The action mechanisms of these natural products are also presented and are wildly varied being apoptosis the most prevalent followed by cell cycle impairment, ROS production, autophagy, necroptosis, and others. The studies reported here confirm the antitumor properties of monoterpenes and their anticancer potential against various types of tumors, as demonstrated in in vitro and in vivo studies using various types of cancer cells and tumors in animal models. The data described serve as a reference for the advancement in the mechanistic studies of these compounds and in the preparation of synthetic derivatives or analogues with a better antitumor profile.Monoterpenes are a group of natural products that have been widely studied due to their therapeutic potential against various pathologies. These compounds are abundant in the chemical composition of essential oils. Cancer is a term that covers more than 100 different types of malignant diseases and is among the leading causes of death in the world. Therefore, the search for new pharmacotherapeutic options applicable to cancer is urgent. In this review, studies on the antitumor activity of monoterpenes found in essential oils were selected, and botanical, chemical, and pharmacological aspects were discussed. The most investigated monoterpenes were carvacrol and linalool with highly significant  Cancer is a leading cause of death worldwide and so an important public health issue. Aging, population growth, and risk factors related to socioeconomic development are the main causes of cancer boost. In 2020, it was estimated that there were 19.3 million new cancer cases and nearly 10.0 million cancer deaths, with female breast, lung, colorectal, prostate, and stomach cancers topping the incidence list [Taxus baccata and the analogs docetaxel (Taxotere) and cabazitaxel (Jevtana) in addition to vincristine and vinblastine (Velban) derived from Catharanthus roseus and their analogues vindesine (Eldisine) and vinorelbine (Navelbine) [Zornia brasiliensis and Guatteria friesiana (W. A. Rodrigues), plants used in folk medicine in Brazil and Colombia, have terpenoids as its main components and are highly cytotoxic and inhibit tumor growth from several types of cancer both in vitro and in vivo [Croton regelianus Muell. Arg., a native plant from Northeast Brazil used in folk medicine, has ascaridole, a monoterpene, as one of the main constituents with high antitumor activity against leukemia, glioblastoma, and sarcomas in mice models [α-pinene, (-)-β-pinene, and limonene from essential oils from Piper rivinoides Kunth were active against tumor cell lines of oral squamous cell carcinoma (SCC9 and SCC25), showing greater selectivity in relation to carboplatin, the drug of choice in the clinic [Several drugs used in cancer treatment are natural products or derivatives of them, such as taxol, isolated from velbine) , 4. In avelbine) , 6. Thusvelbine) . The mixvelbine) –11. The  in vivo , 13. The in vivo , 14. Esse models . Furthere clinic . Thus, aThe present review was based in data search performed in the scientific literature database PubMed, the most accessed for biomedical  literature worldwide. The eligibility criteria and selection of the studies were based on the following keywords: monoterpene, essential oils, antitumoral, anticancer, and cytotoxicity, and covered a period from January 2014 to December 2020, that way, performing an update on our previous review published in 2014 [in vitro and in vivo antitumoral role of essential oil monoterpenes was based on studies indexed in the biomedical database PubMed and as described in Method.This discussion focused on the Chemical structures and names of antitumor monoterpenes are provided in 1,8-Cineole demonstrated promising cytotoxic and proapoptotic properties, which showed cytotoxicity against A2780 cells comparable with that of doxorubicin, enhanced apoptosis in A2780 cell, and caused a dose-dependent increase in preG1 cell cycle events . Furtherα-Thujone has demonstrated several pharmacological effects, such as antitumor, analgesic, and insecticide. It was seen that α-thujone has not shown cytotoxicity to HCT116 and SW620 cells; however, it increased the proliferation of CD3AK immune cells and its cytotoxicity against cancer cells by improving the expression of CD107a, p-Akt, and p-ERK1/2 [α-thujone also exerted effect on GBM (glioblastoma) cells. The observed effects were induction of apoptosis, considerable inhibition of cell motility, induction of oxidative stress, and autophagy in α-thujone-treated tumor cells. Meanwhile, normal astrocytes showed lower sensitivity to α-thujone treatment [reatment .The combination of bornyl acetate with 5-fluorouracil potentiated the anticancer activity of 5-fluorouracil in human gastric cancer (SGC-7901) cells. The results of this combination were induction of apoptosis, DNA fragmentation, G2/M cell cycle arrest, cell morphology alterations, and potentiated effect of cell growth inhibition, indicating a synergistic enhancement in the anticancer activity of 5-fluorouracil .β-Pinene has demonstrated a cytotoxic effect against tumor cells. It was observed in human oral tongue cancer cells that (-)-β-pinene showed cytotoxicity, altered cell morphology, large amounts of pyknotic nuclei, membrane blebs, and cell shrinkage, and caspase inhibitors ZVAD (pancaspase) and ZDEVD (caspase-3) reduced cell death. In addition, (-)-β-pinene also showed selectivity in primary normal human gingival fibroblast [β-Pinene has also been evaluated in association with an already established drug, paclitaxel, in non-small-cell lung cancer cells. The results revealed that the combination of paclitaxel with β-pinene showed a substantial synergistic effect. Furthermore, paclitaxel+β-pinene showed morphological change characteristic of apoptosis like chromatin condensation and fragmentation of the nucleus [(-)-broblast . β-Pinen nucleus .Carvone is a monocyclic monoterpene that has been reported to have antioxidant, antimicrobial, anticonvulsant, and antitumor activities. Regarding the antioxidant effect, it was seen that carvone treatment increased levels of total antioxidant capacity in cultured primary rat neuron cells. In addition, it increased levels of total oxidative stress in primary rat neuron cultures and rat brain NB cell line N2a. There was also a reduction in cell viability rates in both cell types .In addition to the antioxidant effect, carvone also showed antitumor activities. Carvone inhibited proliferation of MCF 7 and MDA MB 231 cells and inhibited the migration of these breast cancer cells. In addition, it induced apoptosis, as was seen by the fragmentation of nuclei and the presence of apoptotic bodies, and it also arrested MCF 7 cells in the S phase of the cell cycle, caused DNA damage and ROS production, and increased levels of p53, Bad, cleaved caspase-3, and cleaved PARP . CarvoneμM. Ye et al. demonstrated that it presented the antitumor capacity to induce cell cycle arrest, suppression of cell migration and invasion, and apoptosis with observation of nuclear fragmentation, chromatin condensation, splitting of the nucleus, increase of Bax and caspase-3, and decrease of Bcl-2 expressions [in vivo and in vitro by preventing the growth of lung cancer cells and inducing apoptosis through mechanisms involving autophagy. There was an increase in Bax and cleaved PARP during treatment, which may be related to induce death of lung cancer cells. Increases were also found in Atg-5, presuming that Atg5 overload may be partially involved in D-limonene-induced apoptosis [Limonene effects have recently been investigated on T24 human bladder cancer cells presenting an IC50 of 9 ressions . D-limonpoptosis .μM, increases intracellular ROS levels and the sub-G1 population, and decreases the migration and invasiveness potential of tumor cells [μM. The approach with this monoterpene significantly increased the Bax/Bcl-2 ratio, positively regulated the expression of p53, and activated caspase-dependent apoptosis by the activation of caspases-3 and -9 [It has recently been demonstrated that thymoquinone induces apoptosis of 786-O human renal carcinoma cells. In more details, it decreases cell viability in a concentration- and time-dependent manner with an IC50 of 3.8–12.9 or cells . Thymoqu3 and -9 .α-pinene is a natural compound that has demonstrated anticancer activity. The α-pinene inhibited liver cancer cell growth. In nude mice with hepatocellular carcinoma, Chk1 and Chk2 levels were upregulated, Cyclin B, CDC25, and CDK1 levels were downregulated, and the average tumor size was significantly smaller in mice treated with α-pinene. The average tumor was significantly lighter than the control, and histology examination of xenografts showed more slow-growing cells and dead cells. α-Pinene also caused marked accumulation of a G2/M population in BEL-7402 cells [α-pinene inhibited the cycle progression at G2 to M phase and markedly increased caspase-3-dependent apoptotic cell death [α-pinene exhibited growth inhibition, G2/M-phase cell cycle arrest, triggered oxidative stress, and induced apoptosis. The cell cycle arrest was associated with downregulated cyclin-dependent kinase 1 (CDK1) and miR-221 levels. There were also upregulated levels of CDKN1B/p27, γ-H2AX, phosphorylated ATM, phosphorylated Chk2, and phosphorylated p53 [α-pinene showed a substantial synergistic effect, showing consequences on cell cycle distributions of A549 cells, in which the percentage of sub-G0/G1-phase cells was decreased on the addition of α-pinene to paclitaxel, and the population of G0/G1 cells was increased presenting morphological change characteristic of apoptosis [α-Pinene also demonstrated protective effect against ultraviolet A-induced cellular damages in human skin epidermal keratinocytes (HaCaT cells). α-Pinene showed a probable antioxidant property, preventing UVA-induced cytotoxicity, generation of ROS, lipid peroxidation, and DNA stand breaks. Furthermore, α-pinene inhibited inflammatory mediators such as NF-κB, TNF-α, and IL-6 expression and also modulated nucleotide excision repair proteins via activation of p53 and p21 that prevent the formation of UVA-induced cyclobutane pyrimidine dimers [The 02 cells . In humall death . It was ated p53 . The compoptosis . α-Pinene dimers .in vivo through combined treatment of DOX with natural borneol [Natural borneol (NB) has been used as a promoter of drug absorption; thus, it was seen that borneol could potentiate the cellular uptake of bisdemethoxycurcumin (possible anticancer activity) . Besides borneol . The eff borneol .in vitro activity of camphene induced apoptosis by the intrinsic pathway in melanoma cells causing endoplasmic reticulum stress. Moreover, there were also release of Ca2þ together with calreticulin and HmgB1, upregulation of caspase-3 activity, and loss of mitochondrial membrane potential. Camphene was also evaluated in vivo, in male C57Bl/6 mice, inhibiting subcutaneous tumor growth of highly aggressive melanoma cells in a syngeneic model [Camphene is a bicyclic monoterpene and a component of essential oils derived from plants such as rosemary, turmeric, pine, and ginger that has high antioxidant, anti-inflammatory, and