MC1R, HERC2, OCA2, TYR, TYRP1, SLC45A2, and some novel candidate genes C1QTNF3, LINC02876, and C1QTNF3-AMACR have not been reported in the GWAS Catalog, with regulatory potential. These results highlight the importance of the assess phototype as a genetic proxy of skin functionality and disease when evaluating open mixed populations.Human pigmentation has largely been associated with different disease prevalence among populations, but most of these studies are observational and inconclusive. Known to be genetically determined, pigmentary traits have largely been studied by Genome-Wide Association Study (GWAS), mostly in Caucasian ancestry cohorts from North Europe, identifying robustly, several loci involved in many of the pigmentary traits. Here, we conduct a detailed analysis by GWAS and Polygenic Risk Score (PRS) of 13 pigmentary-related traits in a South European cohort of Caucasian ancestry . We observed fair phototype strongly associated with non-melanoma skin cancer and other dermatoses and confirmed by PRS-approach the shared genetic basis with skin and eye diseases, such as melanoma (OR = 0.95), non-melanoma skin cancer (OR = 0.93), basal cell carcinoma (OR = 0.97) and darker phototype with vitiligo (OR = 1.02), cataracts (OR = 1.04). Detailed genetic analyses revealed 37 risk loci associated with 10 out of 13 analyzed traits, and 16 genes significantly associated with at least two pigmentary traits. Some of them have been widely reported, such as  Skin, hair, and eye pigmentation are determined by the type and amount of melanin in the basal layer of the epidermis, hair follicles, and the front layers of the iris, respectively ,2,3,4,5.Pigmentary traits are genetically determined, heritable , and witMC1R was associated with red hair, fair skin, or freckles [OCA2 was previously associated with hair and eye color [SLC24A5, linked to pigmentation in zebrafish [TYR was associated with eye color type [Several studies have untangled the genetic component of these traits, identifying the main genes associated with several pigmentary traits. freckles ,16; OCA2ye color ,18; SLC2ebrafish , was alsebrafish ; IRF4 waebrafish ,21 and Tlor type ,23. Howelor type ,26.Ancestry background is important because natural selection shapes the overall genetic architecture of skin pigmentation . The latIn this study, we report a comprehensive analysis of the GCAT cohort, with the identification of phototype associated-diseases in a total of nearly 20,000 subjects and 177 ICD-9 diagnoses, and genome-wide analysis, including structural variants, across 13 pigmentary traits, using polygenic risk scores, in the largest South European population analyzed so far.Graphical abstract and pipeline including all the material and methods can be found in Participants included in this study belong to the GCAT|Genomes for life cohort, a population-based cohort from Southern Europe  . The GCAPigmentary traits were collected for all subjects with a self-report epidemiological questionnaire, during the recruitment . These data included single pigmentation-related phenotypes, comprising: three binary phenotypes for hair color, including red hair , blond hair , and brown hair (brown hair as cases and black hair as controls); eye color ; constitutive skin color ; freckles ; ease sunburn ; ease suntan ; as well as sun habits, such as the use of sunscreen in the exterior ; the use of sunscreen when suntan  and weekly hours spent outside . For theGenetic analysis was conducted in a subsample of the GCAT cohort (GCATcore) that is fully genotyped by SNP-array and imputation. This subsample included 4988 unrelated participants with Iberian ethnicity, determined by self-described ethnicity, and supervised ancestry inference by Principal Components Analysis (PCA), as previously described by Galván Femenía et al. . Genotypp-value (p).Medical diagnoses of the whole cohort were obtained from Electronic Health Records (EHR) of the Public Healthcare System of Catalonia. All diagnostic information (2010-current) is linked to each participant, within the framework of the PADRIS Program as provided by the Agency of Health Quality and Assessment of Catalonia (AQuAS) . We selep < 5 × 10−8) was kept for significance. SNPs were functionally annotated and mapped to the closest gene with SnpEff [Independent genome-wide association analysis (GWAS) for the 13 pigmentary-related traits (as three binary and ten continuous variables). Red hair was analyzed separately from blond and brown color due to its unique genetic architecture. Final analysis included a total of 10,195,351 SNVs and small indels, and 21,620 SVs with a Minor Allele Frequency (MAF) ≥ 0.01 and INFO score ≥ 0.7. Linear and logistic regression models were used for continuous and binary traits respectively using PLINK (version 2.0) , assuminh SnpEff . Manhatth SnpEff . Additioh SnpEff , a gene-−8, linkage disequilibrium threshold r2 = 0.6, and physical distance threshold for clumping 1000 kb), and lead SNPs . Then, LD blocks that were closer than 250 kb were merged into genomic trait-associated loci.Then, for each GWAS, independent genomic loci were identified as described in Watanabe et al. , using GTo further elucidate the biological mechanisms underlying pigmentary traits, we performed a functional enrichment analysis using WebGestalt , which tWe estimated the SNP heritability of each individual trait (h2), and computed pairwise genetic correlations, using LDSC (v1.0.1)  with theA Phenotype Wide association study (PheWAS) was conducted with former identified lead SNPs using the publicly available data PheWeb  that incp-value (p).Polygenic risk scores (PRS) were derived for selected relevant skin and eye diseases, based on disease-phototype association, GWAS results, and available genetic data from public databases. Here we present data for three skin cancer phenotypes , two eye diseases (glaucoma and cataracts), two immune-related dermatoses (psoriasis and vitiligo) and metabolic diseases . We used weights for relevant diseases from the PGS Catalog  giving pDiseases of the skin, and subcutaneous tissue and Neoplasms were the two most represented ICD-9 chapters (71%). The most significantly associated diseases were: Other and unspecified malignant neoplasm of skin , and Other dermatoses . Other prevalent diseases included: Benign neoplasm of skin (ICD-9 code 216), Contact dermatitis and other eczema (ICD-9 code 692), and Erythematous conditions (ICD-9 code 695). Moreover, we found two common conditions associated with fair phototype: Overweight, obesity and other hyperalimentation (ICD-9 code 278) and Essential hypertension (ICD-9 code 401).Among the selected 177 EHR ICD-9 diagnoses, seven diseases were significantly associated with a fair Fitzpatrick scale phototype after Bonferroni correction, and eight were nominally associated . Among tSLC45A2, HERC2, OCA2, IRF4, MC1R, ADAMTS12, RXFP3, TYR, TYRP1, NOX4, AMACR, EXOC2). Noteworthy, SLC45A2, a critical determinant of skin and eye pigmentation, was the most pleiotropic gene, associated with nine pigmentary traits.We carried out a GWAS across > 10M variants with a MAF ≥ 0.01 and INFO score ≥ 0.7 for each individual pigmentary trait, including SNVs and SVs . Single-SLC45A2 (lead rs16891982) was associated with nine traits ; IRF4 (lead rs12203592) show association with six traits ; HERC2 (lead rs12913832) was associated with seven traits  and MC1R (lead rs1805007) presented association with four traits . Pleiotropy, as expected, was evident for the aggregated variables of phototype, which include dependent variables, but it was also higher for skin color, suntan, sunburn, and brown hair; and lower in red and blond hair color and eye color  technique. This allowed us to have an overview of the phenotypic variance explained by the SNPs, as well as to obtain a measure of the shared genetic basis between these traits. The higher heritability was observed for hair color , phototype score , and Fitzpatrick scale . Heritability estimates for red hair  were unreliable due to the low number of cases.p: 1.1 × 10−32). Furthermore, phototype measures correlated with eye and hair measures and ease sunburn were highly correlated with eye color and phototype measures. Regarding habits, sunscreen use in exterior was correlated with sunscreen when suntan and inversely sunscreen when suntan and weekly hours in the exterior. Red hair was highly correlated with sunscreen in, sunscreen when suntan and ease sunburn, However, the low number of red hair cases in the cohort made an accurate estimation difficult (p: 2.88 × 10−6), ease of sunburn and Fitzpatrick scale ; and among brown hair and light hair (meaning brown versus black trait) with phototype score .All the traits showed a high genetic correlation . As expeifficult . Among aHERC2 and SLC45A2, out of the 22 showed associations with skin, hair, eye, and phototype. DPEP1 and SPATA33 were associated with skin, red hair, and phototype. MC1R, TYR, ADAMTS12, AMACR, C1QTNF3, CHMP1A, GAS8, C1QTNF3-AMACR, RXFP3, and SPIRE2 were associated with skin and phototype, and OCA2 and TYRP1 were associated with eye and phototype traits. Six identified genes did not show an association with phototype, being exclusively associated with one trait: LINC02876, DEF8, VPS9D1, and ZNF276 with red hair, SLC25A37 associated with sunscreen, and TRPS1 with skin color. C1QTNF3-AMACR, C1QTNF3 and LINC02876 were not previously reported as pigmentary traits in GWAS Catalog.To further characterize the functional impact of the SNPs identified in the GWAS analysis, we performed a gene-based analysis, with aggregated SNPs into single genes. Then, we tested the association