SFPQ::ZFP36L2 fusion, reported in pediatric T-ALL, deletions of putative suppressors, such as IKZF2 and CSMD1, and amplification of the BCL2 gene. Compared to negative cases, chromothripsis positive T-ALL had a significantly higher level of MYCN expression, and a significant downregulation of RGCC, which is typically induced by TP53 in response to DNA damage. Furthermore we identified mutations and/or deletions of DNA repair/genome stability genes in all cases, and an association with NUP214 rearrangements in 33% of cases.Chromothripsis is a mitotic catastrophe that arises from multiple double strand breaks and incorrect re-joining of one or a few chromosomes. We report on incidence, distribution, and features of chromothriptic events in T-cell acute lymphoblastic leukemias (T-ALL). SNP array was performed in 103 T-ALL , including 38 children and 65 adults. Chromothripsis was detected in 11.6% of all T-ALL and occurred only in adult cases with an immature phenotype . It affected 1 to 4 chromosomes, and recurrently involved chromosomes 1, 6, 7, and 17. Abnormalities of genes typically associated with T-ALL were found at breakpoints of chromothripsis. In addition, it gave rise to new/rare alterations, such as, the  T-cell acute lymphoblastic leukemia (T-ALL), accounts for 15% of pediatric and 20–25% of adult acute lymphoblastic leukemias (ALL). It results from a multistep process leading to the accumulation of multiple genetic abnormalities, that affect cell cycle control, self-renewal capacity, cell differentiation, proliferation and apoptosis, and epigenetic modulation [TAL/LMO, HOXA, TLX3, TLX1, NKX2–1/2–2, and MEF2C [NOTCH, JAK/STAT, RAS/MEK/ERK, PI3K/AKT, WNT), epigenetic modifiers , and/or protein translation  to elicit the leukemia phenotype. Diverse molecular mechanisms, i.e., activating and inactivating mutations, balanced/unbalanced chromosome rearrangements, and/or copy number abnormalities (CNA), underlie gene deregulation [High expression of (onco)genes, coding for transcription factors pivotal for hematopoiesis and/or T-cell development, have been identified by gene expression profile and shown to distinguish unique T-ALL subgroups, i.e., First described by Stephens a et al. . chromotThe advent of high resolution genome analyses, has greatly enhanced the detection of chromothriptic events which appear to be a widespread phenomenon, with an overall prevalence of ∼50% in different human cancer subtypes, such as lung, esophageal, bladder, prostate, and breast adenocarcinomas , 7. ChroIn hematopoietic neoplasms, chromothripsis has been found in B-cell acute lymphoblastic leukemia with iAMP21, myelodysplastic syndromes with complex chromosomal aberrations, acute myeloid leukemia, multiple myeloma, and chronic lymphocytic leukemia –13. T-ALFrom our SNP array study of a large cohort of pediatric and adult T-ALL chromothriptic events emerged in approximately 11% of adult cases, all with an immature phenotype, and provided insight on key underlying biological events.NOTCH1/FBXW7 hot-spot mutations were detected in 60/101 studied. Patients or their parents/guardians gave informed consent for sample collection and molecular analyses, in agreement with the Declaration of Helsinki. The study was approved by the local bio-ethical committee .The investigated T-ALL cohort =103) included 65 adults and 38 children (median age: 24), and had a prevalence of unclassified (38 cases) and immature (39 cases) cases (Supplementary Tables 03 includKaryotyping was done after G-banding with Wright stain and described according to the ISCN (2021). Single nucleotide polymorphism array (SNPa), RNA microarray analysis, Sanger and targeted sequencing are fully described in Supplementary methods . Furthermore, an unequal distribution of copy number abnormalities (CNA) was observed among the main genetic subgroups: cases of the TAL/LMO subgroup had the lowest number of events  while cases belonging to the TLX1 subgroup the highest     than chromothripsis-negative (cth−) ones, the latter including both ETP/near-ETP (media/median 5/5) and non-ETP cases (media/median 5/4) . In the 3 cases with chromothripsis at the long arm of chromosome 6 , a common deleted region (CDR) of 2,7 Mb, at 6q15, involved the lncRNA TSG1, and the ephrin receptor EPHA7. This CDR was also found in 14 cth− cases, with variable interstitial del(6q) . In case no. 3, with a single chromothriptic chromosome 16, the oncosuppressor gene CTCF underwent partial monoallelic loss; CTCF was also deleted in 9 cth− cases. Likewise, chromothripsis of chromosome 17, caused loss of TP53/17p13.1 and of SUZ12/17q11 in 2 cases (nos. 2 and 8)  .Chromothripsis caused focal CNA at new putative oncogenic regions/genes  Fig.  . In case no. 11 (Table SFPQ (1p34.3) and ZFP36L2 (2p21), and produced the SFPQ::ZFP36L2 fusion  cases identified 121 differentially expressed genes (DEG): 65 down- and 56 up- regulated (fold change ≥ ±2.0 and FDR < 0.05) , RNA microarray revealed that 64 DEG (44 down- and 20 up- regulated) distinguished cth+ (=11) from cth− (=22) cases: MYCN   were amongst the top deregulated genes   vs cth− 57) Fig.  and RGCCRGCC. Functional analysis showed the downregulation of nitric oxide related genes  and the upregulation of the NOTCH signaling , as the top enriched pathways in cth+ T-ALL (FDR < 0.1)   or trans- activating abnormalities , in 9 cases (Table TP53 (=3), RB1 (=5), TCF7 (=6), CDKN1B (=6), and the 6q14-q15 region (=7) , as well as of epigenetic modulators, i.e. IDH2 (=1), ASXL1 (=1), DNMT3A (=1), and EZH2 (=3) were identified. Overall, targeted sequencing and SNPa detected 1–2 mutations/deletions of epigenetic modulators in 8 cases (Table The Sophia Myeloid Solution (Sophia Genetics) detected 18 gene variants (range: 1–4 per case), in 10/12 cth+ cases , STK11 (=1), and PTEN (=2 in a single case). Instead, 3 somatic pathogenic/likely pathogenic variants  were detected in 2 cth− T-ALL cases. Five VUS (4 germline and 1 somatic) were also found  detected 16 single nucleotide variants (SNV) (9 in cth+ and 7 in cth− T-ALL cases) with a VAF range from 10.1% to 51.8%  was found in cases nos. 1 and 12; the ATR c.2320del, p.I774Yfs*5 (COSM214499) in cases