antimicrobial activity , 41. Theic model .Carvacrol, when tested on AGS (human gastric adenocarcinomas) and WS-1 , had toxic effects but was more effective in cancer cells. One possible mechanism was apoptosis induced by DNA damage . CarvacrCisplatin (CP) and carvacrol showed dose-dependent cytotoxicity and activated ERK1/2. The MEK inhibitor PD325901 suppressed ERK expression, increased cytotoxicity of carvacrol, increased viability of cells by modulating apoptosis, and increased microtubule-associated protein 1A/1B-light chain 3 beta expression in cisplatin treatment. Cotreatment with cisplatin and carvacrol increased the viability of cancer cells compared to treatment with CP, due to the suppression of apoptosis. MEK inhibition decreased cell viability, without causing changes in apoptosis. The carvacrol also increased cisplatin-induced expression of light chain 3 beta. It induced CP resistance in HeLa cells through ERK1/2-independent suppression of apoptosis and ERK1/2-dependent modulation of autophagy .In vivo, changes in body weight and oxidative stress index were found in plasma and stomach tissues of treated Wistar rats [Other effects were observed, as alteration of soluble factors in HCT-116 and HT-29  . Carvacrtar rats .Citral had an antiproliferative effect on several cancer cells. Apoptosis was observed in human stomach cancer cells ; in prosIn addition to apoptosis, citral also induced cytotoxicity by regulating several genes involved in signaling pathways, inhibiting metastases, inhibiting colony formation, and interrupting migration of colonies of cancer cells . FurtherStudies also have suggested that citral binds to MARK4, inhibiting its enzyme activity, which is associated with the cell cycle and, therefore, cancer. Moreover, it also inhibited proliferation of the breast cancer cell line MCF-7 . It was μg/mL and necroptosis was confirmed by an upregulation of TNF-α pathway and downregulation of caspase-3 and -8 activities. Besides, citronellol at a dose of 50 mg/kg inhibited 80% of subcutaneous tumor growth previously induced by intraperitoneal injection of NCI-H1299 in nude mice. For breast cancer, IC50 was found to be 35 and 80 μM, using two different tumor cell lines, and apoptosis was validated by the loss of cell viability, increase in ROS generation, altered mitochondrial membrane potential, enhanced DNA damage, and modulation of the expression of apoptotic proteins (inhibition of Bcl-2 with upregulation of Bax and caspase-9 and -7) in MCF-7 and MDA-MB-231 cells.Citronellol has been described with antitumor activity against lung  and breaμM [κB, reducing the expression of TNF-α, IL-1β, COX-2, and iNOS [μM and in vivo doses of 50 and 75 mmol G/kg, it inhibits the mevalonate pathway resulting in growth inhibition of A549 cells and in vivo subcutaneous tumor growth besides promotion of apoptosis [μM, geraniol induces apoptosis with the involvement of the mitochondrial pathway, observed by a decrease in Bcl-2 and increase in Bax staining and TUNEL-positive cells, as well as an increase in the mRNA levels of Bax, caspase-3 and -8, cytochrome C, and Fas and a decrease in the Bcl-2 gene [Qi et al. showed that geraniol and geranyl acetate induce apoptosis with upregulation of Bax and downregulation of Bcl-2 expressions, DNA damage, and cell cycle arrest on Colo-205 colon cancer cells, presenting an IC50 of 20 and 30 μM . Howeverand iNOS . On PC-3and iNOS . On lungpoptosis . On Ishil-2 gene .μM, it inhibits the viability of cells and induces cell cycle arrest and apoptosis with a decrease in the expression of p-PI3K, p-AKT, and Bcl-2 and a rise in the expression of Bax [in vitro and in vivo; moreover, it induces a reduction of 55% in mean xenograft subcutaneous tumor weight in a 200 mg/kg dose [μM, linalool induced a concentration of cells in the G1 phase and by cytokine array analysis observed a stimulation of IFN-γ, IL-13, IL-2, IL-21, IL-21R, IL-4, IL-6sR, and TNF-α secretion, suggesting it could also induce Th1 cellular immune response [μM, linalool induces cell cycle arrest facilitating the expression of p53, p21, p27, p16, and p18 [in vitro and in a solid tumor model, linalool appeared to be selectively cytotoxic towards tumor cells in contrast to conventional chemotherapeutic drug, presenting reduction in cell viability, tumor volume, tumor weight, and tumor cell count data; furthermore, it induces apoptosis and cell cycle arrest in tumor cells and extensive necrosis and reduced viable tissue mass in vivo [Linalool has been a major topic of investigation over the last years. Its acts against several types of cancer mainly through induction of cell cycle arrest and oxidative stress. On A549 lung adenocarcinoma cells, linalool suppresses cell growth through cell cycle arrest, and oxidative stress also acts on the mitochondrial membrane potential depolarization and inhibition of cell proliferation, besides preventing cell migration . On OECMn of Bax . On HCT /kg dose . On T-47response . On HepGresponse . On U937 and p18 . On sarc in vivo .μL/mg [μg/mL) and selective (selective index = 400) in A549 lung cancer cells [Perillyl alcohol, its derivative , and biotransformation metabolite (dehydroperillic acid) act against cancer in different ways. Perillyl alcohol presents an IC50 of 1.8 and 2 mM on U87 and U251 glioblastoma cells and an apoptosis induction through a signaling mechanism mediated by Na/K-ATPase . Also, iμL/mg . Regardier cells .μM and observation of DNA fragmentation; besides, the doses of 10 and 20 mg/kg in subcutaneous tumors promoted a reduction in the tumor weight and volume [Terpineol has been described as able to suppress cell migration and induce cell cycle arrest and apoptosis in HepG2 hepatic cancer cells with IC50 of 19.5 d volume . Terpined volume . Terpined volume .  Table μM and apoptosis induction [μM, cell cycle arrest, and mitochondria-related apoptosis with activation of the ROS-JNK/p38 pathway [μM, displaying mitochondrial transmembrane potential depolarization, apoptosis, and a reduction on subcutaneous tumor volume [μM), thymol induced apoptosis and cell cycle arrest with cellular and nuclear morphological changes, phosphatidylserine translocation, mitochondrial membrane depolarization, activation of caspase-3, upregulation of Bax, downregulation of Bcl-2, apoptotic fragmented DNA, and ROS production [μg/mL and promoted ROS production mitochondrial membrane potential loss, caspase-3 activation, DNA damage, and cell cycle arrest [Thymol's antitumor activities have been largely studied over the past years. Elbe et al. have recently investigated thymol's cytotoxic effect on SKOV-3 ovarian, PC-3 and DU145 prostate, MDA-MB-231 breast, and KLN205 lung cancer cells finding IC50 values ranging from 208.36 to 799 nduction , 84. It  pathway . On Cal2r volume . On AGS r volume . On A549oduction . On HCT-oduction . On MCF-e arrest . Altogete arrest , demonste arrest .in vitro and in vivo [μM and a tumor growth inhibition of 69.4% in subcutaneous tumors with a dose of 20 mg/kg. The same group also investigated the effect of cuminaldehyde in A549 lung adenocarcinoma cells finding similar results with an IC50 of 18.33 μM and a subcutaneous tumor growth inhibition of 50% using a dose of 10 or 20 mg/kg.Cuminaldehyde has been shown to possess the ability to inhibit topoisomerase I and II activities and to upregulate lysosomal vacuolation on different cancer cells, exercising antitumor activity both  in vivo , 94. UsiMesosphaerum sidifolium (Lamiaceae) oil, at a dose of 60 mg/kg, induced in vivo cell growth inhibition  on Ehrlich ascites carcinoma model through stimulation of cell cycle arrest [Foeniculum vulgare) and has antioxidant, anti-inflammatory, antinociceptive, antifungal, and acaricidal properties [Fenchone, a major component of e arrest . This bioperties , 97.in vitro, and significantly decrease metastasis promoted by B16F10 cells in vivo, using a 15 mg/kg dose, through inhibition of V-ATPase [κB, and STAT-3 correlated with the suppression of the levels of inflammatory cytokines of TNF-α and IL-6 and biomarkers of oxidative damage (MDA and NO). In addition, a significant increase in caspase-A3 activity and Bax/Bcl-2 ratio indicate that the anti-inflammatory effect and the induction of apoptosis contributed to this activity [Myrtenal has been shown to cause cell death, reduce migration and invasion of B16F0, B16F10, and SkMel-5 melanoma cells V-ATPase . Farrag activity .α-Phellandrene showed antitumor activity, which decreased the cell viability, promoted the cell cycle distribution, significantly increased reactive oxygen species levels, decreased mitochondrial membrane potential levels, increased the necrotic cell number, and increased NO production, LDH leakage, and ATP depletion on human liver cancer cells (J5) [lls (J5) .α-phellandrene upregulated DNA damage-associated genes and DNA fragmentation factor, cell cycle checkpoint genes, and apoptosis-associated genes. In addition, α-phellandrene has downregulated DNA damage-associated gene TATA box binding protein, D19Ertd652c (DNA segment), cell cycle-associated gene cyclin E2, apoptosis-associated gene growth arrest-specific 5, Gm5426 (ATP synthase), and death box polypeptide 33 [It was also seen that ptide 33 .α-PA. α-PA influences the murine WEHI-3 leukemia model in vivo by increasing the percentage of CD3 (T-cell marker), CD19 (B-cell marker), and MAC3 (macrophages) markers but reduced the percentage of CD11b (monocytes) cell surface markers. α-PA increased phagocytosis by macrophages and promoted natural killer cell activity. α-PA also increased B- and T-cell proliferation.In Lin et al. , mice wep-Cymene inhibits MMP-9 expression and increases TIMP-1 production besides inhibiting the ERK1/2 and p38 MAPK signal pathways on HT-1080 human fibrosarcoma cells, presenting a robust 87% inhibition of in vitro invasiveness [p-Cymene is a natural antioxidant found in several plant species and is a constituent of fruits, wines, and spices, such as Origanum vulgare (oregano) and Thymus vulgaris (thyme). This compound has several pharmacological activities, in addition to an antinociceptive effect on cancer pain through inhibitory pathways and modulation of calcium currents [siveness . p-Cymencurrents –106.Mentha [κB signaling pathways [Rotundifolone, also known as piperitenone is a naturally occurring oxygenated monoterpene and represents one of the main components of the essential oil of many species of Mentha , 108. Ropathways .The data described here may have implications for future research, possibly being used as a reference for the advancement in the mechanistic studies of these compounds, as well as in the preparation of synthetic derivatives or analogues with a better antitumor profile. Indeed, chemical modification in natural products is a current approach to improve drug actions against several diseases, including cancer .in vitro and in vivo studies using various types of cancer cells and tumors in animal models. Since the last revision of this subject, several mechanisms of action of these compounds were elucidated, and deeper understanding of the use of theses monoterpenes in in vivo models was gained [κB, TNF-α, IL1-β, COX2, and iNOS. The data described serve as a reference for the advancement in the mechanistic studies of these compounds and in the preparation of synthetic derivatives or analogues with a better antitumor profile.The medicinal properties of essential oils have been evidenced through scientific investigations using experimental models. Considering that monoterpenes are commonly the main constituents of these oils, these natural products should contribute to pharmacological actions. The studies reported in this review confirm the antitumor properties of monoterpenes and their anticancer potential against various types of tumors, as demonstrated in s gained . The mai


