3 fusion is specific for secretory carcinoma (SC) [In the context of salivary gland tumors, oma (SC) . In typioma (SC) . High-groma (SC) , 14. Simoma (SC) .ETV6::NTRK3 fusion, as well as one case with a less common ETV6::RET fusion [ETV6::NTRK3 fusion was reported [In the nasal cavity, three cases of low-grade non-ITAC were reported to harbor the T fusion –17. In areported . Lastly,reported , 20.ETV6::NTRK3 fusion was detected in both cases. We aim to contribute to unraveling the position of this neoplasm in the classification of head and neck tumors.In this report, we present for the first time two cases of primary high-grade sinonasal adenocarcinomas that were initially classified as high-grade sinonasal non-ITAC. Using RNA sequencing, an For conventional microscopy, excised tissues were fixed in formalin, processed routinely, embedded in paraffin (FFPE), cut, and stained with hematoxylin and eosin. Immunohistochemical staining was routinely performed using an automated Ventana BenchMark ULTRA system . The primary antibodies used are summarized in Table https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500.html). In addition, the samples were analyzed using the NGS-based ligation-dependent multiplex RT-PCR assay as described previously [For mutation analysis, TruSight Oncology 500 panel, a comprehensive NGS assay on FFPE samples that identifies fusion transcripts, somatic variants, copy number changes, tumor mutational burden, and microsatellite instability was used. NA libraries were created using the TruSight Oncology 500 Kit (Illumina) following the manufacturer’s instructions , although we used KAPA FragKit  for DNA enzymatic fragmentation. Following the manufacturer’s guidelines, sequencing was conducted using an Illumina NovaSeq6000 sequencer. The OmnomicsNGS analysis program was used for data analysis (DNA variant filtering and annotation) . Reported variants were filtered retaining variations with coding effects, read depths greater than 50, and variants with allelic frequency >10%, with the removal of benign variants according to the ClinVar database. The remaining collection of variations was visually verified in raw data, and probable artifactual variants were eliminated. The list of genes covered by this panel is available at the product website  were observed. Minimal non-neoplastic lymphoplasmacytic infiltration was present in the background in case 1, while case 2 showed more prominent stromal chronic inflammation.The tumors widely infiltrated into the submucosa of the nasal cavity. Both tumors displayed high-grade features, growing mostly in hypercellular solid and dense cribriform nests, often with central necrosis Figs. A-B, 3A-BOn immunohistochemical examination Table , markersSMARCB1, SMARCA2, and SMARCA4 immunoexpression was retained in both cases. Smooth muscle actin, androgen receptor, and Her2 were negative in both cases. Furthermore, case 1 did not stain with the SALL4 and TTF1 antibodies, while case 2 was additionally negative for p16, NOR1, CDX2, and STAT6.ETV6::NTRK3 fusion involving exon 5 of the ETV6 gene and exon 15 of the NTRK3 gene was detected by RNA-sequencing in both cases , with breakpoints at chr12:12022900 and chr15:88483984. The NGS-based ligation-dependent multiplex RT-PCR assay corroborated these results. No pathogenic genetic alterations were revealed by the DNA part of the TruSight Oncology 500 panel.An identical ses Fig. . The refETV6::NTRK3 fusion, we propose the tumors reported herein might represent a subtype of high-grade non-ITAC, distinct from SC.Based on the high-grade morphology with solid, cribriform, and papillary architecture, distinctive immunoprofile with p40/p63, S100, SOX10, and GATA3 positivity, and a complete lack of mammaglobin staining, together with the ETV6::NTRK3 fusion, analogous to the previously characterized low-grade non-ITACs with this genetic aberration [The major consideration in the differential diagnosis should be a HGT of SC, which was described previously in salivary gland SCs . HGT is erration –17. In aerration . Cytokererration .ETV6 rearrangement reported previously [Notably, given the tubular and/or cystopapillary architecture and the immunoprofile with S-100, SOX10, and limited DOG1 positivity, origin in the minor salivary glands of the sinonasal tract could be considered in the cases presented herein, as well as the low-grade non-ITAC cases with eviously –17, and ETV6::NTRK3 fusion, arising in the parotid gland of a 22-year-old male patient, was reported recently [High-grade adenocarcinoma with similar morphology and an recently . The tumrecently .ETV6::NTRK3 fusion was recently interpreted as salivary duct carcinoma with an unusual genetic background [In the same line, a high-grade salivary gland tumor composed of expansile, centrally necrotic nests composed of AR-positive and S100-negative apocrine-type cells with an ckground . This fiETV6-rearranged non-ITACs discussed above.In addition, tumors of a similar morphology, with solid, focally necrotic, as well as micropapillary and cribriform areas, can be noted in previous works that did not include molecular-genetic analysis , 23. HowETV6::NTRK3 fusion. Even though they share a common ETV6::NTRK3 fusion, these high-grade adenocarcinomas might represent a neoplasm distinct from SC by morphology and immunoprofile.In summary, we report 2 cases of a high-grade tumor diagnosed as non-ITAC of the sinonasal region, characterized by overt hypercellularity, largely solid growth pattern with comedo-like necrosis, immunohistochemical positivity for p40/p63, S100, SOX10, and GATA3, with a recurrent


BRAF, NRAS, NF1) or less commonly mutated  genes. Another group of genes frequently mutated in CMs include TERT and ATRX, with known roles in telomere maintenance and chromatin remodeling/epigenetic regulation. Uveal melanoma-related genes  can also be mutated in CMs, albeit infrequently. Additional CM-related mutated genes have increasingly been identified using more comprehensive genetic analyses, awaiting further confirmation in additional/larger studies. As a tumor arising in a partly sun-exposed mucosal tissue, CM exhibits a distinct genomic profile, including the frequent presence of an ultraviolet (UV) signature  and also the common occurrence of large structural variations (distributed across the genome) in addition to specific gene mutations. The knowledge gained from CM genetic studies to date has led to new therapeutic avenues, including the use of targeted and/or immuno-therapies with promising outcomes in several cases. Accordingly, the implementation of tumor genetic testing into the routine clinical care of CM patients holds promise to further improve and personalize their treatments. Likewise, a growing knowledge of poor prognosis-associated genetic changes in CMs  may ultimately guide future strategies for prognostic testing to further improve clinical outcomes (by tailoring surveillance and considering prophylactic treatments in patients with high-risk primary tumors).Conjunctival melanoma (CM) is a rare but aggressive cancer. Over the past decade, molecular studies using rapidly advancing technologies have increasingly improved our understanding of CM genetics. CMs are mainly characterized by dysregulated MAPK and PI3K/AKT/mTOR pathways, driven by commonly mutated ( Conjunctival melanoma (CM) is a rare malignant neoplasm originating from melanocytes in the basal layer of the conjunctival epithelium . CM accode novo cases account for 15–25% of CMs [CMs occur more commonly in sunlight-exposed areas  than in sunlight-protected areas  ,18 Figu. The maj% of CMs ,20,21. L% of CMs . As sugg% of CMs ,24,25,26Recent advancements in the genetics/genomics field have significantly accelerated our molecular and biological understanding of various cancers and facilitated the introduction/application of novel revolutionary treatments, such as targeted therapy (for tumors with specific somatic mutations) and immunotherapy . While CM research has also increasingly benefited from these advancements (as summarized in this review), the genetic aspects of primary CM have yet to be fully understood given the challenges posed by the tumor’s rare occurrence and the paucity of ‘comprehensively analyzed’ large sample collections ,32,33,34The mitogen-activated protein kinase (MAPK) pathway  and the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway are the two highly complex and interconnected biological pathways that are often found dysregulated in CMs . These pBRAF resides at chromosome (chr) 7q34 (https://www.ncbi.nlm.nih.gov/gene/673 (last accessed on 22 July 2023)) and encodes a serine/threonine kinase responsible for activating the downstream MEK, the next kinase in the MAPK (RAS/RAF/MEK/ERK) pathway. Oncogenic BRAF mutations cause constitutive activation of the BRAF protein, which in turn leads to constitutive downstream activation of the MAPK pathway (MEK1/2 and ERK1/2) contributing to tumor growth [r growth ,36.BRAF mutations [BRAF mutations reported to date occur at codon 600 where valine is substituted with glutamic acid  or lysine  or rarely another amino acid; however, mutations affecting other BRAF codons have also been detected. These observations are similar to those reported in skin melanomas, whereas BRAF mutations do not typically occur in posterior uveal melanomas [About one third of CMs have been found to harbor utations ,42,43,44elanomas ,47,48,49BRAF mutations are believed to occur early in CM development, as supported by the observations that they are commonly present in conjunctival nevi and are more frequently found in CMs originating from nevi than PAM [BRAF status of conjunctival nevi in children vs. adults, however, even though children rarely develop CM [BRAF mutations are associated with conjunctival melanocytic lesions, additional factors (cumulative molecular events further activating the oncogenic pathways) are necessary for malignant transformation to melanoma.than PAM ,42,50,51BRAF-mutated CMs appear to arise more often on sun-exposed/bulbar conjunctiva [BRAF-mutated tumors ) and belongs to the RAS gene family. It encodes a GTPase located upstream of BRAF in the MAPK (RAS/RAF/MEK/ERK) pathway and can also activate the PI3K/AKT/mTOR pathway [ pathway .NRAS mutations have been identified in about 20% of CMs [% of CMs ,38,40,59% of CMs ,46,47,49% of CMs .NRAS mutations are also commonly found in conjunctival nevi [NRAS status and tumor origin in CM [NRAS-mutated tumors and more aggressive behavior (increased risk for metastasis and death), which was also noted as a possibility by another recent study [NRAS-mutated CMs [NRAS-mutated tumors are suitable targets for the MEK inhibitors (as monotherapy or in combination with PI3K/mTOR inhibitors), data are currently lacking on the therapeutic use of these inhibitors for advanced or metastasized CMs harboring NRAS mutations [val nevi ; howeverin in CM . A recenin in CM  identifint study . These rated CMs . Althougutations ,57.NRAS and BRAF mutations are predominantly mutually exclusive in CMs [NRAS is the major RAS gene mutated in CM, the activating mutations in other RAS genes (HRAS or KRAS) have also been reported in different studies [Like in skin melanomas, e in CMs ,33,34,40 studies ,30,38.NF1 resides at chr 17q11 (https://www.ncbi.nlm.nih.gov/gene/4763 (last accessed on 22 July 2023)) and encodes a tumor suppressor protein that inhibits RAS and thus functions as a negative regulator of both the MAPK and PI3K/AKT/mTOR