between genes and pigmentary traits using MAGMA software  Figure . After BFunctional enrichment analysis using WebGestalt  revealedSPIRE2 (16:89896057:G:<DEL>) associatedwith red hair , phototype score , Fitzpatrick scale  and also present in ease suntan . Red hair was also associated with three other intronic deletions in GAS8, CHMP1A, and CPNE7. Other significant associated loci were two small intronic deletions associated with eye color in HERC2 (15:28473597:A:<DEL>)  and LURAP1L-AS1 (9:12756780:C:<DEL>) OR = 1.14, CI = 1.09–1.19, p = 2.56 × 10−9), both also associated with phototype score. Moreover, two intronic deletions associated with skin color; a small deletion in BNC2 (9:16876769:G:<DEL>) , and a middle-deletion in SLC45A2 (5:33973937:G:<midDEL>) . In summary, most of the identified SV were deletions (13) and, only suggestively, one insertion (Alu) in chromosome 6 (DST) and one translocation in chromosome 7 (ZNF92) were observed . Amongst the diseases, those belonging to the neoplasm and dermatologic categories were the most frequent ones , and with skin cancer and actinic keratosis. The allele T is associated with fair skin pigmentation, and increasing ease of sunburn, but also increased the risk of melanoma or actinic keratosis , and diseases affecting the eyes, such as cataract or glaucoma. The allele G, associated with fair skin or eye color, decreased the risk of glaucoma and cataract , and with neoplasms and bipolar disorder. Here, the allele A is associated with fair skin color, increased risk of ease of sunburn, and risk of skin cancer and bipolar disorder  analysis with these SNPs and using a PheWeb dataset that contains the results of associations carried out with the UK Biobank data, as described in the Material and Methods section. This analysis revealed that 17 out of the 99 SNPs had at least an association surpassing the suggestive significance threshold in the UKbiobank  an overall increase in DNA damage that overloads the DNA repair machinery , (ii) reIn addition, our study highlighted that the fair skin phototype was also associated vitiligo, cataracts, glaucoma, and psoriasis, and with two common conditions non primarily related with skin color: obesity and essential hypertension. Obesity represents a significant health problem and together with hypertension significantly increases the risks of suffering from other major diseases affecting vital organs as heart, brain, kidney, and other diseases. Several authors suggested that deficiency of vitamin D, may be linked to obesity, hypertension, hyperlipidemia, and diabetes, increasing metabolic and cardiovascular risk ,59, thusMC1R, HERC2, OCA2, TYR, TYRP1, SLC45A2) [ADAMTS12, AMACR, CHMP1A, DPEP1, GAS8, RXFP3, SPATA33 and SPIRE2) [C1QTNF3, C1QTNF3-AMACR and LINC02876 to the studied phenotype. C1QTNF3-AMACR is a conjoined gene [C1QTNF3 (Complement C1q Tumor Necrosis Factor-Related Protein 3) and AMACR  the three of them being associated with skin color and phototype, in the present study. NMDs functions not only as a quality control mechanism targeting aberrant mRNAs containing a premature termination codon but also as a posttranscriptional gene regulation mechanism targeting numerous physiological mRNAs [LINC02876 located in chromosome 17, also known as LINC02876, is a long non-coding RNA, associated with red hair in our study, a trait with a low prevalence in our cohort, making necessary further validation. Some of the identified lead SNPs, mapping IRF4, HERC2, and TYR, were also found to be associated with melanoma or other skin diseases in the UKBiobank. Mutations in the MC1R gene lead to fair skin and red hair in humans, which is also seen with inactivating human POMC gene mutations. The powerful effects of proopiomelanocortine (POMC) and probably Corticotropin-releasing hormone (CRH) on the skin pigmentary, immune, and adnexal systems are consistent with stress-neutralizing activity addressed at maintaining skin integrity to restrict disruptions of internal homeostasis. This CRH/POMC skin system appears to generate a function analogous to the HPA axis that in the skin is expressed as a highly localized response which neutralizes noxious stimuli and attendant immune reactions [When looking at genes, we observed 16 genes associated with one or more pigmentary traits and diseases. Six of them were largely associated with melanogenesis and pigmentary traits, in different ethnic groups (SLC45A2) ,60,61; o SPIRE2) . Our ananed gene  located al mRNAs , revealieactions ,65. AlteSLC45A2, TYR, OCA2 and HERC2), CHMP1A and GAS8, both close to MC1R, associated with red hair [BNC2, a transmission factor of keratinocytes, and SPIRE2 involved in the transport of intracellular vesicles [SEMA6D, a member of the semaphorin family showing a suggestive association with hair greying [CPNE7 associated with skin tanning [LURAP1L-AS1, were interestingly linked to melanin genes. LURAP1L-AS1 is an antisense RNAs, a unique DNA transcript, small, noncoding, and diffusible molecule that complements mRNA, and are recognized as intracellular gene regulators. LURAP1L-AS1 gene overlaps TYRP1, involved in eumelanin synthesis [TYRP1 transcript regulation and putative melanin dysregulation.While most of the GWAS studies have been restricted to SNVs and small indels, our study incorporated a large number of structural variants in the analysis . GWAS anred hair  and hairred hair , respectvesicles ,68, SEMA greying  and CPNE tanning . Unfortuynthesis ,71, and Complementary to single SNP analysis, gene-based analyses provided additional power due to the aggregate effect of multiple SNPs being larger than that of individual SNPs. The functional enrichment analysis revealed an overrepresentation of defective pigmentation diseases involving altered pigment metabolic processes , involved in melanogenesis, or tyrosine metabolism, involving monooygenase activity or the glycolipid binding functions, probably related to melanin, and bile acids biosynthesis. Glycosphingolipid metabolism is enriched in the gene dataset, and glycosphingolipids have been reported essential for a protein-sorting step in the Golgi complex. It has also been suggested that the Hermansky–Pudlak syndrome, a rare disease with associated albinism, has its origin in the altered lipid metabolism . Indeed,Pleiotropy analysis confirmed shared genetics for pigmentary traits, melanoma, and non-melanoma skin cancer. The results suggest a higher risk of skin cancer in individuals with a fair phototype, consequent with a protective effect from UVR damage to the skin and the ability of melanin to absorb from 50% to 75% of UVR, but also could be related to the described pathological role of melanin, linking a defective secretion mechanism to an increase in tumor severity . In addiOur results provide a comprehensive analysis of the genetic basis of pigmentary traits in a large South European population and offer a landscape of their genetic basis for a better understanding of human pigmentation pleiotropy based on the common genetic background to some previously observed associations. Furthermore, our findings support the importance of incorporating population information to adapt personalized care approaches to consider differential risk in general population, being of great importance in modern mixed populations. It is well accepted that natural selection shaped the skin pigmentation phenotype, resulting, in a gradient of variation across populations, directly correlated with absolute latitude ,14,80. U


Nelumbo nucifera Gaertn.). To elucidate the molecular genetic basis of the terpene biosynthetic pathway, terpenes from different parts of the plant, including seeds (S), young leaves (YL), mature leaves (ML), white flowers (WF), yellow flowers (YF), and red flowers (RF), were identified by LC-MS/MS and the relative contents of the same terpenes in different parts were compared. The results indicate that all plant parts primarily consist of triterpenes, with only minor quantities of sesquiterpenes and diterpenes, and there were differences in the terpene content detected in different plant parts. To illustrate the biosynthesis of various terpenoids, RNA sequencing was performed to profile the transcriptomes of various plant parts, which generated a total of 126.95 GB clean data and assembled into 29,630 unigenes. Among these unigenes, 105 candidate unigenes are involved in the mevalonate (MVA) pathway, methyl-erythritol phosphate (MEP) pathway, terpenoid backbone biosynthesis pathway, and terpenoid synthases pathway. Moreover, the co-expression network between terpene synthase (TPS) and WRKY transcription factors provides new information for the terpene biosynthesis pathway.As the largest group of structurally diverse metabolites, terpenoids are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. However, few terpenoid compounds have been identified in plant parts of sacred lotus ( Nelumbo nucifera Gaertn.) and American lotus (Nelumbo lutea Pers.) [Sacred lotus belongs to Nelumbonaceae, which contains only two species, sacred lotus (a Pers.) . Sacred a Pers.) ,3. A lara Pers.) ,6,7,8,9.As the largest group of structurally diverse metabolites, terpenoids are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. Triterpenoids are important plant secondary metabolites with pharmacological effects, such as anticancer, antiviral, and cholesterol lowering. According to the number of rings in the structure, triterpenoids can be classified into monocyclic triterpenes, bicyclic triterpenes, tricyclic triterpenes, tetracyclic triterpenes, and pentacyclic triterpenes, etc. Among them, pentacyclic triterpenoids have received more attention