nos. 4 and 10  Table .Chromothripsis is a widespread phenomenon in human cancers, reported in both solid and hematological tumors , 7, and By applying a whole genome SNP array to a wide range of pediatric and adult T-ALL, we estimated that chromothripsis occurred in ~11% of T-ALL, specifically in 30% of ETP/near-ETP of Adults. Correlation with prognosis and outcome was precluded due to the relatively small cohort of patients and heterogenous treatments.HOXA over-expression and recurrent deletions of TCF7, CDKN1B, RB1, and/or NF1. In addition, abnormalities of epigenetic modulators, typical for immature T-ALL, were detected in 66% of cth+ cases [The genomic profile of chr+ cases was largely overlapping with that of chr- ETP/near ETP ALL, as both subgroups showed a high rate of abnormalities associated with h+ cases .We found that cytogenetics did not reflect the genomic instability of the disease, as chromothripsis was indifferently present in cases with normal and complex karyotypes. Although in T-ALL of patients with Ataxia Telangiectasia the typical pattern of chromothripsis involved acrocentric chromosomes , this grRPL22, TCF7, IKZF1, EZH2, CDKN2AB, ETV6, CDKN1B, TP53, and rearrangement of the HOXA locus, our study provided evidence that it also generated new putative leukemogenic events, broadening the spectrum of oncogenic lesions linked to T-ALL pathogenesis/progression. Although detected in single cases, some of these events deserve attention. For istance, at chromosome 2q34, chromothripsis caused loss of the transcription factor IKZF2 which frequently undergoes intragenic deletions in adult T-cell leukemia/lymphoma (ATL) [CSMD1 (CUB and Sushi multiple domains 1), a tumor suppressor gene at 8p23, which has been associated with poor prognosis in many solid tumors, including breast, gastric, squamous, head and neck, and hepatocellular carcinomas [BCL2, that fits with the well-established upregulation of BCL2 in immature T-ALL [As regards the specific hotspots, chromothripsis must be counted among the molecular mechanisms that generate oncogenic events typical of T-ALL, similarly to what has been observed in other human tumors. Interestingly, besides producing rearrangements and CNA of oncogenes/oncosuppressors recurrently involved in T-ALL, such as ma (ATL) , and tharcinomas , 26 emerre T-ALL .SFPQ::ZFP36L2 fusion in a case assigned to the HOXA subgroup of T-ALL, by RNA expression studies. Interestingly, fusions of ZFP36L2 with different partners, including SFPQ, have been reported in pediatric and adult T-ALL without evidence of subtype-specific oncogenic rearrangements, as in our case [Chromothripsis also produced the our case , 27.ZFP36L2 (zinc finger protein 36-like 2) is a critical factor in hematopoiesis, where it has a role in maintaining self-renewal of early colony forming erythroid progenitors [ZFP36L2 include oncogenes and tumor suppressors, such as NOTCH1, EZH2, MYC, SPI.1, LMO2, LYL1, and TAL1. Interestingly, ZFP36L2 knock-out mice develop T-ALL [SFPQ::ZFP36L2 fusion in T-ALL, especially related to the HOXA subgroup.genitors , 28. In genitors , 30. Tarop T-ALL . OverallMYCN, defects of DNA repair/genome stability genes, and genetic abnormalities at NUP214 nuclear pore complex (NPC) gene.Our study identified three main factors involved in chromosome instability in cht+ T-ALL, in particular, upregulation of MYC/MYCN amplification/overexpression has been reported in mouse models [TP53 inactivation, and MYC/MYCN amplification/overexpression, co-occur in diverse human tumors harboring chromothriptic events [An association between complex genomic rearrangements/catastrophic events and DNA repair defects, inefficient apoptosis, and e models . Likewisc events .MYCN was one of the three most up-regulated genes distinguishing cth+ from cth− ETP/near-ETP ALL cases. MYCN, a negative regulator of NDRG1 (N-myc downstream-regulated gene 1), has a pivotal role in spindle checkpoint, microtubule functioning, and centrosome homeostasis [MYCN in controlling genome stability has been strengthened in transgenic zebrafish, where, mismatch repair, homologous recombination, and base excision repair pathways are typically abolished [MYCN are typically associated with an immature phenotype [MYCN expression and the occurrence of chromothripsis in human immature T-ALL.RNA microarrays showed that eostasis –33. The henotype  but alsoMYCN have been related to both gain/amplification and the L44P hot-spot variant [PRC2 complex, one of the major negative regulators of MYCN, were present in 66% of our cases which harbored SUZ12 deletions and/or EZH2 mutations. The abrogation of the PRC2 activity may explain the high levels of MYCN expression in cth+ ETP-ALL [EZH2 inactivation correlates with an immature signature, transcriptional up-regulation of MYCN, and an increased MYCN-driven replication stress [EZH2 targets.Elevated levels of  variant , 22, 35  ETP-ALL  as obsern stress . In agreMYCN overexpression was associated with abnormalities of DNA repair/genome stability genes in all our cth+ T-ALL cases, consistent with the hypothesis that DNA repair factors play a central role in the onset of chromothripsis [ATR, BLM, BRCA2, MSH3, ERCC4, ERCC6L2, FANCI, FANCM, POLE, POLH, PTEN, RFWD3, STK11, and/or XPC were detected in 7/12 cth+ cases, with ATR and BLM as the only recurrently involved genes. In contrast, variants of ATM and/or TP53, which are closely linked to chromothripsis in human cancers, were not found [RGCC, encoding for a protein which is induced by p53 in response to DNA damage [RGCC behaves as putative oncosuppressor in pediatric B-ALL, multiple myeloma, and other tumor types, where it was found to be epigenetically silenced [As reported in solid tumors , MYCN ovthripsis , 14. Losot found . InteresA damage , 39 was silenced , 41.NUP214, a component of the nuclear pore complex (NPC), which is known to play a pivotal role in preserving the genome integrity, were detected in 33% of cases [SET::NUP214 or SQSTM1::NUP214, as both fusion proteins localize in specific nuclear bodies and do not exert their shuttling activity [Lastly, alterations of of cases , 43. It of cases . This fuactivity .In conclusion, this study provided new insights to the understanding of the genomic complexity of immature T-ALL. In particular, chromothripsis emerged as a frequent mechanism generating major oncogenetic events in a subset of ETP/near-ETP of young adults. A wider case series needs to be studied to estimate whether chromothripsis has a negative clinical impact in T-ALL, as in solid tumors.Supplementary InformationSupplementary Tables