P = 0.019) and overall survival (P = 0.034) than those with non-FAT mutations, and FAT mutations exhibited significantly higher tumor mutational burden (TMB) and microsatellite instability. Notably, FAT mutations had a greater effect on somatic single-nucleotide variation than copy number variation and resulted in more abundant DNA damage repair (DDR) mutations. Further investigation demonstrated that FAT mutations contributed to an inflammatory tumor microenvironment (TME), as indicated by significantly increased numbers of activated CD4 and CD8 T cells, and significantly decreased numbers of mast cell, plasmacytoid dendritic cell, type 2 T helper cell, and high expression of immune-promoting genes. Moreover, biological process antigen processing and presentation, DNA replication, and DDR-related pathways were significantly upregulated in patients with FAT mutations. Collectively, FAT mutations significantly improved the survival of patients with STAD by enhancing tumor immunogenicity  and an inflamed TME, indicating that the FAT family might be a potential prognostic and therapeutic biomarker for STAD.FAT family genes encode protocadherin, which regulates tumor cell proliferation and migration. Although transcriptional levels of FAT family members had been reported in multiple malignant tumors, the association between mutation and prognosis of the FAT family in stomach adenocarcinoma (STAD) has not been investigated. Herein, we performed a multi-omics integrative bioinformatics analysis using genomic and mRNA expression data to explore the role of gene mutations across the FAT family on clinical outcomes of STAD. The results showed that FAT mutations occurred in 174 of 435 (40%) of the samples. Patients with FAT mutations possessed significantly better progression-free survival ( Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cause of cancer death in the world, resulting in over 1,000,000 new cases and an estimated 783,000 deaths in 2018 . UnfortuFN1, SERPINE1, SPARC, ANKRD33, OGN, JAM2, RERG, OLFML2B, ADAMTS1, DNER, LHCGR, NLRP14, OR4N2, PSG6, TTC29, and ZNF568 significantly predicted a poor prognosis of STAD -like domains, and Laminin G-like domains, which frequently mutated across multiple cancer types , 16. At arcinoma , breast arcinoma , and heaarcinoma , but proarcinoma , colorecarcinoma , GC 22)FAT1 was arcinoma . The higis in GC , 25, breis in GC , squamouis in GC , and CRCis in GC . FAT3 mul cancer  and patil cancer . Repressis of GC , CRC  or overall survival (OS). Since the homologs of mutated genes can induce gene functional compensation , we expl1, of whom 435 patients obtained somatic mutations and clinical data, 433 patients had analyzable copy number variation (CNV) data, and 408 patients possessed mRNA expression data (RSEM format).In this study, we used cBioPortal to download clinical and mutational information about patients with STAD from The Cancer Genome Atlas PanCancer studyWe analyzed the whole-exon sequencing data from the 435 primary STAD samples in the TCGA PanCancer study and calculated the mutation frequency in terms of the total number of non-synonymous mutations, including single-nucleotide substitutions (SNVs) and insertion–deletion (indel) mutations. Tumor mutational burden (TMB) was defined as the number of non-synonymous mutations detected at > 1% allele frequency in the coding region of the genome. We determined MSIsensor score > 4% as microsatellite instability (MSI), and MSIsensor score < 4% as microsatellite stability (MSS).q < 0.1. To identify arm and focal-level CNV differences between the two groups, Fisher’s exact test was used for gains and losses, respectively, and a significant difference was determined as P < 0.05.GISTIC version 2.0 was used to identify significantly amplified and deleted regions in this study. The genome doubling (GD) and ploidy data were determined by a previous study . The genA DNA damage repair (DDR) gene list including 275 genes was collected from previously published research , of whicDeconstructSigs package (version 1.8.0) was used to identify mutational signatures within a single tumor sample based on a negative matrix factorization (NMF) algorithm , which r2 (RSEM + 1) for the single sample gene set enrichment analysis (ssGSEA) (The mRNA expression data were transformed by log(ssGSEA)  to deter(ssGSEA) . Genes r(ssGSEA) –43.2. The terms q < 0.05 were considered significant.A total of 408 samples with gene expression profiles were partitioned into two groups according to the mutation status of FAT family genes. The RSEM values were rounded as input data. R package “DESeq2” was used to determine Fold Change from gene expression data between the two groups, and then all log2FoldChange values were used as input to the “clusterProfiler” R package to perform GSEA . The KEGP-value was determined with the log-rank test. Fisher’s exact test was used to detect the proportion of mutually exclusive or co-occurring gene events between the two groups. The difference in continuous variables between the two groups was examined by the Wilcoxon method. Univariate Cox regression was used to assess the association between different variables and PFS or OS, and the results were presented as HRs and their 95% confidence intervals (CIs). All statistical tests were two-sided, and the result with P < 0.05 was considered statistically significant. All analyses and figure drawing were performed or generated using R version 4.0.3.Survival analysis was performed using Kaplan–Meier curves, and the TTN (54%) was the most frequently mutated gene, and FAT4 (22%) and FAT3 (18%) mutations occurred in the top 20 mutated genes  and OS  compared with those without mutations (P = 0.009), higher TMB (P < 0.001), and MSI (P < 0.001) were observed in the FAT-Mut group rather than the FAT-WT group . Mutational landscape in patients with STAD showed that the FAT-Mut group had a higher mutation frequency than the FAT-WT group. The types of mutations were mainly missense and frameshift. Except for TP53, CUBN, CDH1, and ABCA13, the differences in the remaining top 20 genes were statistically significant (KMT2D (12.6%), ZFHX4 (12.9%), TTN (28.3%), MUC16 (19.3%), LRP1B (16.3%), CSMD3 (14.9%), PCLO (12.6%), CSMD1 (12.2%), HMCN1 (11.7%), PIK3CA (10.1%), FLG (12.9%), OBSCN (13.8%), ARID1A (16.1%), SYNE1 (16.1%), PCDH15 (11.5%), RYR2 (11.3%), SPTA1 (10.8%), and DNAH5 (10.8%) were co-occurring events of FAT mutations, and no mutually exclusive genes associated with FAT mutations were found in this study  , signature 6 (associated with defective DNA MMR), signature 15 (associated with defective DNA MMR), and signature 17 (the etiology remains unknown) were all identified regardless of the presence of FAT mutations, but signatures associated with DNA MMR were more abundant in the FAT-Mut group . It is c< 0.001) , suggestn = 172) showed a lower degree of arm-level CNV than the FAT-WT group (n = 261), and such disparity occurred more in losses . Hence, we speculated that a gain of 8p23.1 might be associated with FAT family gene mutations. Furthermore, ploidy, GD, and genome instability index (GII) were evaluated, all of which did not differ significantly between the FAT-WT and FAT-Mut groups . The expression of immune checkpoint genes, such as CD274 and LAG3, in FAT-Mut was significantly higher than that in FAT-WT, whereas CCL2, CXCL12, CXCL14, CD40, ENTPD1, TGFB1, and VEGF-related genes showed a lower expression in the FAT-Mut group . In contrast, adhesion-related pathways  and focal adhesion), MAPK signal, Hedgehog signaling pathway, calcium signaling pathway, and ECM receptor interaction pathway were prominently enriched in the FAT-WT group (ES < 0 and q < 0.05). These results indicated that FAT family genes played an important role in the biological progression of STAD.To further investigate the biological processes affected by FAT family gene mutations in STAD, we performed GSEA to identify differential pathways between the FAT-WT and FAT-Mut groups. As shown in In this study, we determined differences in the somatic mutations, TME, immune-related gene expression profiles, and signaling pathways between FAT mutant and no-mutant groups using 435 STAD samples from the TCGA database. Compared with wild-type FAT, FAT mutations had a significant correlation with a better prognosis. Further investigation found that prolonged PFS and OS induced by FAT mutations might be associated with tumor immunogenicity , activated CD4 and CD8 T cells, the expression of antigen processing and presentation-related genes, and significantly higher expression of immune-related genes. Moreover, the GSEA results showed that FAT mutations upregulated signaling pathways involved in antigen processing, DNA replication, and DDR-related pathways. In summary, these observations illustrated a possible mechanism to improve survival in STAD patients with FAT mutations. This is the first study to report the association between FAT family mutations and clinical outcomes of malignant tumors.FAT2, FAT3, and FAT4 mutations significantly increased the survival time of patients with STAD, respectively. FAT1 mutations had a similar trend, but there was no statistical difference, which may be due to the small sample size of FAT1 mutations. Previous studies have reported that suppressing FAT1 expression inhibited GC cell growth  and contributed to an inflammatory TME, thereby significantly improving the prognosis of patients with STAD, which might be a positive prognostic marker for STAD.https://www.cbioportal.org/study/summary?id=stad_tcga_pan_can_atlas_2018.Publicly available datasets were analyzed in this study. This data can be found here: YW conceived the study and designed the experiments. QW and LC performed the bioinformatic analysis and drafted the manuscript. PL provided insight in methodological approaches and analysis. YW supervised the study. