The signaling lymphocytic activation molecule (SLAM) family receptors were discovered in immune cells for the first time. The SLAM-family receptors are a significant player in cytotoxicity, humoral immune responses, autoimmune diseases, lymphocyte development, cell survival, and cell adhesion. There is growing evidence that SLAM-family receptors have been involved in cancer progression and heralded as a novel immune checkpoint on T cells. Previous studies have reported the role of SLAMs in tumor immunity in various cancers, including chronic lymphocytic leukemia, lymphoma, multiple myeloma, acute myeloid leukemia, hepatocellular carcinoma, head and neck squamous cell carcinoma, pancreas, lung, and melanoma. Evidence has deciphered that the SLAM-family receptors may be targeted for cancer immunotherapy. However, our understanding in this regard is not complete. This review will discuss the role of SLAM-family receptors in cancer immunotherapy. It will also provide an update on recent advances in SLAM-based targeted immunotherapies. There are several cancer therapies available. They are selected for each patient depending on the kind of cancer and the stage of the disease at diagnosis. Chemotherapy, radiation therapy, stem cell transplantation, surgery, and immunotherapy are some treatments .via various strategies, including immune checkpoint inhibitors (ICIs) and adoptive cellular therapies. Clinical trials treating patients with various cancers between 2010 and the present have shown impressive results employing various immunotherapeutic approaches. Immunotherapy-induced anti-tumor responses can persist for a considerable time after the treatment has ceased, unlike responses from therapies that target the tumor directly (Cancer immunotherapy approaches may induce a considerable anti-tumor response. These may be achieved via their immunoreceptor tyrosine-based switch motifs (ITSMs), which are found on both the activating and inhibitory SH2 domain-containing proteins. Evidence indicates that this family’s members are engaged in various physiological and pathological processes, including the regulation of immunological responses and the route of viral entry  family receptors were discovered in immune cells for the first time. In humans and mice, the genes that code for SLAM-family receptors are clustered on chromosome 1 in a 400 kilobase (kb) cluster . Recentlal entry . The disal entry —the XLP al entry . SLAM real entry , 10.Previous studies have reported the role of SLAMs in tumor immunity in a variety of cancers, including chronic lymphocytic leukemia (CLL) , lymphom2The SLAM family of receptors is a group of type I transmembrane receptors that includes SLAMF1 , SLAMF2 (CD48), SLAMF3 , SLAMF4 , SLAMF5 (CD84), SLAMF6 , SLAMF7 , SLAMF8 , and SLAMF9  Table 1 via homotypic or heterotypic cell-cell interactions.Glycosylphosphatidylinositol (GPI) anchor is used by SLAMF2 , 7, 77. 2.1The glycoprotein SLAMF1 or CD150 is found in T, B, natural killer (NK), and dendritic cells DCs; Table 1.In normal immune cells, SLAMF1 is known to recruit and activate intracellular signaling molecules, such as SH2-containing tyrosine phosphatase 2 (SHP-2), phosphatidylinositol 3-kinase (PI3K), and the mitogen-activated protein kinases (MAPKs) p38, upon binding to its ligands , 83. Whi2.2+ cells but not on platelets or red blood cells (SLAMF2 (CD48) is a glycoprotein related to glycophosphatidylinositol expressed on the surface of immune cells such as mature lymphocytes and monocytes and functions as a ligand for SLAMF4 Table 1.od cells , 78. SLAod cells .+ T cell expansion in the small intestine, maintaining homeostasis  superfamily, which belongs to the CD2 subgroup. SLAMF4 is expressed on the cell surfaces of NK cells, T cells, monocytes, eosinophils, myeloid-derived suppressor cells (MDSCs), and DCs Table 1.eostasis .In normal immune cells, the interaction between SLAMF2 and SLAMF4 involves the recruitment of various signaling molecules, including SAP and Src-family kinases such as Fyn and Lyn. Once activated, SLAMF4 signaling leads to downstream activation of MAPK and PI3K/Akt signaling pathways, which are essential for T cell activation and proliferation . In hemaor EAT-2 . If SAP or EAT-2 , 94–97. or EAT-2 .2.3SLAMF3  is expressed in T and B cells, NK cells, and thymocytes Table 1 + T cells. The cytoplasmic tails of SLAM receptors containing phosphorylated tyrosines associate with SHIP, docking protein 1 (DOK1), DOK2, and RAS-GAP, which can potentially take part in these processes  activation and the production of Th2 cytokines , 100. Corocesses . In hemarocesses . In MM, rocesses .2.4+ hematopoietic progenitor cells, monocytes, macrophages, and B/T lymphocytes  is expressed on CD34phocytes Table 1.or cells , 103. SL B cells . In mono B cells . SLAMF5  T cells  and humo T cells .SLAMF5 through SAP binding helps regulate the intensity of T cell receptor (TCR) signals in primary T cell responses, which contributes to p73-mediated apoptosis. This process involves the recruitment of Fyn, which fine-tunes the TCR signal strength. Activated Fyn can impede Itch, a ubiquitin ligase, thereby preventing the degradation of p73 , 108. Ho2.5+ T cell-mediated cytotoxicity  is an immunoglobulin superfamily homophilic cell-surface receptor and a type I transmembrane protein. SLAMF6 has two immunoglobin (Ig)-like extracellular domains and two ITSMs , 62, 110toxicity , 112.via a mechanism that is dependent on SAP, but not on Fyn. According to this mechanism, SAP helps remove SHP1 from the cytoplasmic tail of SLAMF6. This, in turn, leads to stronger signaling from TCR, which eventually results in the expression of genes such as FasL and Bim that mediate the process of restimulation-induced cell death (RICD)  , 108. Fuh (RICD) , 100. Fuh (RICD) . In the  T cells , suggest2.6SLAMF7 is primarily expressed by NK cells, T lymphocytes, activated B cells, and macrophages Table 1.In cells lacking EAT-2 expression, following the homotypic interaction SLAMF7 between immune cells and myeloma cells, SHIP-1 recruitment is occurred, which requires tyrosine residue (Y-284) in the ITSM motif of SLAMF7. Thus, the immune cell is inhibited. In contrast, in cells with EAT-2 expression, following the interaction EAT-2 is mostly recruited to phosphorylated tyrosine residue (pY-304) within the ITSM of SLAMF7, and then PLCγ-1 and PLCγ-2 are recruited to phosphorylated tyrosine (pY-127) on EAT-2, which eventually leads to activate the ERK signaling pathway in immune cells. However, due to the absence of EAT-2 and CD45 expression in myeloma cells, these signaling pathways are revoked –117.2.7SLAMF8  includes an extracellular and transmembrane domain. However, unlike most other SLAM members, it contains no ITSM motifs, resulting in the prevention of SAP-dependent function . SLAMF8 2.8SLAMF9  is found on myeloid cells , plasmac33.1Several studies have expanded current knowledge on SLAMF1 in the pathophysiology of CLL and lymphoma , 118–120+CD180+ B cell CLL to cancer immunotherapies based on CD20 targeted therapy  database reported that the three-gene signature, including SLAMF1/CD27/SELL, could be used as a biomarker to predict the prognosis and personalized immunotherapy for cervical cancer . Notably, the anti-SLAMF2 mAb does not impair normal CD34+ hematopoietic stem cells , overexpression of SLAMF2, CD85, CD45, and PD-1 characterizes a predominantly inhibitory phenotype in circulating and tumor-infiltrating NK cells . In the  T cells . A study T cells .3.3Several studies indicate up-regulated expression of SLAMF3 on MM cells Figure 3in vitro and in vivo  are found in higher numbers in tumor-bearing animals compared to naive controls in syngeneic C57BL/6 mouse models of pancreatic adenocarcinoma and lung carcinoma  expressing SLAMF4 in syngeneic tumor models. Additionally, significant variations in terms of function have been reported between SLAMF4roxidase . Moreoveing mice .3.5. In vitro and in vivo, SLAMF5 inhibition causes CLL cell death  and HSP90 inhibitors . A studyll lysis . In phasll lysis . Elo incll lysis . In the ll lysis . Elo hasll lysis . Table 239-247 peptide can induce antigen-specific CTL against MM cells, lysing tumor cells (Immunogenic peptides from the SLAMF7 antigen effectively activate specific cytotoxic T lymphocyte (CTL) clones. Accordingly, an immunogenic HLA-A2-specific SLAMF7or cells .+ T cells express lower levels of PD-1 and have a reduced capacity to manifest a T cell exhaustion phenotype in this animal model , is not well established. However, a study reported that SAP inhibits PD-1 signaling in T cells by interacting with CD28 . To prev4.3The function of SLAM receptors, including SLAMF1/3/4/6/7, is dichotomous Figure 1SLAMF7 appears to have both inhibitory and stimulatory effects in cancer, depending on the type of cancer and context in which it is expressed. In some types of cancer, such as MM and breast cancer, SLAMF7 has been found to be overexpressed , and itsOverall, further study is required to identify if a given SLAM receptor acts as an activatory receptor or an inhibitory receptor in each cancer type. Functional assays using cancer cells and SLAM receptor-specific antibodies or CAR T cells might be one such strategy. This would aid in determining whether or not SLAM receptor activation enhances the development and survival of cancer cells. Instead, genetic knockdown or knockout approaches might be used to examine the impact of SLAM receptor deletion on the behavior of tumor cells. Eventually, investigating SLAM receptor expression in patient samples might help identify biomarkers and therapeutic targets for various cancers.4.4In the case of targeting SLAM molecules, the potential on-target off-tumor effects could include immune system dysfunction, as SLAM molecules play a crucial role in immune cells function. Not all SLAM receptors are expressed only on normal immune cells. While these receptors have a widespread expression on different immune cells such as T cells, B cells, and NK cells, also these molecules may be expressed in malignant cells. For instance, SLAMF7 is expressed in MM cells . Hence, 5Our knowledge of the role of SLAM-family receptors in cancer immunity has grown during the last several years. There is growing irrefutable proof that receptors belonging to the SLAM family mediate essential functions in immune cells. Studies have indicated that the SAP family of adaptors decides whether a SLAM receptor has a stimulatory or inhibitory function. Additionally, findings have established that SLAMF7 is a valid and safe drug target for the treatment of MM, and results statistically showed significant trends.This article presented an outline of SLAM molecules with a focus on recent and previous findings on the role of SLAM family molecules in treating various cancers. Evidence indicates that some members of this family act as an inhibitory immune checkpoint. Thus, these molecules represent a novel therapeutic avenue to boost antitumoural immunity. Also, these molecules may be used as a predictor of cancer development and aggressiveness. Given recent breakthroughs in current knowledge and our understanding of the role of the SLAM-family receptors in tumor immunity, the receptors within this axis are potential candidates for hematological and solid tumors immunotherapy. However, a significant hurdle limiting the translation of ICIs to clinical therapeutic approaches is the corresponding AEs. For instance, serious AEs were reported in most patients with cancer receiving Elo . OverallSeveral unresolved questions remain to be answered. 1 – Is the expression of SLAM molecules associated with the tumor stage? 2 – Which biomarkers predict the patient’s therapeutic response to SLAM inhibition? 3 – Considering the alterations in the elderly patients’ immune system, is the therapeutic response of this patient population to the inhibition of the SLAM molecule similar to other patients? 4 – Is there a possibility of any immune-related AEs associated with SLAM inhibition?Several emerging therapies have been used in association with SLAM molecules. One of these novel paradigms is CAR T cells directed against SLAMF3 and SLAMF7. Clinical trials assessing the effects of these CAR T cells on progression-free survival and overall survival might be an intriguing research direction in solid and liquid tumors. mAb directed against SLAMF7, such as HuLuc63 or Elo, has emerged as a powerful tool in the armamentarium against MM Figure 4PF and A-AD conceptualized the study. PF, ShamM, ShabM, and HN drafted the manuscript. MA and A-AD revised the article critically for important intellectual content. MA and A-AD supervised the study. All authors contributed to the article and approved the submitted version.