Gastric cancer (GC) ranks as one of the most life-threatening malignancies worldwide, and over one billion new cases and 783,000 deaths were reported last year. The incidence of GC is exceptionally high in Asian countries. Multiple oncogenic signaling pathways are aberrantly activated and implicated in gastric carcinogenesis, leading to the malignant phenotype acquisition. G-protein-coupled receptor (GPCR) signaling is one of them, and the aberrant activation of GPCRs and G proteins promotes GC progression. The activated GPCRs/G proteins might serve as useful biomarkers for early diagnosis, prognostic prediction, and even clinically therapeutic targets. This review summarized the recent research progress of GPCRs and highlighted their mechanisms in tumorigenesis, especially in GC initiation and progression.G-protein-coupled receptors (GPCRs) belong to a cell surface receptor superfamily responding to a wide range of external signals. The binding of extracellular ligands to GPCRs activates a heterotrimeric G protein and triggers the production of numerous secondary messengers, which transduce the extracellular signals into cellular responses. GPCR signaling is crucial and imperative for maintaining normal tissue homeostasis. High-throughput sequencing analyses revealed the occurrence of the genetic aberrations of GPCRs and G proteins in multiple malignancies. The altered GPCRs/G proteins serve as valuable biomarkers for early diagnosis, prognostic prediction, and pharmacological targets. Furthermore, the dysregulation of GPCR signaling contributes to tumor initiation and development. In this review, we have summarized the research progress of GPCRs and highlighted their mechanisms in gastric cancer (GC). The aberrant activation of GPCRs promotes GC cell proliferation and metastasis, remodels the tumor microenvironment, and boosts immune escape. Through deep investigation, novel therapeutic strategies for targeting GPCR activation have been developed, and the final aim is to eliminate GPCR-driven gastric carcinogenesis. Gastric cancer (GC) is a substantial global health burden, accounting for the fifth most commonly diagnosed cancer and the third leading cause of fatal malignancies worldwide. Incidence rates are markedly increased in Eastern Asia, especially in Mongolia, Japan, and Korea, which are strongly associated with various predisposing and etiological factors, according to several migrant studies ,2. Most Since G protein-coupled receptors (GPCRs) were reported in cellular transformation in 1986, emerging evidence shows that these membrane-embedded receptors regulate many biological processes and are crucial targets against several human malignancies . This reGPCRs comprise over 800 members accounting for about 4% of human genes. They have various structures and signal transduction. Based on their specific characteristics, GPCR members are further classified into different subgroups and participate in various physiological processes, whereas the aberrant expression and abnormal activation of GPCRs are associated with tumor progression.B), metabotropic glutamate receptors (mGluRs), and a calcium-sensing receptor (CasR). The frizzled/taste family (class F) includes frizzled and smoothened proteins that can be activated by the lipo-glycoproteins of the Wnt and Hedgehog families [GPCRs have seven transmembrane α-helices (TM1-7) that connect the N-terminal extracellular domain (ECD), three extracellular and intracellular loops: ECL1-3, ICL1-3, and the C-terminus . They arfamilies ,9,10.Thrilling technologies, such as X-ray crystallography, cryo-electron microscopy (cryo-EM) ,12, and By binding to various extracellular ligands, GPCRs change their conformation, activate G-coupling proteins, and couple with other proteins, such as β-arrestins and GPCR kinases (GRKs). However, the determinants of GPCR selective binding have not been fully understood. Fundamentally, the G-protein barcode determines the selectivity between GPCRs and G proteins, which contain variable residues on the conserved positions and can be recognized by different GPCRs . G prote2+ levels and activate protein kinase C (PKC) by converting phosphatidylinositol 4,5-biphosphate (PIP2) into inositol 1,4,5-triphosphate (PIP3) and diacylglycerol (DAG). Additionally, the Gβγ subunits trigger the PI3K-γ/PI3K-β to catalyze the conversion of PIP2 to PIP3, which is a direct stimulator of AKT. The Gα12/13 and Gαq family members regulate a group of Rho GEFs, which harbor an RGS homology domain and activate Rho GTPase. As shown in When the activated GPCRs bind with the heterotrimeric G proteins, the Gα subunits dissociate from Gβγ after the exchange of GDP with GTP on the Gα proteins. This process releases the heteromeric G proteins from the GPCRs and retains the plasma’s GTP-bound Gα and Gβγ subunits . One GPCβ-arrestins undergo conformational changes when recognizing the GRK-phosphorylated GPCRs. Then, they enhance the process of desensitization, internalization, and clathrin-mediated endocytosis of the activated GPCRs. As scaffold proteins, β-arrestins facilitate GPCR-stimulated signal transduction. As one of the most prominent and earliest examples, the GPCR-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation is a β-arrestin-dependent and G protein-independent signaling . The genGPCRs are sophisticated dynamic machines rather than static on-and-off switches. When they are engaged with different ligands, receptors, and regulatory partners, they may exhibit specific conformations and undergo subcellular distributions. Exploring the dynamic nature of GPCRs is vital to elucidate the mechanisms underlying allosteric modulation, biased agonism, oligomerization, and sustained and compartmentalized signaling. These mechanisms