pathways. Loss-of-function/inactivating NF1 mutations are therefore associated with increased RAS activity, which in turn leads to overactive MAPK and PI3K/AKT/mTOR signaling.NF1 mutations [NF1 mutations have been reported to date, mostly nonsense or frameshift, thus resulting in the loss of function of this tumor suppressor gene/protein [NF1 mutations can co-exist with either NRAS or BRAF mutations in CMs, albeit infrequently [NF1 mutations [About one third of CMs have been found to harbor utations ,32,34,38/protein . Like inequently ,28,31,38utations ,60.NF1 mutations and clinicopathological features (or prognosis) of CM to date [NF1 mutations appear to occur more frequently in CMs associated with UV signature  and higher mutational load [NF1-mutated tumors [Although the data are currently limited, no association has been reported between  to date ,28,38. Lnal load , suggestd tumors ,38.KIT resides at chr 4q12 (https://www.ncbi.nlm.nih.gov/gene/3815 (last accessed on 22 July 2023)) and is also known as c-KIT because of its initial identification as the cellular homolog of feline sarcoma viral oncogene v-kit. KIT encodes a receptor tyrosine kinase (RTK), belonging to a large family of transmembrane proteins that are capable of activating several downstream pathways (including MAPK and PI3K/AKT/mTOR pathways) upon stimulation by their ligands [ ligands .KIT mutations have been found in CMs to date, resulting in constitutive KIT activation and overactive MAPK and PI3K/AKT/mTOR signaling [KIT mutations do not commonly occur in CMs [KIT overexpression is more frequently observed, probably due to other events such as copy number alterations affecting the KIT locus [Various oncogenic ignaling . Like inr in CMs ,38,40,61IT locus ,40,61.BRAF and NRAS mutations, suggesting mutual exclusivity between them [KIT-mutated CMs are more frequently observed in Asian populations where BRAF-mutated CMs occur less frequently [BRAF and NRAS mutations, KIT mutations can co-exist with NF1 mutations in CMs [CMs harboring activating mutations and/or gains of the KIT gene/locus appear to lack both een them ,40,61. Iequently . Like BRs in CMs ,30,57,62s in CMs ,63.KIT mutations in other (sun-protected) mucosal melanomas, a recent study [KIT status and survival in CM to date [KIT-mutated tumors are suitable targets for c-KIT inhibitors. It remains unknown, however, whether these inhibitors would be beneficial to CM patients with KIT-mutated or KIT-amplified tumors [Consistent with the common occurrence of nt study  has note to date ,29, KIT-d tumors ,29,58,62PTEN resides at chr 10q23 (https://www.ncbi.nlm.nih.gov/gene/5728 (last accessed on 22 July 2023)) and encodes a tumor suppressor protein that negatively regulates PI3K and thus functions as an AKT/mTOR pathway inhibitor. Loss of PTEN activity  therefore leads to increased PI3K activity and overactive PI3K/AKT/mTOR signaling.Like skin melanomas, CMs can demonstrate reduced/lost PTEN expression and upregulation of the mTOR pathway . Uveal mPTEN mutations have been reported in more recent studies [PTEN mutations can also contribute to the PTEN loss observed in CMs. While PTEN mutations appear to be usually mutually exclusive with NRAS mutations, they often co-occur with BRAF or KIT mutations [In addition to the copy number alterations initially found to cause the PTEN loss in CMs ,40, inac studies ,28,29,31An association between PTEN loss and CM pigmentation was noted in one previous study , which dTERT, ATRX, and DAXX) involved in telomere maintenance [In addition to the MAPK and PI3K/AKT/mTOR pathways, telomere maintenance and chromatin remodeling play important roles in CM development and progression as summarized below. In order to gain an unlimited proliferation potential and immortality, cancer cells need telomere maintenance, which can be achieved either by the activation of telomerase reverse transcriptase (TERT) or by the induction of alternative lengthening of telomeres (ALT) pathway (associated with ATRX or DAXX loss) . About 1ntenance ,67. Growntenance ,37,59,68ntenance ,32,34.TERT resides at chr 5p15 (https://www.ncbi.nlm.nih.gov/gene/7015 (last accessed on 22 July 2023)) and encodes the catalytic protein subunit of telomerase (a ribonucleoprotein polymerase), which is responsible for adding short repetitive sequences to the ends of chromosomes to maintain the telomere length. In normal somatic cells, telomeres undergo progressive shortening with successive cell divisions due to repressed telomerase expression, which in turn limits the cells’ replicative capacity, leading to senescence. Abnormal telomerase expression/activity can therefore prevent telomere depletion with successive replications, protecting chromosomes from degradation and making cells ‘immortal’.TERT promoter (TERTp) mutations occurring at the same hotspots (in about 35–40% of cases) [BRAF or NRAS mutations and often display a typical UV signature [TERTp mutations can cause increased TERT expression (via de novo transcription factor binding), thus enabling neoplastic cell survival and immortality. It remains to be determined, however, whether other genetic and/or epigenetic mechanisms also contribute to increased TERT expression/activity in CMs [Like skin melanomas, CMs frequently harbor f cases) ,37,59,68ignature ,37. TERTy in CMs .TERTp mutations can also be found in PAM lesions (with atypia), but not in conjunctival nevi, implying a role of increased TERT expression/activity in malignant transformation [TERTp mutations have also been linked to non-PAM-derived CM [TERTp mutations [ormation ,68. Howerived CM  in additrived CM ,68, suggutations ,68,69.TERTp mutation and the development of metastasis, indicating its prognostic significance. The presence of TERTp mutation may also have therapeutic significance given that the telomerase and reverse transcriptase inhibitors represent future therapeutic options for TERTp-mutated tumors [Recent CM studies ,37 have d tumors .ATRX resides at chr Xq21 (https://www.ncbi.nlm.nih.gov/gene/546 (last accessed on 22 July 2023)) and encodes a member of the SWI/SNF family of chromatin remodeling proteins that plays a role in alternative telomere lengthening (through homologous recombination) and in epigenetic regulation (via DNA methylation). Inactivating ATRX mutations and loss of ATRX protein are frequently observed in cancers that use the ALT pathway for telomere maintenance, including mucosal melanomas [elanomas ,71.ATRX mutations were initially detected in small CM series subjected to comprehensive genetic analyses (identified in ~20–60% of cases) [ATRX-mutated tumors upon further analysis of a subset of these tumors.f cases) ,34 and lf cases) . Furtherf cases)  functionATRX mutations often co-occur with NF1 mutations and less commonly co-exist with NRAS or BRAF mutations [ATRX-mutated CMs appear to frequently harbor mutations in genes involved in histone modification and epigenetic regulation  [ATRX mutations also appear to co-occur with TP53 mutations in CMs and other mucosal melanomas [ATRX mutations) and TERT activation (via TERT amplification or TERTp mutations) often show mutual exclusivity in various cancers [utations ,32,34. Felanomas . By cont cancers , awaitinATRX-mutated CMs appear to arise more commonly in non-sun-exposed areas and show association with less aggressive behavior indicating prognostic significance [ATRX mutations may also have therapeutic significance as CMs harboring these mutations would be resistant to anti-telomerase therapies but confer sensitivity to PARP inhibitors [The observation of ATRX loss and ALT positivity in both intraepithelial and invasive components of CMs suggests their early occurrence in CM evolution . ATRX-muificance . ATRX muhibitors .GNAQ (at 9q21) or GNA11 (at 19p13) and later events involving EIF1AX (at Xp22) or SF3B1 (at 2q33) or BAP1 (at 3p21) [https://www.ncbi.nlm.nih.gov/gene/1964 (last accessed on 22 July 2023)) while SF3B1 is involved in RNA splicing (https://www.ncbi.nlm.nih.gov/gene/23451 (last accessed on 22 July 2023)). BAP1 is a deubiquitinating enzyme (https://www.ncbi.nlm.nih.gov/gene/8314 (last accessed on 22 July 2023)) that functions as a tumor suppressor protein involved in chromatin remodeling, transcription regulation, DNA damage response, and cell death [Major genetic drivers identified in uveal melanoma include early events activating at 3p21) ,73,74. Gat 3p21) ,75. EIF1ll death ,76.BAP1, SF3B1, and GNAQ/11 have also been detected in CMs, albeit less frequently, including both uveal melanoma-related hotspot mutations and other mutations [BRAF, NRAS, KIT, NF1, and ATRX [While CM has an overall distinct genetic profile, overlapping more with that of skin and mucosal melanomas than that of uveal melanoma, mutations affecting uveal melanoma-associated genes utations ,38,77,78and ATRX ,28,29,31The presence of uveal melanoma-related hotspot mutations in CMs has been linked to advanced disease and propensity for metastasis and death, indicating an overall poor prognosis similar to that seen in other mucosal melanomas harboring these mutations ,78,79,80ATM at 11q22 (encoding a cell cycle checkpoint kinase that regulates multiple proteins) [TP53 at 17p13 (encoding a well-known tumor suppressor protein with diverse functions) [CDKN2A at 9p21 (encoding tumor suppressor proteins involved in cell cycle regulation) [FBXW7 at 4q31 (encoding a tumor-suppressor protein involved in ubiquitin-mediated oncoprotein degradation) [TET2 at 4q24 (encoding a methylcytosine dioxygenase involved in epigenetic regulation) [SETD2 at 3p21 (encoding a histone methyltransferase involved in epigenetic regulation) [IDH1 at 2q34 (encoding an isocitrate dehydrogenase involved in metabolism) [CBL at 11q23  [ALK at 2p23 (encoding a receptor tyrosine kinase) [MET at 7q31 (encoding a receptor tyrosine kinase) [CTNNB1, ACSS3, PREX2, APOB, RYR1/2, SYK, NOTCH3, CHEK2, KMT2A/C, ARID2, FAT4, RB1, APC, as well as some MAPK/MAP2K/MAP3K genes, PIK3CA/B/G genes, additional RTK genes , and some HDAC and additional SETD genes) [BRAF, NRAS, NF1), those found in tumors lacking these known driver mutations, such as the recently reported mutations in CTNNB1, ARID2, TET2, TP53, RB1, RUNX1, TSC2, CDKN2A, CIC, and some MAPK genes [Recent CM studies have identified mutations in additional genes using either unbiased whole genome or exome next-generation sequencing (NGS) or targeted NGS of large panels of cancer-related genes ,31,32,34roteins) ,28,32, Tnctions) ,31,32,77ulation) ,28,31, Fadation) ,32, TET2ulation) ,32, IDH1abolism) ,31, CBL  kinase) ,28. AddiIdentification of a genomic mutational pattern dominated by C > T substitutions (at dipyrimidine sites), including a subset of CC > TT substitutions, is indicative of UV light-induced DNA damage , which is usually associated with increased tumor mutational burden (TMB) .Tumor genomic profiles  are known to differ between the two major melanoma groups—epithelium associated  vs. non-epithelium associated  melanomas—as well as among the subtypes within each group ,82,83. IDue to UV light-absorbing effects of the cornea and lens, ocular melanomas occurring in areas with different levels of sunlight exposure  melanomas vs. posterior uveal melanomas) demonstrate corresponding differences in the presence of UV signature and the levels of TMB ,72,82. AInterestingly, both UV-driven and non-UV-driven CMs appear to harbor various large structural variations ,82, whicIn addition to specific gene mutations, various chromosomal aberrations and