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thymus roseus were screened for their plant growth promotion (PGP), and the applicability of potent strains as bioinoculant has been evaluated. Regarding PGP traits, the percentage of strains were positive for the siderophore production (84%), auxin synthesis (69%), diazotrophs (76%), phosphate solubilization (79%), and production of lytic enzymes , lipase (62%), protease (61%), chitinase (34%), and displayed antagonistic activity against Verticillium dahliae (74%) in vitro. The inoculation of strain XIEG05 and XIEG12 enhanced plant tolerance to salt stress significantly (p < 0.05) through the promotion of shoot, root development, and reduced the activities of antioxidant enzymes , compared with uninoculated controls in vivo. Furthermore, inoculation of strain XIEG57 was capable of reducing cotton disease incidence (DI) symptoms caused by V. dahliae at all tested salt concentrations. The GC-MS analysis showed that many compounds are known to have antimicrobial and antifungal activity. Our findings provide valuable information for applying strains XIEG05 and XIEG12 as bioinoculant fertilizers and biological control agent of cotton under saline soil conditions.Bacterial endophytes are well-acknowledged inoculants to promote plant growth and enhance their resistance toward various pathogens and environmental stresses. In the present study, 71 endophytic strains associated with the medicinal plant  On a global scale, salinization of soil is one of the most representative environmental stressors and is a major factor that decreases agricultural productivity and causes a loss of land area of about 2000 ha per day, contributing to 1–2% agricultural soil losses every year worldwide, resulting in decreased crop yield up to 70%, mainly in arid and semi-arid regions, causing a loss of US $27.3 billion per year ,5,6,7. AEndophytic bacteria are ubiquitous in nature, residing inside all parts of plant tissues of most ecosystems and completing their life cycle, mostly in a facultative or obligate symbiotic relationship ,11,12. ELamiaceae) and is well known worldwide for its ability to synthesize high quantities of essential oils, which have been widely used in pharmaceutical and food applications  × 100  × 100 .g for 15 min at 4 °C. The supernatant was used for the determination of enzyme activities. The activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were measured using assay kits , following the manufacturer’s instructions  [Cotton leaf extracts were used to measure antioxidant enzymes as described by . Cotton er 2021) . All expChlorophyll content was quantified by using the Leaf Chlorophyll Meter (SPAD 502 Plus). Readings were taken on the uppermost fully expanded leaf with a visible collar during vegetative growth, and from the ear leaf (12 leaves per treatment) as suggested ,62.Streptomyces albidoflavus (XIEG24), Curtobacterium flaccumfaciens (XIEG29), and Nocardiopsis alba (XIEG57) with V. dahliae in 500 mL−1 of broth medium at 28 °C for 20 days with agitation speed at 180 rpm in triplicate. Cells were collected by centrifugation at 6000 rpm for 15 min. The cell-free supernatant was mixed with an equal volume (1:1) of ethyl acetate by vigorous shaking for 60 min at room temperature and allowed to separate. The organic solvent phase was evaporated at 43 ± 1 °C under vacuum, using a rotary evaporator . The ethyl acetate extract was dissolved in 5 mL of Tris-Cl buffer  and analyzed using gas chromatography/mass spectrometry (GC-MS) [An antibiosis experiment was conducted by co-cultivation of strains  (GC-MS) ,53.−1, and the split mode was 5:1. The GC was fitted with a quadrupole mass spectrometer with an Agilent model 5975 detector. The MS conditions were as follows: ionization energy, 70 eV; electronic impact ion source temperature, 230 °C; quadrupole temperature, 150 °C; scan rate, 3.2 scans/s; mass range, 40–800 µ. The compounds were identified based on the match with their mass spectra and retention indices according to NIST/Wiley 275 library . The Relative abundance of each feature was calculated from the Total Ion Chromatogram (TIC) computationally [GC-MS analysis of the endophytic actinobacterial cell-free extracts was performed using a gas chromatograph  equipped with a split-splitless injector, an Agilent model 7693 autosampler, and an Agilent HP-5MS fused silica column . Injecting volume was 0.8 µL, and heating from 50 to 300 °C at 10 °C/min, followed by 10 min at 300 °C. The injector was maintained at 280 °C. Helium was used as the carrier gas, at 1.0 mL mintionally . https://www.R-project.org/, accessed on 31 March 2020.The data represent mean of values represent an average of 10–12 10–12 replicates ± standard error (SE) calculated by MS Excel. One-way ANOVA was used to compare the means of root length, root fresh weight, shoot length, shoot fresh weight, SOD, POD, CAT, and Chlorophyll content for each salt concentration separately, and Tukey’s HSD post-hoc test was used for multiple comparisons at α = 0.05. Statistical analyses were conducted in R (R Core Team). R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL Streptomyces, Mycobacterium, Nocardiopsis, Saccharopolyspora, Micrococcus, Curtobacterium, Alloactinosynne ma, Williamsia, and Pseudonocardia genera. Similarly, most of the tested strains showed nitrogenase activity (76%) based on growth on both NFC and Ashby’s free nitrogen medium, including Streptomyces, Saccharothrix, Saccharopolyspora, Nocardiopsis, Alloactinosynnema, Pseudonocardia, and Mycobacterium genera  displayed inhibitory activity with the percentage of inhibition ranging from 12.5 to 42.0% against Verticillium dahliae Kleb in vitro  was observed for strain XIEG24, which belonged to Streptomyces albidoflavus, followed by Nocardiopsis alba XIEG57 (30.3%), and Curtobacterium flaccumfaciens XIEG24 (29.1%)  . In lighStreptomyces luteus (XIEG05), Nocardiopsis dassonvillei (XIEG12), and Alloactinosynnema album (XIEG20) were positive for at least six plant-beneficial traits in vitro including IAA production, phosphate solubilization, nitrogenase activity, siderophore, and at least production of two hydrolytic enzymes were selected to test their plant growth stimulation properties in pot experiments with cotton plants under salt stress (To explore the plant growth-promoting (PGP) effects in conditions of salt stress. Three strains, t stress .p < 0.05) increased cotton root and shoot length and total fresh weight at different salt concentrations, while strain XIEG20 showed slightly increased, compared to the uninoculated controls. In addition, notably that these results indicated that all tested strains in this study have a different response in terms of cotton growth at certain salt concentrations.Our results in this pilot study showed that cotton growth under different NaCl concentrations was strongly impaired by salinity when cotton was not inoculated with selected endophytic actinobacterial strains, evidenced by decreased root and shoot length and total fresh weight compared to non-stressed control . On the p < 0.05) by 16.5, 23.9, 34.4, and 25.1% at 50, 100, 150, and 200 mM NaCl, respectively, compared to the uninoculated controls (p < 0.05) of the growth rate by 50.9, 43.3, 20.5, and 25.8% at 50, 100, 150, and 200 mM, respectively, compared to the uninoculated control , Nocardiopsis dassonvillei (XIEG12), and Alloactinosynnema album (XIEG20), which showed plant growth stimulation under saline conditions (50–200 mM NaCl) on cotton plant physiological parameters involved in defense systems against oxidative stress to cope with salinity stress. The results pertaining to the effects and alleviating of salt stress on antioxidant enzymes activities by all tested strains are reported in  like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). To minimize the deleterious effects of ROS, we investigated the effect of three endophytic actinobacterial strains, orted in .p < 0.05) of SOD enzyme by 15.7, 11.5, 12.4, and 15.6%, at 50, 100, 150, and 200 mM NaCl, respectively, compared with salt treatments without application of endophytic actinobacterial strains (The superoxide dismutase (SOD) activity increased with increasing salinity levels. The highest SOD concentration observed under the salinity level at 50, 100, 150, and 200 mM NaCl was 36.6, 45.4, 49.4, and 64.8%, respectively, as compared to non-stressed control A. Howeve strains A.p < 0.05) reduced the peroxidase activity at 50, 100, and 150 mM NaCl by 8.5, 16, 7.8%, respectively, in comparison to the uninoculated controls. However, this effect was not significant at 200 mM NaCl.In the leaves of cotton, the activity of POD was positively and progressively correlated with the salinity level. POD content increased by 12.0, 25.9, 60.0, and 83.3% at 50, 100, 150, and 200 mM NaCl, respectively, as compared to non-stressed control B. MoreovThe level of CAT activity gradually increased during all stages of the salt stress treatments. In particular, the results showed that the CAT activity increased due to salinity stress by 33.5, 82.5, 126.3, and 210% at 50, 100, 150, and 200 mM NaCl treatments, respectively, as compared to non-stressed controls C. Whereap < 0.05) in chlorophyll at 50, 100, and 150 mM NaCl by 15.3, 8.2, and 18.7% at 50, 100, and 150 mM NaCl, respectively, while a slight increase was observed in 200 mM NaCl treatment in comparison to the uninoculated controls. In the present study, the data presented in The results pertaining to the effect of salt stress on chlorophyll contents in the presence and absence of tested endophytic actinobacterial strains are shown in D. Our reStreptomyces albidoflavus (XIEG24), Curtobacterium flaccumfaciens (XIEG29), and Nocardiopsis alba (XIEG57) were chosen for a pot experiment under greenhouse conditions after evaluating their ability for antagonistic activity in vitro to test their ability to suppress cotton disease caused by V. dahliae under salt stress in vivo. About 12 days after fungal inoculation, the first signs of aerial wilt symptoms developed on Verticillium-inoculated cotton appeared as blotchy, consisting of chlorotic areas between the main veins of the lower leaves on the first and second mature leaves, and twig dieback that ultimately browned of leaves and defoliated of the non-inoculated cotton at different salt concentrations. The application of V. dahliae on cotton (uninoculated control) increased the disease severity index (DSI) up to 43.7, 52.0, 60.4, and 67.5% at 50, 100, 150, and 200 mM NaCl, respectively, as compared to the uninoculated controls , Curtobacterium flaccumfaciens (XIEG29), and Nocardiopsis alba (XIEG57) with V. dahliae were studied by GC-MS  with V. dahliae showed 72 peaks (Curtobacterium flaccumfaciens (XIEG29) with V. dahliae, the GC-MS resolved several features in the extracts: 72 compounds in total  with V. dahliae, , siderophores (84%), phosphate solubilization (79%), and nitrogenase activity (76%). The findings of our study are parallel to some of the previous investigations which reported that endophytes produced several plants promoting traits including the production of phytohormones, siderophores, phosphate solubilization, nitrogenase activity, and cell-wall-degrading enzymes ,68,69,70%, sideroStreptomyces atrovirens XIEG05 showed the strongest stimulation of root length and root weight as compared to the controls. Likewise, the highest increase in the shoot length and shoot weight was obtained by inoculation of Alloactinosynnema album strain XIEG12, compared to the controls. Accordingly, we observed that the selected salt-tolerant endophytic actinobacterial strains improved the symbiotic performance of cotton under saline conditions. Data obtained in Figure 2 showed that all selected endophytic actinobacteria in this investigation demonstrated a significant role in plant growth activity and displayed increased physiological parameters such as plant biomass and plant length at different salt