convey novel insights into drug discovery.1 muscarinic receptor in the CNS, which has been stopped because it fails to improve recognition and increases adverse effects like diarrhea [Allosteric ligand binding sites in GPCRs are potential new targets for modulating GPCR functions and improving drug selectivity. These modulators augment  or reduce  the affinity and efficacy of endogenous agonists . The disdiarrhea ,31.Another ligand-receptor dynamic is biased agonism, a mechanism in which the active conformational states of the receptors are stabilized by some ligands, resulting in distinct cellular signaling profiles . There aReceptor oligomerization conveys much more diversities in the function and physiological roles of GPCRs. However, unlike the oligomer tyrosine kinase receptors and ion channels, the formation of GPCR multimers remains controversial ,37. It hCompartmentalized signaling may partly explain why the GPCRs can activate a typical profile of secondary messengers and kinases. In addition to locating the membrane, the GPCRs might be desensitized and undergo β-arrestin-mediated endocytosis and intracellular signaling ,44,45. NBased on the recent pan-cancer analysis, GPCR signaling was among the 55 pathways most significantly mutated. Mutations and the aberrant expression of GPCRs and G proteins contribute to various diseases, including neurodegenerative, reproductive, immunological, and metabolic disorders, as well as cancers and infectious diseases ,50. The HCRT2, P2RY12, LPAR4, and GPR174, frequent mutations in the DRY motif may result in constitutive activation due to conformational changes in TM3, TM5, and TM6 [1B- and β2-adrenoceptors, which may account for lower agonist potency in transducing the downstream IP1 and cAMP signaling, respectively [GPCRs are mutated in over 20% of all sequenced samples ,56. Unli and TM6 . Mutatioectively . Understectively .GRM1-8, class C) [LPAR1-6), sphingosine-1-phosphate (S1P) receptors (S1PR1-5), and muscarinic receptors . However, most adhesion receptors are orphan receptors with unknown ligands and physiological functions [GPR98 and GRM3 were two of the most frequently mutated genes, with 27.5% and 16.3% mutation rates. GRM3 mutants selectively mediate the MEK signaling that contributes to tumor growth in melanoma, acting as an indicator for patient stratification and precision medicine [GPR98 (21.25%), TSHR (13.90%), and BAI3 (13.62%), while CELSR1 (11.20%), EDNRB (8.14%), and GPR45 (5.09%) account for the three most frequently mutated GPCRs in GC. However, the functional roles of these mutant GPCRs in GI cancers remain unknown.The large and ever-grossing body of sequencing by pan-cancer analysis suggests that the frequently mutated GPCR families are adhesion-related GPCRs, such as the glutamate metabotropic receptors (class C) , lysophounctions ,61. GPCRmedicine . MutSig2HRH2), exhibit significant copy number variations (CNVs) in tumors. Several broad-type GPCRs are universally overexpressed throughout the GI tract, regulating digestive and pathophysiological processes [Besides mutations, GPCRs, like chemokine and histamine receptors  database. GNAS aberrations widely occur in tumors originating from the pituitary (28%), pancreas (12%), thyroid (5%), colon (6%), and a few other locations [GNAS, indicating that the stabilization of the intramolecular hydrogen bond network (H-bond network) plays a pivotal role in mutation-mediated constitutive activation [GNAS are responsible for initiating and progressing multiple types of GI cancer, such as colon neoplasia, GC, and pancreatic adenocarcinomas (PDAs). In colon cancer, the GNAS can mediate the tumorigenesis of inflammatory factors by stimulating the Gs-Axin-β-Catenin pathway axis [GNAS mutations were tightly associated with deep submucosal invasion and increased tumor size by activating the Wnt/β-catenin pathway [GNAS mutations (~41–75%) suppressed the PKA-mediated SIK and reprogrammed lipid metabolism in the precursor of PDAs [As oncogenic drivers in multiple prevalent cancers, many G proteins are considered part of the cancer-associated gene panels routinely employed by a wide range of clinical oncology studies. MutSig2CV analysis indicates that ocations . Previouocations ,71 and Gocations , might bocations . The distivation . These away axis . In the  pathway . Besides of PDAs ,78.GNAQ and GNA11 mutations were less studied in tumors than GNAS, these mutations were well-established in Sturge-Weber syndrome [Q209L contributed to MAPK pathway activation [R183Q [Although syndrome  and leptsyndrome , and alssyndrome ,82. The tivation  and exhin [R183Q . The actn [R183Q . Furthern [R183Q , nasophan [R183Q , breast n [R183Q , squamoun [R183Q , and coln [R183Q . Interesn [R183Q . Large-sn [R183Q ,93.GNB1 and GNB2. The gain-of-function alterations of these proteins can disrupt the interactions of Gα-Gβγ and constitutively stimulate the downstream signaling effectors, conferring resistance to targeted kinase inhibitors [GNB2 Arg52Leu in familial cardiac arrhythmia condition, Gly77Arg in neurodevelopmental disorder, and monoallelic missense variants in developmental delay/intellectual disability (DD/ID) [The cDNA library screening distinguished the functionally relevant mutations of the Gβ proteins hibitors ,95. Rece (DD/ID) . Emergin (DD/ID) . For exa (DD/ID) .BCR-ABL fusion protein, ETV6-ABL1, JAKV617F, and BRAFV600K, to enhance the drug resistance of the corresponding kinase inhibitors [Some mutations occur in oncogenic kinase alterations, such as hibitors ,98. NeveGPCRs play hierarchical roles in many signaling networks. Dysregulations of the GPCRs extensively exist in tumor progression, metastasis, and immune response reprogramming. In recent years, aberrant GPCR members have been emerging in GC studies. This section will outline the updated findings of the GPCR signaling pathway in GC .Mounting evidence has unveiled the multilayered crosstalk between GPCRs and proliferation- and apoptosis-related signaling circuits. The representative ones involve EGFR transactivation, MAPK cascades, the PI3K-AKT-mTOR pathway, and the Hippo signaling pathway ,135.H. pylori boosted the expression of interleukin-8 (IL-8), which promoted cell proliferation by inducing EGFR transactivation [2-EP4 pathway induction, the ubiquitous overexpression of the EGFR ligands and Adams have been identified in mouse gastric tumors [2-induced uPAR expression has also been implicated in the activation of Src, c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (Erk), and p38 mitogen-activated protein kinase (p38 MAPK) [GPCRs share many similarities with the tyrosine kinase receptors, such as EGFR and the MAPK/ERK signaling pathways , in regutivation . Due to c tumors . PGE2-in38 MAPK) ,143. GPC38 MAPK) . Proteas38 MAPK) .PI3K is stimulated by the activated RTKs or GPCRs, ultimately leading to the synthesis of PIP3 and the recruitment of oncogenic effectors such as the serine/threonine kinase AKT. The PH domain in AKT permitted its binding with PIP3, contributing to the membrane accumulation and subsequent phosphorylation at T308 and S473 by PDK1 and mTORC2 . Even th12/13, Gαi/o, and Gαq/11 or suppress YAP/TAZ by binding with Gαs. However, which of these that Rho is involved with has not yet been identified; it is also unclear how the actin cytoskeleton regulates Lats1/2 phosphorylation. Emerging findings have revealed how specific GPCRs may fine-tune YAP/TAZ in given cellular surroundings [12/13 and stimulating YAP [GNAQ (encoding for Gαq) and GNA11 (encoding for Gα11), respectively [The canonical Hippo pathway kinase cascade is a critical tumor suppressor pathway, and its dysregulation has been widely implicated in organ size modulation and carcinogenesis . The coroundings . The trioundings ,152. Theting YAP , connectting YAP ,155, metting YAP ,157, andting YAP ,159. Theting YAP . The GPCting YAP . As suchectively . As a reBesides the above signaling circuits, other pathways have also been linked to GPCR-mediated oncogenicity in GC. These pathways involve the Notch pathway , hedgehoMetastasis is how cancer cells establish ‘bench-heads’ in other organs or anatomical sites instead of the initial lesion, and it is responsible for more than 90% of cancer-related mortality  Figure A. The moEpithelial-to-mesenchymal transition (EMT) is regarded as the initial step for invasion, featuring a loss of cell polarity and integrity and the acquisition of motile mesenchymal characteristics. The pathologic activation of the EMT program is primarily executed by transcription factors  and microRNAs, ultimately resulting in the accumulation of the genes associated with mesenchymal properties, such as vimentin, fibronectin, and N-cadherin . Though H. pylori infection, as shown in previous studies [Although chemokines are tiny polypeptides (8-14kDa), they display pleiotropic effects in cancers. The chemokine system comprises nearly 50 chemokines that bind to 20 different chemokine receptors or four atypical chemokine receptors (ACKRs) . This su studies ,182,183. studies ,185.Chemokine receptors have attracted considerable attention due to their involvement in GC metastasis. A notable correlation was found between CCR7 expression and gastric carcinoma lymph node metastasis via stepwise regression analysis ,187. Str2aR) or GPR30 could also be observed in GC [2R), S1P2R, muscarinic acetylcholine receptor 3 (M3R), P2Y receptors (P2YR), and γ-Aminobutyric acid receptor A (GABAA), thus contributing to the invasion and metastasis in GC [Many other GPCRs also govern the development of GC invasion. For example, GPER1 inhibition blocked EMT in GC cells by inhibiting the PI3K/AKT pathway . Similared in GC ,123. In is in GC ,121,128.is in GC .H. pylori was reported to be crucial during the invasion by upregulating cyclooxygenase-2 (COX-2) through ATF2/MAPK stimulation. The COX-2 inhibitor or EP2 receptor antagonist repressed angiogenesis and tumor invasion via the uPA system, which is a determinant factor in transforming the zymogen plasminogen into plasmin for degrading the ECM constituents [H. pylori’s consistent infection manipulated a variety of extracellular proteases [BM, a specialized extracellular matrix (ECM), plays a critical role in normal epithelium tissue architecture. BM disruption is a must for cancer cells leaving the primary location, controlled by the balance between the expression of MMPs and their tissue inhibitors (TIMPs) ,199. Thetituents . Furtherroteases , but theroteases .1R is the first reported GPCR in blood vessel formation. Furthermore, the intrauterine death of Edg1 ablation mice happened mainly due to abnormal angiogenesis [H. pylori-induced VEGF upregulation was activated through p38 MAPK COX2-PEG2-EP2/4 signaling [Angiogenesis is the process of vessel splitting from pre-existing vessels and is essential for tumorigenesis and progression, especially for those solid tumors exceeding 1–2 mm in diameter, as it provides oxygen and nutrients ,204. Manogenesis ,208. Theogenesis . Moreoveignaling . Other oignaling .TME acts as a unique niche populated by multiple cell types , ECM, and diverse secreted factors (such as exosomes and microRNAs) ,213. TheIt is noteworthy that chemokines and chemokine receptors can be ubiquitously expressed in tumor cells, immune cells, and stromal cells . AlternaOn the other hand, chemokine signaling is also involved in the formation of immune-suppressive TME, where tumors evolve to escape recognition and clearance. This process has been largely linked to the infiltration of diverse protumor immune cell populations, such as regulatory T (Treg) cells, the M2 macrophages, monocytic myeloid-derived suppressor cells (M-MDSCs), and granulocytic (or PMN-) MDSCs ,225. CCLPTGER1-4). PGs, especially the PGE2, were produced by the cyclo-oxygenases COX-1and COX-2, the inhibitors ) of which have been utilized to comfort pains and reduce the incidence of a broad range of cancer types. The role of PGE2 has been extensively studied for inducing inflammation by stimulating other signaling pathways, including the Toll-like receptor (TLR)/MyD88 pathway [H. pylori may suffer from severe gastritis due to lacking suppressing macrophage cytokine secretion and cellular infiltration [Other GPCRs are also involved in the regulations of immune responses. For example, prostaglandin (PG) production can mediate inflammation