because of more types and functions ,11,12,13Salvia officinalis Linn. [Cinnamomum camphora (L.) Presl. [Salvia guaranitica St. Hil. [Despite the scientific and industrial interest in sacred lotus, terpenoid components have not been identified. Moreover, the genes involved in terpenoid biosynthesis were been completely studied in aquatic botany. In recent years, RNA sequencing (RNA-Seq) and metabolite analysis were developed as powerful tools for identifying genes involved in the biosynthesis of various secondary metabolites in higher plants, including is Linn. , Cinnamo) Presl. , and SalSt. Hil. .N. nucifera, and the molecular genetic basis of terpenoid biosynthesis pathways is still unveiled. Considering the specific characteristics of sacred lotus, it was of interest to profile the terpenoid biosynthesis and key genes related with the terpenoid metabolic pathway. In this study, the contents and compositions of terpenoids in seeds, young leaves, mature leaves, and flowers with different colors were analyzed with LC-MS/MS. In addition, the key genes and transcription factors involved in terpenoid biosynthesis were screened by RNA-seq. We believe these results will shed light on the mechanism of terpenoid biosynthesis in sacred lotus.As one of the best-known medicinal plants, few terpenoid compounds have been identified in plant parts of For unveiling the distribution of terpenoids in flowers with different colors, the widely planted cultivars with a red flower (Jinlinghuodu), white flower (Baiyinlian), and yellow flower (Jinsenianhua) were selected. In order to compare terpenoids in various plant parts, the seeds, young leaves, and mature leaves from Taikonglian 36 were harvested A. In totTo identify genes involved in terpenoid biosynthesis in sacred lotus, 18 RNA libraries were prepared and analyzed. In total, 249,600,000 reads were obtained from these 18 libraries (accession number: PRJNA857167). After filtering the original data, clean reads were obtained for subsequent analysis . Additio−5 in the public databases: NR (NCBI non-redundant protein sequences), KOG (euKaryotic Ortholog Groups), PFAM (Protein family), GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes). In summary, 76,070 unigenes were successfully annotated by at least one database and 18,045 unigenes shared annotation in all databases , metabolic pathways (2194 unigenes), and plant–pathogen interaction (534) were the three richest pathways. In addition, 122 unigenes were mapped to the ‘Metabolism of terpenoids’, including ‘Terpenoid backbone biosynthesis’ , ‘Sesquiterpenoid and triterpenoid biosynthesis’ , and ‘Diterpenoid biosynthesis’  .To fully explore potential DEGs in various plant parts, 22,336 DEGs were identified by comparing the eleven groups . The total number of DEGs, up-regulated DEGs, and down-regulated DEGs in each group are presented in The 50 GO-Terms with the lowest q-value in the enrichment analysis results were selected and a column chart of the enrichment items was drawn A. Among According to the KEGG pathway enrichment analysis, biosynthesis of secondary metabolites (1750) and metabolic pathways (3205) were the two richest pathways. In addition, 99 DEGs were positioned as “terpenoid metabolism”, including “Terpenoid backbone biosynthesis” , “sesquiterpene and triterpene biosynthesis” , and “diterpene biosynthesis”  B.AACT2 and HMGS2 exhibited the highest mRNA level in S, while HMGR3 was the highest expressed in ML  was positively correlated with five terpenoids, including Triacetate (lmjp04533), 12,13-Dihydroursolic acid (Zmjp01361), Progesterone (Lmqp010784), 2α,3α,23-trihydroxyolean-12-en-28-oic acid (Smpn009230), and Madasiatic acid (Hmjn003948), with correlation coefficients varying from 0.83 to 0.97. Gibberellin 2 beta-dioxygenase (GA2-5), 1,8-cineole synthase (CINS3), and ent-kaurene oxidase (KO) were positively correlated with the same three terpenoids. 24,30-Dihydroxy-12(13)-enolupinol (pmn001700) was positively correlated with nine TPS genes and negatively correlated with four TPS genes. Beta-amyrin synthase  exhibited a strong positive correlation with 24,30-Dihydroxy-12(13)-enolupinol (pmn001700) and 12,13-Dihydroursolic acid (Zmjp013616).As key enzymes in the synthesis of terpenoids, 49 DEGs encoding terpenoid synthase (TPS) were found, including 15 genes involved in sesquiterpene and triterpene synthesis (ko00909), 26 genes involved in diterpene synthesis (ko00904), and 8 genes involved in monoterpene synthesis (K07385 and K15095) . CorrelaGERD2, GA2-2, GA2-7, and KAO2-2, which was positively correlated with GERD3, (+)-neomenthol dehydrogenase , AMYS3, and AMYS4. WRKY72 (LOC104596936) was closely related to 15 TPS genes. Among them, WRKY72 was positively correlated with NEOD5, AMYS1, AMYS2, and AMYS3, and the Pearson correlation coefficient exceeded 0.9. By contrast, WRKY72 was negatively correlated with GA2-2, GA2-3, GA2-4, GA2-7, gibberellin 3-beta-dioxygenase (GA3-5), GERD2, and ent-kaurenoic acid hydroxylase (KAO2-2). WRKY17 (LOC104603161) was positively correlated with AMYS1, AMYS3, and AMYS4. Transcription factors (TFs) usually control the expression of TPS genes by activating or repressing their promoters, thereby regulating the accumulation of terpenoids. In total, 47 DEGs were annotated as WRKY transcription factors, 27 of which were highly correlated with 15 terpenoids . A co-exAACT1 and FPPS1 in white flower.To verify the reliability of the RNA-Seq data, the expression of 12 unigenes was detected by qRT-PCR . The expHMGR1, HMGR2, and HMGR4 exhibited the highest expression in YL, with the lowest expression in ML  GPPS.SSU in transgenic tobacco plants [Salvia sclarea L. hairy roots was increased by engineering the GGPPS and CPPS [GPPS, two FPPS, and three GGPPS. We found that the expression of FPPS was higher in young leaves than in seeds and mature leaves, which is consistent with the results detected for the metabolic content of triterpenoids. It has been shown that the GGPPS transcript was mainly concentrated in the above-ground parts, such as leaves, flowers, or fruits, and the expression patterns of different GGPPS homologous genes in the same plant were various [GGPPS2 and GGPPS3 identified in this study are consistent with this conclusion.IPTS are the key enzymes that connect the upstream MVA and MEP pathways with downstream isoprenoid biosynthesis branch points of different structures. In recent years, GPPS, GGPPS, FPPS, and CPPS, as the key enzymes in the biosynthesis of isoprenoid compounds, were identified in higher plants. In addition, most studies showed that the transcription levels of these genes were closely related to the content of corresponding terpenoids ,39,40,41o plants  and the and CPPS . In this various ,49,50,51A. thaliana, tomato, rice, and tobacco [As a key enzyme playing an important role in the synthesis of terpenoids, the TPS genes were identified in  tobacco ,54,55,56 tobacco ,58. In t tobacco ,59,60,61The widely planted cultivars “Taikonglian 36”, “Jinsenianhua”, “Jinlinghuodu”, and “Baiyinlian” were planted in pools at Henan University of Technology, with a photoperiod of light for 16 h and dark for 8 h. Sacred loti were cultivated to the full-bloom stage. The materials including seeds (Taikonglian 36), young leaves (Taikonglian 36), mature leaves (Taikonglian 36), and blooming flowers with red (Jinlinghuodu), white (Baiyinlian), and yellow (Jinsenianhua) were freeze dried in liquid nitrogen and kept at −80 °C.http://www.anpel.com.cn/, accessed on 5 September 2021) before UPLC-MS/MS analysis. Chromatography and mass spectrometry were performed with the assistance of Wuhan Metware Biotechnology Co., Ltd.  as the method described [Biological samples were freeze dried using a vacuum freeze dryer (Scientz-100F) and crushed using a mixer mill  with a zirconia bead for 1.5 min at 30 Hz. Then 100 mg of lyophilized powder was dissolved with 1.2 mL 70% methanol solution, and vortexed 30 s every 30 min 6 times in total. The samples were refrigerated 4 °C overnight and centrifugated at 12,000 rpm for 10 min. The extracts were filtrated  and RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina®  following manufacturer’s recommendations. The cDNA libraries were sequenced on the Illumina sequencing platform by Metware Biotechnology Co., Ltd. .Total RNA was extracted using the Total Plant RNA Extraction Kit . RNA concentration and purity were determined using NanoPhotometerhttps://www.ncbi.nlm.nih.gov/genome/?term=AQOG01, accessed on 25 September 2021) [https://cloud.metware.cn, accessed on 3 January 2022), and Pearson correlation coefficient greater than |0.8|.The original data were filtered to remove reads with adapters. HISAT version 2.1.0  was used to construct the index and compare clean reads to the reference genome (er 2021) , and Strer 2021) . We useder 2021) ,66 was u−△△CT method [The key DEGs in the terpenoid synthesis pathway were selected to validate their expression levels by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Synthesis of cDNA for qRT-PCR was performed with First Strand cDNA Synthesis Kit . The primers were synthesized by Tsingke Biotechnology Co., Ltd.  . The reaT method . At leasIn this study, 21 terpenoids were detected in sacred lotus and 105 genes involved in terpenoid synthesis were identified. WRKY, an important transcription factor, is highly correlated with the TPS gene family and might play an important regulatory role in the terpenoid synthesis process. This study illustrated the metabolic diversity of terpenoids in sacred lotus and provided a global overview of the gene expression profiles related with terpenoid biosynthesis in sacred lotus.