Pelvic radiotherapy is used to treat a range of cancers. Radiotherapy can damage surrounding, non-cancerous tissue and organs, causing long-term problems, including bowel symptoms such as bleeding, pain, and incontinence. The provision of support and treatment for those affected as well as shared decision making regarding treatment should be informed by a solid understanding of the prevalence, nature, and severity of symptoms. We conducted a systematic review of population-based studies presenting patient-reported bowel symptoms to synthesize evidence on symptom prevalence and severity following pelvic radiotherapy. Multiple different bowel symptoms have been reported, and prevalence varies from 1% (bleeding) to 59% . We found substantial variation in the reported methods and few data pertaining to cancers other than prostate. Our review supports the view that bowel symptoms are a significant problem following pelvic radiotherapy and highlights limitations of the evidence base that should be addressed in future research. n = 39; gynecological, n = 6) reporting 19 datasets were included. Studies were methodologically heterogeneous. Most frequently assessed was bowel function . Also assessed was urgency, diarrhea, bleeding, incontinence, abdominal pain, painful hemorrhoids, rectal wetness, constipation, mucous discharge, frequency, and gas. Prevalence ranged from 1% (bleeding) to 59% . In total, 10 papers compared radiotherapy with non-cancer comparators and 24 with non-radiotherapy cancer patient groups. Symptom prevalence/severity was greater/worse in radiotherapy groups and symptoms more common/worse post-radiotherapy than pre-diagnosis/treatment. Symptom prevalence varied between studies and symptoms. This review confirms that many people experience chronic bowel symptoms following pelvic radiotherapy. Greater methodological consistency, and investigation of less-well-studied survivor populations, could better inform the provision of services and support.Pelvic radiotherapy can damage surrounding tissue and organs, causing chronic conditions including bowel symptoms. We systematically identified quantitative, population-based studies of patient-reported bowel symptoms following pelvic radiotherapy to synthesize evidence of symptom type, prevalence, and severity. Medline, CINAHL, EMBASE, and PsychINFO were searched from inception to September 2022. Following independent screening of titles, abstracts, and full-texts, population and study characteristics and symptom findings were extracted, and narrative synthesis was conducted. In total, 45 papers (prostate,  The term pelvic radiation disease has been applied to a group of chronic symptoms that may arise following pelvic radiotherapy ,2. TheseAcute bowel symptoms often arise during treatment and, for many, resolve within three months of the cessation of radiotherapy . ChronicTreatment options for post-radiotherapy bowel symptoms remain limited and are not without risk ,8. NovelThis systematic review aimed to identify data on chronic bowel symptoms reported by survivors following pelvic radiotherapy. Specific objectives were to synthesize data on symptom type, prevalence, and severity; temporal patterns in symptoms post-treatment; and survivor factors associated with increased prevalence and/or severity of symptoms. We focused on data from survivors recruited from population-based sampling frames to reduce the influence of selection biases on eligible studies.This review was registered (CRD42021274083) with the Prospective Register for Systematic Reviews (PROSPERO) and conducted and reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines .Systematic searches were carried out in Medline, the Excerpta Medica Database (EMBASE), American Psychological Association (APA) PsychINFO, and the Cumulative Index of Nursing and Allied Health Literature (CINAHL). The search, developed with input from a medical librarian, covered three concepts: radiotherapy, pelvic cancer, and bowel symptoms . No dateAfter de-duplication, titles and abstracts were screened independently by two reviewers . Full texts of papers considered potentially eligible by reviewers one or two were obtained and assessed, again by two reviewers, against the inclusion criteria. Discrepancies were resolved by discussion, with the third reviewer (LS) consulted in the event of disagreement.A paper was eligible if it was a primary, peer-reviewed research article, available in English which reported the results of a population-based quantitative study that included at least 100 survivors exposed to pelvic radiotherapy, this was to ensure focus on studies large enough to provide some confidence in precision of findings. Papers had to report the prevalence and/or severity of chronic symptoms  and theA paper was excluded if (1) the study population was participating in a clinical trial, as survivors may have experienced more intensive follow-up or intervention than constitutes routine care; (2) bowel symptoms were based on data abstracted from clinical records or clinician’s assessment; (3) outcomes were not collected in a standardized way across all participants; (4) it reported qualitative results only or a case study; (5) the study included survivors treated with different modalities, and it was not possible to disaggregate the results for irradiated survivors; (6) it was not possible to disaggregate chronic from acute symptoms; (7) the study was of colorectal cancer survivors due to the problem of distinguishing effects of radiotherapy from the effects of the cancer and/or associated surgery on the bowel; or (8) data for small sub-groups of irradiated survivors could not be readily combined to achieve a sample size ≥ 100. Criteria 1–6 were specified a priori, and criteria 7 and 8 were added following initial scoping searches. Data extraction from eligible papers was conducted by one reviewer onto a structured form and checked by another (AB and IB) with discrepancies resolved by discussion. We extracted data describing study characteristics , population characteristics , and findings  or univariate comparisons with groups not exposed to pelvic radiotherapy ; and any comparisons of prevalence/severity between subgroups defined by survivor characteristics).Quality appraisal used an adapted 10-item version of the Methodological Index for Non-Randomized Studies (MINORS) tool . Each itPapers reporting results from survivors drawn from the same data source(s) and treated/diagnosed during the same period were regarded as a single study reporting on the same dataset. Papers using the same data source but covering different periods of diagnosis/treatment were considered separate studies.Heterogeneity with respect to cancer sites, symptoms assessed, data collection tools, and assessment time points precluded meta-analysis. Instead, narrative synthesis was undertaken . We presFollowing de-duplication, 9031 citations underwent title and abstract screening ; 79 weren = 5 datasets; n = 20 papers) [n = 4 datasets of which n = 1 reported with New Zealand; n = 5 papers) [n = 2 datasets; n = 2 papers) [n = 2 datasets; n = 6 papers) [n = 2 datasets; n = 6 papers) [n = 1 dataset; n = 1 paper) [n = 1 dataset reported together; n = 2 papers) [n = 1 dataset; n = 1 paper) [n = 1 dataset; n = 2 papers) [n = 1 [n = 1 [n = 4 [Papers reported on datasets from nine countries: USA ( papers) ,56,57,59 papers) ,44,51,54 papers) ,58, Swed papers) ,24,25,55 papers) ,45,46,471 paper) , Ireland papers) ,27, Finl1 paper) , and Nor papers) ,34. A to1 [n = 1 , mixed g1 [n = 4 ,24,25,55n = 12 datasets) reported cross-sectional analyses [n = 9 datasets; all prostate) reported longitudinal analyses [n = 23 papers) [n = 4) [n = 2) [n = 2) [n = 2) [n = 1) [n = 1) [In total, 22 papers  ,38,43,54 [n = 2) ,40, Euro [n = 2) , EORTC Q [n = 2) , EORTC Q [n = 1) . Thirtee [n = 1) ,52,53,55n = 26 papers) [n = 19) [n = 16) [n = 10) [n = 8) [n = 6) [n = 5) [n = 3) [n = 3) [n = 2) [n = 2) [n = 1) [n = 1) [The most frequently reported bowel outcome was a bowel function score n =  papers [ papers) ,57,58,59[n = 19) ,52,53,54[n = 16) ,50,52,55[n = 10) ,48,50,55 [n = 8) ,53,55, d [n = 6) ,49,50,58 [n = 5) ,49,50, p [n = 3) ,49,50, r [n = 3) ,49,50, c [n = 2) ,23, muco [n = 1) , and gasn = 8 with a comparison group) were scored as high-quality papers [Nine papers  to 15 years. The prevalence of symptoms six months post-treatment/diagnosis ranged from 1% for bleeding  to 46% fAll ten papers ,53,54,59Overall, papers suggested a peak in the prevalence of most symptoms at six months (compared with baseline) and some decrease thereafter . Exceptin = 2 prostate; n = 3 gynecological) reported comparisons of irradiated populations with non-cancer comparators [n = 2 gynecological) with a non-radiotherapy group (surgery or no treatment) diagnosed with the same cancer as the irradiated group [Five papers  ,47,48,59A summary of findings by symptom is presented in Bowel function was reported as a score or a prevalence (sometimes at a stated degree of bother or problem) of experiencing bowel dysfunction.n = 20 papers) [Bowel function score: Twenty-six papers  reported on bowel function score ,57,58,59 papers) ,56,57,59 papers) ) to 90.3 papers) ).Two papers  ,28 preseFossa et al.  comparedVan de Poll-Franse et al.   ,52,53,54Seven papers from three prostate datasets compared pre- and post-treatment ,35,48,53Fossa et al.  reportedBergmark  reportedSixteen papers  from six datasets  reported on urgency ,50,52,55Three papers  ,26,55 coTen papers  ,34,53,55All four papers  which compared incontinence in irradiated survivors with non-cancer comparators reported higher prevalence in the cancer group. This was statistically significant in the three gynecological papers. Two prostate papers from different datasets, and one gynecological paper, reported higher prevalence of fecal incontinence in irradiated compared to non-irradiated patients. This was statistically significant in the prostate papers.Two papers  ,53 reporSeven papers  ,50,53,58One prostate paper compared diarrhea prevalence pre- and post-radiotherapy and reported a statistically significant increase of 7.6 percentage points .Three papers  compared radiotherapy with other treatments ,50,53. DTwo gynecological papers from two datasets reported worse/higher prevalence of diarrhea in radiotherapy and non-radiotherapy groups ,58; statFive papers  reported on abdominal pain or painful bowel movements ,28,49,50One prostate paper reported a higher prevalence of symptoms at six months post-treatment compared with the baseline  .Two papers reported data from the same prostate dataset ,50. The One prostate paper comparing a radiotherapy and a non-cancer group reported higher prevalence in the radiotherapy group  .Two high-quality prostate papers from one dataset reported no statistical difference in the prevalence of pain in a radiotherapy group versus a surgery group ,50. BergThree papers from one prostate dataset  ,49,50 reTwo of these papers  ,50 also Three papers ,49,50  compared radiotherapy and surgery groups ,50 reporDrummond et al.  found stTwo papers  ,55 from One paper  reportedOne gynecological paper  identifiThis systematic review aimed to identify and synthesize population-based data on the type, prevalence, and severity of bowel symptoms following pelvic radiotherapy, temporal trends in prevalence/severity, and associations with survivor characteristics. In total, 45 eligible papers reported on 12 distinct symptoms, though 7 were reported in fewer than 5 papers. The most commonly