structural variations  have also been detected in CMs as summarized below. Copy number alterations (CNAs) have been well studied in uveal melanomas, and some are strongly implicated in prognosis and metastatic outcome, such as loss of chromosome 3 and gain of 8q or 6p . The CNABRAF, NRAS, and TERT) are observed in a subset of the tumors driven by those [KIT (4q12), CCND1 (11q13), CDKN1A (6p21), RUNX2 (6p21) and others , and the deletions of NF1 (17q11), TP53 (17p13), CBL (11q23), CDKN2A/2B (9p21), ASNS (7q21), and HLA-A (6p22) [Numerical chromosomal aberrations such as polyploidy or aneuploidy are observed in CMs, while the structural chromosomal aberrations reported to date include the gains at 1q, 3p, 6p, 7p/q, 8p/q, 11p/q, 12p, 13q, 14p, 17q, and 22q, and the losses at 1p, 3q, 4q, 6q, 8p, 9p/q, 10p/q, 11q, 12q, 15p, 16p/q, 17p, 19p/q, and 21p ,40,41,89by those ,40. OtheA (6p22) ,33,40,41A (6p22) .BRAF/NRAS-wildtype tumors [BRAF-mutated tumors whereas the losses at 10q are found more commonly in this tumor group [BRAF-mutated tumors include those located at 10q11-23  and at 10q26 . While the 10q loss can also be observed in NRAS-mutated tumors, its more frequent presence in BRAF-mutated tumors supports the hypothesis that these tumors acquire additional event(s) to activate the PI3K-AKT pathway in addition to the MAPK pathway during their evolution [Like in skin melanomas, the frequency of CNAs in CMs appears to vary depending on the genetic background and has been shown to be relatively higher in e tumors ,40. Howeor group ,40. The volution .NEURL1, SUFU, PDCD4, and C10orf90) were reported to be associated with increased tumor thickness, lymphatic invasion, and metastatic spread of CM [While most CNAs identified in CMs to date do not seem to have any clinical/prognostic value, recurrent deletions occurring at 10q24-26  in addition to specific gene mutations. Commonly observed mutations indicate the major roles of dysregulated MAPK (RAS-RAF-MEK-ERK) and PI3K/AKT/mTOR pathways as well as the telomere maintenance and chromatin remodeling/epigenetic regulation mechanisms in CM pathogenesis. While uveal melanoma-related driver genes are infrequently mutated in CMs, an increasing number of other  genes have been implicated by a growing number of CM studies using more comprehensive genetic analyses . While our knowledge of CM genetics has significantly improved over the last years, the studies that used high-throughput approaches  are currently scarce and small in size, thus future studies using these comprehensive approaches in larger cohorts are warranted to uncover CM’s genomic landscape with its full complexity.Although rare in incidence, CM is a highly recurrent and potentially deadly cancer, and there is currently no consensus on the treatment of locally advanced or metastatic disease. Ongoing advances in our understanding of the genetic and molecular mechanisms involved in CM pathogenesis are therefore important as they increasingly yield new therapeutic targets and options ,92,93,94NRAS, TERT, or uveal melanoma-related genes/hotspots and recurrent deletions affecting 10q24-26) although awaiting validation in additional/independent studies. This in turn may inform future strategies for prognostic tumor genetic testing to further improve clinical outcomes in CM patients (by tailoring surveillance and considering prophylactic treatments in those with high-risk primary tumors).Unlike uveal melanomas, little is known about the prognostic implications of genetic changes observed in CMs, therefore predictive genetic testing is not currently standard practice in CM management. However, as tumor genetic testing becomes more accessible and larger CM datasets become available, our knowledge of poor prognosis-related genetic changes has also been advancing (i.e., mutations affecting


GRB2-associated binder 1 (GAB1) is a docking protein pivotal in linking multiple stimuli to various intracellular signaling pathways. Embryonic development is disrupted in GAB1-deficient mice, and oncogenic mutations have been noted in cancer cases. In numerous types of cancer, high GAB1 expression levels correlate with a poor prognosis. Studies reveal that GAB1 significantly influences cellular transformation by changes in proliferation, evasion of apoptosis, metastasis, and angiogenesis—all fundamental processes in cancer development. Furthermore, GAB1 is implicated in the resistance/sensitivity to antitumor treatments, thus establishing its potential as an anticancer therapy target.GRB2-associated binder 1 (GAB1) is the inaugural member of the GAB/DOS family of pleckstrin homology (PH) domain-containing proteins. Upon receiving various stimuli, GAB1 transitions from the cytoplasm to the membrane where it is phosphorylated by a range of kinases. This event recruits SH2 domain-containing proteins like SHP2, PI3K’s p85 subunit, CRK, and others, thereby activating distinct signaling pathways, including MAPK, PI3K/AKT, and JNK. GAB1-deficient embryos succumb in utero, presenting with developmental abnormalities in the heart, placenta, liver, skin, limb, and diaphragm myocytes. Oncogenic mutations have been identified in the context of cancer. GAB1 expression levels are disrupted in various tumors, and elevated levels in patients often portend a worse prognosis in multiple cancer types. This review focuses on GAB1’s influence on cellular transformation particularly in proliferation, evasion of apoptosis, metastasis, and angiogenesis—each of these processes being a cancer hallmark. GAB1 also modulates the resistance/sensitivity to antitumor therapies, making it a promising target for future anticancer strategies. Drosophila and Suppressor Of Clear (Soc1) in Caenorhabditis elegans [C. elegans, Egl-15 [GAB1 gene is located on chromosome 4q31.1 in humans [GRB2-associated binding 1 (GAB1) is the inaugural member of the GAB family of docking proteins, which includes GAB1, GAB2, and GAB3 in mammals, Daughter Of Sevenless (DOS) in  elegans . Histori elegans ; GAB2 wa elegans , and GAB elegans . DOS was elegans ,9. Soc1 , Egl-15 . These p, Egl-15 . However, Egl-15 . The GABn humans . Two ison humans ,13. Howen humans . Unlike n humans . In addin humans ,17.GAB1 through its SH2 domains by in vitro kinase assays [GAB1 is phosphorylated at tyrosine residues in response to a wide variety of stimuli including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), and insulin and stem cell factor (SCF); cytokines such as erythropoietin (EPO), interleukin-6 (IL-6), interferon-α (IFN-α), interferon-γ (IFN-γ), interleukin-3 (IL-3), and thrombopoietin (TPO); and various immune stimuli such as anti-IgM or F(ab’)2 and lysophosphatidic acid (LPA) . These pe assays . Similare assays . It has e assays .Analogously, GAB1 is also phosphorylated in response to different stresses such as oxidative stress, fluid shear stress and ultraviolet (UV) light ,23,24. IGAB1 recruitment from the cytoplasm to activated membrane receptors can be direct or indirect . The dirAs mentioned, GAB1 can channel signaling from the external stimulus to the PI3K/AKT pathway. Activation of this pathway is triggered by the direct interaction of GAB1 with the SH2 domain of the p85 regulatory subunit of PI3K kinase ,33. MutaAmong other proteins that bind to GAB1, the role of the SHP2 protein, which interacts with the phosphotyrosine residues Y627 and Y659 located at the GAB1 C-terminal end, is noteworthy . The GABAs described above, GAB1 is phosphorylated in response to EGF and is involved in PI3K/AKT and MAPK pathway signaling. GAB1 has been shown to be strictly required in EGF-induced activation of the PI3K/AKT signaling pathway by associating it with the p85 subunit of PI3K ,50. FurtOn the other hand, GAB1 has been described to contain potential binding sites for the SH2 domain of phospholipase C γ (PLCγ), CRK, CRK-like (CRKL), and other proteins such as P21-activated kinase 4 (PAK4) or some members of the partitioning defective (PAR) complex ,53,54,55In addition to the functions of GAB1 as a molecular integrator of different signals, GAB1 also performs other functions. GAB1 acts as a molecular switch that allows the interconnection of different cell signaling pathways such as the connection between the EGFR/PI3K/AKT pathway and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway in glioblastoma cells. This interconnection has been determined to be exerted through the GAB1–SHP2 complex using the mutant version of GAB1 unable to bind SHP2 (Y627F). Furthermore, in this interconnected system of cell signaling pathways, the phosphatase domain of SHP2 exerts a negative regulatory effect on GAB1 phosphorylation and, therefore, on NF-kB activity . AdditioAll these findings point to GAB1 as a platform for integrating external signals to trigger and amplify their emergent signaling. Consequently, these results suggest the presence of sophisticated regulatory mechanisms. Since the PH domain of GAB1 is involved in the interaction with PIP3 present in biological membranes and these are products of PI3K kinase action, it has been reported that the lipid phosphatase and tensin homologue (PTEN) or SH2 domain-containing inositol 5-phosphatases 1/2 (SHIP1/2) could negatively influence the GAB1 membrane localization and thus lead to its inactivation ,60. FurtAbelson) fusion has been reported in perineurioma, angiofibroma, and solitary fibrous tumors [GAB1 is involved in diverse biological processes such as cell proliferation, survival, invasion and migration, cell differentiation, angiogenesis, and inflammation ,68,69,70s tumors . Also, es tumors . GAB1 ges tumors . Furthers tumors ,77,78,79s tumors ,81,82. Is tumors ,84,85,86This review focuses on GAB1’s influence on cellular transformation particularly in proliferation, evasion of apoptosis, metastasis, and angiogenesis—each of these processes being a cancer hallmark. GAB1 also modulates the resistance/sensitivity to antitumor therapies.Xenopus laevis oocytes depends on GAB1. The Met-binding domain, the pleckstrin homology domain, and the PI3K and SHP2 binding sites are required to perform this function. Furthermore, this GAB1-dependent regulation of cell cycle progression is also observed in the case of the FGF receptor [Alteration of cell cycle control and apoptosis evasion are two hallmarks of cancer. In addition, another typical feature of tumor cells is uncontrolled proliferation ,88. It hreceptor . Suppresreceptor ,91. Alsoreceptor . GAB1 doreceptor . GAB1 ovreceptor . Moreovereceptor . In hepareceptor . The absreceptor . HCK-medreceptor .Fibroblasts’ oncogenic transformation leads to morphological changes that allow uncontrolled proliferation and progression even in the presence of inhibitory signals such as cell–cell contact. The Tpr-Met and epidermal growth factor receptor 2  oncoprotein has been reported to induce oncogenic transformation, and this mechanism has been demonstrated to require GAB1 involvement ,97,98. OGAB1 suppression increased apoptosis in hilar cholangiocarcinoma, chondrosarcoma cell lines by modulating the B-cell lymphoma 2 (BCL-2)/ Bcl-2 Associated X-protein (BAX) axis, and pancreatic carcinoma through microRNA-383 regulation. It has been also depicted that GAB1 leads to partial apoptosis induction in HCT116 colorectal cancer cells through miR-5582-5p modulation ,101,102.3 [Angiogenesis is a complex multistep process in which new blood vessels are produced from pre-existing ones. This complex biological process is orchestrated by the interaction of a wide