concentrations. The obtained results are in harmony with many previous studies which suggested that the application of endophytic actinobacteria can increase the growth of different crops such as wheat, rice, lettuce, tomato, pepper, canola, and bean during exposure to salt stress [Based on the obtained results, the growth of cotton under salt stress was strongly impaired with increasing salinity when cotton was not co-inoculated with selected endophytic actinobacterial strains. In this regard, in response to salinity stress, the maximum decrease recorded in the root, shoot length, and root, shoot weight of cotton were 32.1, 31.5, 29.5, and 33.3%, respectively, at all salt concentrations . Contrart stress ,83,84,85Pseudomonas fluorescens Ms-01 on wheat grown under salt stress decreased the activity of SOD, which improves the defense pathway. In the present experiment, the highest chlorophyll pigments were observed at all salt concentrations via application of strain XIEG20 as compared to uninoculated plants (Ocimum basilicum L.).Our study showed that the co-cultivation with strain XIEG05 and XIEG12 enhanced cotton growth and salinity tolerance, especially in the development of primary root length, by regulating cell division and differentiation. Apparently, all tested strains appear to influence the root system architecture via root developmental changes, which might enhance plant tolerance to salt stress by the stimulated root system and could explain the enhanced capacity of the plant by solubilizing mineral nutrients and facilitating their availability to plants to acquire and utilize more nutrients which are in agreement with the findings of ,87,88. Od plants D. Our reV. dahliae can survive in the soil for a long time in dormant forms and causes wilt diseases and crop losses of varying severity, as well as natural ecosystems [Verticillium wilt disease start with yellowing, followed by chlorosis and necrosis of leaves [Streptomyces albidoflavus (XIEG24), Curtobacterium flaccumfaciens (XIEG29), and Nocardiopsis alba (XIEG57) the portion of plants that exhibited disease symptoms decreased at a different level of salt treatments (Nocardiopsis. sp produces different volatile organic compounds (VOCs) as a biofungicide. Moreover, endophytic actinobacteria associated with medicine plants may play an important role in suppressing the growth of fungal pathogens and can minimize the challenges imposed by phytopathogens including the genus Streptomyces, which are well known for their ability to synthesize different secondary metabolites that play an important role in the biological control of plant disease [Controlling Verticillium wilt is challenging since the pathogen osystems . The sigf leaves . In the eatments . These o disease ,64,72,99V. dahliae, were tentatively identified as compounds with known antimicrobial, antiphrastic, antitumor, and anticancer properties. Data obtained in  are known as antimicrobial compounds such as Dibutyl phthalate [To further understand the underlying mechanism, we conducted Gas-Chromatography/Mass-Spectrometry (GC-MS) analysis to identify the antimicrobial compounds produced by antagonistic strains. Although it is hard to judge whether the components identified by GC-MS are from bacteria or fungi; however, the analysis of obtained data revealed that co-cultures of strains XIEG24, XIEG29, and XIEG57, with ained in A showed hthalate , Bicyclohthalate .Curtobacterium flaccumfaciens (XIEG29), the GC-MS resolved several antimicrobials compounds in the extracts as major peaks such as p-Xylene, Benzene, 1,3-dimethyl-, Benzaldehyde [For strain aldehyde , Phenylealdehyde , and alsaldehyde ; Diethylaldehyde , 9-Octadaldehyde , Pyrroloaldehyde , 1-Docosaldehyde , and Nonaldehyde  ; the major peaks in -, and Nonacos-1-ene [In this study, about 81 compounds were identified by GC-MS for strain peaks in D showed os-1-ene , and 13-os-1-ene . OverallT. roseus produced multi-plant growth-promoting substances including IAA, solubilized phosphate, fixed nitrogen, production of siderophores, and produced lytic enzymes  in vitro. Overall, the greenhouse experiments in this study showed that in response to high salinity stress, inoculation of the selected endophytic actinobacterial strains Streptomyces luteus (XIEG05) and Nocardiopsis dassonvillei (XIEG12) were significantly (p < 0.05) enhanced cotton growth promotion at different salinity levels (50–200) mM compared to un-inoculated cotton. In addition, each tested strain in this study had a different response in terms of cotton growth at certain salt concentrations. In the present study, the activities of antioxidant enzymes  were positively and progressively increased correlated with the salinity level, while the reverse was true for the co-inoculation of cotton with tested endophytic actinobacterial strains as compared with NaCl treatments. Our study revealed that endophytic actinobacterial associated with medicinal plant Streptomyces albidoflavus (XIEG24), Curtobacterium flaccumfaciens (XIEG29), and Nocardiopsis alba (XIEG57) could slow disease development, and the expression of signs was delayed compared to the control plantlets under pathogen-challenged at different salt concentrations. Although all tested endophytic actinobacterial strains conferred some degree of Verticillium wilt resistance, the distribution of disease grades varied dramatically at different salt concentrations compared to uninoculated controls. In addition, most compounds revealed by GC-MS were mainly fatty acid esters, phenols, alkanes, alkenes, and aromatic chemicals and have been reported to have antibacterial and antifungal activity. Moreover, the inoculation of cotton with tested endophytic actinobacterial strains resulted in a drastic increase of the chlorophyll synthesis. Noticeably, the results indicated that the three selected antagonistic endophytic actinobacterial strains T. roseus and their interactions with cotton under stressful environments. Moreover, these results support the development of natural products that may minimize the need for the application of chemical fertilizer and fungicides, which would be an environmentally friendly approach and preserve biological resources in a sustainable agricultural system. Further research is needed for future applications in cotton plant growth promotion and crop productivity, as well as to verify the biological control efficacy of selected endophytic bacteria against V. dahliae in the field experiment.In summary, our results provide insights about plant beneficial traits of endophytic bacteria associated with the medicinal plant


Seriola dumerili) is a gonochoristic fish with no sexual dimorphism in appearance, making sex identification difficult. Piwi-interacting RNAs (piRNAs) function in transposon silencing and gametogenesis and are involved in various physiological processes, including sex development and differentiation. Exosomal piRNAs can be indicators for the determination of sex and physiological status. In this study, four piRNAs were differentially expressed in both serum exosomes and gonads between male and female greater amberjack. Three piRNAs  were significantly up-regulated and piR-dre-332 was significantly down-regulated in serum exosomes and gonads of male fish, compared to female fish, consistent with the serum exosomal results. According to the relative expression of four marker piRNAs derived from the serum exosomes of greater amberjack, the highest relative expression of piR-dre-32793, piR-dre-5797, and piR-dre-73318 in seven female fish and that of piR-dre-332 in seven male fish can be used as the standard for sex determination. The method of sex identification can ascertain the sex of greater amberjack by blood collection from the living body, without sacrificing fish. The four piRNAs did not show sex-inclined expression in the hypothalamus, pituitary, heart, liver, intestine, and muscle tissue. A piRNA–target interaction network involving 32 piRNA-mRNA pairs was generated. Sex-related target genes were enriched in sex-related pathways, including oocyte meiosis, transforming growth factor-beta signaling pathway, progesterone-mediated oocyte maturation, and gonadotropin releasing hormone signaling pathway. These results provide a basis for sex determination in greater amberjack and improve our understanding of the mechanisms underlying sex development and differentiation in the species.The greater amberjack ( Seriola dumerili) is economically valuable worldwide and mainly inhabits the deep sea environment, and is characterized by fast growth, good taste, and high nutritional value [2) ratio in the serum is not effective for young adults with very low blood hormone levels [The greater amberjack  [Exosomes are 30–150 nm extracellular vesicles with a lipid bilayer structure and contain various nucleic acids, proteins, and lipids ,21,22. Eilaevis) ,35. In tt-test, p > 0.05; There were no significant differences in body length or weight between the two groups in greater amberjack  with a tea-tray-like structure by transmission electron microscopy (TEM) G. The naarticles H. As detarticles . The molhttp://www.ncbi.nlm.nih.gov/genbank/, accessed on 23 November 2021) databases, miRBase database, and piRBase database, and annotated as various non-coding RNAs, including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), Cis-reg, other Rfam RNA, gene, microRNAs (miRNAs), and piRNAs . The numbers of clean reads  in the male group and female group were 23.98–24.62 M and 24.41–24.69 M, respectively. These clean reads were aligned with the Rfam database, GenBank (d piRNAs A. There p < 0.05 and |log2 fold change (FC)| > 2, of which 76 were upregulated and 38 were downregulated in the male group compared to the female group  enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The target genes were associated with 7633 GO terms and 155 KEGG pathways. The top 30 GO terms  included cell differentiation and sex-related terms A. The KEp < 0.05); the results for these seven piRNAs were consistent with the piRNA profiles obtained by small RNA sequencing (p < 0.05) (p < 0.05)  in serum exosomes were chosen as candidate signature piRNAs for verification by real-time quantitative polymerase chain reaction (qPCR), with TPM expression levels exceeding 100 in at least one of six sequenced samples A. Six piquencing . Further < 0.05) . The hig < 0.05) . Further < 0.05) . Therefop > 0.05). The expression levels of piR-dre-32793 did not differ significantly between sexes in hypothalamus, pituitary, heart, and muscle samples (p > 0.05) but were significantly lower in the liver and intestine in male fish than in female fish (p < 0.05).The four candidate piRNAs were evaluated by qPCR in six other tissue types, including the hypothalamus, pituitary, heart, liver, intestine, and muscle . The expression levels of the four piRNAs in six tissue samples from male and female fish were not consistent with the results obtained for the gonads or serum exosomes . There wadcy9 (adenylate cyclase 9) and pik3cb  were targeted by more than one piRNA. PiR-dre-332 was a hub in these networks. In addition, both piR-dre-332 and piR-dre-5797 targeted adcy9, and both piR-dre-332 and piR-dre-73318 targeted pik3cb.Four