reported symptom was bowel function (often measured using EPIC-26), but even for this single symptom several different measures of function were reported .Prevalence of problems varied by symptom, ranging from 1% for bleeding  to 59% (n = 7) and marked by low prevalence. Previous studies have reported higher prevalence of these particular bowel symptoms [Bleeding and fecal incontinence are particularly problematic or distressing to cancer survivors ,61 and psymptoms . These dsymptoms . Furthersymptoms , it may In the papers we reviewed, there was little systematic documentation of temporal trends in symptom prevalence post-treatment, and studies varied greatly in the time-points at which they assessed outcomes. Some papers suggested a peak in symptom prevalence around six months ,33, and Previous, small, non-population-based studies have reported on survivor characteristics that may be risk or protective factors for bowel problems post-radiotherapy . A largeWe selected studies for review that used population-based sampling frames and patient-reported outcome measures. This was intended to increase the homogeneity and comparability of included studies. However, we found marked heterogeneity across multiple dimensions, including symptoms assessed, data collection tools used, comparator groups included, assessment time points, and cancer site treated . Additionally, radiotherapy regimens varied over time and institution. This heterogeneity precluded a statistical combination of study findings in a meta-analysis. However, our review highlights some specific gaps that might be addressed through future data collection and research to build a more consolidated evidence base.Most of the 45 eligible papers from 19 datasets pertained to prostate cancer . There were fewer papers and only two datasets pertaining to gynecological cancer survivors . There is a need for more population-based studies of gynecological cancers, for which the extent of damage to surrounding organs might be greater than for prostate cancer due to treatment usually involving irradiation of a larger area. There is also a need for more studies of less-researched cancers such as bladder cancer, the treatment of which, with radiotherapy, is also associated with bowel symptoms , but forThe potential for learning from future studies would be improved through greater consistency in data collection methods across studies, including in the timing of data collection relative to treatment, the choice of outcomes, and data collection instrument. Regarding the latter, EPIC-26, though widely used, has possible shortcomings, including poor construct validity . An alteOur review did not consider the lived experiences of survivors with post-radiotherapy bowel problems. Evidence  suggests chronic bowel problems may have a substantial impact on peoples’ quality of life, but symptoms are under reported, under-recognized, and under-treated by health professionals ,72,73. TThis review confirms that, following pelvic radiotherapy, cancer survivors may live, sometimes long-term, with a variety of chronic bowel symptoms. Worldwide, there are more than eight million survivors of prostate, endometrial, and cervical cancer , many of


To date, data on the efficacy of targeted therapies for mucosal melanoma (MM) are limited. In this study, we analyzed genetic alterations according to the primary site of origin, which could provide clues for targeted therapy for MM.We conducted a retrospective cohort study of 112 patients with MM. Targeted sequencing was performed to analyze genetic aberrations. Kaplan–Meier analysis was conducted with the log-rank test to compare the significance among subgroups.BRAF (17%), KIT (15%), RAS (15%), TP53 (13%), NF1 (12%), SF3B1 (11%), GNA11 (7%), GNAQ (5%), and FBXW7 (4%). A large number of chromosomal structural variants was found. The anatomic sites of esophagus and small bowel were independent risk factors for progression-free survival  and overall survival . Casitas B-lineage lymphoma (CBL) mutants showed significantly poorer PFS and OS. In contrast, MM patients who received immune checkpoint inhibitors (ICIs) had a significantly more favorable OS .In total, 112 patients with MM were included according to the anatomic sites: 38 (33.9%) in the head and neck, 22 (19.6%) in the genitourinary tract, 21 (18.8%) in the anorectum, 19 (17.0%) in the esophagus, 10 (8.9%) in the uvea, and 2 (1.8%) in the small bowel. The most significantly mutated genes included Our findings reveal the genetic features of patients with MM, mainly across six anatomic sites, offering a potential avenue for targeted therapies.The online version contains supplementary material available at 10.1186/s12935-023-03032-3. Primary mucosal melanomas (MM), arising from melanocytes in mucosal tissues lining the head and neck, respiratory, gastrointestinal, and urogenital tracts, account for 26% of all melanomas in Asian populations . AtypicaBRAF/MEK tyrosine kinase inhibitors improves patient survival [BRAF mutations are less frequent in MM and are susceptible to resistance, resulting in a lower effectivity of BRAF/MEK inhibitors [BRAF, RAS, KIT, NF1, and SF3B1 have been identified as significantly mutated genes [Importantly, MM is markedly different from cutaneous melanoma in terms of biological aspects. Recently, whole-genome and exome sequencing have revealed the molecular landscape and potential oncogenic drivers of MM, which are characterized by greater copy number (CN) variations, structural variations (SVs), and a lower tumor mutation burden (TMB) . Targetesurvival . Howeverhibitors . Immune hibitors . Althouged genes , there rTo further explore the molecular profiles of MM, we performed targeted next-generation sequencing (NGS) of 112 MM patients to explore the clinical significance of genetic characteristics potentially providing molecular targets and individualized treatments and provide a better mechanistic understanding of MM biology across the anatomic sites of MM.A total of 112 MM samples were collected from patients treated or consulted at the Sun Yat-sen University Cancer Center  between April 2007 and April 2022. Patients with cutaneous melanoma that had metastasized to the mucosa were excluded. The staging standard that we used followed the newest proposal for all anatomical sites published in April 2022 . For allFormalin-fixed and paraffin-embedded (FFPE) tissue blocks were assessed using hematoxylin-eosin (H&E) staining, and regions containing minimum of 20% tumor cells in unstained sections were selected for microdissection and subsequent experiments. Genomic DNA was extracted from tumors and patient-matched normal tissues or white blood cells using a QIAamp DNA FFPE Tissue Kit  according to the manufacturer’s instructions . The extAs described previously , we usedP-value of < 0.05. All the statistical analyses were performed using IBM SPSS V.25.0 .TMB comparison among the different subsets of patients was performed using the Wilcoxon rank-sum test. The Kaplan–Meier method was used to estimate overall survival (OS) and PFS, and differences were compared using the log-rank test. Cox proportional hazards regression analyses were used to evaluate the independently predictive factors of each biological and clinical features associated with OS and/or PFS. Statistical significance was defined as a two-tailed In total, 112 patients with MM who underwent tumor sequencing were identified: 86 patients (76.8%) with primary tumors, 17 (15.2%) with metastatic tumors, and 9 (8.0%) with recurrent tumors. A total of 51 patients underwent 295-gene panel sequencing, and 61 underwent 1021-gene panel sequencing for further analysis , KIT , MYC , RAS , TP53 , NF1 , SF3B1 (12/112 11%), TERT , GNA11 , CBL , GNAQ  and FBXW7  , LRP1B , MET , PRKDC , NOTCH4 , CCND3 , and FGF3/4/19 . Germline mutations were observed in two cases  with BRCA2 (p.W1692Mfs*3) and ATM (c.331 + 5G > A).Additionally, previous studies reported that other genes are frequently mutated in melanoma and other cancer types , 12–14, TERT and ATRX.Next, we observed that MM had a large number of chromosomal SVs, including somatic CN changes, fusions, and the telomere length-associated genes MYC , KIT , DAXX , KDR , NBN , CDK4 , MDM2 , PDGFRA , CCND1 , MDM4 , CCND3 , FGF19 , MET , FGF3 , FGF4 , FANCE , IRF4 , CDK6 , BRAF , NOTCH1 , and NOTCH2 . We also found several CN variations and the co-occurrence of KIT, KDR, PDGFRA, FGF3/4/19, FANCE, DAXX, and NOTCH clustered in the same chromosomal segment in one case, whereas CN deletions were detected in 14 cases. For example, CIC and PTEN deletions were more frequent in three and two cases, respectively.Somatic CN amplification was observed in 143 genes from 85 different chromosomal regions in 73 patients , among which TERT mutations were identified in 11 cases , all with TERT promoter mutations containing c.146 C > T , c.124 C > T , c.-124 C > T , and c.-58-u68_-58-u66delCCCinsTCT . Most ATRX variations had single nucleotide alterations or small fragment deletions. Loss of function was only detected in six cases . Interestingly, none of the cases had mutations in either TERT or ATRX, indicating they are mutually exclusive in MM, similar to those observed in gliomas [Finally, highly prevalent  gliomas .P < 0.001; OS: HR = 5.26, 95% CI = 2.51–11.03, P < 0.001) and CBL mutations  were independently risk factors for survival. In addition, we found that mitotic activity with ≥ 10/mm2  was an independent risk factor for OS. There were no associations between other factors and PFS or OS , whereas no V600 mutations were observed. Mutations in other genes, such as KIT, RAS, and NF1, were found in the primary sites of the head and neck, esophagus, anorectum, and genitourinary tract in our cohort. Notably, we found that SF3B1 mutations as recurrent genetic events in MM were more common in esophageal and uveal melanomas, which is similar to that reported in recent studies [GNAQ and GNA11 occurred in the majority of uveal melanomas found in this study, which is consistent with the findings of previous research [Consistent with previous reports, which revealed that acral melanoma and MM had limited DNA mutational burden , 22–24, efficacy . Neverth studies –31. In aresearch , 32.MYC amplification, which exemplified their role in MM pathogenesis and was in line with the results of other investigations [MYC expression is associated with aggressive biological behavior in cancers [KIT, PDGFRA, MDM2, and MDM4) and cell cycle genes, such as CDK4/6 and CCND1, were found in this study, similar to those reported in previous studies [PTEN loss correlates with poor survival in stage III melanoma [TERT and inactivation of ATRX commonly and exclusively occurred in MM, which has been reported previously, indicating the importance of telomerase activation or alternative telomere lengthening mechanisms in reducing length in MM [A previous whole-genome study showed that the SV load had the feature of complexity and diversity in MM . Not surigations , 33, 34. cancers . Additiomelanoma . In thisth in MM .A cohort of 466 Chinese patients with melanoma showed that MM was more aggressive and was associated with shorter survival than cutaneous lesions . MM arisCBL mutations in patients with MM; these were risk factors for poor OS and PFS, which are infrequent in published melanoma exome studies but frequent in desmoplastic melanoma [CBL mutations occur in myeloid malignancies and have been associated with poor prognosis [CBL mutants drive PI3K/AKT signaling and provide a rationale for therapeutic targets in myeloid malignancies [CBL mutations can activate the innate immune system to restrain cancer metastasis and improve the sensitivity to immunotherapy [CBL mutations might be promising targets for MM immunotherapy.We identified melanoma . Recurrerognosis . Ebert egnancies . Anotherotherapy . Finallyotherapy . Taken tOur study had some limitations. First, the sample size was small owing to the rarity of this disease, especially small bowel melanoma. Second, for molecular profiling in MM, we used targeted NGS, which only targeted genomic regions of partial genes and, therefore, could not reveal novel pathogenic point mutations, rearrangements, or epigenetic changes. Thus, the whole-exome sequencing in a larger cohort is required to comprehensively depict the molecular profile of MM. A better understanding of the molecular mechanisms is required to explore additional avenues for immunotherapy in MM.CBL mutations in MM are potential targets. Further studies are warranted to elucidate the mechanisms that link CBL mutations to immunotherapy responses, which may provide a rationale for immunotherapy.Our study demonstrates the molecular landscape of Chinese patients with MM based on targeted sequencing. Our finding on the genetic characteristics of MM among different anatomic sites reveal that Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6