variety of mediators, as well as the involvement of different cell types . In 19713 ,105. The3 ,88. Foll3 . Tumor a3 . Among a3 .Tumors engrafted in GAB1-ecKO mice have been shown to have a substantially lower level of capillary density, as well as a marked decrease in tumor weight and volume . It has The role of GAB1 in the promotion and control of physiological angiogenesis is well known . HoweverThe presence of metastases is often responsible for high cancer mortality . Tumor cEpithelial–mesenchymal transition (EMT) is the process by which epithelial cells lose polarity and adhesion to acquire mesenchymal cell characteristics. The acquisition of these mesenchymal features depends on a wide variety of intracellular signaling networks such as the MAPK pathway, PI3K/AKT, and others. In relation to EMT, the final convergence of these cell signaling pathways is the expression of EMT-inducing transcription factors such as SNAIL, SLUG, XEB1, and TWIST, among others. This process is the underlying mechanism for the development of tumor metastasis ,122. HigRecent studies indicate that different elements of the tumor microenvironment such as the extracellular matrix, fibroblasts, endothelial cells, and different immune populations can modulate the migratory and invasive capabilities of cancer cells . In mostThe clinical efficacy of cancer treatment has increased thanks to the contribution of numerous studies and the increasing discovery of new targeted therapies. However, cancer is a very heterogeneous and dynamic disease, so as treatment progresses, treatment efficacy may be lost or even lead to a complete loss of therapeutic response following the emergence of resistance mechanisms . GAB1 mRNA and protein levels. Elevated GAB1 expression levels in ibrutinib-treated CLL cells have been associated with augmented tonic activation of pAKT, implying cellular relocalization signals and cell survival. Thus, GAB1 inhibitors reduce cell migration and increase cell apoptosis individually or in combination with ibrutinib by altering the FOXO1–GAB1–pAKT axis [GAB1 has been described to be involved in alectinib resistance mediated by activation of the HGF/MET signaling pathway in anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer . It has AKT axis . On the AKT axis .Besides the use of chemotherapy as a treatment for cancer patients, radiotherapy is also an effective therapeutic option to treat some tumor types . GAB1 isAs mentioned above, GAB1 acts as a signal integrator of multiple signaling pathways and is deregulated in many cancers. Considering the GAB1 involvement in the different hallmarks of cancer, this protein could be a potential therapeutic target to treat different tumor types. Recently, selective inhibitors of GAB1 have been developed. These new compounds, GAB-001 and GAB-004, target the PH domain of GAB1 by inducing a conformational change that prevents its phosphorylation. They have only been tested in breast cancer cell lines MDA-MB231 and T47D, showing potent cytotoxicity . HoweverGAB1 behaves as a protein integrator of multiple cell signaling pathways regulating cell survival and proliferation, angiogenesis, and cell migration and invasion, facilitating cellular transformation, thus playing a crucial role in tumorigenesis. GAB1 also mediates resistance/sensitivity to various chemotherapeutic drugs and radiotherapy. GAB1 expression is altered in a wide variety of tumors and is associated with poor patient prognosis in several tumor types. GAB1 inhibitors have a good prospect in cancer therapy. Therefore, GAB1 could be considered a potential therapeutic target for cancer treatment. However, further pre-clinical studies and clinical trials are needed for the clinical application of GAB1 inhibitors in specific tumor contexts.


Inflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal (GI) disorder characterized by intestinal inflammation. The etiology of IBD is multifactorial and results from a complex interplay between mucosal immunity, environmental factors, and host genetics. Future therapeutics for GI disorders, including IBD, that are driven by oxidative stress require a greater understanding of the cellular and molecular mechanisms mediated by reactive oxygen species (ROS). In the GI tract, oxidative stressors include infections and pro-inflammatory responses, which boost ROS generation by promoting the production of pro-inflammatory cytokines. Nuclear factor kappa B (NF-κB) and nuclear factor erythroid 2–related factor 2 (Nrf2) represent two important signaling pathways in intestinal immune cells that regulate numerous physiological processes, including anti-inflammatory and antioxidant activities. Natural antioxidant compounds exhibit ROS scavenging and increase antioxidant defense capacity to inhibit pro-oxidative enzymes, which may be useful in IBD treatment. In this review, we discuss various polyphenolic substances , phenolic compounds including thymol, alkaloids such as berberine, storage polysaccharides such as tamarind xyloglucan, and other phytochemicals represented by isothiocyanate sulforaphane and food/spices , as well as antioxidant hormones like melatonin that target cellular signaling pathways to reduce intestinal inflammation occurring with IBD. Lesions of CD may occur in the small or large intestine but most commonly involve the colon and rectum as discontinuous areas of transmural inflammation. In contrast, UC affects only the colon and rectum continuously, with inflammation restricted to the mucosa . The cliIn vitro and in vivo studies have demonstrated that the activation of the transcription factor hypoxia-inducible factor (HIF) functions as a warning signal in several murine disease models. For example, HIF-1, which is increasingly stabilized in inflammatory lesions, protects against inflammation and IBD by triggering the transcription of several genes that allow the intestinal epithelial cells to operate as an efficient barrier (Nrf2 overexpression was reported to improve UC (Scientific evidence that increased levels of reactive oxygen species (ROS) but decreased levels of antioxidants contribute to disease pathogenesis establishes a link between ROS and IBD , 5. The  barrier . While H barrier Figure 1 barrier . A decre barrier , while Nprove UC .2The intestinal mucosa in people with IBD  is typically infiltrated with numerous inflammatory cells, including neutrophils, macrophages, and lymphocytes , 10. Unc2•–), hydroxyl radical (·OH), hydroperoxyl radical (HO2·), nitric oxide (NO), and singlet oxygen (1O2)  . Furtheren (1O2) . ROS anden (1O2) –17. OS ien (1O2) . This re3Canine chronic inflammatory enteropathies (CIE) refer to a group of intestinal disorders characterized by persistent or recurrent gastrointestinal (GI) signs and variable intestinal inflammation , 20. TheThere are few clinical studies evaluating the role of OS in dogs with CIE. In one case-control study, colonic lavage analytes as markers of mucosal inflammation were compared between healthy dogs and dogs with biopsy-confirmed idiopathic IBD . PolyethSeveral other studies have investigated serum biomarkers of OS in dogs with CIE at diagnosis and in response to medical treatment. In one study, trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of the plasma (FRAP), total thiol concentrations, and paraoxonase-1 (PON1) activity were evaluated in serum to determine the antioxidant response in dogs with idiopathic IBD. Additionally, ferrous oxidation-xylenol orange (FOX), thiobarbituric acid reactive substances (TBARS), and ROS concentrations in serum were determined . The meaDifferences in systemic phospholipids were reported in another study involving dogs with idiopathic IBD and FRE . OverallWhile most studies have investigated the pathomechanisms of OS in chronic gastroenteritis, other studies have evaluated the role of antioxidants in acute enteropathy and in mitigating OS induced by surgery. In one study, a comparison of the OS status of dogs with uncomplicated acute diarrhea (AD) was compared to healthy controls . Both coSummarizing, different metabolomic studies in dogs with CIE also show disturbances in serum and fecal metabolites reflective of OS at diagnosis. Notably, disturbances in lipid metabolism appear to be a common denominator across multiple studies. Moreover, treatment using a hydrolyzed diet with or without different dietary supplements improves several different measures of OS in most animals showing clinical remission. Given the importance of these metabolites in mediating chronic intestinal inflammation, additional well-designed and sufficiently powered clinical trials in dogs with CIE are warranted.4The most prevalent antioxidant signaling pathway is the Kelch-like epichlorohydrin-related protein-1 (Keap1)/Nrf2-ARE signaling pathway , 34. As in vivo  and inducible nitric oxide synthase (iNOS) by inflammatory cytokines leads to the production of superoxide and nitric oxide by intestinal epithelial cells (IECs), neutrophils, and macrophages . IECs geBoth ROS and RNS have been implicated in IBD pathogenesis, with a particular role in CD initiation and progression . With inThese ROS promote cell damage and harm tissue integrity by preventing the accumulation of antioxidant defenses in host cells. For example, oxidative damage is observed in the intestinal tissues and peripheral blood leukocytes of patients with CD . MoreoveChronic NF-ĸB stimulation promotes cellular infiltration and mucosal inflammation by increasing the transcription of proinflammatory cytokines  and by degrading the intestinal barrier through increased apoptosis of intestinal epithelial cells Figure 3TGF-β, TNF-α, IL-2, IL-6, IL-8, MMP9, Intercellular Adhesion Molecule 1 (ICAM-1/CD54), STAT1, and STAT3)  system . To ensud STAT3) .6Oxidative/nitrosative stress is a significant pathophysiologic factor that plays a role in the initiation and progression of IBD. Overproduction of ROS is stimulated, and consequently, OS is triggered during inflammation because of the large number of cytokines and chemokines secreted by inflammatory cells. Considering this, therapeutic interventions incorporating substances possessing antioxidant and anti-inflammatory properties may be considered. Multiple antioxidant therapeutic strategies are being investigated because of the importance of OS in the pathophysiology of IBD to eliminate ROS, enhance AOE activities, and inhibit abnormal redox signaling Figure 26.1Flavonoids, phenolic acids, lignans, and stilbenes are examples of polyphenols, a family of phytochemicals found in many plant diets. A growing body of research has demonstrated that natural polyphenols effectively mitigate the severity of intestinal inflammation and OS in the early stages of IBD , 85–89. 