signature piRNAs  were used to construct piRNA–target interaction networks. The sex-related interaction networks included 32 piRNA–target genes pairs , and theThe mechanisms underlying sex determination in fish are complex and diverse, involving various regulatory factors ,37. StudDanio rerio) [Cyprinus carpio) [Oreochromis niloticus) [Salmon salar L.) [Scophthalmus maximus) [Diplodus puntazzo) [Paralichthys olivaceus) [C. semilaevis [D. puntazzo [O. niloticus [Differentially expressed piRNAs were identified in the serum exosomes of greater amberjack, as in the testis or/and ovary of other teleosts, including zebrafish (o rerio) , common  carpio) , Nile tiloticus) ,41, Atlaalar L.) , turbot maximus) ,44, sharuntazzo) , Japanesivaceus) ,47,48, amilaevis ,35,49. Ipuntazzo  to 296,7S. salar L. [S. maximus [P. olivaceus [Piwil1 and piwil2 were specifically highly expressed in the testis and ovary but were expressed at low or negligible levels in other tissues, including the brain, fin, skin, muscle, liver, vertebrae, intestine, eye, heart, spleen, kidney, and gill [In this study, four signature piRNAs  were differentially expressed in serum exosomes and gonads between male and female greater amberjack at one year of age. Furthermore, the expression patterns of these four signature piRNAs  in serum exosomes and gonads were not consistent with those in six other tissues , showing that these piRNAs have tissue-specific sex-inclined expression; furthermore, expression patterns in the serum were more similar to those in the gonads than to those in other tissue types. Tissue-specific expression of piRNAs has also been found in other fish, including salar L. , S. maxi maximus ,44, and livaceus ,48. Piwiand gill ,44,47,48Furthermore, the four signature piRNAs and their target genes were related to in utero embryonic development, oocyte meiosis, the TGF-β signaling pathway, progesterone-mediated oocyte maturation, and the GnRH signaling pathway. In the hypothalamus–pituitary–gonad axis in fish, GnRH secreted from the hypothalamus regulates gonadotropin (Gn) secretion from the pituitary, thus regulating sex hormone secretion and synthesis from the gonads ,51,52,53smc1a, espl1, anapc4, ccnb1, ppp2r5d, adcy9, ppp2r5b, pik3cb, itpr3, cul1, camk2a, camk2b, cdc25b, pik3r3, fbxo43, itpr2, map2k1, pde3b, and fbxw11) were involved in oocyte meiosis or/and progesterone-mediated oocyte maturation, suggesting that the piRNAs are male-inclined and their target genes are generally female-inclined.The four signature piRNAs and their target genes were involved in sex-related pathways. Oocyte meiosis and progesterone-mediated oocyte maturation were female-inclined pathways. Furthermore, the four piRNAs and most of their target genes  to modulate ovarian development in the giant freshwater prawn (Macrobrachium rosenbergii) [Adcy9 may be simultaneously modulated by the down-regulation of piR-dre-332 and up-regulation of piR-dre-5797 to participate in progesterone-mediated oocyte maturation; this will be further investigated in subsequent studies. cyclase ,59,60, c cyclase ,62. In anbergii) . Adcy9 mPik3cb was a target gene of piR-dre-332 and piR-dre-73318. Pik3cb encodes p110β catalytic subunits of PI3K, a significant regulator of cell growth, differentiation, survival, proliferation, migration, and metabolism [Pik3cb is highly expressed in obese individuals during pregnancy and can increase insulin sensitivity in offspring, which is beneficial for the metabolic health of offspring after weaning [pik3cb showed low expression in the chronic sleep restriction group, and it was related to the prolactin (PRL) signaling pathway; the expression of pik3cb could be impacted by chronic sleep restriction to interrupt the Pi3k/Akt signaling pathways, further inhibiting spermatogenesis [tabolism ,66,67,68 weaning . In addiogenesis .The four piRNAs  were not only significantly differentially expressed between male and female greater amberjack but also targeted many sex-related target genes, suggesting that the four piRNAs are very promising biomarkers for sex determination. However, it is still unclear how these piRNAs interact with their target genes to regulate gonadal differentiation and development, and further research should focus on the regulatory mechanisms of these piRNA–target pairs.The greater amberjack specimens were collected from Zhangzhou, Fujian, and used as serum donors. The greater amberjack experiments were approved by the academic ethics committee of Guangdong Ocean University and were carried out under the relevant guidelines of the Laboratory Animal Center of Guangdong Ocean University.Three male  and three female  one-year-old greater amberjack were evaluated. The appearance of males was similar to that of females. The gonadal tissues of each fish were collected and placed in a centrifuge tube with 4% paraformaldehyde. Blood samples were collected from each fish and added to a centrifuge tube on ice for centrifugation.To accurately identify the sex of greater amberjack, the gonadal samples were used to make paraffin sections for hematoxylin-eosin staining ,71. The g for 10 min (4 °C). Serum samples of approximately 1 mL were isolated. Subsequently, exosomes from the serum samples were isolated following the instructions provided with Total Exosome Isolation Reagent . The mixtures of serum samples from each fish and Total Exosome Isolation Reagent (2:1) were placed overnight at 4 °C and then centrifuged at 10,000× g for 1 h (4 °C). The supernatant was discarded and the centrifuge tube was inverted on filter paper for 2 min. The isolated exosomes were resuspended in 50 μL of phosphate-buffered saline (PBS) and stored at −20 °C for further identification and RNA extraction.Blood samples were maintained on ice for several hours and centrifuged at 3500× 2O) was dripped on the exosomes on the copper mesh at room temperature for 5 min and absorbed by filter paper, and this process was repeated once. After drying at room temperature, the exosomes were observed under a transmission electron microscope . Images of exosomes were captured using a digital camera  [For TEM, 5 μL of each exosome sample with PBS was fixed to a copper mesh at room temperature for 5 min. PBS around the exosomes was absorbed by filter paper . The exo, Japan) .For a NTA, 20 μL of exosomes was diluted with PBS to 1000 μL and mixed homogeneously with a vortex mixer. The Malvin Nanosight NS300  was used for the quantification and size analysis of diluted exosomes, repeated three times for 30 s each time ,49. Datag at 4 °C for 5 min to obtain the supernatant [For western blotting (WB), 100 μL of exosomes was lysed with 200 μL of RIPA Lysis Buffer with protease inhibitor  and phosphatase inhibitors (Roche) at 4 °C for 15 min. The lysed exosomes were centrifuged at 12,000× ernatant . The supernatant . At roomernatant ,76,77. ATotal RNAs were isolated from serum exosomes using AG RNAex Pro Reagent  and the quality and concentration were evaluated using the Agilent Bioanalyzer . High-quality RNA from three female  and three male greater amberjack  were sequenced separately. Six small RNA libraries were constructed and used for Illumina HiSeq sequencing .http://www.ncbi.nlm.nih.gov/genbank/, accessed on 23 November 2021) databases, miRBase database [6 [p < 0.05 and |log2 FC| > 2 as thresholds, and the p-value for each piRNA was calculated using the DEG algorithm [Raw reads  were obtained by base calling  and were used for further quality control. To obtain clean reads, reads with adapter sequences were filtered using cutadapt (version 1.14)  and the database ,83, and database . The disabase [6 , where Nlgorithm  in the Dlgorithm  with the2 FC of the candidate signature piRNA was evaluated. The expression levels of the candidate signature piRNAs were assayed by qPCR, using oni-let-7a as an internal reference.Differentially expressed exosome piRNAs in the serum were chosen as candidate signature piRNAs with the following standards. (1) Differentially expressed piRNAs are sex-related. (2) The expression levels of each piRNA in three samples from the male group were higher or lower than those of three samples from female group. (3) In the six samples, the piRNA expression level of at least one sample was higher than 100. The logCandidate signature piRNAs were selected for qPCR with serum exosomal samples  for validation. When qPCR results were consistent with the sequencing results, piRNAs were selected for qPCR analysis of additional serum exosomal samples . Those piRNAs with 100% consistency across analyses were selected for qPCR of gonadal samples , an important organ showing primary sexual characteristics. Finally, piRNAs whose expression patterns were consistent with the serum piRNA sequencing results were selected for qPCR analyses of other tissues, including the hypothalamus, pituitary, heart, liver, intestine, and muscle samples , i.e., organs without primary sexual characteristics, to evaluate tissue specificity. The verified piRNAs were considered signature piRNAs.−ΔΔCt method [Total RNAs from serum exosomes were isolated using AG RNAex Pro Reagent , while total RNAs from seven other tissue types  were isolated using TRIzol Reagent . Total RNAs from the serum exosomes (10 ng) and other tissues (400 ng) from the male group and female group were used for reverse transcription. The specific stem-loop primers for reverse transcription and specific quantitative primer pairs of the candidate signature piRNAs and the internal reference (oni-let-7a) were designed using miRNA Design (Version 1.01) and Primer3 (Version 0.4.0) shown in . Accordit method . The relhttp://www.cytoscape.org/, accessed on 30 September 2022).Based on the results of qPCR validation and the KEGG pathway enrichment analysis, the validated signature piRNAs and their predicted targets were selected to construct a piRNA–target interaction network. The piRNA–mRNA pairs expressed in the serum of greater amberjack were selected from the sex-related pathways, and their interaction networks were constructed using Cytoscape (Version 3.5.1) (p-value < 0.05 was recognized as a significant difference.Statistical analyses were performed using SPSS 25.0. The independent samples t-test was used to evaluate differences between the male group and female group. Parameters, including body length, body weight, and qPCR results, are shown as the mean ± SD. A In this study, piRNA profiles of serum exosomes from greater amberjack were characterized to screen sex-inclined piRNAs as molecular markers for sex determination in the species. Four piRNAs that were differentially expressed in the serum exosomes and gonads between male and female greater amberjack were identified as signature piRNAs for sex determination, and their sex-related target genes were enriched in sex-related pathways, including oocyte meiosis, the TGF-β signaling pathway, progesterone-mediated oocyte maturation, and the GnRH signaling pathway. This work provides biomarkers for the identification of the sex of greater amberjack based on piRNAs from serum exosomes and provides insight into the interactions between piRNAs and sex-related target genes in the regulation of sex development and differentiation in greater amberjack.