Insights into flavor formation during fruit ripening can guide the development of breeding strategies that balance consumer and producer needs. Cherry tomatoes possess a distinctive taste, yet research on quality formation is limited. Here, metabolomic and transcriptomic analyses were conducted on different ripening stages. The results revealed differentially accumulated metabolites during fruit ripening, providing candidate metabolites related to flavor. Interestingly, several key flavor-related metabolites already reached a steady level at the mature green stage. Transcriptomic analysis revealed that the expression levels of the majority of genes tended to stabilize after the pink stage. Enrichment analysis demonstrated that changes in metabolic and biosynthetic pathways were evident throughout the entire process of fruit ripening. Compared to disease resistance and fruit color genes, genes related to flavor and firmness may have a broader impact on the accumulation of metabolites. Furthermore, we discovered the interconversion patterns between glutamic acid and glutamine, as well as the biosynthesis patterns of flavonoids. These findings contribute to our understanding of fruit quality formation mechanisms and support breeding programs aimed at improving fruit quality traits. Solanum lycopersicum) serves as a primary model system for studying climacteric fleshy fruit development and ripening [Solanum lycopersicum var. cerasiforme), as one of the tomato subspecies, are widely planted in China and are favored by consumers due to their intense flavor. Along with apples, grapes, and bananas, cherry tomatoes are among the four priority fruits supported by the Food and Agriculture Organization of the United Nations. Compared to the “big-fruit” tomato, cherry tomatoes exhibit higher levels of sugar (~44% higher), organic acids (~5% higher), and volatile organic compounds [Improving fruit flavor while maintaining high yields, disease resistance, and long postharvest shelf life poses a significant challenge for breeders ,2. To acripening . Cherry f sugar ~4% higherc acids ~% higher,ompounds . This maThe ripening process of red-fruited-clade tomato fruit can be divided into five stages: the mature green stage, the breaker stage, the pink stage, the light-red stage, and the red ripe stage. These stages are characterized by physiological and biochemical changes that ultimately lead to alterations in the appearance, texture, flavor, and aroma of the fruit ,7. Key mACC, ACS, and ACO genes involved in direct ethylene synthesis [CNR [PSY1 [OG [PG [PL [PME [E8 [LIN5 [PAR1, and PAR2 [To date, numerous key genes related to fruit ripening in tomatoes have been identified, including ynthesis ,15,16,17sis [CNR , PSY1 [1NR [PSY1 ,20, and PSY1 [OG  influenc [OG [PG ,23,24, P [PG [PL ,26, and [PL [PME  affectin[PME [E8 , LIN5 [2E8 [LIN5 , PAR1, aand PAR2  influencand PAR2 . Howeverand PAR2 . The comOmics approaches, such as metabolomics and transcriptomics, can contribute to our understanding of the genetic, hormonal, and metabolic networks that regulate tomato fruit development and ripening ,33. InteIn order to investigate the metabolic changes that occur during fruit ripening, we conducted a quantification of metabolites at three different stages of ripening: mature green (MG), pink (Pk), and red ripe (RR). A representative picture of the fruits is shown in During the MG-Pk stage, a total of 71 differentially accumulated metabolites (DAMs) were detected b, with 5Similarly, during the Pk-RR stage, a total of 79 DAMs were detected b, with 3Overall, across all three stages, a total of 24 DAMs were detected b. Among To investigate the expression patterns of genes during fruit ripening, we conducted whole-transcriptome sequencing at three distinct ripening stages. A total of 47.85 Gb of raw sequences with a read length of 150 bp was generated. From this dataset, we retained 68,677,640 high-quality reads, achieving an average alignment rate of 94.8% . UtiliziA total of 2169 differentially expressed genes (DEGs) were identified in fruits between the MG stage and Pk stage, including 680 upregulated genes and 1489 downregulated genes a. Gene OSubsequently, in fruits transitioning from the pink (Pk) stage to the ripe red (RR) stage, we identified a total of 1165 DEGs. Among these DEGs, 165 genes were upregulated, while 1000 genes were downregulated d. The GOAFF, known for promoting locule gel formation and improving firmness and solids content at low expression levels [PAR2, which enhances the accumulation of 2-phenylethanol and reduces the accumulation of 2-phenylacetaldehyde, thereby influencing aroma and flavor [AFF at the Pk-RR stage. However, the expression of several genes related to flavor showed continuous changes from the Pk stage to the RR stage. For example, ADH2, which encodes alcohol dehydrogenase and affects the aroma derived from the lipoxygenase pathway [The comparison between the MG-Pk and Pk-RR stages revealed only 356 common DEGs, indicating distinct physiological and biochemical responses before and after the pink stage of fruit ripening. We focused on analyzing well-characterized genes involved in color, texture, and flavor from the DEGs g. Interen levels , showed d flavor , display pathway , was conThere were 80 KEGG terms commonly enriched in both the MG-Pk and Pk-RR stages, mainly involving metabolite biosynthesis and various metabolic pathways. These findings reflect the continuous variations in abundant metabolites during the fruit ripening process . FurtherTo understand patterns linking the transcriptome and metabolome, correlations were calculated between the abundance of DEGs and DAMs. A total of 5332 pairwise correlations were identified, involving 1289 genes and 97 metabolites. Among these correlations, the top ten metabolites displayed the highest number of correlations with 934 genes , includip-value = 8.83 × 10−3). This observation suggests that alterations in fruit flavor are accompanied by a wide range of changes in metabolite composition, while fruit color may not exert a dominant influence on flavor. Additionally, none of the DAMs were significantly correlated with disease resistance genes, while two of eight firmness-related genes (ERF.D7 and TBG6) were found to be correlated with five or more metabolites  and Solyc11g011380 , along with the downregulation of Solyc03g083440 , indicating a reciprocal conversion between glutamic acid and glutamine during fruit ripening. Specifically, during the MG-Pk stage, glutamic acid is converted to glutamine, while during the Pk-RR stage, glutamine is converted back to glutamic acid. Furthermore, the conversion from glutamic acid to gamma-aminobutyric acid and then to succinic acid semialdehyde was found to occur during the Pk-RR stage, and the corresponding genes, Solyc11g011920 , Solyc01g005000 , and Solyc12g006470 , showed a lower expression level at this stage.To investigate the dynamic changes in fruit flavor during ripening, we performed an integrative analysis of the transcriptome and metabolome. Previous studies have demonstrated a significant association between glutamic acid and overall fruit flavor intensity and consumer preference . MeanwhiSolyc01g096670  showed a negative correlation with 5-O-caffeoylshikimic acid accumulation and a positive correlation with cholorogenic acid. Interestingly, both metabolites interacted with caffeoyl-CoA and displayed opposite accumulation profiles, suggesting a potential competitive relationship involving Solyc01g096670 in their biosynthesis. Moreover, when comparing the expression profiles of the DEGs Solyc02g083860 , Solyc11g013110 , and Solyc03g115220  with the accumulation profile of the dihydrokaempferol-kaempferol-quercetin pathway, we observed a closer resemblance, indicating that this pathway predominantly operates during fruit ripening, rather than the dihydrokaempferol-taxifolin-quercetin pathway. These findings contribute to our understanding of the mechanisms underlying the formation of fruit quality.In the context of the flavonoid biosynthesis pathway, we identified nine DEGs and nine DAMs, providing a comprehensive view of the changes in flavonoid metabolism . SpecifiThe formation of fruit flavor is a complex and multifactorial process that is not yet fully understood. In this study, we aimed to gain a comprehensive understanding of fruit ripening in cherry tomatoes by integrating transcriptome and metabolome data. By identifying DAMs and DEGs, we were able to unravel the patterns of metabolite accumulation and gene expression during fruit ripening. And the integrative analysis provided valuable insights into the development of improved fruit flavor and tomato breeding.ACO1, ACO3, ACO5, ACS2, and ACS4, promotes ethylene accumulation, leading to physiological and biochemical changes [PSY1 [TGB6 [FUL1 [PG2a [qFIS1 [SlERF.D7 [Solyc11g011300 [ADH2 [CGT [Fruit flavor development is typically associated with the ripening process . Previou changes . Moreovees [PSY1  contribuY1 [TGB6  leads toB6 [FUL1 , PG2a [4L1 [PG2a , qFIS1 [a [qFIS1 , SlERF.DSlERF.D7 , and Sol1g011300 , showed 00 [ADH2  and CGT DH2 [CGT , promoteSolyc01g096670, which encodes the p-coumaroyl quinate/shikimate 3′-hydroxylase, has been reported to be involved in the biosynthesis of both 5-O-caffeoylshikimic acid and chlorogenic acid [Solyc01g096670 promotes the accumulation of chlorogenic acid while suppressing the accumulation of 5-O-caffeoylshikimic acid during fruit ripening  were found to be correlated with five or more metabolites  were idabolites . TherefoSolanum lycopersicum var. cerasiforme cv. Yu-nu) were cultivated in Pu-dang village, Xindian town, Fuzhou city, China, during the spring of 2020. The cultivation was carried out using soil-less techniques in greenhouses. Water-soluble fertilizers were applied, and the nutrient supply was closely integrated with the irrigation system. No supplemental lighting was utilized, relying solely on natural sunlight for plant growth. Samples were collected at three distinct stages: mature green , pink , and red ripe . Immediately after harvesting, the fruits were frozen in liquid nitrogen for preservation. Biological