through its cognate GPCR EP1-EP4 , and 54% of GPCRs are orphans that are under-exploited. Machine learning approaches may be employed for predicting the interaction between immersed compounds and GPCRs based on established high-quality structural models . With thMore efficient drug delivery systems with enhanced solubility and stability, lower dosages, and less toxicity have been developed, such as nanomaterials, nanocarriers, nanoconjugation, and nanoencapsulation techniques ,252. FurGPCRs govern multiple signaling pathways and regulate GC development in various aspects. The heterogeneous and complicated characteristics of GPCRs contribute to GC heterogeneity and result in the current untimely diagnosis and inefficiency of therapeutic applications. Not only can GPCRs transduce the extracellular changes to the intracellular signaling circuits, but the conformational changes of GPCRs can also continuously influence intracellular events. Aberrant GPCR activation and mutated GPCRs/G proteins can fuel cancer cell proliferation, migration, invasion, angiogenesis, and metastasis. In addition, the dysregulation of GPCRs affords advantages for immunosuppressive TME and drug resistance to malignancies.Although much progress has been made on novel biomarker identification and molecular mechanism investigation, the current GPCR-based diagnosis and therapy in GC are far from clinically available. It is urgent that GPCR signaling-targeted therapy be developed. In future studies, several issues need to be addressed. First, as the mutation rates of GPCRs and G proteins are prominent in some cancer types, the development of small molecules that target the driver mutations is urgent. Second, because of the heterogeneity of the cancer cells and tumor microenvironment, we need to comprehensively appraise the activation of GPCR signaling and its crosstalk by using cutting-edge techniques such as scRNA-seq or scDNA-seq. Last but not least, more preclinical models based on patient-derived samples, such as organoids or xenografts, need to be developed to evaluate the efficacies and side effects of the screened drugs. With the deep investigation of the molecular mechanisms of GPCR signaling and the multicenter clinical trials, more therapeutic strategies will be delivered for targeting GPCR signaling, which will benefit GC patients.


Salmonella enterica serovar Enteritidis is a significant health threat. Salmonella enterica serovar Enteritidis SE3 was isolated from soil at the Subaé River in Santo Amaro, Brazil, a region contaminated with heavy metals and organic waste. Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing were used for de novo hybrid assembly of the Salmonella SE3 genome. This approach yielded 10 contigs with 99.98% identity with S. enterica serovar Enteritidis OLF-SE2-98984-6. Twelve Salmonella pathogenic islands, multiple virulence genes, multiple antimicrobial gene resistance genes, seven phage defense systems, seven prophages and a heavy metal resistance gene were encoded in the genome. Pangenome analysis of the S. enterica clade, including Salmonella SE3, revealed an open pangenome, with a core genome of 2137 genes. Our study showed the effectiveness of a hybrid sequence assembly approach for environmental Salmonella genome analysis using HiSeq and MinION data. This approach enabled the identification of key resistance and virulence genes, and these data are important to inform the control of Salmonella and heavy metal pollution in the Santo Amaro region of Brazil.In Brazil,  Salmonella spp., is a global threat to human health  using the NEBNext Fast DNA Fragmentation and Library Preparation Kit  following the manufacturer’s recommendations. The library quality was assessed using the Agilent 2100 Bioanalyzer equipment, and the paired-end DNA sequencing was carried out in the Illumina HiSeq 2500 platform. After sequencing, the raw read quality was assessed using the FastQC v0.11.5 software  were downloaded from the NCBI Genbank database in July 2022. Genomes with more than 500 contigs were removed, and contigs smaller than 500 bp were removed from the remaining genomes. Genome quality was evaluated with CheckM v.1.0.13 (52.17%) ,54. HoweSalmonella SE3 genome revealed it was located within the properly classified cluster of S. enterica. During taxon analysis we identified 159 genomes with incorrect taxonomic classification, highlighting that it is important to confirm identity prior to undertaking phylogenetic analyses. Phylogenetic analysis of the Salmonella SE3, revealed the core genome was composed of 2137 genes and the accessory genome comprised 3390 shell genes and 69,352 cloud genes. This indicates Salmonella SE3 has an open pangenome with a diversity of unique genes. A study by Chand et al. [S. enterica, and concluded that the genus Salmonella displays an open pangenome, comprising a reservoir of 10,775 gene families. Of these 2847 constituted the core gene families, 4657 were dispensable or accessory gene families, and 3271 strain-specific gene families. Park et al. [Salmonella enterica subsp. enterica was open. However, it is important to note that pangenome size is heavily influenced by the properties of the genomes used and variation would likely result in inconsistencies, and secondly, newly described genes are often included which results in open pangenomes [The pangenome analysis of d et al.  undertook et al.  construcSalmonella SE3 identified genes potentially involved in resistance to aminoglycosides, fluoroquinolones, macrolides, a monobactam (golS), nitroimidazole (msbA), tetracycline and related drugs (mdfA), and cephalosporins. Other studies of Salmonella isolates from southern Brazil have also reported tetracycline (mdfA) and aminoglycoside (aac(6’)-Iaa) resistance genes, in addition to other genes such as aac(3)-Iva, aph(3”)-Ib, aph(4)-Ia, aph(6)-Id, tet(34) and tet(A) [S. enterica have been described [aadA, aadB, aacC, aphA, strAB), β-lactams , chloramphenicol , inhibitors of the folate pathway , and tetracycline , none of these resistance genes were detected in our study. The antimicrobial resistance gene profile of d tet(A) ,59,60,61escribed , such asSalmonella pathogenic