Members of the innate immune system, innate lymphoid cells (ILCs), encompass five major populations  cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer cells) whose functions include defense against pathogens, surveillance of tumorigenesis, and regulation of tissue homeostasis and remodeling. ILCs are present in the uterine environment of humans and mice and are dynamically regulated during the reproductive cycle and pregnancy. These cells have been repurposed to support pregnancy promoting maternal immune tolerance and placental development. To accomplish their tasks, immune cells employ several cellular and molecular mechanisms. They have the capacity to remember a previously encountered antigen and mount a more effective response to succeeding events. Memory responses are not an exclusive feature of the adaptive immune system, but also occur in innate immune cells. Innate immune memory has already been demonstrated in monocytes/macrophages, neutrophils, dendritic cells, and ILCs. A population of decidual NK cells characterized by elevated expression of NKG2C and LILRB1 as well as a distinctive transcriptional and epigenetic profile was found to expand during subsequent pregnancies in humans. These cells secrete high amounts of interferon-γ and vascular endothelial growth factor likely favoring placentation. Similarly, uterine ILC1s in mice upregulate CXCR6 and expand in second pregnancies. These data provide evidence on the development of immunological memory of pregnancy. In this article, the characteristics, functions, and localization of ILCs are reviewed, emphasizing available data on the uterine environment. Following, the concept of innate immune memory and its mechanisms, which include epigenetic changes and metabolic rewiring, are presented. Finally, the emerging role of innate immune memory on reproduction is discussed. Advances in the comprehension of ILC functions and innate immune memory may contribute to uncovering the immunological mechanisms underlying female fertility/infertility, placental development, and distinct outcomes in second pregnancies related to higher birth weight and lower incidence of complications. A complex array of immunological interactions takes place in the uterine environment that is crucial for reproductive success, contributing to tissue homeostasis and renewing, placental development, and maternal tolerance –3. ImmunAll innate immune cells, except basophils, are eventually present in the uterus under physiological conditions –12. Exce)  cells  , 19. Rec) .+ T lymphocytes. ILC1s and Th1 cells play a key role in immune responses to intracellular pathogens (mainly viruses) and tumors; ILC2s and Th2 cells fight against large extracellular parasites and respond to allergens; ILC3s and Th17 cells combat extracellular microbes such as bacteria and fungi. ILCs also contribute to tissue homeostasis and remodeling as well as metabolic regulation (NK and ILC precursors develop independently through the divergence of common lymphoid progenitors. ILCs are considered innate counterparts of adaptive T lymphocytes that lack recombination activating gene (RAG)-dependent antigen receptors. ILC1s, ILC2s, ILC3s, and ILCregs mirror the function and cytokine profile of Th1, Th2, Th17, and regulatory T cells, respectively. Similarly, NK cells are considered counterparts of cytotoxic CD8gulation , 21–23. gulation , they ar−CD56dimCD16+ NK cells present high cytotoxicity, whereas CD3−CD56brightCD16− NK cells display low cytotoxicity and strong immunomodulatory properties ; dNK2 ; dNK3 cells . HLA-C molecules on trophoblast cells can be recognized by both activating KIR2DS1 and KIR2DS4 as well as inhibitory KIR2DL1, KIR2DL2, and KIR2DL3 receptors expressed by dNK1 cells. dNK3 cells in turn contain KLRB1 and TIGIT, which may bind to CLEC2D and PVR present in trophoblast cells, indicating the capacity of these cells to cooperate during pregnancy. Furthermore, molecules produced by dNK1 , dNK2, and dNK3 (ANX1) have anti-inflammatory properties inhibiting immune activation at the maternal-fetal interface . Further+ NK cells are also present in the human endometrium, and increased levels of these cells are associated with infertility  cells and CSF1+CD59+KIR+ dNK cells in women with unexplained recurrent pregnancy loss , suggesting their importance in immune regulation , 53, 54.e system . In a si- ILC1s in the human uterus. While some studies report their presence , ST-2, an IL-33 receptor subunit, and IL-17RB, an IL-25 receptor subunit , 63. ILC+ILC2s were described in both mouse and human uteri. In mice, these cells are located in the myometrium and increase during pregnancy  NKp44: NKp44+ and NKp44– ILC3s. In mice, the ILC3 population is sub-divided into NKp46– and NKp46+ cells. Each subtype produces different sets of cytokines. NKp44– ILC3s secrete IL-17 and TNF-alpha, whereas NKp44+ ILC3s produce IL- 8, IL-22, and GM-CSF (+ ILC3s expressing the chemokine receptor 6 (CCR6) and its ligand CCL20 also produce IL-17A and IL-22 and promote the accumulation at the inflammatory site of these cells and attraction of others, such as memory CD4+ T cells, regulatory Th17 cells, B cells, and dendritic cells (DCs) (ILC3s express the surface marker CD117 and the transcription factor retinoic-acid-receptor-related orphan nuclear receptor γ t (RORγt). These cells mirror the features of Th17 lymphocytes regarding marker expression and cytokine output. ILC3s and ILC2s express major histocompatibility complex class II (MHCII) and can process and present antigens MHCIId GM-CSF , 71, 72.ls (DCs) , 74.via IL-22 secretion  to differentiate, LTi cells do not. LTi cells have a distinctive transcriptome and express CCR6, CCR7, CXCR5, CXCR6, IL-7Ra, LTα1β2, and RANKL at the cell surface , 85. Durnd CD30L , 87. IL-olecules . CCL21 solecules .+CD117+) as well as in pregnant and non-pregnant mouse uterus , ID3, and SOX4. TGF-β signaling is essential for their survival and expansion. ILCregs contribute to the resolution of intestinal inflammation by suppressing the activation of ILC1s and ILC3s of IL-10 . Populatof IL-10 –92. WhetThe traditional concepts characterizing innate and adaptive responses have been recently challenged. Innate immune cells present features previously attributed do adaptive immune cells, including clonal-like expansion, contraction, development of memory, and recall responses , 93, 94.+NKG2C+ NK cells in humans and Ly49H+ NK cells in mice expanded in response to murine CMV (MCMV) infection. After subsequent viral challenge, memory Ly49H+ NK cells triggered a recall response protecting against infection  leads to epigenetic remodeling of the conserved non-coding sequence (CNS) 1, an enhancer in the IFNG locus, increasing its accessibility to transcription factors and IFNγ expression  and STAT  , molecules related to immune cell survival and functions. NK cells deficient in this miRNA present impaired effector and memory properties . The devription)  to promoCellular metabolism partakes in trained immunity by mediating epigenetics and other cellular processes. Metabolites can influence epigenetic features, conversely, epigenetic mechanisms contribute to maintaining cellular metabolism. When cells first recognize external stimuli, signaling cascades lead to elevated metabolism. The resulting metabolites can function as signaling molecules, cofactors, and substrates to modulate chromatin-modifying enzymes and direct transcriptional processes , 116. Fovia inhibition of mTOR or activation of AMPK in a process dependent on ATG3 (Autophagy Related 3) amplifies the formation of memory NK cells  and BNIP3L (BNIP3-like) mediate the removal of dysfunctional mitochondria and reactive oxygen species through mitophagy promoting cell survival. Autophagy induction NK cells . Virus-irocesses .+ hematopoietic stem cells residing in the human endometrium is committed to the differentiation of NK cells, as observed in vitro upon stimulation with a blend of stem cell factor, FMS-like tyrosine kinase ligand, IL-7, IL-15, and IL-21 or co-cultivation with decidual cells   . CD94-NKptor B1) , 147. LIptor B1) . NKG2Chio models . IFNγ iso models . The prognancies . These cAlthough the acquisition of innate immune memory may have positive effects on pregnancy, one may speculate that impairments in the establishment of innate immune memory or the development of memory with inhibitory actions against pregnancy can be related to reproductive complications. Pregnancy induces long-term epigenetic memory in maternal T and NK cells characterized by differential methylation patterns of genes related to their differentiation and functions. These changes are impaired in preeclamptic women, indicating that alterations in innate and adaptive memory are associated with pregnancy diseases . The stiSo far, the implications of microbiota, pathogens, and vaccination on innate immune memory and its association with reproduction have been poorly interrogated. Chronic exposition of NK cells to both endogenous and exogenous ligands can modulate their memory properties . Such movia innate immune cells and their memory properties. HCMV infection appears to be a priming factor in the development of pregnancy-trained NK cells. Comparisons between nulligravidae and multigravidae women that were HCMV-seronegative and -seropositive revealed increased percentages of LILRB1+NKG2C+ memory NK cells only in those previously infected by HCMV . RD-D is receiving a Master’s grant from the German Academic Exchange Service (DAAD).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objective: To describe two novel heterozygous splicing variants of the CSF1R gene responsible for CSF1R-microglial encephalopathy in two unrelated Han Chinese families and further explore the relationship between the pathological and neuroimaging findings in this disease.Methods: The demographic data, detailed medical history, and clinical manifestations of two unrelated Han families with CSF1R-microglial encephalopathy were recorded. Some family members also underwent detailed neuropsychological evaluation, neuroimaging, and genetic testing. The probands underwent whole-exome sequencing (WES) or next-generation sequencing (NGS) to confirm the diagnosis. The findings were substantiated using Sanger sequencing, segregation analysis, and phenotypic reevaluation.Results: Both families presented with a dominant hereditary pattern. Five of 27 individuals (four generations) from the first family, including the proband and his sister, father, uncle, and grandmother, presented with cognitive impairments clinically during their respective lifetimes. Brain magnetic resonance imaging (MRI) depicted symmetric, confluent, and diffuse deep white matter changes, atrophy of the frontoparietal lobes, and thinning of the corpus callosum. The proband’s brother remained asymptomatic; brain MRI revealed minimal white matter changes, but pseudo-continuous arterial spin labeling (pCASL) demonstrated a marked reduction in the cerebral blood flow (CBF) in the bilateral deep white matter and corpus callosum. Seven family members underwent WES, which identified a novel splice-site heterozygous mutation (c.2319+1C>A) in intron 20 of the CSF1R gene in four members. The proband from the second family presented with significant cognitive impairment and indifference; brain MRI depicted symmetric diffuse deep white matter changes and thinning of the corpus callosum. The proband’s mother reported herself to be asymptomatic, while neuropsychological evaluation suggested mild cognitive impairment, and brain MRI demonstrated abnormal signals in the bilateral deep white matter and corpus callosum. NGS of 55 genes related to hereditary leukodystrophy was performed for three members, which confirmed a novel splice-site heterozygous mutation (c.1858+5G>A) in intron 13 of the CSF1R gene in two members.Conclusions: Our study identified two novel splicing mutation sites in the CSF1R gene within two independent Chinese families with CSF1R-microglial encephalopathy, broadening the genetic spectrum of CSF1R-microglial encephalopathy and emphasizing the value of pCASL for early detection of this disease. In 1936, van Bogaert and Nyssen described a Belgian family who presented with adult-onset orthochromatic leukodystrophy and dementia, whose histopathological characteristics included diffuse myelin and axonal loss, accompanied by pigmented macrophages and termed the condition pigmentary orthochromatic leukodystrophy (POLD) . In 1984CSF1R) gene has been recently identified in some families with pathological diagnoses of the above-mentioned conditions (CSF1R gene encodes the CSF1R protein (a transmembrane tyrosine kinase receptor), which is mainly expressed in the microglia in the brain and mediates the proliferation, differentiation, and survival of the microglia (CSF1R mutation can result in amino acid changes in the tyrosine kinase domain (TKD) and microglial dysfunction, culminating in rapidly progressive dementia  and cause various brain dysfunctions, including early-onset cognitive impairment, behavioral changes, parkinsonism, seizures, and depression , multiple sclerosis, frontotemporal dementia, and other leukoencephalopathies. Herein, we reported two novel splicing mutation sites (c.2654+1G>A and c.1858+5G>A) in the intron of the CSF1R in two unrelated Han Chinese families and analyzed the relationship between the pathological and neuroimaging characteristics, with the aim of expanding the genetic and radiological spectrum of CSF1R-microglial encephalopathy.Since the discovery of orldwide . The majorldwide . PresentThe study was approved by the Institutional Review Board of Beijing Tiantan Hospital of Capital Medical University (KY-2021-028-01). All individuals involved in this study provided written informed consent for genetic analysis before enrollment.