triplicates were utilized for all experimental procedures.Cherry tomatoes  at Biozeron Biotechnology Co., Ltd., located in Shanghai, China. In brief, the tissues were lyophilized and pulverized, and 200 mg frozen powder was weighed and extracted with 0.6 mL 2-Chloro-L-phenylalanine . After ultrasonic and centrifugal treatment, 300 μL supernatant was collected and filtered through a 0.22 μmol membrane filter. Then, the filtrate was transferred into the detection vial and used to perform LC-MS analysis. An amount of 20 µL filtrate from each tested sample was mixed to create a quality control (QC) sample, which was used to correct the deviation in the mixed sample analysis results and any errors caused by the analytical instrument itself. And LC-MS analysis was performed on the remaining tested samples® HSS T3  column maintained at 40 °C. The temperature of the autosampler was 8 °C. Gradient elution of analytes was carried out with 0.1% formic acid in water (A2) and 0.1% formic acid in acetonitrile (B2) or 5 mM ammonium formate in water (A3) and acetonitrile (B3) at a flow rate of 0.25 mL/min. Injection of 2 μL of each sample was performed after equilibration. An increasing linear gradient of solvent B (v/v) was used as follows: 0~1 min, 2% B2/B3; 1~9 min, 2~50% B2/B3; 9~12 min, 50~98% B2/B3; 12~13.5 min, 98% B2/B3; 13.5~14 min, 98~2% B2/B3; and 14~20 min, 2% B2-positive model .Chromatographic separation was accomplished in a Thermo Vanquish system equipped with an ACQUITY UPLCm/z 81-1000 for full scan at a mass resolution of 70,000. Data-dependent acquisition (DDA) MS/MS experiments were performed with HCD scan. The normalized collision energy was 30 eV. Dynamic exclusion was implemented to remove some unnecessary information in the MS/MS spectra.The ESI-MSn experiments were executed on a Thermo Q Exactive Plus mass spectrometer with spray voltages of 3.8 kV and −2.5 kV in positive and negative modes, respectively. Sheath gas and auxiliary gas were set at 30 and 10 arbitrary units, respectively. The capillary temperature was 325 °C. The analyzer scanned over a mass range of https://www.metaboanalyst.ca/, accessed on 5 July 2023). Differentially accumulated metabolites (DAMs) were identified using a combination of the t-test and the orthogonal projection to latent structures–discriminant analysis (OPLS-DA) model. Metabolites with a p-value less than 0.05 and a variable influence on projection (VIP) value greater than or equal to 1 were considered as DAMs. The OPLS-DA model analysis .Data processing and metabolite identification were performed using MetaboAnalyst 5.0  according to the manufacturer’s instructions and genomic DNA was removed using DNase I . Then, RNA quality was determined using 2100 Bioanalyser  and quantified using ND-2000 . High-quality RNA samples  were used to construct the sequencing library.Total RNA was extracted from the 500 mg frozen powder using TIANGENRNA-seq transcriptome libraries were prepared following the TruSeqTM RNA sample preparation Kit from Illumina , using 1 μg of total RNA. Briefly, messenger RNA was isolated with polyA selection using oligo(dT) beads and fragmented using fragmentation buffer. cDNA synthesis, end repair, A-base addition, and ligation of the Illumina-indexed adaptors were performed according to Illumina’s protocol. Libraries were then size-selected for cDNA target fragments of 200–300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase  for 15 PCR cycles. After quantification via TBS380, paired-end libraries were sequenced with Illumina NovaSeq 6000 sequencing .Quality control measures were applied to the sequencing data using fastp software (version: 0.21.0) . AlignmeDifferential expression analysis was performed using the R package edgeR (version: 3.40.2) . Genes sFor Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, the TBtools software (version v1.1.20) was employed .In the subsequent analysis, an integrative approach was employed to explore the correlation between metabolite accumulation and gene expression.p-value less than 0.01. This analysis aimed to identify highly systematic changes occurring during the ripening stage.Correlation analysis between metabolite accumulation and gene expression was performed using R software (version: 4.2.2). The significance of correlations was determined by setting a threshold with an absolute value of correlation coefficient greater than 0.95 and a Tomato fruit ripening is a complex and systematic process, accompanied by the cessation of fruit development and abundant changes in metabolites. In this study, we identified differentially accumulated metabolites during the fruit ripening process, providing candidate metabolites related to flavor. Notably, key flavor-related metabolites already reached a stable level prior to the MG stage. Identification of differentially expressed genes revealed that the expression levels of the majority of genes tended to stabilize after the pink stage, and changes in metabolic and biosynthetic pathways occurred throughout the entire process of fruit ripening. Compared to disease resistance and fruit color genes, genes related to flavor and firmness might have a broader impact on the accumulation of metabolites. Additionally, we discovered interconversion patterns between glutamic acid and glutamine, as well as the biosynthesis patterns of flavonoids. These findings offer a systematic perspective on the accumulation of metabolites and changes in gene expression during the ripening of cherry tomato cv. Yu-nu. This study provides insights into the understanding of flavor formation mechanisms and can contribute to future breeding programs in cherry tomatoes, ultimately enhancing their flavor characteristics and quality.


Under isoflurane anesthesia, rat blood pressure (BP), heart rate (HR), and left ventricular (LV) hemodynamics were measured using Millar pressure transducers. The LV myocardium and brain cortex were analyzed by proteomics, bioinformatics, and western blot to explore the molecular mechanisms underlying GBP-induced cardiac dysfunction. In the first experiment, we found that i.v. GBP significantly decreased BP, HR, maximal LV pressure, and maximal and minimal dP/dt, whereas it increased IRP-AdP/dt, Tau, systolic, diastolic, and cycle durations . In the second experiment, we found that chronic GBP treatment resulted in hypotension, bradycardia, and LV systolic dysfunction, with no change in plasma norepinephrine. In the myocardium, we identified 109 differentially expressed proteins involved in calcium pathways, cholesterol metabolism, and galactose metabolism. Notably, we found that calmodulin, a key protein of intracellular calcium signaling, was significantly upregulated by GBP in the heart but not in the brain. In summary, we found that acute and chronic GBP treatments suppressed cardiovascular function in rats, which is attributed to abnormal calcium signaling in cardiomyocytes. These data reveal a novel side effect of GBP independent of the nervous system, providing important translational evidence to suggest that GBP can evoke adverse cardiovascular events by depression of myocardial function.Gabapentin (GBP), a GABA analogue, is primarily used as an anticonvulsant for the treatment of partial seizures and neuropathic pain. Whereas a majority of the side effects are associated with the nervous system, emerging evidence suggests there is a high risk of heart diseases in patients taking GBP. In the present study, we first used a preclinical model of rats to investigate, firstly, the acute cardiovascular responses to GBP (bolus  Gabapentin (GBP) is a 3,3-disubstituted derivative of gamma-aminobutyric acid (GABA). It is recommended as a first-line treatment for chronic neuropathic pain, particularly in diabetic neuropathy and post-herpetic neuralgia , and as 2+ channels (VGCCs) GBP binding assay and α2δ-null mice, Fuller-Bicer et al. [2+ influx and subsequent intracellular signaling transduction [Despite its structural similarity to GABA, GBP cannot bind to GABA receptors or affect the neuronal uptake or degradation of GABA, suggesting that the effects of GBP are not mediated by interacting directly with GABA receptors or with high-affinity Nasporters . Insteadhic pain . VGCCs acurrents ,30. By befficacy ,31. In aefficacy  is relatr et al.  demonstrr et al. . Actuallsduction ,34.V1.2 channel subunit, mediates excitation-contraction coupling (ECC), a highly coordinated process whereby membrane depolarization leads to contraction [2+ ions enter the cytosol via the CaV1.2 and trigger the release of massive amounts of intracellular Ca2+ from the sarcoplasmic reticulum (SR) into the cytosol via the ryanodine receptor , known as Ca2+-induced Ca2+ release (CICR) [2+ ions released from SR bind to troponin-C and enable actin-myosin cross-bridging and sliding of the myofilaments, resulting in sarcomere shortening and myocardial contraction [V1.2 and RyR but also CaM. CaM is a Ca2+ binding protein, which crosstalks with both CaV1.2 and RyR, forming a triangular interaction to regulate Ca2+ behavior and kinetics [V1.2 are believed to be reciprocal. CaV1.2 opens RyR, defined as orthograde signaling, and RyR can prevent CaV1.2 inactivation, defined as retrograde signaling [2+-bound and Ca2+-free states can bind and regulate both RyR and CaV1.2. RyR activity is increased by CaM at low Ca2+ concentrations  but inhibited by CaM at high Ca2+ concentrations , whereas CaV1.2 can be inactivated and facilitated by CaM via its binding to channel’s C-terminal domain as a Ca2+ sensor [2+ signaling which we believe underlies the subsequent cardiac dysfunction. Indeed, it has been found that hyper-activated Ca2+/CaM signaling contributed to pathology in several cardiac diseases, including heart failure, cardiac hypertrophy, and arrhythmia [2+ signaling dysfunction in GBP-induced cardiac side effects and will open novel avenues for treating in the future.Cardiomyocyte VGCCs, the Catraction . During e (CICR) . These Ctraction . CICR iskinetics . On one ignaling ,40. On t+ sensor . One of rhythmia ,42. Alth2+ signaling pathways within cardiac myocytes plays a pivotal role in these effects.In conclusion, the present study revealed that both acute and chronic treatments with GBP in normal adult rats had a significant adverse effect on global cardiovascular function, including hypotension, bradycardia, and reduced LV systolic/diastolic function. This is the first study to provide direct evidence demonstrating inhibition of GBP on cardiac contractility. Given the observed substantial increase in myocardial CaM protein expression in the GBP-treated rats, we speculate that a disruption in Ca