islands were identified in Salmonella SE3 which is relatively high compared that reported for other Salmonella isolates. A S. enterica serovar Typhimurium isolate, ms202, from a patient in India possessed six Salmonella pathogenicity islands: SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, and SPI-11 [Salmonella Choleraesuis strains provided by the Brazilian Salmonella reference laboratory of the Oswaldo Cruz Foundation (FIOCRUZ-RJ), which receives Salmonella isolates from diverse isolation sources and regions of the country. Pathogenicity islands SPI-1, -2, -3, -4, -5, -9, -13, -14 and CS54 island were detected in five strains and SPI-11 in four strains. The majority of these SPI, with the exception of SPI 4 and SPI 11, were also detected in Salmonella SE3. SPI-1 and SPI-2 are known to be involved in the invasion of intestinal epithelial cells and survival and replication within phagocytic cells, respectively, through the formation of type 3 secretion systems, SPI-5 is associated with fluid secretion and inflammatory response and SPI-3, -4, -11, -13, -14 and CS54 are associated with Salmonella survival and adaptation to stresses within macrophages [Ten d SPI-11 , but in d SPI-11  analyzedrophages .Salmonella SE3. Some of these virulence genes are also found in other serovars of Salmonella. Borah et al. [Salmonella isolates recovered from humans and different species of animals. Among the 88 isolates, some virulence genes such invA, sipA, sipB and sipC were detected irrespective of the serovar, and these were also detected in Salmonella SE3. fepA was also present in a high percentage (64.7%) of isolates belonging to Salmonella serovars Enteritidis, Weltervreden, Typhi, Newport, Litchfield, Idikan and Typhimurium, as well as Salmonella SE3 and. Other virulence genes were present in varying percentages among the Salmonella serovars studied by Borah et al. [sopB (86.36%), sopE2 (62.5%), pefA (79.54%) and sefC (51.14%); of these genes only sefC was not detected in Salmonella SE3. The virulence genes identified in Salmonella SE3 are involved in several different processes, such as the invA gene usually codes for a protein in the inner bacterial membrane that is responsible for the invasion of intestinal cells of the host [fepA gene encodes outer membrane receptor protein FepA, which participates in iron transport and plays a role in infection colonization in Salmonella [Salmonella SE3 chromosome. Upon secretion the SipB, SipC, and SipD proteins are thought to form a complex in the eukaryotic membrane that is required for translocation of the remaining effectors into the host cell cytoplasm [Salmonella SE3 and is the plasmid-encoded fimbrial major subunit antigen of Salmonella Typhimurium [Salmonella plasmid-encoded fimbrae have been found to mediate adhesion to mouse intestinal epithelium [In total, 144 potential virulence genes were identified in h et al.  investigh et al.  such as the host ,68. The lmonella . T3SS-1 ytoplasm . PefA ishimurium . Salmoneithelium .arsC, encoding arsenate reductase, was found in the genome of Salmonella SE3. Arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite which is extruded from the cell [Salmonella SE3 was isolated from the soil of Subaé River where heavy metal concentrations were above reference values [Mytella charruana) gathered from the same region also contained lead, arsenic and cadmium in concentrations above reference values [The gene the cell ,73. Thise values . In addie values . Carvalhe values  also detSalmonella SE3, including CRISPR-Cas type IE, CBASS type I, and RM type I and III systems. Similar antiviral systems and subtypes were identified by the PADLOC and DefenseFinder tools, except for AbiU and RM type II which were only identified by PADLOC. Most bacteria, including Salmonella, possess multiple antiviral defence systems that protect against infection by phages and mobile genetic elements [Several virus defence systems were detected in elements .Salmonella SE3 genome, two were intact, and five were incomplete. By comparison, in S. enterica Typhimurium ms202 nine prophages were detected, two were intact, five were incomplete and two were questionable [Salmonella SE3 had not only Salmonella prophage sequences (e.g. phage RE-2010) but also prophages annotated as belonging to closely related genera Shigella (phage POCJ13) and Escherichia (phage 500465-2), which may indicate horizontal gene transfer or polyvalent phages. A previous study has reported that phage populations in S. enterica contribute to horizontal gene transfer, including virulence and virulence-related genes within the subspecies [Salmonella phages may uncover the receptor-interaction mechanisms between phages and hosts which may lead to improving phage therapy as an option for the treatment or control of Salmonella.Seven prophages were detected in the tionable . Moreovebspecies ,77,78,79Salmonella isolates could be a key determinant for better control of salmonellosis. Our study showed the effectiveness of a hybrid sequence assembly approach for environmental Salmonella genome analysis using HiSeq and MinION data. Salmonella SE3 was determined to belong to a monophyletic clade containing 23 S. enterica strains of serovar Enteritidis. The hybrid genome assembly enabled mobile genetic elements, genomic islands, Salmonella Pathogenicity Islands, antiviral systems, antimicrobial resistance genes, virulence genes, and prophages to be identified in Salmonella SE3. Furthermore, a gene encoding heavy metal resistance, arsC, was detected. These data are important to inform the control of Salmonella and heavy metal pollution in the Santo Amaro region of Brazil. Salmonellosis is a healthcare issue around the world, so genomic analysis of