6.1.1The polyphenol resveratrol  is found in grapes, soybeans, berries, nuts, and pomegranates, among other natural sources . In rodeMPO is responsible for tissue damage in IBD and is inhibited effectively by resveratrol and its derivatives . InhibitSIRT-1) gene and increases the activity of NF-κB. By activating SIRT-1 and down-regulating NF-κB activation, RSV plays a vital role in the regulation of inflammation that mediates colitis and colon cancer  encoding glutamate cysteine ligase, which is the rate-limiting enzyme in glutathione synthesis  levels and increased SOD and CAT activities in tissues , 39, 49.ynthesis . It has ynthesis . Oral deynthesis . Antioxiynthesis . Curcumiynthesis –158  tyrosine phosphorylation and the TLR4/MyD88/PI3K signaling pathways (++-independent NOS stimulated by cytokines (Quercetin (QCT), a member of the flavonols (a subclass of flavonoids), is a polyphenolic molecule found in plants and has demonstrated anti-inflammatory, antioxidant, and antitumoral effects. Several studies have shown that quercetin can suppress the expression of inflammatory mediators and cytokines such as COX-2, NO, NF-kB, prostaglandin E2 (PGE2), iNOS, TNF-α, IL-1β, and IL-6 that are generated through the LPS-TLR4 pathway  -response element is critical for the action of quercetin. Through its interaction with C/EBPβ, quercetin has the potential to inhibit the recruitment of co-activators. Quercetin also suppresses apolipoprotein B (apoB) expression by inhibiting the transcription of C/EBPβ -dependent signaling pathways . Epigallin vitro. It appears that the powerful antioxidative potential of EGCG is responsible for its anti-inflammatory effects in the DSS-murine model of colitis. Lipid peroxidation occurs during IBD inflammation when ROS are not neutralized, altering the permeability and selectivity of the cell membrane and the activity of transmembrane transporters, receptors, and enzymes (The combination of EGCG and piperine (piperine for enhancing the bioavailability of EGCG) significantly decreased weight loss, improved the clinical course, and increased overall survival in the DSS-murine model of colitis . Reduced enzymes . DSS-col enzymes   have been studied extensively , 178–181in vivo and in vitro models. FA-97 activates Nrf2 and promotes its nuclear translocation to increase expression of its downstream target proteins HO-1 and NAD(P)H:quinone oxidoreductase (NQO-1), which reduces ROS. It also inhibits the NF-κB and AP-1 signaling to suppress the expression of pro-inflammatory cytokines IL-1, IL-6, TNF-α, and IL-12 (FA-97 (caffeic acid phenethyl ester 4-O-glucoside) is a new synthetic CAPE derivative shown to attenuate body weight loss, colon length shortening, increased colonic inflammatory cell infiltration, and pro-inflammatory cytokine production . While Fnd IL-12 , 99.6.1.6The flavonoid compound luteolin, found in various plant extracts, has been shown to have anti-inflammatory, antioxidant, anti-metastasis, and apoptosis-inducing properties. Luteolin has been reported to reduce LPS-stimulated expression of NF-κB, TNF-α, and ICAM-1 and TBK1-kinase activity via the MyD88-independent signaling pathway . LuteoliLPS-induced inflammatory responses are controlled at the transcriptional level through the MAPK and NF-κB pathways , 184. To6.1.72O2- or LPS-treated IEC-6 cells (Xanthohumol (XN), a compound derived from hop plants, is a prenylated chalcone with, antioxidant, anti-cancer, and anti-inflammatory activities via inhibition of TLR4/MD-2 complex . Downstr-6 cells .2O2- or LPS-treated IEC-6 injury, possibly by the interaction between the α, β-unsaturated carbonyl moiety of XN and Cys99 in IKKβ, and thus by its ability to inhibit the IKKβ/NF-κB signaling pathway. Additionally, XN inhibited the activation of the canonical NF-κB pathway, as evidenced by the downregulation of NF-κB target genes, like A1a, A20, Bcl-xL, and c-myc  is composed of approximately 90% proanthocyanidins, including oligomers (74.8%), dimers (6.6%), trimers (5.0%) and tetramers (2.9%) . InhibitNutritional and pharmacological dosages of proanthocyanidins protect against endotoxin (LPS)-induced intestinal inflammation, OS, and intestinal permeability in colitic rats . Notably6.1.11in vitro. In murine colitis, anthocyanins extracted from the Chinese plant Dioscorea alata L  and the oxidative DNA-damage marker 8-hydroxydeoxyguanosine (8-HdG) . In anot6.1.12Silybum marianum), has been shown to have anti-inflammatory and antioxidant properties. Silymarin decreases inflammation by inhibiting NF-κB pathways and by optimizing the redox balance in the cell through activating AOEs and non-enzymatic antioxidants via Nrf2 activation (Silymarin (SM), an extract from milk thistle , a monoterpene phenol derivative of cymene . Thymol Along with improving barrier function, thymol can decrease the production of ROS and the expression of pro-inflammatory cytokines following stimulation with LPS . Thymol 6.26.2.1Berberis aristata, has been used for decades to treat intestinal parasites and enteropathogenic diarrhea due to its bactericidal activity, its ability to inhibit protozoan growth, and also to inhibit enterotoxin-induced intestinal electrolyte secretion , a nanofiber extracted from tamarind, is a novel antioxidant that prevents DSS-induced colitis in mice , 125. TX6.46.4.1Sulforaphane, found in cruciferous vegetables, contains a wide range of antibacterial, antioxidant, anti-inflammatory, and immunomodulatory properties , 226. Tr6.56.5.1Flaxseed oil is an herbal product with a high α-linolenic acid content, and it has been shown to reduce colonic damage in DSS-induced colitis by modulating inflammatory factors, oxidative state, and the cecal microbiota imbalances. DSS-colitic rat colon showed low SOD activity and GSH levels, increased MPO activity and MDA levels, and flaxseed oil dose-dependently alleviated this condition. Compared to the DSS-treated group, flaxseed oil treatment for six weeks raised SOD activity and GSH levels while decreasing MDA levels and MPO activity  Supple.6.5.2Ginger rhizomes, a rich source of many active compounds, including gingerols, shogaols, gingediols, zingerone, dehydrozingerone, gingerinone, and diarylheptanoids, have a wide range of anti-inflammatory, analgesic, antioxidant, and anti-cancer effects , 229. S-In mice with DSS-induced colitis, ginger alleviates the pathological lesions and reduces the expression of IL-6 and iNOS . This stGinger extract exhibited the antioxidant effects in IL-1β-mediated OS in human C28I2 chondrocyte cells by stimulating the expression of several AOEs, decreasing IL-1β-induced ROS production, LPx, and by reducing apoptosis   Supple.6.6There is growing recognition that the endocrine system plays an important role in the pathogenesis of IBD through various mechanisms . HormoneOne study involving 1,203 females  reported increased symptoms during their menstrual cycle. Symptoms were comparable among CD and UC cohorts except for pregnant women, where symptoms worsened . Women w6.6.1Estrogens have been shown to increase AOEs by upregulating their expression , 245. ThIncreased expression of tight junction proteins was a mechanism by which progesterone and estrogen facilitated wound healing and epithelial barrier function in intestinal epithelial cells, as reported using 2D cell lines and IBD patient-derived inflammatory organoid models . These s6.6.2Glucagon decreased iNOS expression and plasma levels of nitrite/nitrate in LPS-treated rats . Glucago6.6.3Glucagon-like peptide-1 (GLP-1) and GLP-2 are important enterohormones that mediate local and systemic effects (gut-brain-periphery axis), contribute to glucose homeostasis, and modulate GI functions such as the intestinal absorption of lipids and antioxidant defense . The effActivation of the GLP-1 receptor has anti-inflammatory, antioxidant, and anti-apoptotic effects, which include a reduction of the pro-inflammatory actions of advanced glycation end products  and of tThe enterotrophic peptide hormone glucagon-like peptide 2 (GLP-2) was shown to abrogate OS and improve intestinal antioxidant capacity by reducing LPS-induced increases in intestinal IL-1β and oxidized glutathione levels  and incrGiven the effects of GLP-1 and GLP-2 in maintaining intestinal homeostasis, including cross-talk with the immune and central nervous systems , 274, thReduced levels of serum motilin and gastrin, together with lower pro-inflammatory cytokines and reduced tissue NF-κB and COX-2 levels, and increased serum somatostatin, vasoactive intestinal peptide, and tissue SOD, NO, and MDA levels were associated with genistein- and daidzein-rich fermented soy (shuidouchi) in an experimental animal model . Similar6.6.4The presence of glucocorticoid receptors in endothelial and inflammatory cells –243, as 6.6.5Thyroid hormones are associated with the oxidative and antioxidative status of an organism because of their role in regulating oxidative metabolism and in the formation of ROS –55, 281.6.6.6A study was conducted to investigate the impact of administering Corticotropin releasing hormone (CRH) to induce psychosocial stress in DSS-treated colitic mice, specifically by examining the potential enhancement of autophagy in intestinal macrophages. The inflammatory challenges associated with IBD led to increased autophagy in both intestinal macrophages and murine bone marrow-derived macrophages, and these effects were further increased by CRH .6.6.7IBD is distinguished by symptoms such as reduced appetite, inadequate nutrition, changes in body composition, and the enlargement of mesenteric white adipose tissue (mWAT) , 286. Ad6.6.7.1Leptin serves as a regulator of diverse immune and inflammatory reactions in addition to its metabolic and endocrine roles. Leptin has the ability to initiate activation and alter the pattern of cytokine production, favoring a Th1 response by promoting the release of IL-2 and IFN-γ while inhibiting the secretion of IL-4 . Additio6.6.7.2in vitro studies have shown that IL-6 reduces adiponectin levels  in the intestinal epithelial cells, macrophages, and T cells of mice with experimental IBD demonstrates immunoregulatory effects and potentially plays a role in the prevention of intestinal inflammation , 304. Th6.6.9-), RO2•, and singlet oxygen, and decreased expression of COX-2 and iNOS and NF-kB activation are all antioxidant effects of melatonin  has several unique scavenging effects making it an exceptionally potent direct free radical scavenger and multifunctional antioxidant , 309. Meelatonin , 319, pol damage . Animal amage (•  SuppleSupple-, amage (• .By modulating autophagy and Nrf2 signaling pathways, melatonin slows the progression of colitis-associated colon cancer in mice. In this colon cancer model, melatonin decreased the levels of inflammatory markers IL-6, IL-17, TNF-α, NF-κB, COX-2 and STAT3, as well as reduced DNA damage and OS indicated by reduced TBARS and increased GSH levels . Another7Oxidative stress plays a crucial role in the pathogenesis of IBD and is closely associated with the development of intestinal inflammation in humans and animals. Therapeutic strategies involving natural antioxidant products have shown benefit in numerous human studies and animal models. An improved comprehension of the free radical biology mediating GI disease is essential for developing effective future treatments to reduce intestinal inflammation and improve the quality of life in affected individuals. Also, hormonal intervention holds potential importance in the context of IBD and cannot be disregarded. In this review, we have discussed the major antioxidant and anti-inflammatory properties of various phytochemicals and hormones in IBD. We have also discussed significant evidence-based observations, including the results from clinical trials using natural antioxidants or their modified formulations. Based on the existing scientific evidence, it appears likely that future therapies will include antioxidants with standard treatments or even as an alternative medical option in humans and animals with IBD.Writing—original draft preparation, revisions, figures, review, and editing DKS, Writing—review, and editing RMH, BP, AP, VKY, DW, AEJ. All authors contributed to the article and approved the submitted version.