Gout represents a metabolic ailment resulting from the accumulation of monosodium urate crystals within joints, causing both inflammation and, harm to tissues. The primary contributor to gout’s emergence is an elevated presence of serum urate, which is under the regulation of kidney and, gut urate transporters. Mitigating this risk factor is crucial for averting gout’s onset. Several treatments rooted in TCM and related active compounds have demonstrated efficacy in managing gout, skillfully regulating serum uric acid (UA) levels and curbing inflammation’s progression. This analysis compiles key foundational research concerning the molecular signaling pathways and UA transporters linked to gout, under the regulation of TCM. The focus includes individual botanical drug, active compounds, and TCM formulations, which have been consolidated and examined in this overview. The primary keywords chosen were “gout, hyperuricemia, gouty arthritis, traditional Chinese medicine, Chinese botanical drug, medicinal botanical drug, and natural plant”. Various relevant literature published within the last 5 years were gathered from electronic databases, including PubMed, Web of Science, CNKI, and others. The findings revealed that TCM has the capacity to modulate various signaling pathways, including MAPK, NF-κB, PI3K/Akt, NLRP3 and JAK/STAT. Additionally, it impacts UA transporters like URAT1, GLUT9, ABCG2, as well as OATs and OCTs, thereby contributing to gout treatment. TCM helps maintain a balanced inflammatory interaction and facilitates UA excretion. This study enhances our understanding of TCM’s anti-gout mechanisms and introduces novel perspectives for establishing the clinical significance and future prospects of TCM-based gout treatment.  Gout represents a painful type of arthritis triggered by the accumulation of monosodium urate (MSU) crystals within synovial fluid and other bodily tissues in people with hyperuricaemia. Intermittent flaring condition of gout often bring along unforeseeable discomfort, swelling of joints, reddening, and damage to joints as a result of recurrent flare-ups . The enhGout presents a multifaceted pathological sequence, encompassing diverse underlying factors. These include urate accumulation , hindran(Phellodendron chinense C.K.Schneid. (Coptis chinensis Franch. (Atractylodes lancea (Thunb.) DC.  DC. , Achyrana Blume. , Smilax ra Roxb. , Eucommies Oliv. , Ermiao es Oliv. , Simiao es Oliv. , Sanmiaoes Oliv. , Wuling es Oliv.  and Erdies Oliv.  demonstrIn clinical practice, the core strategy for effective treatment can be considered as two aspects in early-onset and intermittent of gout: 1) in early-onset of gout: urate-lowering therapy to hyperuricemia, which relate to dissolute MSU crystals and prevent long-term of gout flares; and 2) in intermittent of gout: anti-inflammatory therapy to intermittent episodes of severely painful arthritis (gout flares) caused by deposited MSU crystals . Thus, tOne of the most extensively studied and vital inflammasomes is the NOD-like receptor thermal protein domain-associated protein 3 (NLRP3). This vital component comprises the sensing molecule NLRP3, coupled with apoptosis-associated speck-like (ASC) adaptors, caspase activating recruitment domain, and recruitment caspase-1. Activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) prompts the upregulation of NLRP3 expression. Following this, it combines and forms an intricate configuration with an adapting protein, ASC, and procaspase-1. Caspase-1 is subsequently activated through the autolysis of procaspase-1. This sequential process subsequently triggers the handling and liberation of interleukin (IL)-1β, IL-18, along with alarm signals. Additionally, it leads to the initiation of pyroptotic cell demise . PathogeScutellaria baicalensis Georgi. This compound exhibits neuroprotective, antioxidant, anti-inflammatory, and anti-tumor properties  W.T.Wang, has demonstrated a noteworthy ability to reduce the levels of NLRP3, caspase-1, and IL-1β in the paw tissues of mice with MSU-induced acute gout. It also diminishes proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-18, and thereby alleviating gout-related pain and swelling. This compound acts therapeutically by inhibiting the activation of the NLRP3 inflammasome and the subsequent caspase-1 formation  and reducing p65 within the nucleus. Additionally, it lessens NF-κB DNA-binding activity in synovial tissues of rats experiencing inflammation induced by MSU . Furthermore, it inhibits the nuclear translocation of p65 and IκB, thus effectively mitigating the symptoms of acute gout arthritis (Curcuma Longa L. hinders the degradation of IκBα and phosphorylation of NF-κB subunits (p50 and p65). This dual effect leads to decreased expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, as well as cyclooxygenase-2 (COX-2) and PGE2. As a consequence, a protective impact of curcumin is exerted against gout by alleviating the inflammatory response via modulation of the NF-κB signaling pathway , myeloid differentiation factor 88 (MyD88), as well as several key proteins involved in the NF-κB pathway, including phospho-IKBα, IKBα, phospho-p65, and p65. This led to the inhibition of the TLR4/MyD88/NF-κB pathway’s activation triggered by MSU, ultimately providing relief from gout symptoms  . It alsoPhosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT) are upstream elements of the NF-κB signaling cascade, pivotal in the initiation and release of various inflammatory factors . ElevateCoptis chinensis Franch. diminishes the levels of cleaved poly ADP-ribose polymerase (PARP), cleaved-caspase-3, Bax, cytochrome C (cytC), and apoptosis-inducing factor (AIF). Concurrently, it elevates the protein levels of Bcl-2 and p-Bad, thereby offering protection against oxidative stress, mitochondrialapoptosis, and renal inflammatory harm triggered by hyperuricemia. This safeguarding effect is achieved by modulating the PI3K/AKT signaling pathway , PI3K, and mTOR. It effectively safeguards against hyperuricemia-induced kidney damage by activating the PI3K/AKT signaling cascade , and the signal transducer and activator of transcription (STAT) signaling pathway is composed of tyrosine kinase-related receptors called JAK, and STAT , This paPhellodendron chinense C.K.Schneid., has exhibited the capacity to diminish the expression of phospho-JAK2, phospho -STAT3, and suppressor of cytokine signaling 3 (SOCS3). Moreover, it lessens renal concentrations of TNF-α, IL-1β, and IL-6 in mouse models of hyperuricemia, thereby mitigating renal impairment. This is achieved through the inhibition of the JAK-STAT signaling pathway’s activation and the attenuation of the inflammatory response triggered by UA  and hepatitis, is capable of reducing the expression of phospho-JAK2 and phospho-STAT3. It also operates to hinder the JAK2/STAT3 signaling cascade within synovial tissues, thereby alleviating ankle joint swelling and gout-induced inflammation in rats (Liriodendron chinense (Hemsl.) Sarg., a component of TCM employed to expel “wind and dampness”, has been observed to impede the advancement of hyperuricemic nephropathy. This is achieved by curbing the activation of the JAK2/STAT3 signaling cascade, lessening the influx of inflammatory agents, and diminishing the buildup of UA within the kidney , comprising extracellular signal-regulated kinases (ERK1/2), Jun N-terminal kinase (JNK), and P38, play crucial roles in the onset of gouty arthritis episodes. Serving as precursor kinases, mitogen-activated protein kinase kinase 4/7 (MKK4/7), MKK3/6 andmitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) govern the activation of JNK, P38, and ERK1/2, respectively . Recent Dioscorea polystachya Turcz., the total saponin fraction exhibits the ability to ameliorate synovial tissue conditions. Additionally, it diminishes the protein expressions of phospho-MEK1/2, phospho-ERK1/2, phospho-JNK, and MKK4 in rats induced with MSU crystals. This process effectively mitigates inflammation in gout arthritis by inhibiting the MAPK signaling pathway , has been found to widespread use in TCM for various forms of arthritis  Decne., and Terminalia chebula Retz., exhibits the ability to alleviate ankle joint swelling and diminish TNF-α and IL-1β concentrations within MSU-induced mice air pouch lavage fluid. This is accomplished by restraining the levels of p-p38/p38 through the modulation of the MAPK signaling cascade, thus effectively mitigating gout-related inflammation , organic anion transporter 4 (OAT4/SLC22A11), OAT10/SLC22A13, and urate anion transporter 1 (URAT1/SLC22A12) . ATP BinAn additional path for UA elimination occurs within the intestines. ABCG2 is a pivotal regulator of urate excretion primarily in the gastrointestinal tract , signifyCurrently, it has been widely proven that TCM can effectively reduce UA in clinical practice. The multi-target function of TCM simultaneously affects multiple UA transporters, achieving the reduction of UA and the protection of the kidneys. Various herbal extracts used in TCM and natural bioactive compounds have been demonstrated to be beneficial in treating gout .This comprehensive analysis has substantiated that Chinese medicine monomers, individual herbal remedies from Chinese tradition, and formulations of TCM offer significant assistance in gout management by impacting the expression and function of urate transporters. These encompass GLUT9, URAT1, OATs, and ABCG2, as well as their associated regulatory cascades like NLRP3, NF-κB, JAK/STAT, PI3K/AKT, and MAPK pathways. However, there are still several limitations must be considered in the gout therapy using TCM.As we reviewed, these botanical drugs have shown positive anti-gout effects. Yet it is worth noting that the therapeutic effect of inhibiting inflammatory signaling pathways and uric acid transporters on gout has only been experimentally validated in cells and animals. Due to the differences between species, the therapeutic effects of some plant drugs with anti-inflammatory and reducing uric acid effects at the cellular and animal levels may not be satisfactory in the human, so more in-depth research is needed.Another point to consider is that more studies are needed to confirm whether TCM can be effective targets for gout treatment. In recent literature, a compelling association between UA transporters and signaling pathways has come to light. UA drives excretion through the activation of signaling cascades involving NLRP3, PI3K/Akt and MAPK, leading to the augmentation of PDZK1 and ABCG2 expression, thereby mitigating the effects of hyperuricemia . The PI3Notably, studies on the regulatory mechanisms the therapeutic applications of small molecule metabolites in gout by regulating protein functions are particularly lacking, which may be a weak direction for understanding the role in the pathogenesis and treatment of gout. Utilizing a metabolomics approach, researchers have employed a strategy to unveil the underlying metabolic mechanisms and plausible targets associated with TCM. Likewise, the interplay between metabolites and receptors offers an avenue to investigate potential therapeutic targets and the regulatory function of TCM within signaling pathways, facilitating internal signaling communication for addressing gout. A prime illustration lies in the distinctive involvement of purine metabolism in both inflammatory responses and immune system modulation in the context of gout. Notably, purinergic receptors, including P2X1, P2X7, P2Y2, and P2Y14, are found expressed across a wide spectrum of immune cells . ATP stiAt present, we are engaged in clinical experimental investigations focusing on the role of biomarkers influenced by protein targets in the initiation of gout. Simultaneously, we are exploring the potential of utilizing TCM-regulated proteins to modulate metabolic pathways for gout management in mouse models. Furthermore, by combining and synthesizing the results of multiple studies that investigate TCM’s inhibitory effects on signaling pathways like NLRP3, along with its positive effects on gout, we are optimistic about substantial progress in the field of anti-gout TCM. This advancement involves a direct emphasis on proteins and biomarkers in the approaching phase, potentially leading to a smooth transition into clinical application and improving the current scenario of gout detection, prediction, and therapy.