-weighted imaging (DWI), T1-contrast enhancement, angiography, and pseudo-continuous arterial spin labeling (pCASL).Information on sex, age, age of onset (if applicable), and clinical manifestations were collected from all members of both unrelated families. Three individuals from the first family and two individuals from the second family underwent MRI using 3.0-T devices with the following sequences: T1-weighted imaging (T1WI), T2-weighted imaging (T2WI), fluid-attenuated inversion recovery (FLAIR), diffusionABCD1, ARSA, CSF1R, EIF2B2, GJC2, PEX1, POLR3A, POLR3B, PSAP, TREX1, EIF2B1, EIF2B3, EIF2B4, EIF2B5, PLP1, GFAP, ASPA, MLC1, HEPACAM, YP27A1, GBE1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, LMNB1, SLC17A5, FUCA1, FAM126A, GALC, L2HGDH, DARS2, EARS2, SUMF1, GJA1, RNASET2, HSD17B4, ACOX1, SCP2, ALDH3A2, SOX10, MFSD8, HSPD1, MPV17, PEX10, PEX13, PEX5, PEX6, POLR1C, SDHA, SDHB, TMEM187, NOTCH3, and HTRA1) were conducted to discover the causative variants in the patients from the first and second families, respectively. Briefly, genomic DNA was isolated from peripheral leukocytes using the DNA Isolation Kit . Subsequently, the qualified DNA samples were fragmented into 200–300 base pairs (bp). DNA libraries were prepared using the KAPA Library Preparation Kit KR0453  following the manufacturer’s instructions. The libraries were estimated with the Qubit dsDNA HS Assay kit  and finally sequenced on the Illumina NovaSeq 6000 platform  using paired-end 200-bp reads. The sequencing data were filtered and aligned with the human reference genome (GRCh37/hg19) using the BWA Aligner (http://bio-bwa.sourceforge.net/). The quality scores were estimated. Consensus calling for single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels) was performed using the Genome Analysis Toolkit. Each of the called variants was annotated using ANNOVAR (annovar.openbioinformatics.org/en/latest/) using several public databases . The annotation content was composed of the variant position, variant type, allele frequency, and conservation prediction, which can help locate mutations corresponding to diseases.Whole-exome sequencing (WES) and a hereditary leukodystrophy-targeted next-generation sequencing (NGS) panel . The SNPs and indels occurring in the exons and canonical splice sites were analyzed using multiple computational software programs to predict the effects of these variants. The Professional Version of the Human Gene Mutation Database (HGMD) was searched to determine if the variants were reported previously. Finally, the selected variants were classified according to the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines  ≥ 1%, and ExAC hom AC ≥ 3. The variants obtained idelines . Sanger CSF1R mutation was detected in four of the seven members who underwent genetic testing, including the proband, his sister, brother, and son. The brother (45 years) and son (17 years) of the proband had the same CSF1R mutation but remained asymptomatic.The first family is a nonconsanguineous family of Han Chinese origin that spanned four generations and consisted of 27 members. The family pedigree is depicted in The proband was a 43-year-old man with a high school education. He first experienced dizziness and constant numbness in the distal portion of the bilateral upper limbs at the age of 39 years. He developed cognitive and psychiatric impairment 5 months after onset, characterized by executive dysfunction, attention deficit, and indifference. The above-mentioned clinical symptoms gradually worsened, and he developed speech problems, including slow speech and anomia and weakness in the bilateral lower limbs by the age of 41 years. The cognitive decline and indifference were exacerbated at 42 years of age, and the slow and slurred speech and speaking problems also progressed. Furthermore, he developed gait disturbance and rigidity and required a wheelchair for mobility. He was admitted to Beijing Tiantan Hospital in November 2020. He denied having a history of medication or substance abuse. There was no medical history of hypertension, diabetes mellitus, coronary heart disease, and traumatic brain injury. Neurological examination demonstrated mild dysarthria and non-fluent aphasia; impaired cognition with respect to orientation, memory, calculation, comprehension, and executive ability; and left facial nerve and hypoglossal nerve palsy. The muscle strength of all four extremities was grade 4 with slightly increased muscle tone. A postural tremor was absent. Hyperreflexia was observed in the knees and ankles bilaterally, with a bilateral positive Babinski sign. Both upper limbs exhibited superficial hypoesthesia. He also walked with a slow and shuffling gait. Poor bilateral finger–nose and heel–knee coordination was also observed.The proband’s Mini-Mental State Examination (MMSE) score was 15/30, with substantial deterioration in orientation, calculation, and executive ability. The Montreal Cognitive Assessment (MoCA) score was 6/30 with obvious deficits in orientation, memory, calculation, comprehension, and executive ability. The results of laboratory tests, including routine blood analyses; liver, renal, and thyroid function; ion concentration; erythrocyte sedimentation rate; homocysteine, vitamin B, and C-reactive protein levels; and immunological biomarkers of rheumatic disease were unremarkable. He also tested negative for anti-aquaporin-4, anti-myelin oligodendrocyte glycoprotein, and autoimmune encephalitis-related antibodies . Brain MRI revealed relatively symmetric confluent and diffuse white matter hyperintensities that markedly affected the bilateral periventricular areas, corona radiata, and corpus callosum; prominent frontal and parietal lobe atrophy; and thinning of the corpus callosum . GadolinCSF1R gene located on chromosome 5q32 that consisted of a splicing c.2654+1G>A mutation in intron 20 (https://www.ncbi.nlm.nih.gov/sra/PRJNA780236). These mutation sites were not found in the ExAC, Human Gene Mutation Database, or 1000 Genomes Project. Mutation Taster, PROVEAN, and FATHMM-indel predicted that mutation c.2833_2835delGAG was a polymorphism , neutral , and neutral , respectively. However, Mutation Taster, FATHMM-XF, and CADD predicted that mutation c.2654+1G>A was deleterious , pathogenic , and deleterious , respectively. dbscSNV classified the splicing c.2654+1G>A mutation as “disease-causing,” with an adaptive boosting score of 0.999 and random forest score of 0.934. Mutation Taster, NetGene2, and Human Splicing Finder predicted that the mutation might alter the WT donor site, with a high probability of interrupting normal splicing. Thus, the splicing c.2654+1G>A mutation was predicted to be possibly pathogenic (PVS1+PM2), while the indel mutation (c.2833_2835delGAG) was of uncertain significance (PM2+PM4), according to the ACMG/AMP guidelines. The c.2654+1G>A mutation was not found in the Genome Aggregation Database, while the c.2833_2835delGAG mutation was present in this database. There are six individuals carrying this indel mutation, from 40 to 80 years old, none of whom have any clinical symptoms. Therefore, we speculated that the splicing mutation (c.2654+1G>A) in intron 20 was more significant than the indel mutation (c.2833_2835delGAG) in this family and disrupted the function of CSF1R, resulting in CSF1R-microglial encephalopathy.The results of WES demonstrated a novel heterozygous mutation in the ntron 20  and an intron 20 . These dCSF1R mutation after collating the clinical manifestations and neuroimaging and genetic findings , but the free triiodothyronine and tetraiodothyronine levels were normal.Brain MRI revealed confluent and diffuse signal abnormalities in the bilateral periventricular areas, corona radiata, and corpus callosum, and prominent thinning of the corpus callosum .CSF1R gene  accompanied by leukodystrophic loci or spots of demyelination  in the white matter , and eventually causes demyelination, axonal loss and swelling, which precedes white matter degeneration. This finding is in line with the notion that CSF1R mutation-induced leukoencephalopathy is a primary microglial disorder and reemphasizes the feasibility of the nomenclature “CSF1R-microglial encephalopathy.”Interestingly, both probands from the two unrelated families exhibited bilateral persistent hyperintensity in the periventricular white matter on DWI. Previous studies also showed that hyperintense signals on DWI sequences could persist for several months to years in patients with findings . The meclination , which ie matter . Inhibite matter . It is se matter . The selCSF1R mutations in 2012, conditions such as HDLS, ASLP, brain malformations, and skeletal dysplasia have been considered as part of the varied phenotypic spectrum of a unified disease  in intron 20 and (c.1858+5G>A) in intron 13 of CSF1R as the cause of CSF1R-microglial encephalopathy, and firstly highlighted the potential of the pCASL technique to enhance the clinical diagnostic accuracy of CSF1R-microglial encephalopathy, further broadening the genetic and radiological spectrum of this condition. Neurologists should consider CSF1R-microglial encephalopathy when a patient exhibits early-onset rapid progressive dementia with symmetrical changes and persistent diffusion restriction in the deep white matter because the genetic spectrum of mutations is yet to be fully elucidated. Future functional research is needed to elucidate the pathogenicity of these two novel intronic mutations of CSF1R. Prospective studies should be developed to identify the sensitivity and specificity of pCASL technique to diagnose the CSF1R-microglial encephalopathy.In conclusion, we developed the concept “CSF1R-microglial encephalopathy” that encompasses the genetic, pathological, and clinical characteristics and their respective relationships to describe the various phenotypic spectrum of a unified disease due to