Ferroptosis is a form of regulated cell death characterized by iron overload, overwhelming lipid peroxidation, and disruption of antioxidant systems. Emerging evidence suggests that ferroptosis is associated with pregnancy related diseases, such as spontaneous abortion, pre-eclampsia, gestational diabetes mellitus, intrahepatic cholestasis of pregnancy, and spontaneous preterm birth. According to these findings, inhibiting ferroptosis might be a potential option to treat pregnancy related diseases. This review summarizes the mechanisms and advances of ferroptosis, the pathogenic role of ferroptosis in pregnancy related diseases and the potential medicines for its treatment. Cells are the fundamental organizing unit of life. Cell death, is therefore of critical importance in diverse aspects of mammalian development and homeostasis. Ferroptosis is a form of regulated cell death, coined in 2012. Generally, it is mainly characterized by iron overload, overwhelming lipid peroxidation, and disruption of antioxidant systems, particularly depletion of glutathione peroxidase 4 (GPX4) . MountinMammalian lipid bilayers consist of up to 62% of unsaturated fatty acids of which 35% are polyunsaturated fatty acids (PUFAs) . However2+ and H2O2 (Fenton reaction), subtract hydrogen from lipid to form lipid radicals (L•) , which then interacts with adjacent PUFAs to form lipid hydroperoxide (LOOH), and many electrophilic species such as malondialdehyde (MDA), and 4-hydroxynonenal (4HNE) (The non-enzymatic phospholipid (PL) autoxidation is iron-dependent lipid peroxidation. Hydroxyl radicals, produced by the interaction of Feals (L•) . After tl (4HNE) .in vivo model of acute renal failure, 12/15- LOX deletion cannot eliminate the cell death of GPX4 knockout mouse , a dioxygenase containing non-heme iron, has six isoforms in humans: 15-LOX-1, 15-LOX-2, 12-LOX-1, 12-LOX-2, E3-LOX, and 5-LOX . Althougut mouse . Therefout mouse . Similarut mouse . POR, idut mouse .2+ acts as a cofactor of LOXs or prolyl hydroxylase , which can be recognized by transferrin receptor-1 (TfR1) in the cell membrane. And then, Fe3+ endocytosis in endosomes, where it is reduced to Fe2+ by six-transmembrane epithelial antigens of the prostate 3 (STEAP3). Finally, Fe2+ is transported to the cytosolic labile iron pool via divalent metal transporter 1 (DMT1)  through the Fenton reaction . What’s roxylase , which aroxylase . Consequ1 (DMT1) . Fe2+ alroptosis . It can roptosis . But ferroptosis . Ferroporoptosis , resistiThe SLC7A11-GSH-GPX4 axis is a classical signaling pathway of ferroptosis . GlutathThe ferroptosis suppressor protein 1 (FSP1)–NAD(P)H–coenzyme Q10 (CoQ10) pathway, acting in parallel to the SLC7A11-GSH-GPX4 pathway, is a potent suppressor of lipid peroxidation and ferroptosis . FSP1, kFerroptosis is associated with a great number of diseases. Multiple genes and signaling pathways, associated with lipid and iron metabolism, and antioxidant systems, play a role in inhibiting ferroptosis, providing new potential therapeutic drugs for these diseases .PUFAs, substrates of lipid peroxidation, are responsible for ferroptosis. Selectively bis-allylic deuterated PUFA, suppressing ferroptosis induced by RSL3 and erastin , is a pr2+ overload in intracellular iron pools, like endoplasmic reticulum (ER), may trigger ferroptosis H–CoQ10 pathway, acting in parallel to the GPX4 axis, is a powerful antioxidant system in membrane structures . Idebeno2+ overload was a key mediator of ferroptosis , a ferroptosis inhibitor, decreased the mortality rate of trophoblasts  and high glucose (HG). Furthermore, it was found that HL and HG can induce GDM in pregnant rats, leading to the damage of rats’ placenta  is common during pregnancy and is increasing in prevalence globally . The incplacenta . The expplacenta . Consequvia the Fenton reaction. As a result, oxidative damage leads to the injury and ferroptosis of pancreatic β–cell in GDM  is a complication, most occurs in the third trimester, in 0.3%–15% of pregnancies in various populations . It is cExcessive ferroptosis occurred in spontaneous abortion rat model with low levels of GSH, GPX4 and increased levels of TFR1, ACSL4 and MDA . Some evvia significantly increasing GSH levels (2D3 is related to PRDs, which may result from ferroptosis, such as spontaneous abortion, GDM and PE (Trophoblast ferroptosis may provide a useful therapeutic target for pregnancy-related diseases. Quercetin, as an antioxidant, can significantly promote trophoblast invasion during early pregnancy H levels . AdditioH levels . Iron chH levels . SimilarH levels . ThiazolH levels . A recenH levels , indicatH levels . Thus, SH levels . CoQ10 iH levels . FurtherH levels . TherefoM and PE . VitaminM and PE . CertainFerroptosis is a form of regulated cell death involving lipid metabolism, iron metabolism and antioxidant system, regulated by multiple genes and signaling pathways. PRDs are mainly associated with placenta dysfunction due to trophoblasts injury and death. Recently, an increasing number of experimental studies are exploring role of ferroptosis in PRDs in order to provide new potential therapeutic drugs and therapeutic targets for it. However, there are numerous problems that have not been elucidated on the association between ferroptosis and pregnancy related diseases. Firstly, the exact molecular mechanism of transplacental iron transport is not clear, though much work has been done on it. Secondly, ferroptosis is a form of cell death that is associated with lots of signaling pathways, like hypoxia signaling , AMP-act


CβS, MTHFR, NOS3, ACE1, CYBA, HAP, ACP1, GSTT1, GSTM1, and CYP1A1. Notably, the ACE1 gene emerges as a prominent factor with the presence of the I allele offering protection against HPV infection. The association of NOS3 with HPV infection is perceived with the 4a allele showing a protective effect. The presence of the GSTT1 null mutant correlates with increased susceptibility to HPV infection, HSIL and ICC, and ICC. This study also uncovers intriguing epistatic interactions among some of the genes that further accentuate their roles in disease modulation. Indeed, the epistatic interactions between the BB genotype (ACP1) and DD genotype (ECA1) were shown to increase the risk of HPV infection, and the interaction between BB (ACP1) and 0.0 (GSTT1) was associated with HPV infection and cervical lesions. These findings underscore the pivotal role of four oxidative stress-related genes in HPV-associated cervical lesions and cancer development, enriching our clinical understanding of the genetic influences on disease manifestation. The awareness of these genetic variations holds potential clinical implications.Human papillomavirus (HPV) infection is a necessary but not sufficient factor for the development of invasive cervical cancer (ICC) and high-grade intraepithelial lesion (HSIL). Oxidative stress is known to play a crucial role in HPV infection and carcinogenesis. In this study, we comprehensively investigate the modulation of HPV infection, HSIL and ICC, and ICC through an exploration of oxidative stress-related genes:  Cervical carcinoma, a malignancy of the uterus, accounted for approximately 3.1% of all malignant tumors in 2020, ranking as the fourth most prevalent cancer among women and posing a significant global public health challenge . FactorsCβS 844ins68 allele has been observed to normalize homocysteine and folate levels in individuals with the MTFHR C677T variation [Cystathionine beta-synthase (CβS) and methylenetetrahydrofolate reductase (MTFHR) are pivotal enzymes in homocysteine metabolism. Homocysteine, an antioxidant and redox regulator, is associated with aminothiol profiles that are indicative of oxidative stress-related pathologies . CβS medariation .NOS3 gene have been found, one of the most important with clinical importance and associated with variations in the NO levels is the variable number of a 27 bp tandem repeat (VNTR) in intron 4 [NOS3 post-transcriptionally by altering the formation of a small interfering RNA (siRNA). In vitro studies have shown higher siRNA levels in endothelial cells containing five copies (4b), determining lower NOS3 mRNA levels [Nitric oxide synthase (NOS) converts L-arginine into L-citrulline to produce nitric oxide (NO), a central player in reactive nitrogen species (RNS) regulation. NO’s interactions with unpaired electrons generate ROS, such as peroxide nitrite (ONOO–), and its oxidation products may lead to cellular toxicity . Crosstaintron 4 . There aA levels .ACE I/D-variation consists of the insertion or deletion (I/D) of a 287 bp fragment in intron 16. The D allele has been associated with higher ACE activity, accounting for 47% of the total phenotypic variance of serum enzyme levels [Angiotensin-converting enzyme (ACE1) plays a role in oxidative stress by mediating the production of angiotensin II, a potent oxidative stress mediator. Angiotensin II activates membrane NAD(P)H oxidases in vascular smooth muscle cells to produce ROS, such as superoxide and hydrogen peroxide, which are then involved in pleiotropic effects . ACE I/DCYBA gene causes a functional nonconservative substitution from histidine-72 to a tyrosine residue that decreases its activity [The NOX enzyme is a membrane-associated, multi-protein that produce O2- for host defense and other functions . The genactivity .2O2, and heme, per se, can act as a pro-oxidant [Hp1 and Hp2), and the way their gene products interact produces three main phenotypes of haptoglobin—Hp1.1, Hp2.1, and Hp2.2—which may have an impact on the strength of the interaction with hemoglobin; Hp 2.2 is biologically less-effective in binding free hemoglobin [Although haptoglobin is a late acute-phase protein of inflammation, it also binds to free hemoglobin released during intravascular hemolysis. Free hemoglobin can catalyze OH formation from H-oxidant ,34. Two moglobin .GSTM1/T1 results in loss of enzyme activity [Glutathione S-transferases (GSTs) play a vital role in detoxifying electrophilic compounds by generating free radicals. The genetic variation in the genes encoding GST enzymes is, therefore, crucial to regulating oxidative stress. The null genotype of activity .ACP1) is a gene that encodes a low molecular weight phosphotyrosine phosphatase (LMW-PTP), which presents two main enzymatic activities: phosphoprotein tyrosine phosphatase and flavin mononucleotide phosphatase. The ACP1 genotype was found to directly correlate to glutathione reductase activity [ACP1: ACP1*A, ACP1*B, and ACP1*C. The ACP1 alleles differ on single-nucleotide polymorphisms (SNPs), which affect both the total enzymatic activity and the ratio between isoforms F/S [Acid phosphatase locus 1 . DNA extracts were obtained between 1996 and 2003 and are now categorized as archived samples.A total of 308 women infected with HPV , and 44 with low-grade squamous intraepithelial lesions (LSIL)) from the Portuguese Oncological Institutes in Lisbon and Oporto were included in this study. All women had cytologic and histopathologic analyses performed. HPV detection was performed using polymerase chain reaction  variation, was performed. The amplicons were visualized in an agarose gel with 420 bp representing the allele with an additional VNTR (b) repeat and 393 bp representing the allele without an additional VNTR repeat (a).ACE1 Genotyping—rs4646994An amplification with PCR, flanking the I/D (insertion–deletion) variation, was performed. The amplicons were visualized in an agarose gel with 319 bp representing the allele with the deletion (D) and 597 bp representing the allele with the insertion (I).CYBA Genotyping—rs4673RsaI allowed for the facilitated genotyping: CC-348 bp; CT-348, 188, and 160 bp; TT-188 and 160 bp.An amplification with PCR, flanking the C-242T variation, was performed. An amplicon