Increasing evidence shows that lncRNAs, an important kind of endogenous regulator, are involved in the regulation of follicular development and female fertility, but the mechanism remain largely unknown. In this study, we found that SDNOR, a recently identified antiapoptotic lncRNA, is a potential multifunctional regulator in porcine follicular granulosa cells (GCs) through RNA-seq and multi-dimension analyses. SDNOR-mediated regulatory networks were established and identified that SOX9, a transcription factor inhibited by SDNOR, mediates SDNOR’s regulation of the transcription of downstream targets. Functional analyses showed that loss of SDNOR significantly impairs GC morphology, inhibits cell proliferation and viability, reduces E2/P4 index, and suppresses the expression of crucial markers, including PCNA, Ki67, CDK2, CYP11A1, CYP19A1, and StAR. Additionally, after the detection of ROS, SOD, GSH-Px, and MDA, we found that SDNOR elevates the resistance of GCs to oxidative stress (OS) and also inhibits OS-induced apoptosis. Notably, GCs with high SDNOR levels are insensitive to oxidative stress, leading to lower apoptosis rates and higher environmental adaptability. In summary, our findings reveal the regulation of porcine GCs in response to oxidative stress from the perspective of lncRNA and demonstrate that SDNOR is an essential antioxidative lncRNA for maintaining the normal state and function of GCs. Based on these interactions, the PPI network was established and visualized by using Cytoscape v3.7.2 software. In addition, the Cytohubba and MCODE package functions were utilized to identify the hub genes (nodes with top 5% higher degree) and different modules within the PPI network.The SDNOR-mediated DEmiRNA–DEmRNA and DETF–DEmiRNA regulatory networks were constructed as described previously . In brie−∆∆CT method. GAPDH and U6 were selected as loading controls to normalize the expression level of coding and non-coding genes, respectively. The primers used here are listed in RT-qPCR was performed to confirm the accuracy of RNA-seq analysis and the regulatory interactions among different molecules. Briefly, the total RNA from GCs under different conditions was reverse-transcribed into cDNA by using HiScript III RT SuperMix . RT-qPCR reactions were performed by using AceQ qPCR SYBR Green Master Mix  with three independent biological replicates on an ABI PlusOne System . The expression levels of genes of interest were calculated using the 2Western blot assays were performed as previously described . The priChIP assays were performed as described previously . In brietreatment-ODcontrol)/ODcontrol. Each group has at least six independent replicates. For cell cycle measurement, GCs after treatment were first collected and re-suspended using 75% cold ethanol overnight. Then, the ethanol was replaced with PBS containing 5 μL PI and 20 μL RNase in a dark room for 20 min. Subsequently, cells were sorted by flow cytometry  and analyzed using Flowjo software . GC apoptosis was detected by using an Annexin V-FITC/PI Apoptosis Detection kit  as previously described [To detect the states of GCs under different conditions, the proliferation, viability, cycle, and apoptosis were analyzed as previously described . Brieflyescribed . In brie2O2 for 2 h. ROS levels were detected using a cell counting machine with an excitation wavelength of 488 nm. GCs under different conditions were collected, and the activity of superoxide dismutase (SOD) was analyzed using the WST-8 method under 450 nm wavelength , the activity of glutathione peroxidase (GSH-Px) was measured using NADPH-mediated colorimetric method at 340 nm wavelength , and the level of malondialdehyde (MDA) was detected using the TBA method under 532 nm wavelength .For reactive oxygen species (ROS) measurement, an ROS detection kit  was used. Briefly, GCs after treatment were submerged in the FBS-free DMEM/F12 medium with 10 μM DCFH-DA for 30 min at 37 °C in a dark room. After incubation, GCs were washed with FBS-free medium three times and then treated with 150 μM HAfter transfection for 12 h, the cell culture medium was replaced with an FBS-free medium for another 24 h, which was then collected for steroid hormone quantification. To analyze the concentration of 17β-estradiol (E2) and progesterone (P4) within the follicular fluid and the cell culture medium mentioned above, enzyme-linked immunosorbent assays (ELISAs) were performed by using E2 (#AR E-8800) and P4 (#FR E-2500) detection kits obtained from Beijing North Institute of Biotechnology Co., Ltd . Briefly, samples were diluted five times and transferred into an ELISA plate for 30 min at 37 °C. Then, 50 μL enzyme reagent was added and incubated for 30 min at 37 °C. Subsequently, the reagent was removed, and 100 μL developer was added and incubated in a 37 °C dark room for 10 min. Finally, the optical density (OD) value of each sample was detected under 450 nm wavelength after adding 50 μL reaction termination buffer. The sensitivity of the detection kit was 0.1 pg/mL for E2 and 0.045 ng/mL for P4. Each group contained at least three independent samples. KpnI and XhoI restriction enzyme sites, which was termed pcDNA3.1-SOX9. To analyze the effects of miR-29c on the 3′-UTR activity of its candidate targets, the 3′-UTR fragments with the wild-type or mutant miR-29c-responsive elements were synthesized and cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector between XbaI and SacI restriction enzyme sites. All the plasmids constructed in this study were verified by Sanger sequencing. For dual-luciferase activity assays, GCs were collected after transfection for 24 h, and the luciferase activities of each sample were analyzed by using a dual-luciferase detection system . The relative luciferase activity of each sample was calculated and normalized as the ratio of Firefly/Renilla. The overexpression plasmid of SDNOR (pcDNA3.1-SDNOR) was prepared in our previous study . For SOXTo analyze the morphometric features of GCs under different conditions, high-resolution images were obtained using an Odyssey Imaging System (LI-COR Biosciences) after treatment for indicated times. Specifically, cells with clear, smooth edges, and no obvious serration, breaks, or vacuoles were considered to have better membrane integrity. Inversely, cells that have no clear and smooth edge, or normal cell morphology, but that have multiple vacuoles inside, exhibit shrinkage, and are even broken were considered seriously damaged. The numbers of abnormal GCs were counted under a microscope, and five detection fields of each well were randomly selected for each sample. The statistical analyses were conducted with three independent samples per group.t-test and one-way analysis of variance (ANOVA) followed by S-N-K post hoc tests were utilized for significance evaluation. Significance between groups is denoted as * p < 0.05 and ** p < 0.01. The experiments in this study were conducted in three independent replicates with at least three independent samples, and the data are shown as mean ± S.E.M. The statistical analyses, including significance calculation, Pearson correlation, and simple linear regression, were performed using IBM SPSS Statistics v26.0 , GraphPad Prism v8.0 , and RStudio v4.1. An unpaired two-tailed Student’s Sus scrofa genome assembly 11.1 (Sscorfa 11.1) with a two-iteration mapping strategy | ≥ 1 and adjusted FDR < 0.05, 311 (108 down- and 203 upregulated) and 385 (192 down- and 193 upregulated) DEmRNAs were identified in GCs after knockdown and overexpression of SDNOR, respectively (p < 0.05) involved in the regulation of cell state (apoptosis and proliferation) and function (estrogen synthesis) were identified, including TGF-β, Wnt, TNF, and hormone synthesis pathways (p < 0.05), including 4 cell component (CC) terms, 6 molecular function (MF) terms, and 11 biological process (BP) terms, were identified , namely Akt3, IL6, ACTA2, PTPN11, NOS3, PRKCG, GNAL, GNA13, and CXCR5, were considered as hub genes. In addition, three significantly enriched modules were identified with the MCODE package function; they were termed modules I, II, and III  and 17 highly involved GO terms (p < 0.05) were identified, indicating that they may play important roles in reproduction, stimulus response, RNA degeneration, and cellular processes via multiple crucial pathways, such as AMPK, Wnt, PI3K-Akt, FoxO, and mTOR. miRNAs, another important class of non-coding RNA, are essential downstream functional factors of lncRNAs . Thus, wZNF614, the expression levels of other target genes were all dramatically inhibited in miR-29c-overexpressed GCs  [CEBPB and FZD4) [COL4A1, PTPN11, and INHBE) [Until now, only a few lncRNAs have detailed functional annotations due to the lack of omics exploration which was mainly utilized for the identification of omics changes and crucial factors during physiological and pathological processes ,46. Herend JUND) ,47,48, fnd FZD4) ,50, and d INHBE) ,52,53, id INHBE) ,54, (b) d INHBE) , (c) lncd INHBE) . In thisSOX9 is regulated by non-coding RNAs and cytokines [SOX9 transcription in breast cancer cells by recruiting PolII and raising H3K4me3 levels [SOX9 expression [SOX9 in porcine GCs.It has been reported that TFs mediate the functions of lncRNAs in several essential biological processes, such as the lnc-ISIR/IRF3 axis in autoinflammation , the lncytokines ,66,67. Apression , we specPhenotype is determined by the interactions between genes and the environment. Recent studies have shown that environmental stressors in the sow breeding industry increase ROS accumulation, break redox balance, and cause oxidative stress in the ovary, which further leads to GC apoptosis, follicular atresia, low fecundity, and infertility ,69. Sow In summary, we demonstrate that SDNOR, a recently identified lncRNA, is essential for the normal state and function of porcine GCs. Mechanistically, SDNOR alters the transcriptome of GCs partially through TFs, such as SOX9. Interestingly, SDNOR also functions as an antioxidant and suppresses oxidative stress-induced GC apoptosis. Importantly, GCs with high SDNOR levels have higher resistance to oxidative stress. Our findings identify an endogenous functional lncRNA for the regulation of GC state and follicular development and also provide a potential biomarker for the selection and breeding of sows with strong antioxidative activity and environmental adaptability.


Enterobacterales, of which Escherichia coli (86.44%) was the dominant host, followed by Salmonella (8.57%) and Klebsiella pneumoniae (3.48%). Salmonella ST11 and K. pneumoniae ST11 were common and mostly from humans. IncF33 plasmids were found worldwide but prevalent in Chinese farm animals, predominantly carrying antibiotic resistance genes such as blaCTX-M-55, fosA3, blaTEM-1B, rmtB, and floR. Comparative genomics analysis of 103 complete IncF33 plasmid sequences showed highly similar backbones except for 16 lacking partial backbone fragments. Variable regions were diverse, containing various antibiotic resistance genes, insertion sequences, or other plasmid fragments. They can be roughly divided into two sublines based on the production of different CTX-M enzymes. Similar IncF33 plasmids from different countries were identified. Some early IncF33 plasmids lacked part of their leader regions, which showed over 99% homology with early F2:A-:B- plasmids, indicating that leader regions of IncF33 likely came from F2:A-:B- plasmids. In addition, IncF33 plasmids cointegrating with other types of plasmids to form new cointegrate plasmids are increasing, making them more efficient in their dissemination and persistence in Enterobacterales, which could pose a significant threat to global public health.To comprehensively understand the formation and evolution of IncF33 plasmids, a global collection of whole-genome sequences of 863 strains positive for IncF33 plasmid replicons was analyzed. The results showed that the IncF33 plasmids were mainly identified in Enterobacterales in China, and they are important vectors for transmitting several clinically important antibiotic resistance genes. The study revealed that the IncF33 plasmid is mainly prevalent in China animal-derived Escherichia coli and has the potential for cointegration and intercontinental dissemination. Therefore, it is crucial to enhance surveillance and control measures to limit the spread of IncF33 plasmids and their associated antibiotic resistance genes.Plasmids that capture multiple antibiotic resistance genes are spreading widely, leading to the emergence and prevalence of multidrug-resistant bacteria. IncF33 plasmids are a newly emerged plasmid type highly prevalent in animal-source  Enterobacterales are of great concern. The horizontal spread of antibiotic resistance genes (ARGs) is the main reason for the acquisition of resistance in Enterobacterales. Of these, plasmids are crucial vehicles for the effective transmission of ARGs.The emergence and spread of multidrug-resistant bacteria pose a serious threat to global public health and have attracted increasing attention on a worldwide scale . ParticuEnterobacterales distinguished by PCR-based replicon typing . Subsequently, we performed a statistical analysis of the isolation source, geographical location, year of isolation, and bacterial host of 103 IncF33 plasmids and 863 strains carrying IncF33 plasmid replicons. The results showed that IncF33 plasmid replicons were identified in 41 countries, including those in Asia , Africa , South America , North America , Europe , and