NUCB2 and Fat Mass and Obesity Gene (FTO) polymorphisms, BMI, Eating Disorders (EDs), and pathological behaviors. Nesfatin-1 secretion was evaluated both in normoxic fully differentiated 3T3-L1 mouse adipocytes and after incubation under hypoxic conditions for 24 h. Omental Visceral Adipose tissue (VAT) specimens of 11 obese subjects, and nesfatin-1 serum levels’ evaluation, eating behaviors, NUCB2 rs757081, and FTO rs9939609 polymorphisms of 71 outpatients seeking treatment for EDs with different Body Mass Index (BMI) were studied. Significantly higher levels of nesfatin-1 were detected in hypoxic 3T3-L1 cultured adipocytes compared to normoxic ones. Nesfatin-1 was highly detectable in the VAT of obese compared to normal-weight subjects. Nesfatin-1 serum levels did not vary according to BMI, sex, and EDs diagnosis, but correlations with grazing; emotional, sweet, and binge eating; hyperphagia; social eating; childhood obesity were evident. Obese subjects with CG genotype NUCB2 rs757081 and AT genotype FTO rs9939609 polymorphisms had higher nesfatin-1 levels. It could represent a new biomarker of EDs comorbidity among obese patients.Nesfatin-1 is a new anorexigenic neuropeptide involved in the regulation of hunger/satiety, eating, and affective disorders. We aimed to investigate nesfatin-1 secretion in vitro, in murine adipose cells, and in human adipose fat samples, as well as to assess the link between circulating nesfatin-1 levels,  NUCB2) protein after post-translational cleavage by specific convertases, PC2 and PC3/1 [Obesity is one of the most common nutritional disorders, and its medical, psychological, social, and economic effects have a significant burden on healthcare resources , leadingnd PC3/1 ,16. NUCB2 by PCs in other potential sites produces the nesfatin-2 (residues 85–163) and nesfatin-3 (residues 166–396) forms, but only nesfatin-1 or the full-length NUCB2 has the potential role to induce satiety [NUCB2 is proteolytically transformed into nesfatin-1, it can stimulate anorexia [NUCB2 gene have been associated with susceptibility to obesity [The cleavage of  satiety . Furtheranorexia . Polymor obesity . Nesfatin-1 is abundantly secreted both in central and peripheral regions. It has been detected in several regions of the hypothalamus and demonstrated to play key roles in the control of food intake, while it seems to be modulated by starvation and refeeding ,20. As nFTO) were also assessed. Finally, circulating nesfatin-1 plasma levels were evaluated in human subjects across a range of BMI and were compared to eating behaviors and affective symptoms.To our knowledge, this is the first study that compared nesfatin-1 levels according to eating behaviors and psychopathological features among patients at different BMI ranges, with or without an eating disorder. We aimed to investigate differences in NUCB2/nesfatin-1 levels in human VAT and in serum of obese patients compared to normal and underweight controls. Second, we tested the nesfatin-1 secretion profile in in vitro models of fully differentiated 3T3-L1 adipocytes. In order to estimate the link between obesity development and nesfatin-1 levels, polymorphisms of NUCB2/nesfatin-1 and the Fat mass and obesity gene  for 24 h [2). At the end of incubation, the chamber was opened in the anaerobic glove box (flushed with N2) to avoid reoxygenation. Cell viability was tested by 3--2,5-diphenyl tetrazolium bromide assay, to evaluate the absence of cytotoxicity after exposure to hypoxia, as described previously [3T3-L1 murine preadipocytes were cultured and differentiated as previously described . Cells wfor 48 h . As a sufor 24 h . Controleviously . Omental VAT specimens were obtained from 11 consecutive, unrelated, obese subjects, during elective bariatric surgery, and 6 normal-weight controls from patients undergoing open abdominal surgery. Each specimen was immediately snap-frozen in liquid nitrogen and stored at −80 °C until protein extraction. All the operated subjects were nonsmokers and had not taken any medications for at least 6 months before the study. Criteria of exclusion were secondary obesity, genetic syndromes of insulin resistance, psychiatric disorders, Type 1 or Type 2 diabetes, previous gestational diabetes mellitus, hepatic or renal impairment, presence of malignancies or rheumatologic disease, and treatment with medications affecting glucose tolerance. 2, 1 mM CaCl2, 1,5% NP-40, 2 mM EDTA) supplemented with the protease inhibitor cocktail, PMSF (2 mM), and phosphatase inhibitor. The final concentration in the extracts was determined using the Bradford colorimetric assay (Biorad). Proteins (20 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane (Immobilon-PSQ 0.2 µm Millipore ISEQ00010), and blotted for 2 h. Blotted membranes were treated with blocking solution  with 5% w/v nonfat dry milk, and then incubated overnight at 4 °C with anti NUCB2/nesfatin-1 , or anti beta Actin antibodies. Anti-rabbit  or anti mouse IgG horseradish conjugates  were used as secondary detection antibodies. Immune complexes were visualized by enhanced chemiluminescence  and autoradiographic films, and the density of specific bands was measured using the ImageJ software program. Total protein extracts were prepared from cells and tissues as previously described . Briefly2 based on the World Health Organization (WHO) [A total of 71 consecutive outpatients seeking treatment for EDs were recruited at the University “Magna Graecia” of Catanzaro, Unit of Psychiatry, from September 2017 to July 2019. Participants were considered eligible if they were: 18–65 years old; drug-naïve; diagnosed with AN restrictive type or BED according to DSM-5, suffering from obesity without an ED in comorbidity or normal-weight healthy controls without any psychiatric diagnosis according to DSM-5; able to answer a self-reporting questionnaire; able to understand the process in which they were involved. Obesity was defined as BMI > 30 kg/mon (WHO) . For eacWere measured using.A portable stadiometer  and a balance scale (Seca 761) were used to measure participants’ height and weight and calculate BMI. Subjects were asked to wear light indoor clothing and no shoes. Standing height was measured to the nearest 0.1 cm and body weight to the nearest 0.1 kg at 8.00 a.m. BMI was classified according to the WHO guidelines . -The 21-item self-report Beck Depression Inventory (BDI-II)  to evalu-The Binge Eating Scale (BES)  is usefu-The State-Trait Anxiety Disorder (STAI) . This 40Trained psychiatrists interviewed participants by means of the Structured Clinical Interview for the DSM-5 (SCID-5-CV)  and the Venous blood collection was performed at around 08.00 a.m. after overnight fasting. Following venous blood withdrawal, blood was collected in pre-cooled laboratory EDTA tubes containing aprotinin  to prevent protease activity. Tubes were then centrifuged at 4 °C for 10 min at 3000 g , and plasma aliquots were stored at −80 °C until use. Plasma nesfatin-1 was measured using a commercial enzyme-linked immunosorbent assay, and the Mybiosource human NES1 (Nesfatin-1) ELISA kit, according to the manufacturer’s instructions. The assay displayed a detection sensitivity of 9.38 pg/mL, a detection range of 15.63–1000 pg/mL, and a coefficient of variation < 10%. The antibody used in this ELISA was raised against nesfatin-1 and recognized both nesfatin-1 and the full-length protein precursor NUCB2 containing the epitope. DNA was extracted from whole blood using commercial DNA isolation kits . rs757081NUCB2/nesfatin-1 and rs9939609 FTO genotype calls were determined with the TaqMan allelic discrimination assay . Template DNA was amplified, and fluorescence was detected on a BioRad CFX-96 Thermal Cycler . Genotyping quality was tested by including a blinded duplicate in each 96-well assay. The average agreement rate of duplicate samples was >99%.The data are reported as means, standard deviations (SD), frequencies, and percentages (%). Generalized linear model (GLM) analysis was conducted to compare the main effects of ED diagnosis and BMI categories and the interaction between EDs, BMI, and eating behaviors on plasma nesfatin-1 levels controlling for BES, BDI-II, and STAI. EDs diagnosis included four levels , while BMI consisted of five progressive levels . Forward stepwise linear regression analysis served to establish variables associated with nesfatin-1 considering BMI, age, sex, childhood obesity, BDI-II, and BES scores as independent variables. Two-tail Spearman correlations were run between eating behaviors and nesfatin-1 circulating levels. Statistical analysis was performed with the Statistical Package for Social Sciences version 21 .2) [3T3-L1 murine differentiated adipose cells have been shown to secrete nesfatin-1 protein . To esta2)  and compn = 6; mean BMI = 23.6 ± 1.5 kg/m2) and obese patients . Our data showed that nesfatin-1 was undetectable in the VAT of normal-weight individuals, while a significant amount of this protein was observed in all (n = 11) obese patients , 16 normal weight (BMI 18.5–24.9 kg/m2), while 14 had class I (BMI 30–34.9 kg/m2), 6 class II (BMI 35–39.9 kg/m2), and 21 class III obesity (BMI > 40 kg/m2). For statistical purposes, patients with obesity were split into two groups: Obesity 1, corresponding to class I ; Obesity 2, corresponding to class II / III . No differences in sex distribution were observed upon BMI categories ; instead, patients with AN and BED were overrepresented among females . A total of 71 subjects, aged 36.4 ± 12.7 years, were included in the current cross-sectional study. p = 0.130), EDs diagnosis , or sex .Average levels of circulating nesfatin-1 are summarized in Although no significant correlations emerged between nesfatin-1 and EDs diagnosis, inverse correlations with binge eating, grazing, emotional and sweet eating, and positive correlations with hyperphagia and social eating were evident . Finally, a series of linear regression analyses was run to ascertain variables associated with nesfatin-1 considering BMI, age, sex, childhood obesity, BDI-II score, and BES score as independent variables . Higher NUCB2/nesfatin-1 gene, and for FTO , a known gene involved in the regulation of fat mass, adipogenesis, and body weight [p = 0.303). To search for further interrelationships between nesfatin-1 and the obese status, we analyzed circulating nesfatin-1 levels in obese patients genotyped for the most prevalent genetic polymorphisms for the y weight . NUCB2 rp = 0.018) on nesfatin-1 plasma levels  is linked to childhood adiposity, while the less frequent GG genotype (Q338E) could be considered as protective against excessive adiposity gain, providing further evidence for the role of NUCB2 in the determination of human adiposity. To note, even if our data did not reach significance, their trend is in line with this study. We also examined the possible correlation between nesfatin-1 serum levels and the FTO gene variants. SNPs of the FTO gene are intensely associated with obesity [FTO gene plays an important role in fat metabolism, and the FTO risk allele carriers display lower fat cell lipolysis compared with the others [FTO and obesity development remains unclear. Some studies reported that the FTO gene may exert its effects by altering body composition and gaining adiposity [FTO gene and BMI emerged on nesfatin-1 serum levels. As a further step forward in the understanding of potential bonds between nesfatin-1 and BMI, we also evaluated plasma levels of nesfatin-1 and rs757081 polymorphism. In this regard, many studies have, in fact, tried to explain the link between the  obesity . Recentl obesity  posited d intake . They ann et al.  independ obesity . Among t obesity ,67. The  obesity . The FTOe others . The mecdiposity ,70. Our Despite this evidence, contradictory results have been reported regarding the association between BMI and circulating nesfatin-1 levels ,32,33,34We also described that higher circulating nesfatin-1 could be linked to childhood obesity. Recent studies agreed with these results . In partObese patients with higher scores of the BES, probably affected by BED, have shown lower levels of nesfatin-1, so nesfatin-1 could be related to the comorbidity of EDs in obese patients. In this regard, lower plasma nesfatin-1, by altering the satiety signaling, could be the trigger and the maintaining determinant of binge behaviors. Moreover, coherently, low circulating nesfatin-1 has been defined as a facilitating factor for the development of obesity . It is nFew studies have been conducted to evaluate nesfatin-1 plasma levels in patients with EDs ,39,75. WSeveral limitations should be noted, prior to proceeding with the conclusions. First, the sample size limited the power in this study, and due to its exploratory nature, no corrections for multiple testing were made, thus increasing the likelihood of significant results. Second, the presence of nesfatin-1 in the adipose tissue of patients with or without BED was not verified, and it would be interesting to understand if nesfatin-1 levels also vary in relation to the reduction in binge behaviors. Overall, future investigations on nesfatin-1 on EDs need to ensure that such confounders are evaluated and described, and if possible, accounted for in statistical analyses. Limitations of cross-sectional studies obviate conclusions regarding causality.Future studies with a longitudinal design could be useful to elucidate the variability in nesfatin-1 levels according to other variables, including the determination of nesfatin-1 in CSF in patients with varying degrees of BMIs.The newly identified neuropeptide nesfatin-1 is characterized by a wide range of regulating activity in the brain and in other peripheral sites. Increasing evidence supports it as a new, potentially significant factor involved in the pathogenesis of mental disorders. To summarize, based on our results, in adipose tissue, nesfatin-1 abundance varied in relation to BMI. Higher levels were found in the VAT of obese patients with respect to normal-weight controls. Circulating nesfatin-1 levels could be influenced by other important variables. Our results showed that eating behaviors could play a role in determining plasma nesfatin-1 concentration rather than affective symptoms or BMI. An inverse correlation emerged with dysfunctional eating behaviors, such as binge eating, grazing, emotional and sweet eating, and craving for carbohydrates, frequently described in obese patients with BED, and a positive correlation with hyperphagia and social eating, described among obese patients without EDs.In this regard, nesfatin-1 could be identified as a potential biomarker of comorbidity of EDs among obese patients.