of 348 bp was visualized in an agarose gel. Subsequent restriction with HAP Genotyping—1/2Hap genotyping was determined using the Hp phenotype (Hp–Hb complexes) with PAGE electrophoresis and peroxidase staining that followed a modified version of Linke’s method (1984) .ACP1 Genotyping—A/B/CBsh136I and MspA1I facilitated genotyping: Bsh136I generated two fragments of 225 bp and 175 bp when alleles A or B, while MspA1I generated two fragments of 328 bp and 72 bp for alleles B and C.An amplification with PCR, flanking the A/B/C variation, was performed. An amplicon of 400 bp was visualized in an agarose gel. Subsequent restriction with GSTT1 and GSTM1 Genotyping—1/0GSTT1 gene, 230 bp represented the presence of the GSTM1 gene, and the 157 bp band served as the positive control for the amplification. A null allele was identified by the absence of the corresponding gene amplicon and the presence of the control amplicon.An amplification with multiplex PCR, flanking each gene, was performed. The amplicons were visualized in an agarose gel, where 480 bp represented the presence of the CYP1A1 Genotyping—rs4986884|rs1048943|rs1799814MspI facilitated genotyping: two fragments of 200 bp and 140 bp identified allele C, while the absence of restriction identified allele T. An amplification with PCR, flanking the A4889G (rs1048943) variation and C4887A (rs1799814), was performed. An amplicon of 204 bp was visualized in an agarose gel. Restriction with BsrDI identified two fragments of 149 and 55 bp for allele A (rs1048943), while the absence of restriction identified allele G. Restriction with BsaI identified two fragments of 139 and 65 bp for allele C (rs1799814), while the absence of restriction identified allele A.An amplification with PCR, flanking the T5639C (rs4986884) variation, was performed. An amplicon of 340 bp was visualized in an agarose gel. Subsequent restriction with p-value < 0.005 after correction for multiple testing using the Bonferroni adjustment.All statistical tests were conducted using SPSS 28.0 software. Group differences were assessed using Pearson’s chi-square or Fisher tests. When more than 20% of cells had an expected count inferior to 5, correction for chi-square was conducted using Fisher’s Exact test (2 × 2 tables) or the Monte Carlo simulation method for chi-square (other tables). Statistical significance was defined as a CβS, MTHFR, NOS3, ACE1, CYBA, HAP, GSTT1, GSTM1, ACP1, and CYP1A1).We commenced our analysis by investigating the association between HPV infection and ten distinct genes . Notably, due to the inability of our GSTM1/GSTT1 genotyping methodology to distinguish between 1/1 and 1/0, this gene was excluded from the codominant analysis.Among the genes studied, only NOS3 (p = 0.003) and ACE1 (p = 0.002) exhibited distinct allele distributions. In the case of NOS3, allele 4a demonstrated a protective factor . Regarding ACE1, allele I displayed a protective effect .For a clearer understanding of the risk or protective effects of each allele, we examined the allele distribution between the two populations whenever feasible . NotablyACP1, which comprises more than two possible alleles, two distinct analyses were conducted: one contrasted a single genotype against all others, and the other grouped the genotypes based on functional characteristics. The genotypes were aggregated according to fast/slow ratio ((AA and AB and BB) < (AC and BC)), fast isoform concentration ((AA and AC) < (AB and BC) < (BB)), slow isoform concentration ((AA and AB) < (BB and AC) < (BC)), or total activity ((BB and AB)/(AA and BC and AC)). These isoforms possess differing catalytic and molecular properties, potentially serving distinct cellular functions [In cases where feasible, we evaluated the genotype distribution using the dominant, overdominant, and recessive models . Notablyunctions ,46,47.ACE1 and GSTT1 genes. In the case of ACE1, the presence of allele I (II and ID) were associated with protection . Conversely, the GSTT1 null allele (0/0) was considered a risk factor .The genotype distributions varied for the GSTT1 in this disease context. Once again, the genotype of GSTT1 exhibited significant associations with ICC  and HSIL and ICC   or the most severe phenotypes, namely high-grade squamous intraepithelial lesion (HSIL) plus ICC. Analyzing the genotype distribution within the lesions further underscored the influence of 5–5.366) .We also tested for differences in the genotype distributions between LSIL, HSIL, and ICC, but found no significant results .ACP1(BB)-ACE1(DD)-epistatic interaction when compared to each individual genotype -ACP1(BB) compared to OR = 2.131, CI 1.373–3.309 for ACE1(DD) and no risk for ACP1(BB)). A similar trend was noted for the epistatic interaction ACP1(BB)-GSTT1(0.0) in HPV infection -GSTT1(0.0) compared to no risk for ACP1(BB) and OR = 2.691, CI 1.510–4.794 for GSTT1(0.0)). This last epistasis was also present when the analysis was applied to the population with the most severe lesions (HSIL and IC), in this case an OR = 9.455 (CI 2.615–34.187) was much higher than the risk of having only GSTT1(0.0) , while the BB genotype from ACP1 presented no risk alone.Subsequently, we delved into the most promising epistatic interactions, including those between homozygous genotypes or genotypes featuring the presence of one of the alleles . NotablyIn this study, our investigation revealed associations between HPV infection or cervical lesions and four of the ten candidate genes under consideration.ACE1, where the I allele emerged as a protective factor. The allele’s protective effect could be attributed to its connection with a lower ACE activity and subsequent reduction in ROS levels given that angiotensin II activates NAD(P)H oxidases [ACE1 expression in the tumor microenvironment compared to healthy tissues [The gene most consistently associated with HPV infection was oxidases ,28. Prevoxidases ,49. Rega tissues , a highe tissues , and a l tissues . AlthougNOS3 and HPV infection. Other studies have indicated that lower siRNA levels in endothelial cells carrying five copies (4a) result in elevated NOS3 mRNA levels [NOS3 that favors NO production may be an advantage for HPV clearance. Our findings align with this concept as the 4a variation emerged as a protective factor against HPV infection, suggesting that genetic variations favoring NO production may aid HPV clearance. However, the role of NO in carcinogenesis is complex, as its consequences are dose-dependent and time-sensitive. While low NO levels promote cell proliferation and anti-apoptotic responses, high levels induce cycle arrest, apoptosis, and senescence through oxidative and nitrosative stresses [NOS3 with cancer, phytochemical-induced apoptosis has been linked to the NO signaling pathway activation in cervical cancer [Our study also revealed an association between A levels . As a viA levels . NotablyA levels . In addiA levels ,57,58. NA levels ,59,60,61stresses ,62. Althl cancer .GST genes, the corresponding proteins play a crucial role in the mechanisms against oxidative stress. The homeostasis of a powerful antioxidant, glutathione, is essentially regulated by GST activity. In addition, GST proteins are responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. GST-null genotypes result in the loss of the enzyme’s ability to bind genotoxic substrates, leading to a decreased detoxification and an increased tumorigenesis [GSTT1-null genotype with HPV, lesions, and cancer. Also, de Carvalho et al. [GSTT1-null genotype in uterine cervix adenocarcinoma, while the GSTM1-null variant was not implicated in this disease. However, meta-analysis studies revealed that the GSTM1-null variant caused cervix lesions, especially among HPV infection in Indian and Chinese populations [GSTM1 or double mutants with cervical cancer or precursor lesions; GST-null mutants have been reported to be associated with oncologic diseases in general, though [Concerning the igenesis ,65. Our o et al.  verifiedulations ,68. Alsoulations . Our res, though .CβS and MTHFR did not exhibited associations with HPV infection or major lesions. Meta-analyses, other reviews, and original papers have been describing associations of this MTHFR variant with several types of cancers, such as ovarian cancer, breast cancer, colorectal cancer, bladder cancer, lung cancer, childhood acute lymphoblastic leukemia [CβS, our bibliographic survey did not reveal any study of this gene in HPV infection, cervical cancer, or precursor lesions. Nevertheless, this variant has been associated with breast cancer and exhibits altered expression in various cancer forms [Both leukemia ,74,75,76leukemia ,79,80,81leukemia ,83,84,85er forms ,87.ACP1 gene’s relevance. However, the results presented in this work are puzzling since no relevant result concerning functionality was achieved, and the BB genotype was found as a risk factor for HPV infection and severe lesions if in epistasis. Notably, the BB genotype in epistasis with DD (from ACE1) or 0.0 (from GSTT1) indicated risks for these phenotypes. This underscores the interplay of genetic backgrounds in disease contribution. Although lacking prior associations with HPV infection or cervical lesions, the ACP1 genetic variation has been linked to other cancer types [Our investigation highlighted the er types ,89. Curier types .CYBA gene revealed no association with HPV infection, cervical lesions, or cancer. However, CYBA’s involvement has been previously verified in cervical cancer [The l cancer , and thel cancer . This val cancer ,94.HAP was not implicated in tumorigenesis or HPV infection. However, it is known that Haptoglobin’s role in binding free hemoglobin contributes to oxidative damage prevention [Hp 1 allele, particularly in homozygosity, had an increased risk of developing invasive neoplastic lesions. A study carried out at the University of Ghana also obtained similar results, concluding that individuals with the Hp 2.2 phenotype had a lower risk of developing cervical cancer compared to Hp 1.1 individuals [In our study, evention . Regardievention , Mahmud evention  reportedividuals . There iividuals . Nowadayividuals .CYP1A1 gene. There is at least one study able to associate CYP1A1 with susceptibility to HPV infection [Our investigation did not reveal significant associations for the nfection , and allnfection ,107,108.The discrepancies between our study and others could reflect unique genomic features of the Portuguese population. Being one of the oldest countries in Europe, Portugal identity results from the combination of the histories of Iberian tribes, Celtic peoples, the Roman Empire, Germanic kingdoms, Muslim invasions, Christian reconquer, exploration of the “New World”, and emigration and immigration fluxes. Indeed, the Portuguese population has a history of miscegenation with other populations, resulting in a unique genomic background.As limitations to this work, we consider the lack of information on HPV genotyping, sexually transmitted diseases , diet on folic acid, tabaco (immune suppressor), sex hormone intake, and medication with ACE inhibitors. These factors could potentially act as confounders, though gathering such information from archival samples is challenging.In conclusion, our study underscores the importance of four oxidative stress-related genes in HPV infection, cervical lesions, and invasive cervical cancer. While governmental vaccine programs are mitigating this issue in some regions, underdeveloped countries still grapple with HPV-related public health challenges. Further research, especially focusing on functional aspects, will be essential to unravel the molecular mechanisms involved. The clinical significance of these genetic variations should not be overlooked, as they contribute to disease susceptibility in this context.