Oceania . Of note, both IncF33 plasmids  and whole-genome sequences of strains  positive for IncF33 plasmid replicons are mainly distributed in China  plasmids with typical IncF33 plasmid backbones, some of which have fragments of other plasmid types inserted into variable regions; (ii) cointegrated plasmids formed by the fusion of a complete IncF33 plasmid and a complete plasmid of another type; (iii) complete in China .Enterobacterales. E. coli strains  are the dominant host, followed by Salmonella  and K. pneumoniae . IncF33 plasmids are also sporadically identified in other Enterobacterales, such as Escherichia fergusonii, Escherichia albertii, Citrobacter freundii, Enterobacter cloacae, Enterobacter hormaechei, Klebsiella variicola, Serratia marcescens, and Shigella flexneri. Furthermore, IncF33 plasmids were identified in Enterobacterales isolates from various origins, including animals, humans, food, and the environment. Except for 83 strains for which no source information was found, of the remaining 780 strains, the most common source was animals , followed distantly by humans , food , and the environment . Animal isolates were predominantly from poultry  and swine   . These rE. coli isolates to 157 distinct sequence types (STs) except for 77 isolates with novel STs, and ST10  was most frequently detected  and K. pneumoniae ST11  were the most frequently detected  and 82.35% (14/17), respectively  and blaCTX-M-65 , fosfomycin resistance gene fosA3 , aminoglycoside resistance genes rmtB , aph(3'')-Ib , aph(6)-Id , and aph(3')-IIa , amphenicols resistance gene floR , quinolone resistance gene oqxAB , tetracycline resistance gene tet(A) , and sulfonamide resistance gene sul2   . Furtherce genes repA2/copB-repA1-repA4) that control plasmid replication; leading region contained genes  related to plasmid maintenance and stability; conjugative transfer region contained genes  responsible for pilus synthesis and assembly, DNA transfer, aggregate stability, surface exclusion, and regulation of transfer region gene expression  and transfer region genes (traW to traI) were also frequently deleted, possibly due to a recombination event. The leading genes and partial transfer regions genes of pLSH-KPN148-2 were inverted compared with those of pHN7A8. Similarly, pHNAH9 and pIncFII_L111 lacked a large part of leading and transfer regions, which might also result from multiple recombination events  and p477Kp (LN897475) plasmids from Bolivia were highly similar to China epidemic plasmid pHN7A8 (JN232517), and there were only nine single nucleotide polymorphisms (SNPs) between p397Kp and pHN7A8 (MW298658) from bovine in Canada, unnamed plasmid (CP066367) from humans in the United States, and unnamed plasmid (CP062214) from water in New Zealand were all highly similar to the pHN7A8 plasmid backbone , GCA_002517365.1 , GCA_012809895.1 , and GCA_014286695.1 . For convenience, the GenBank accession numbers of the strains are tentatively referred as their plasmids in subsequent analysis. Linear comparison of four plasmids with pHN7A8 plasmid backbone showed that the group II intron was not detected in all four plasmids, and plasmid GCA_014286695.1 possessed the same backbone as plasmid pHN7A8 except for pemK which was truncated by IS1294. In addition, no ARGs were identified in plasmid GCA_014286695.1. The other three plasmids were all missing an approximately 3.6 kb segment containing the tir, pemI/K, ycdA, and ycdB genes. Plasmid GCA_002517365.1 also lost most of the leading region genes and putative genes downstream of this segment, including stbB/A, ycgA/B, ssb, and parB, etc.  database. We found that tir-pemI/K-ycdA-ycdB was presented in F2:A-:B- plasmids (CP038385 and CP025139) from E. coli isolates from humans in Iran in 1963 and E. coli isolates from humans in Brazil in 1974, respectively. Linear comparisons of these two plasmids with pHN7A8 plasmid backbone showed that the tir-pemI/K-ycdA/B-stbB/A-ycgA/B-ychA-klcA-ycjA segment showed 99% identity to the corresponding segment of pHN7A8, but the entire conjugative transfer region genes differ (88% coverage and 96% identity) from those of pHN7A8, with lower homology to the corresponding genes of pHN7A8, such as traM, traJ, trbD, traN, trbA, and traS genes. These findings suggested that early IncF33 plasmids, such as GCA_000460335.1, GCA_002517365.1, and GCA_012809895.1, may have acquired the tir-pemI/K-ycdA-ycdB segment or a larger segment of the leading region from the F2:A-:B- plasmids, giving them a more complete leading region that facilitates their efficient persistence among Enterobacterales and widespread dissemination in the environment, and eventually evolved into the more prevalent IncF33 plasmid lineage , and several other major clusters included clusters B (n = 4), C (n = 3), D (n = 4), E (n = 4), and F (n = 3). The clustering results showed that the variable regions of IncF33 plasmids are highly diverse.Clustering analysis of the variable region sequences of 103 IncF33 plasmids using CD-HIT program (version 4.8.1) showed that the variable region sequences of all plasmids were assigned to 65 clusters, of which 59 clusters were represented by single or two plasmids. The remaining six clusters comprised three or more plasmids. Cluster A contained the largest number of plasmids (JQ432559) from a porcine-derived E. coli isolate in China in 2008 and F2:A-:B- plasmid CP020340 from a human-derived Shigella flexneri isolate in China in 2007 (aac(6')-Ib-cr resistance module [IS26-hp-aac(6')-Ib-cr-∆IS26-IS26] in CP020340 was not found in these IncF33 plasmids. This observation may be associated with the recombination between the two copies of IS26 in the same orientation that could lead to the insertion or loss of the resistance module. At the same time, a segment found in these IncF33 plasmids and CP020340, containing three IS26 elements, truncated intl1, and the fosA3 resistance module, was identical to that of pHK23a with opposite orientation. In addition, blaCTX-M-3 was found in plasmid pHK23a and CP020340 with two SNPs compared to blaCTX-M-55 in those IncF33 plasmids.Plasmids pAH01-3, pT224A, pHNMC02, pHNBC-6, and pHNGD4P177 possessed variable regions similar to that of F2:A-:B- plasmid pHK23a  was absent in pCREC-591_2, which might be the result of a recombination event between intl1∆ and orf1. In addition, plasmids pHNMPC43 and pHNMPC51 lacked the fosA3 module and one IS26 element compared with pAH01-3, possibly due to the recombination events between IS26 elements.Plasmids pCREC-591_2, pHNMPC43, and pHNMPC51 possessed similar variable regions to pAH01-3, but a segment (26 or IS1294 (1-∆Tn2-IS26-intl1∆) was identical to segments in the cluster A plasmids with a size of 2.8 kb.Compared with cluster A plasmids, the variable region of cluster B plasmids acquired segments from other types of plasmids and became more complex. Briefly, it was divided into six regions by ISr IS1294 . The firCR2 elements, incomplete transposon TnAO22, and resistance genes such as floR, ∆tetA/tetR, strA/∆strB, and sul2. This segment showed 99% identity to IncA/C plasmid lacking IS5075-TnA022 regions such as pSH11G0791  and pST56-1 .The second segment (~15 kb) consisted of IS26 in the same orientation and included 10 putative open reading frames (ORFs) and an IncN1 pABWA45_3-like plasmid segment (resP-CUP6-Iterons I-∆repA). The second part (~18 kb) was bounded by IS26 and IS1294 in the opposite orientation. It contained two regions; the first region (~15 kb) was highly homologous to that of IncI1 plasmid pCombat13F7-3 (CP019248), which was carried by an E. coli strain of human origin in 2004 from China. The second region (~2.9 kb) contained four putative ORFs and was found in F-:A13:B- plasmids, such as pK245 , pKp2177_2 , and p08EU827_2 . Therefore, this ~18 kb hybrid part might have been generated, owing to the recombination between an IncFIA-like fragment and an IncI1 fragment.The third segment contained two parts. The first part (~15 kb) was bounded by two copies of ISddp3-bis-repX-pir-stbD/E) showed 98% identity to IncX1 plasmid pOLA52 (EU370913) lacking repX gene. The generation of this segment might involve the insertion and transposition of multiple IS elements, including IS26, IS1294, and IS50. The fifth segment contained aph(3')-IIa, ∆ble, and oqxAB. The oqxAB was embedded in composite transposon Tn6010 (IS26-oqxA-oqxB-oqxR-IS26). The last segment (~10 kb) was identical to the segments of the cluster A plasmids and contained blaTEM-1b, rmtB, fosA3 resistance module, and blaCTX-M-55.The fourth segment  of cluster B plasmids but in an opposite orientation. The variable regions of pHNFKD271 and pHN04NHB3 may have been generated from pHK23a-like (cluster A-like plasmid) multidrug resistance region (MRR) by ISbination .26-mediated homologous recombination.Compared with pHNFKD271 and pHN04NHB3, the IncN replication region was not found in plasmid p103-2-4, probably due to the ISintl1, three putative ORFs, and two IS26 elements. The second fragment was an IncN segment without IS5075 insertion but with an opposite orientation. The third fragment contained the IncN replication region, IncI1 segment, IncFIA-like segment, IncX1 segment, composite transposon Tn6010, and the second IncN1 segment. In addition, the variable regions of pZY-1 and pM1023-4Ar.4 were related to pECB11 but with the deletion of different quantities of putative ORFs in the IncN segment , the fosA3 module, and the resistance region bounded by IS1294 or IS26. BLASTn analysis indicated the high similarity (95% coverage and 99% identity) of pHN7A8 MRR to plasmid ps12177-CTX (CP101349) recovered from human origin in Henan Province, China, in 2006  steps. The second subline includes cluster_EF-like plasmids producing CTX-M-65, which have a significantly different structure. Moreover, IncF33 plasmids carrying the variable region producing CTX-M-55 are more dominant, and their sources are more diverse. On the other hand, IncF33 plasmids carrying the variable region producing CTX-M-65 are more frequently found in human and chicken sources. Interestingly, only IncF33 plasmids carrying blaCTX-M-55 have been identified in pig and environmental sources, while blaCTX-M-65 has not been found. This may be related to the fact that cluster_ABCD-like variable regions carry more insertion fragments, including replicon fragments of other types of plasmids, which may facilitate the spread of plasmids carrying this type of variable region across different hosts .In recent years, the IncF33 plasmids have generated cointegrates with various plasmids, among which, we observed two different types of cointegrates. One was formed by insertion of fragments from other plasmid types into the variable regions of complete IncF33 plasmids (type I). The other resulted from fusion of complete IncF33 plasmids with plasmids of other types, mediated by homologous recombination involving IS elements or transposons (type II). The former  was more common than the latter  within our collection of 103 IncF33 plasmids , analyzing only typical IncF33 plasmids. However, it is known that IncR-IncF33 cointegrate plasmids lack complete conjugative transfer regions and thus cannot spread horizontally. They disseminate primarily via ST11 K. pneumoniae clones  and was used to match the GenBank assembly database. Whole-genome sequences of strains with matches identical to the replicon sequence were downloaded for subsequent analysis. To ensure the reliability of the results, study data were cleaned based on the following methods. (i) The low-quality sequences were excluded from the study. (ii) Since the IncR-IncF33 cointegrated plasmids carrying blaKPC genes were formed by insertion of IncF33 plasmid fragments (lacking most conjugative transfer regions) into IncR plasmid backbones, their backbones remained IncR plasmids rather than typical IncF33 plasmids, which did not align with our research aims. Therefore, the strains and plasmids carrying blaKPC genes were excluded from this study. (iii) Duplicate sequences uploaded by the same institution simultaneously were excluded from this study. Finally, the whole-genome sequences of 863 strains were obtained. Similarly, the IncF33 plasmids were collected from the GenBank nucleotide database and were cleaned using the same methods. Finally, the complete sequences of 292 plasmids were obtained, including 103 plasmids negative for blaKPC genes. In addition, we designed an automated Python program to extract information about the strains and plasmids.The sequence of the IncF33 plasmid replicon was downloaded from PubMLST . Annotation was performed using the RAST server , Prokka The multilocus sequence typing (MLST) were identified using ABRicate 0.9.8 . ARGs an1, IS1294, etc.) boundaries in the variable regions and the locations are all downstream of the pemI/K addiction system; thanks to this, we can easily distinguish it in an artificial way. After removing the variable region of the IncF33 plasmid, we use the remaining sequences as the input to the panaroo software, with the --core_threshold parameter set to 0.95, and finally obtain the backbone sequence of the IncF33 plasmid.Almost all IncF33 plasmids have obvious insertion sequence (IShttps://www.snapgene.com/). CD-HIT (https://github.com/tseemann/snippy) on the core gene alignment. Pairwise SNPs between plasmids were counted from the core SNP alignment with snp-dists 0.6.3 (https://github.com/tseemann/snp-dists).The plasmid backbone and variable region sequences were compared using Easyfig  and Snap. CD-HIT  was used