Cancers harboring serine threonine kinase (STK11) alteration or SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily B, member 1 (SMARCB1) mutation are conventionally considered as treatment-refractory to immune checkpoint inhibitors or chemotherapy, respectively. However in the present report, we demonstrated a case of dedifferentiated non-small cell lung cancer, characterized by STK11 loss (due to promoter loss) mutation co-mutated with SMARCB1 deficiency mutation, has achieved significantly partial response to neo-adjuvant treatment with pembrolizumab and platinum doublet regimen. Our case highlighted that either STK11 loss, or SMARCB1 deficiency mutation, might not be used to select patients for PD-(L)1 blockade therapy or chemotherapy, respectively. SKT11 loss accompanied with SMARCB1 deficiency mutation may benefit from immunotherapy combined with chemotherapy. Serine threonine kinase  is considered as one of tumor suppressor genes, which encodes for a serine/threonine 11 that activates AMP-activated protein kinase (AMPK), hence being capable of regulating cell response to DNA damage, tumor growth, and energy metabolism . There hSWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily B, member 1  is also a tumor suppressor gene located in chromosome 22q11.2, expressed in the nucleus of almost all normal tissues of the body , 10. SMAHerein, we reported a rare case of dedifferentiated lung cancer harboring STK11 loss and SMARCB1 deficiency, due to p.S67X mutations, which presented nearly complete response to neo-adjuvant treatment with pembrolizumab and platinum doublet. Subsequently, he received radical left upper lobectomy, and still being disease free status.th, 2022 with chest distress and slight pain on left chest for approximately three months. He denied smoking or alcohol history, nor any other medical or family disease history. Chest CT on June 6th, 2022 showed a large space-occupying lesion (size as 8.8 × 5.1 cm) in the superior lobe of left lung, which was closed to mediastinum, presented as obstructive pneumonia, accompanied with vein and artery in left superior pulmonary involvement (via next-generation sequencing (NGS) was performed using tissue sample  showed STK11 loss, due to the loss of promoter (frequency as 30.67%) and SMARCB1 deficiency, due to p.S67X*10.74% mutations, with a tumor mutational burden (TMB) of 6.85 mutations per megabase, as well as PD-L1 tumor proportion score (22C3) being less than 1%. Based on those, the patient was diagnosed as dedifferentiated lung cancer, with vein and artery in left superior pulmonary involvement, along with metastasis on mediastinal lymph nodes, which was staged as IIIB  according to the criteria of American Joint Committee on Cancer (AJCC) 8th edition. After the discussion by multi-disciplinary team , regimen of chemotherapy combined with anti-PD-1 agent was adopted as palliative or neo-adjuvant treatment, which depended on the response status during the drug administration. In addition, maintenance therapy with anti-PD-1 agent should also be taken into consideration after disease control if necessary. Exactly, combined treatment with chemotherapy and immunotherapy has already been recommended as standard choice in advanced NSCLC patients in absence of sensitive targetable mutations. The patient thereby was started on pembrolizumab , and carboplatin/nab-paclitaxel  as initial treatment. After three cycles′ treatment, restaging chest CT scans showed significant improvement on the primary lesion, as well as metastatic mediastinal lymph nodes , which happened after the first cycle exposure of the combined regimen. At the time of manuscript preparation, the patient continues to receive mono-therapy of pembrolizumab, and still maintained an ongoing disease free status.A 61-year-old Chinese man was admitted to hospital on June 6Herein in the present report, we demonstrated a case of dedifferentiated non-small cell lung cancer, characterized by STK11 loss and SMARCB1 deficiency, due to p.S67X mutations, which has been conventionally considered as treatment-refractory, has achieved significantly partial response to neo-adjuvant treatment with pembrolizumab and platinum doublet regimen. To the best of our knowledge, the present case was the first one to report the co-alteration of STK11 loss and SMARCB1 deficiency mutation. Results of the present case highlighted that biomarkers including loss, or SMARCB1 deficiency mutation, might be probably predictive to response to PD-1 blockade therapy.P < 0.0001; KEAP1 HR = 2.05, P < 0.0001) and OS  survival to immunotherapy among KRAS mutant lung adenocarcinoma (P = 0.0001)) or KEAP1 (KEAP1-mutant 12.0 months versus KEAP1-wild 24.4 months (P = 0.005)) mutations 1 therapy, the issue is far from settled. An exploratory analysis from the trial KEYNOTE-042 revealed that pembrolizumab monotherapy was associated with improved OS when compared with platinum-doublet regardless of STK11 and KEAP1 mutational status . Just beIn the present report, we demonstrated a case of dedifferentiated non-small cell lung cancer, characterized by STK11 loss and SMARCB1 deficiency mutation that conventionally been considered as treatment-refractory, has achieved significantly partial response to neo-adjuvant treatment with pembrolizumab and platinum doublet regimen. Our case highlighted that either STK11 loss, or SMARCB1 deficiency mutation, might not be used to select patients for PD-(L)1 blockade therapy or chemotherapy, respectively. In addition, awareness and recognition of the co-mutations of STK11 loss and SMARCB1 deficiency mutation may help sharpen focus on effective therapeutic development, and expand our understanding on the potential mechanism, as well as pathological significance of the rare co-mutations.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethical Committee of People′s Hospital of Quzhou. The patients/participants provided their written informed consent to participate in this study.JC, conceptualization, methodology, software, writing- original draft preparation, software, and validation. JW, data curation, visualization, investigation, supervision, writing-reviewing, and editing. All authors contributed to the article and approved the submitted version.