Bph15 gene, known for its ability to confer resistance to the brown planthopper , has been extensively employed in rice breeding. However, the molecular mechanism by which Bph15 provides resistance against BPH in rice remains poorly understood. In this study, we reported that the transcription factor OsWRKY71 was highly responsive to BPH infestation and exhibited early-induced expression in Bph15-NIL (near-isogenic line) plants, and OsWRKY71 was localized in the nucleus of rice protoplasts. The knockout of OsWRKY71 in the Bph15-NIL background by CRISPR-Cas9 technology resulted in an impaired Bph15-mediated resistance against BPH. Transcriptome analysis revealed that the transcript profiles responsive to BPH differed between the wrky71 mutant and Bph15-NIL, and the knockout of OsWRKY71 altered the expression of defense genes. Subsequent quantitative RT-PCR analysis identified three genes, namely sesquiterpene synthase OsSTPS2, EXO70 family gene OsEXO70J1, and disease resistance gene RGA2, which might participate in BPH resistance conferred by OsWRKY71 in Bph15-NIL plants. Our investigation demonstrated the pivotal involvement of OsWRKY71 in Bph15-mediated resistance and provided new insights into the rice defense mechanisms against BPH.The  Nilaparvata lugens Stål) is a major rice (Oryza sativa) pest and a representative piercing-sucking insect. The most effective strategy for managing BPH is the cultivation of insect-resistant rice varieties (Bph14 encodes a nucleotide-binding and leucine-rich repeat (NLR) protein, which interacts with OsWRKY46 and OsWRKY72, ultimately leading to the activation of downstream defensive genes in rice (Bph6 encodes a unique leucine-rich repeat (LRR) protein that is specifically localized within the exocyst complex and exhibits interactions with OsEXO70E1 and OsEXO70H3. This interaction promotes protein secretion and strengthens the plant cell wall , encodes three lectin receptor kinases (OsLecRK1-OsLecRK3) that are located on the plasma membrane and function as receptors in plant immunity , thereby initiating pattern-triggered immunity (PTI). However, the precise molecular mechanisms underlying the functions of Bph15 and Bph3 remain poorly elucidated.The brown planthopper  (OsWRKY70 in response to BPH attack has been demonstrated to result in a reduction in gibberellic acid (GA) production in rice, leading to an increased susceptibility to BPH  represent a substantial assemblage of TFs, characterized by a discernible affinity for the W-box sequence situated within the promoter regions of their respective target genes . The infOsWRKY89 . However the BPH . Additioer (SSB) . The acty to BPH . In geneOsWRKY28, OsWRKY62, OsWRKY71, and OsWRKY76 are encompassed  in rice  was found to possess the resistance gene Bph15 in the Taichung Native 1 (TN1) background. This particular line, Bph15-NIL, was employed as a control in the study due to its demonstrated resistance. In contrast, the varieties TN1 and Nipponbare (NIP) were selected as susceptible rice controls. The BPH insects, which were reared on the TN1 variety, were utilized for plant feeding during the research conducted at Wuhan University in China.As previously stated by wrky71 mutants were generated using CRISPR-Cas9 technology  genotyping technology, and these plants were subsequently cultivated for bioassay purposes  fragment of OsWRKY71 (accession no. AY676927) was amplified from Bph15-NIL  were introduced to infest the seedlings. The plant condition was assessed and assigned a resistance score ranging from 0 to 9, with 9 indicating the highest susceptibility and 0 indicating the highest resistance level, as explained in previous research .The assessment of BPH insect performance on rice encompassed the utilization of assays to quantify BPH weight gain and weight gain rate. BPH was introduced to seedlings at the four-leaf stage after the planting of each line’s seeds in a plastic cup with a diameter of 10 cm. Newly emerged female BPH insects were weighed and enclosed in parafilm sachets, which were then affixed to the leaf sheath of a seedling at the four-leaf stage. The BPH instars were allowed to feed on the rice crops for 48 hours. After this feeding period, each insect was removed from the sachet and reweighed. The BPH weight gain was determined by measuring the difference between the initial and final weights of the BPH insect. Furthermore, we computed the weight gain rate, which is determined by dividing the weight gained by the insect’s initial weight before feeding. For each cultivar or line, a minimum of three replicates, each containing 10 insects, were utilized.Bph15-NIL and wrky71 mutant wrky71-5-9 were harvested for 0 (uninfested control), 3, and 24 HAI (hours after BPH infestation). All treatments were terminated at the same time, as previously described .Stems of three-leaf stage escribed . The Bphhttps://www.omicstudio.cn/tool.Using the Illumina paired-end RNA-seq technique, transcriptome sequencing was performed, resulting in a total of 305 million paired-end reads with a length of 2×150 bp. Following the application of Cutadapt, the reads underwent filtration and purification, resulting in 42 Gbp of reads with high quality. Subsequently, the cleaned reads were aligned to the rice reference genome using HISAT2. The estimation of transcript expression levels was conducted using the StringTie and Ballgown software, and the expression abundance was determined by calculating the FPKM values. The DESeq2 software was employed for performing differential expression analysis between distinct groups. Genes displaying a p-value below 0.05 and an absolute fold change exceeding 2 were categorized as differentially expressed genes (DGEs). Furthermore, the DGEs underwent subsequent analysis to ascertain the enrichment of functions associated with Gene Ontology (GO) and KEGG pathways. The bioinformatics analysis was carried out utilizing the OmicStudio tools accessible at Bph15-NIL, wrky71-5-9, and TN1 were collected after BPH infestation for 0, 3, 6, 12, 24, and 48 HAI. All treatments were terminated simultaneously. The tissue expression profile samples included different rice tissues: stem and leaf of 10 days seedling; stem, sheath, and leaf at the heading stage; stem, sheath, and leaf at 7 days after pollination. The TRIzol reagent (Invitrogen) was employed for the extraction of total RNA. Subsequently, the PrimeScript RT Reagent Kit (Takara) was utilized to perform reverse transcription of 1 μg of total RNA from each sample into cDNA. The qRT-PCR analysis was performed using the QuantStudio™ 5 (Applied Biosystems) and gene-specific primers .OsWRKY71 increased in response to BPH feeding in the Bph15 introgression line . The expression level of OsWRKY71 in the Bph15-NIL was significantly higher than in TN1 at 0, 3, and 6 HAI, but this expression pattern was reversed at 12 and 24 HAI  among seven comparisons: R0_S0, R3_S3, R24_S24, R0_R3, R0_R24, S0_S3, and S0_S24  response, jasmonic acid (JA) response, gibberellin metabolic, bacterium defense, virus defense, and herbivore response  terms identified in this study suggest that the response to BPH feeding in both the NIL rice Figure 5wrky71 mutant and Bph15-NIL plants upon exposure to BPH infestation. Notably, these pathways encompassed plant-pathogen interaction, plant hormone signal, autophagy, et al.  and KD (sensitive to BPH), and its involvement in BPH antixenosis resistance , a member of the EXO70 family, which exhibited significant up-regulation in the R0_R3 and R0_R24 comparisons, but down-regulation in the R3_S3 comparison  (LOC_Os11g37540), Pigm (LOC_Os06g17900), and RGA2 (LOC_Os04g30530), was observed in the R_S comparisons  in all three rice lines. However, the expression levels of these genes were higher in the wrky71 mutant compared to Bph15-NIL plants  in the activation of PTI for plant immunity   . In thisckground Figure 7ell wall . NotablyBph6, Bph9, and Bph14   , therebyd Bph15)  to gain ical map  could beOsWRKY71 plays a critical role in the Bph15-mediated BPH resistance in rice. This investigation significantly enhances our comprehension of the molecular mechanisms underlying Bph15-mediated BPH resistance. Further investigations should be conducted to successfully clone and identify all resistance genes in the Bph15 locus.In summary, the most significant finding to emerge from this study is that https://ngdc.cncb.ac.cn) with the accession number CRA011634 (https://ngdc.cncb.ac.cn/gsa/browse/CRA011634). The raw sequence files were deposited in the GSA database of the National Genomics Data Center (XL: Investigation, Writing – original draft, Writing – review & editing. JZ: Investigation, Writing – original draft, Writing – review & editing. XS: Investigation, Writing – review & editing. JY: Investigation, Writing – review & editing. LZ: Investigation, Writing – review & editing. JH: Investigation, Writing – review & editing. BD: Investigation, Writing – review & editing. WL: Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.