The aim of this study was to investigate the expression levels of sensory receptors, inflammatory proteins, and pro-apoptotic proteins in the urothelium of non-Hunner’s interstitial cystitis (NHIC) bladders of patients with different clinical and cystoscopic phenotypes. The urothelia from the bladders of 52 NHIC patients were harvested. The expression of sensory receptors, including TRPV1, TRPV4, TRPA1, H1-receptors, and sigma-1 receptors; the inflammatory proteins p38 and tryptase; and the pro-apoptotic proteins, such as caspase-3, BAD, and BAX in the urothelium, were investigated using immunohistochemistry and Western blotting. We compared the expression levels of these proteins in NHIC subtypes according to IC symptom scores, visual analog scores of bladder pain, maximal bladder capacity, glomerulation grades, and combined maximal bladder capacity and glomerulations after cystoscopic hydrodistention. The expression levels of TRPV1, TRPV4, sigma-1, P38, tryptase, caspase-3, and BAD were significantly increased in the urothelium of IC/BPS patients compared with the expression levels in the controls. TRPV1 was significantly associated with IC symptom severity. However, no significant differences in sensory receptor expression in the IC/BPS bladders with different bladder conditions were detected. Inflammatory and pro-apoptotic protein expression levels in the urothelium were similar among the IC/BPS subgroups. This study concluded that IC/BPS patients with frequency and bladder pain complaints have higher levels of urothelial sensory receptors, and inflammatory and pro-apoptotic proteins. The expression levels of these sensory receptors, inflammatory proteins, and pro-apoptotic proteins are not significantly different among IC/BPS bladders with different conditions. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disorder of the urinary bladder characterized by bladder pain and urinary frequency, urgency, nocturia, and sterile urine . The cliPatients with IC/PBS exhibit predominant bladder pain or urgency frequency symptoms. Under cystoscopic hydrodistention, patients have reduced MBC under anesthesia, mild to severe glomerulations, and/or Hunner’s lesions, but some patients do not show these features . AlthougThe urothelium expresses many receptors typical of primary sensory neurons  V1, TRPA1, and purinergic receptor P2X3), and neurons have high levels of sigma-1 receptors, which are involved in the modulation of urological pain. The sigma-1 receptor had been found to modulate somatic pain and be involved in the bladder pain in the cystitis . UrothelThe purpose of this study was to investigate the expression of sensory receptors , inflammatory markers (p38 and tryptase), and pro-apoptotic markers , and BcL-2-associated X (BAX)) in the bladder urothelium of patients with non-Hunner’s IC (NHIC)/BPS with different cystoscopic phenotypes and histopathological subtypes. This study provides evidence that the pathophysiology of IC/BPS in patients with different cystoscopic IC features is related to altered sensory receptor expression in the bladder mucosa. The results of this study are clinically relevant in the decision-making for treatment strategies for patients with IC/BPS.The study included 52 female patients with IC/BPS and 6 controls. The mean age of patients with IC/BPS was significantly younger than the age of the controls. The immunohistochemical staining of the location of the sensory receptors, TRPV1, TRPV4, TRPA1, P2X3, H-1, and sigma-1 receptors, is shown in In the correlation analysis among all IC/BPs patients and controls, TRPV1 negatively associated with bladder volume (r = −0.294) and bladder compliance (r = −0.313), TRPV4 positively associated with voiding detrusor pressure (r = 0.278), and P2X3 negatively associated with the first sensation of bladder filling (r = −0.261). However, H-1 and sigma-1 did not correlate with any of the VUDS parameters. (Data not shown).Only TRPV1 expression was significantly associated with the O’Leary–Sant symptom score (OSS), interstitial cystitis symptom index (ICSI), and interstitial cystitis problem index (ICPI). No significant associations of the other sensory receptors with IC symptom severity were detected. Significant positive correlations between TRPV1 and BAD, P2X3 and BAD, sigma-1 and caspase-3, and sigma-1 and BAD were detected. Negative correlations were detected between TRPV4 and tryptase, H-1 and P38, and H-1 and tryptase . The scaThe expression levels of the sensory receptors, TRPV1, TRPV4, and sigma-1; the inflammatory proteins, P38 and tryptase; and the pro-apoptotic proteins, caspase-3 and BAD, were significantly increased in the urothelium of IC/BPS patients compared with the expression levels in the urothelium of the controls. The expression levels of several of these sensory receptors and inflammatory proteins significantly correlated with each other. However, the expression of the sensory receptors did not significantly differ in the IC/BPS bladders with different bladder conditions, such as MBC, glomerulation, and bladder pain severity. The sensory receptor expression levels were significantly associated with reduced first sensation of bladder filling and bladder capacity, and TRPV1 was significantly associated with IC symptom severity. Nevertheless, the inflammatory and pro-apoptotic protein expression levels in the urothelium were similar among IC/BPS patients with different bladder conditions.The bladder urothelium expresses many receptors typical of primary sensory neurons , and neurons express high levels of sigma-1 receptors ,19. The Multiple TRP channels  are expressed in the bladder with specific tissue distributions in the lower urinary tract. These TRP channels are implicated in bladder disorders, including IC/BPS. The TRPV4 channels are strong candidates for mechanosensors in the urinary bladder, and TRPV4 antagonists are promising therapeutic agents for OAB . TRPV1 aIC/BPS symptoms include urinary urgency, frequency, and pain, indicating a key role for hypersensitivity in bladder-innervating sensory neurons. This hypersensitivity translates to increased sensory input and activation in the spinal cord, which may underlie the symptoms of bladder hypersensitivity and pain experienced in IC/BPS patients . These sOur recent study demonstrated that patients with IC/BPS can be stratified according to the cystoscopic hydrodistention findings. The clinical characteristics, symptoms, urodynamic parameters, and findings of cystoscopic hydrodistention are distinct between patients with HIC and NHIC . Among tPositive bladder histopathological findings are associated with a smaller bladder capacity, denuded urothelium, increased inflammation, reduced urothelial proliferative function, and increased bladder sensation ,24,25. TPsychological  stressors exacerbate the symptoms of IC/BPS. Stress and urinary urgency are significantly related . In our The limitation of this study is the different mean age between the IC/BPS patients and control women. It is not easy to obtain normal bladder tissues from women with completely normal urinary tract function which is confirmed using video urodynamic study. We cannot be certain that the differences in the sensory receptors of IC/BPS patients are not caused by age. However, a previous study revealed that the ageing process increases the sensory receptors and neuropeptide expressions from the bladder urothelium, which results in increased afferent mechanosensitivity and increased smooth muscle contractility. Therefore, the higher age in the control women might not affect the results of this study ,29. In aBladder biopsies from IC/BPS female patients involved in our previous studies were analyzed for this study ,15. BladAll patients had not been treated with intravesical therapies such as botulinum toxin A or platelet-rich plasma. Patients with IC/BPS underwent endoscopic cold-cup biopsies of the bladder wall, including the mucosa and submucosa, during intravesical treatment, such as botulinum toxin injections or platelet-rich plasma injections, after cystoscopic hydrodistention. The diameter of each specimen was approximately 2 mm. Bladder biopsies from NHIC subjects were obtained from the posterior wall near the glomerulation hemorrhage. Bladder biopsies from HIC patients were obtained from sites with relatively intact mucosa near Hunner’s lesions. Endoscopic electrocauterization of the biopsy sites was performed to prevent bleeding. Histopathological analysis of the IC/BPS bladder urothelium was performed as previously reported . BladderUrinary bladder biopsy specimens from patients with IC/BPS were embedded in OCT medium and stored at −80 °C. Four 5-μm sections per specimen were obtained using a cryostat and collected on new silane III-coated slides . The sections were post-fixed in acetone at −20 °C, blocked with rabbit serum, and incubated overnight at 4 °C. Immunohistochemical staining of biopsies from NHIC bladders was performed using primary antibodies to TRPV1, TRPV4, TRPA1, P2X3, H1-receptor, sigma-1 receptors, tryptase, p38, caspase-3, BAD, and BAX. GAPDH was used as a normalizing protein for quantification. The immunohistochemistry study was performed using the UltraVision Quanto Detection System HRP DAB . Slides were first treated with hydrogen peroxide block reagent (ThermoScientific) and rinsed with PBS. Following that, Ultra V Block reagent (ThermoScientific) was used to block nonspecific binding. The slides were subsequently incubated with primary antibodies including TRPV1 , TRPV4 , TRPA1 , P2X3 , H-1 , and sigma-1 . The Primary Antibody Amplifier Quanto (ThermoScientific), HRP Polymer Quanto (ThermoScientific), DAB Quanto Chromogen, and DAB Quanto substrate (ThermoScientific) were used to visualize. The procedures were in accordance with our previous study. The methodology is described in previous studies ,31,32. WThe bladder biopsy specimens from the IC/BPS and control patients were homogenized in liquid nitrogen and then lysed for 10 min on ice using Hytra tissue protein extraction reagent . The extraction solution was supplemented with a protease inhibitor cocktail  and a phosphatase inhibitor cocktail (Roche Diagnostics). Proteins were separated using electrophoresis on 10% Tris-glycine gel. After gel electrophoresis, the protein blots were transferred to 0.2-μm PVDF membranes using 3% skim milk as blocking buffer for 1 hour, then adding TRPA1 , TRPV1 , TRPV4 , P2X3 , P38 ,Tryptase , Caspase3 , BAX , BAD , H-1 , sigma-1  as primary antibodies and GAPDH  as positive control. The membranes were incubated overnight at 4 °C; after incubation, they were washed with TBST 4 times each for 10 min. The secondary antibody  was then applied. The membranes were finally probed with enhanced chemiluminescence reagent  and exposed to X-ray films. The scanned film after gel electrophoresis was quantified using a gel documentation system . GAPDH was used as normalizing protein for the quantification. All of the bladder samples from all patients were analyzed using identical techniques. [t-test, and among multiple IC/BPS subgroups and the controls were analyzed using the Kruskal–Wallis test (non-parametric ANOVA). Effect size was calculated using SPSS, and Cohen’s d and Eta squared values were presented for 2-group and multiple group comparisons, respectively, in the statistical analysis. The magnitudes of effect size were 0.20, 0.50, 0.8, and 1.2 for small, moderate, large, and very large effects in the t-test, respectively [p values of <0.05 were considered statistically significant.Descriptive statistics was expressed as means ± standard deviation (SD) or percentages. Differences in expressions of sensory receptors between IC/BPS and controls were analyzed using Student’s ectively . In multectively . The posSensory receptors , inflammatory proteins (tryptase and P38), and pro-apoptotic proteins (caspase-3 and BAD) are significantly elevated in the bladders of IC/BPS patients. Elevated sensory receptor levels are associated with lower bladder sensation (P2X3), smaller volume (TRPV1), and lower bladder compliance (TRPV4). Although TRPV1 expression is significantly associated with IC symptom severity, these sensory receptors, inflammatory proteins, and pro-apoptotic protein levels are not significantly different among IC/BPS bladders with different conditions. IC/BPS patients with frequency and bladder pain complaints have higher urothelial sensory receptor, inflammatory, and pro-apoptotic protein expression levels, which might be downstream of chronic inflammation.


Immune checkpoint inhibitors (ICIs) are being used in patients with various advanced malignancies, and patient outcomes have improved considerably. Although ICIs can effectively treat tumors, 30–60% of patients experience immune-related adverse events (irAEs). Autoimmune encephalitis (AE) is a rare irAE that has become a novel topic in neuroimmunology and has received increasing attention in recent years. Herein, we report a rare case of GAD65-antibody–associated AE after metastatic small cell lung cancer treatment with pembrolizumab. The patient received IVIg therapy for AE and continuous pembrolizumab therapy without suspension of tumor treatment. At 1 year follow-up, both the patient’s AE symptoms and tumors were stable. We consider that the treatment of ICI-associated AE should be more individualized with prudent decision-making and should balance the tumor progression and AE treatment. In addition, we have also comprehensively reviewed the literature of ICI-associated AE, and summarized the clinical features, treatment, and prognosis of AE caused by ICI, thus broadening our understanding of the neurological complications caused by ICI. Immune checkpoint inhibitors (ICIs) are a class of monoclonal antibodies that target regulatory immune checkpoint molecules that inhibit T cell activation. ICIs can enhance T cell-mediated anti-tumor immunity and promote immune-mediated tumor cell clearance by blocking co-inhibitory signaling pathways ,2,3. TheICIs can be divided into monoclonal antibodies against CTLA-4 and monoclonal antibodies against PD-1/PD-L1 depending on the pathways they act on . MonocloCommon irAEs include skin rash, itching, colitis, hepatitis, and all types of endocrine diseases . NervousReferences for the review were identified by searching English literature in the PubMed database published from 2016 to 2022, using the search terms : “immune checkpoint inhibitor”, “autoimmune encephalitis”, “immune-related adverse events”, “anti PD1”, “anti PDL1”, and “anti CTLA4.” The inclusion criteria are as follows: patients with (1) encephalitis symptoms identical to classical AE, regardless of whether neuronal autoantibodies were detected; (2) any AE symptoms associated with classic tumor neuronal autoantibodies; or (3) symptoms associated with autoantibodies against synaptic receptors or other neuron cell surface proteins after ICI treatment. Patients were excluded if neuronal autoantibodies were detected before ICI treatment .2O), nucleated cell count 2 × 106/L, protein 514.45 mg/L, chloride 128 mmol/L, and sugar 2.96 mmol/L. Cranial magnetic resonance imaging (MRI) showed evidence of mild cerebellar atrophy  revealed two round and low-density lesions with increased fluorodeoxyglucose (FDG) metabolism on the right liver lobe, enlarged lymph nodes with FDG hypermetabolism in the retroperitoneal area and the space between the right kidney and pancreas. The size of lesions on the right lobe of the liver were 1.3 × 1.1 cm and 1.34 × 1.44 cm. Considering the liver and lymph node metastases, on the 3rd and 25th of February 2021, the patient received pembrolizumab therapy. On 15 April 2021, he developed slurred speech and an unsteady gait. The patient was again treated with pembrolizumab on the 29 of April 2021, and his symptoms progressed significantly and his modified Rankin Scale (mRS) score deteriorated to 4. Neurological examination revealed dysmetria on the finger-to-nose test, positive Romberg’s sign, ataxic gait, and nystagmus. The cranial nerves were intact and there was no nuchal rigidity. The patient had normal muscle strength, muscle tone, and tendon reflexes. Cerebrospinal fluid (CSF) analysis showed normal opening pressure  . The patThe incidence of irAEs in the nervous system after ICI treatment is 2% to 6% ,22, of wBy reviewing the literature, we found 50 cases of ICI-associated AE; of which, 28 and 22 cases were with and without autoantibodies, respectively. Patients’ age ranged from 19 to 81 years, with a mean age of 61.5 years, with a male-to-female ratio of 1:1. The patients had lung cancer 20), melanoma (12), renal cancer (6), pleural mesothelioma (3), Hodgkin’s lymphoma (2), ovarian cancer (1), breast cancer (1), endometrial cancer (1), uterine cancer (1), Merkel cell carcinoma (1), thymic carcinoma (1), and urothelial carcinoma (1). Thirty-nine patients were explicitly prescribed with ICI-specific drugs: Nivolumab (15), ipilimumab/Nivolumab (11), Pembrolizumab (7), Sintilimab (1), Durvalumab (2), Dostarlimab (1), and Atezolizumab (2). After the use of ICI, the patients presented with AE. The time from ICI use to AE symptom onset ranged from 4 days to 18 months . Examination of CSF showed increased an cell number in 21 patients, increased protein in 35 patients, and a positive oligoclonal band (OCB) in 7 patients. Twenty-eight patients were found to have positive autoimmune antibodies in the cerebrospinal fluid or serum, including Ma2 Ab (10), GAD Ab (7), Hu Ab (3), NMDAR Ab (4), SOX1 Ab (2), Ri Ab (1), GABAbR Ab (1), and CASPR2 Ab (1) . Hu Ab a, melanomThis table summarized the reported cases of ICI-associated AE . We noticed that among the reported cases, only seven cases were related to the GAD65 antibody , rangingThe mechanisms of ICI-associated AE may be due to the following reasons: (1) The immune response induced by ICI may cross-react with CNS autoantigens; (2) AE is closely associated with tumors . ICI mayIn addition to AE, ICI can also lead to other neurological complications. Neurological syndromes caused by ICI are mainly peripheral neuropathies, including myasthenia gravis , GuillaiImmune-related adverse events are classified into five grades . Due to This is the first case of pembrolizumab-associated AE with GAD65 and SOX1 antibodies, characterized by progressive cerebellar ataxia. It should be noted that this patient received IVIg therapy for AE and continuous pembrolizumab therapy without suspension of tumor treatment. At present, the treatment of ICI-associated AE needs to be more individualized with close monitoring, evaluation, and prudent decision-making. Finally, the mechanisms, clinical features, and treatment of ICI-associated AE are a new concept in the field of neuroimmunology, and further studies are needed.