Yet, thymopoiesis can be transiently or permanently damaged contingent on the types of systemic stresses encountered. The thymus also undergoes a functional decline during aging, resulting in a progressive reduction in naïve T cell output. This atrophy is evidenced by a deteriorating thymic microenvironment, including, but not limited, epithelial-to-mesenchymal transitions, fibrosis and adipogenesis. An exploration of cellular changes in the thymus at various stages of life, including mouse models of in-born errors of immunity and with single cell RNA sequencing, is revealing an expanding number of distinct cell types influencing thymus functions. The thymus microenvironment, established through interactions between immature and mature thymocytes with thymus epithelial cells (TEC), is well known. Less well appreciated are the contributions of neural crest cell-derived mesenchymal cells, endothelial cells, diverse hematopoietic cell populations, adipocytes, and fibroblasts in the thymic microenvironment. In the current review, we will explore the contributions of the many stromal cell types participating in the formation, expansion, and contraction of the thymus under normal and pathophysiological processes. Such information will better inform approaches for restoring thymus functionality, including thymus organoid technologies, beneficial when an individuals’ own tissue is congenitally, clinically, or accidentally rendered non-functional.The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from the 3 Positielection . Thereinelection , 34]. Thelection , 36. Finelection , 32, 37.election , 39. S1Prd PP -derived mesenchymal cells (Mes) and endothelial cells are two key cell types essential for thymus formation, establishing the thymus vasculature. The vasculature serves as the entry site for T lymphocyte precursors or early thymus progenitors (ETPs), generated in fetal liver and bone marrow, to the thymic cortex. Endothelial cells form a cushion around the developing arterioles and veins, with the perivascular space comprising collagen fibers. Perivascular cells include pericytes and vascular smooth muscle cells (VSMCs) . The thyrd PP . Mes relrd PP , 7. Fibrrd PP , 42. Mulrd PP . Dysregurd PP . A key rrd PP . Second,rd PP , 45. Extrd PP , 46–48. rd PP . Epidermrd PP , 50. Third PP , 52. Therd PP .PDGFRA, COLEC11, C7, GDF10), Fibroblast type 2  and VSMCs (ACTA2) (ALDH1A2), which is needed for retinoic acid (RA) production, a morphogen that supports TEC development  involves erythropoietin-producing hepatocellular carcinoma (EPH) ligand-EPH receptor (EPHR) coupled Mes and TEC signaling . The Mes (ACTA2) Table 2  (ACTA2) . Fibroblelopment . Fb2 areelopment . Notablyelopment . The antelopment .. In adult tissues, endothelial cells regulate thymocyte homing via the lymphotoxin β receptor (LTβR)  . The ligr (LTβR) . This ispression . It is lpression .Psmb11) and PLET1 , proteases , proteasome complex components , peptide transporters required for MHC class I peptide presentation , many keratins and Cd83 , generating peptides that are bound by MHC class I to support the positive selection of CD8+ T cells  and CREB binding sites. Notably, TAp63 participates in TEC homeostasis  precursors , 64. Thend PLET1 . Immaturnd PLET1 Table 1.nd PLET1 . The firnd PLET1 , 67. Latnd PLET1 –70. The 1  as well as the progression of thymocytes from the DN to DP stages of thymopoiesis . CCL9, CCL21, CCL25, CCL12, positively regulated by FOXN1, provide directional cues for developing thymocytes  gene. AIRE enforces the expression of “tissue-restricted” proteins that eliminate autoreactive T cells , suggesting either the presence of some parathyroid TECs or the existence of a bi-functional TEC. The 6th epithelial subset (E-6) expresses Nkx2.1, which may be a thyroid or parathyroid precursor cell type. ScRNA sequencing of human thymuses obtained at gestational weeks 19 and 23 (e14.5-e16.5 in murine embryos) and postnatal samples from 6-day old newborn (e20) and an infant (10 months) reveals 3 major epithelial groups broken down into 9 subclusters . One cluster is the cTEClo, which is rapidly proliferating based on Ki67+ expression. The second cluster is the cTEChi. mTECs are also evident in the embryonic tissues at this developmental stage, and split into 3 distinct subgroups partly defined by the levels of HLA class II. These are mTEClo (CLDN4 and lower levels of HLA class II), mTEChi , and corneocyte-like mTECs  , muscle-like myoid , and myelin+ epithelial cell markers  , reveal 6 distinct epithelial subsets Table 2.clusters . Two cluT1, IVL) . Cells i in mice . Another10, MPZ) , 57. A mThe various TEC subsets constitute the basic thymic microenvironment. They provide cytokines, chemokines, multiple molecular signals, and cell-to-cell interactions to control thymocyte proliferation, differentiation, and selection. This process leads thymocytes from immature to mature, and in turn, TECs themselves also get maturation from bi-potent epithelial progenitors to functional cTECs and mTECs. The added complexity of many different subsets suggests distinct roles for each in the formation and regeneration of the thymus at different stages.FOXN1 contribute to diminished thymopoiesis  is principally expressed on cTECs and mTECs, with ligand mediated signaling supporting TEC survival and functionality  syndrome, or those who had diabetic embryopathies  (6 cells), an abnormal CD4/CD8 ratio < 1 and the use of murine cell lines. ATOs can, however, reveal whether a patient with probable in-born error of immunity would need a bone marrow or thymus transplant. For example, patients with mutations in genes affecting stromal cell subsets  will retain HSPCs capable of thymus development  expressing the Notch ligands Delta-like ligand 1 (DLL1) or DLL4, with both supporting T cell development to the SP stage by engaging Notch on immature thymocytes , 175. DLs (HSPC) . HSPCs cs (HSPC) . While ts (HSPC) –179. Gros (HSPC) –179. Whielopment , 179. Th National Institutes of Health grant R21AI144140 (NvO).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


CARD10) gene. CARMA3 can form a CARMA3-BCL10-MALT1 complex by recruiting B cell lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), thereby activating nuclear factor-κB (NF-κB), a key transcription factor that involves in various biological responses. CARMA3 mediates different receptors-dependent signaling pathways, including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). Inappropriate expression and activation of GPCRs and/or RTKs/CARMA3 signaling lead to the pathogenesis of human diseases. Emerging studies have reported that CARMA3 mediates the development of various types of cancers. Moreover, CARMA3 and its partners participate in human non-cancer diseases, including atherogenesis, abdominal aortic aneurysm, asthma, pulmonary fibrosis, liver fibrosis, insulin resistance, inflammatory bowel disease, and psoriasis. Here we provide a review on its structure, regulation, and molecular function, and further highlight recent findings in human non-cancerous diseases, which will provide a novel therapeutic target.Caspase recruitment domain and membrane-associated guanylate kinase-like protein 3 (CARMA3) is a scaffold protein widely expressed in non-hematopoietic cells. It is encoded by the caspase recruitment domain protein 10 ( CARD11 and is mainly expressed in lymphoid and hematopoietic tissues, and CARMA2 is encoded by CARD14 and expressed in mucosal and skin tissues, while CARMA3 is encoded by CARD10 and is widely expressed in tissues other than hematopoietic cells  lengths are 4111nt and 3099nt respectively. It encodes a 1032-amino acid protein, which comprises five functional domains: a caspase recruitment domain (CARD) domain, a coiled-coil (CC) domain, a PDZ domain, an SH3 domain, and a guanylate kinase-like (GUK) domain , was discovered in 2001 . The CAR) domain , as show) domain . Close t) domain . CC doma) domain , 17. The) domain . The PDZ) domain .CARD10 through negative feedback . Knockdown of CARMA3 reduces thrombin-induced gene expression and NF-κB signaling, while overexpression can effectively rescue miR-181b-mediated inhibition of NF-κB target genes, such as VCAM-1, ICAM-1, and E-SELECTIN  reportedly play important roles in regulating CARMA3 levels . It inhifeedback , and miRfeedback . Silencitic skin . miR-181SELECTIN .CARD10 transcription (CARD10 (-1.5 kb and -7kb). In immature granulocytes from CEBPE-knockout mice, the expression of CARMA3 was significantly lower than that at the same stage of differentiation in wild-type mice  family of transcription factors, regulates cription , as showype mice .via its deubiquitylation activity  and vascular endothelial growth factor (VEGF) are known to induce NF-κB activation , 45, 46.via the C-terminal GUK domain and blocks downstream activation of IRF3 by preventing the formation of MAVS oligomerization. In the late stages of virus infection, CARMA3 is degraded by the proteasome, releasing MAVS to form high-molecular-weight aggregates and activate IRF3  stage, tumor status and grade, metastasis, and poor prognosis –59. CARM pathway . In lung pathway . Moreove pathway . Similar pathway , colorec pathway , 58, ova pathway , 61, and pathway –64. In cOver the past decades, a lot of studies have indicated the importance of CARMA3 in non-cancer diseases. Here, we illustrate the role of CARMA3 in non-tumor diseases from two aspects: CBM complex and CARMA3 mutations.Inflammation is a key characteristic in the pathogenesis of cardiovascular diseases, including atherosclerosis, hypertension, and abdominal aortic aneurysms . In the Alternaria alternata, the total number and percentage of type 2 innate lymphoid cells (ILC2) in the lungs were reduced, suggesting that CARMA3 mediates the accumulation of ILC2 in the lungs after allergen exposure, which may be one mechanism by which type 2 cytokine production is attenuated in the lung and airway inflammation in CARMA3-deficient mice , which are essential for the pathogenesis of insulin resistance, are increased in the serum of obese people. Deletion of BCL10 in mice eliminates hepatic NF-κB activation induced by a high-fat diet, resulting in mitigated inflammation and protection from insulin resistance .Ang II is involved in tissue-repairing process by mediating inflammatory cell infiltration, myofibroblast proliferation, and collagen synthesis after liver injury . But in CARMA3 is widely expressed in various tissues such as the heart, lung, liver, intestine, and kidney . HoweverLactobacillus acidophilus, a probiotic, could eliminate PAF-induced inflammation in IECs by blocking PAF-induced NF-κB activation and IL-8 production. In addition, Lactobacillus acidophilus culture supernatant (LA-CS) can attenuate the PAF-induced elevation of BCL10 mRNA and protein levels, BCL10 promoter activity, BCL10 interaction with MALT1, and PAF-induced ubiquitination of IKKγ , CBM signalosomes activate the NF-κB signaling pathway triggered by platelet-activating factor (PAF) and play a pivotal role in IL-8 production . Subsequ of IKKγ . These rIn normal human epidermal keratinocytes (NHEKs), CARMA2 is more investigated, it is expressed in keratinocytes and its mutations increase the risk of inflammatory skin disease . RecentlCARD11 and CARD14 have been studied extensively. CARD11 mutations are linked to lymphoproliferative disorders and immunodeficiencies, whereas CARD14 variants are associated with several inflammatory skin disorders  in Dutch and German populations (CARD10 rs9607469 and CARD10 rs9610775 are associated with primary open-angle glaucoma and amnestic mild cognitive impairment, respectively (CARD10 polymorphism, Hang et al. were the first to identify two in-frame deletions in CARD10 among 101 cases of ovarian endometriosis, which may cause the disease (CARD10, which is implicated in rare diseases, repeated infections, Crohn’s disease, allergic diseases, and other disorders (CARD10 mutations, rs9610775 and R420C are missense variants and are located in exons 4 and 7 of CARD10 respectively. Both mutations alter the CARMA3 CC domain, which is associated with BCL10 aggregation and activation, suggesting that the rs9610775 and R420C mutations affect CBM assembly and the subsequent NF-κB activation (CARD10 mutations on NF-κB signaling. However, Said et al. found that the NF-κB p65 level is increase in the AIH patients with CARD10 polymorphism rs6000782, suggesting that the CARD10 polymorphism rs6000782 may cause disease by affecting NF-κB signaling and the expression levels of downstream genes (CARD10 mutations on the NF-κB signaling pathway.Over the past few years, mutations in isorders , 84. Howulations , which Mulations . Howeverulations . This diectively –90. Apar disease . Luo et isorders . Among ttivation . To dateam genes . FurtherAll the functions of CARMA3 in non-cancer diseases were summarized in CARMA3 is widely expressed in a variety of tissues  and playCurrent studies on the regulation of CARMA3 mainly focus on the transcriptional level, with no investigations on phosphorylation, ubiquitination, and acetylation of CARMA3 proteins. To further understand the contribution of CARMA3 in diseases, its regulation at the protein level is worthy of investigation. Furthermore, CARMA3-dependent signal transmission plays a fundamental role in pathophysiology of various diseases, indicating that generating molecular tools, such as novel inhibitors specific to CARMA3 and small peptides target to CBM signalosome, would be immeasurable in treating CARMA3-related disorders.WX and ZJ conducted the review, and ZG, WX, and YZ drafted the manuscript. All the authors have written, reviewed and approved the manuscript.