This study illustrated the liver protection mechanism of ACP from the perspective of autophagy activation. ACP suppressed the inflammatory injury of KCs, and decreased the cell apoptosis rate. After LTG and LC3 staining, ACP promoted lysosomal production, increased LC3 expression, activated autophagy, and suppressed the expression of NLRP3 and inflammatory factors. Under the electron microscope, ACP accelerated the production of autophagosomes. After simultaneous treatment with 3-MA and ACP, the effect of ACP on resisting KC injury decreased, the expression of NLRP3 was up-regulated, and autophagy was suppressed. As discovered in the mouse model of liver injury, ACP inhibited the ALT and AST levels, promoted the occurrence of autophagy, reduced NLRP3 expression and alleviated liver injury.ACP activates autophagy to induce NLRP3 degradation, thus suppressing inflammatory response in liver injury and exerting the liver protection effect, which is one of the mechanisms of action of ACP. At present, the incidence of liver injury shows an increasing trend year by year, and it involves several pathological types, like drug-induced injury, alcoholic liver injury and immune injury commonly seen in clinic . InflammAutophagy is a lysosome activation-dependent degradation pathway, which is important for regulating protein and nucleic acid degradation. It is found in liver injury that, the autophagy level was up-regulated in acute phase, which exerts the protective effect , 7, but 2 conditions. After cell passage, the cell viability was tested by trypan blue reagent, and KCs at logarithmic phase were harvested for experiments. KCs were then divided into DMSO (control), LPS and ACP groups. Cells in LPS group were treated with 1 mg/L LPS to induce inflammatory response, while those in ACP group were pretreated with high-dose (15 mg/L) and low-dose (5 mg/L) ACP for 6 h, and then exposed to LPS to induce inflammatory response.The mouse KCs  were cultured in the RMPI 1640 complete medium supplemented with 10% FBS in the incubator under 37° C and 5% COIn the assay to verify the mechanism of ACP through activating autophagy, KCs were divided into DMSO, LPS, ACP and 3-MA+ACP groups. Cells in ACP group were treated with 15 mg/L ACP, those in 3-MA+ACP group were pretreated with the autophagy inhibitor 3-MA (25 μM) to suppress autophagy and intervened with 15 mg/L ACP for 6 h before experiments.Cells were inoculated into the 96-well plates, the serum-free medium was changed after cell adherence, and LPS was then added to induce for 12 h. Later, each well was added with 10 μl CCK-8 solution to incubate for 4 h, and the absorbance (OD) value was detected at 450 nm. Meanwhile, the blank medium was set for background absorption correction.Cells were inoculated into the 6-well plates and grouped after adherence according to the aforementioned method. After LPS induction for 12 h, the adherent and suspension cells were collected, washed with pre-chilled PBS twice, and centrifuged at 3000 rpm/min for 30 min. After suspension with Binding Buffer, cells were stained with 5 μl Annexin V-FITC for 5 min in dark and then with 5 μl PI in dark for another 5 min. After washing with PBS, cells were loaded for test using the cell apoptosis detection kit .The levels of inflammatory factors IL-1β, IL-18 and TNF-α in the medium were determined in cell assays. In brief, cells were inoculated into the 12-well plates and cultured with serum-free medium after adherence. After LPS induction for 12 h, the cell medium was collected. Afterwards, the suspension cells and cell debris were removed, and the supernatant was collected to detect the inflammatory factor levels by the standard curve method in line with the ELISA kit  instructions. The results were expressed as ng/ml.Detection of inflammatory factors in tissues and serum. The peripheral blood samples were centrifuged to collect the upper layer serum, meanwhile, liver tissues were grinded with liquid nitrogen, lysed with 1.0 ml NP-40 lysate on ice for 30 min and the supernatant was collected for protein quantification according to the methods in cell experiments.The levels of LC3 and NLRP3 in cells were detected by IF staining. Briefly, the sterile slides were added into the 6-well plates. After LPS treatment, KCs were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.2% Triton X-100, blocked with 2% BSA, and incubated with monoclonal antibodies against LC3 and NLRP3  under room temperature. Later, cells were labeled and incubated with the fluorescence-labeled IgG antibody . In NLRP3 staining, the cell nuclei were stained with DAPI. After washing twice with PBS, DAPI was added and cells were observed under the fluorescence microscope.After adherence, KCs were treated with LPS for 6 h, incubated with 200 μl lysosome-labeled probe  for 30 min, and washed with fresh medium thrice. Then, PBS was added dropwise to cover the cells, which showed green fluorescence after lysosome staining under the fluorescence microscope.After adherence, KCs were treated with LPS for 6 h, fixed with 2.5% glutaraldehyde, dehydrated with ethanol, and embedded with Epon812 epoxy. Afterwards, the sample was sliced into ultra-thin sections using the LKB III type ultra microtome. Then, sections were double-stained with uranyl acetate and lead citrate, and the changes in ultrastructure were observed with the EM420 transmission electron microscope.KCs were collected after LPS induction for 12 h. washed and lysed with 1.0 ml NP-40 lysate  for 30 min on ice. Then, the mixture was diluted with 5x loading buffer to 20 μl, and boiled to denature the proteins. Afterwards, proteins were separated by SDS-PAGE, and transferred onto the PVDF membranes. Membranes were then blocked with 5% skimmed milk powder, then incubated with TBST-diluted monoclonal antibodies against LC3, P6 and NLRP3 overnight at 4° C, and later with HRP-IgG . Finally, the sections were detected by chemiluminiscence, the OD was analyzed using Image Pro-Plus 6.0 software, and the results were expressed as the OD ratio of target protein to GAPDH.To detect proteins in tissues, proteins were extracted and detected according to the methods in ELISA.The SPF C57BL/6 mice were randomly divided into Control, D-GalN/LPS (D/L) and ACP groups. Mice in ACP group were given intragastric administration of ACP at 5 mg/kg and 15 mg/kg once a day for 7 consecutive days. While mice in Control and D/L groups were given intragastric administration of equivalent volume of normal saline once a day. At 24 h after the last administration, mice in D/L and ACP groups were intraperitoneally injected with 1000 mg/kg D-GalN  and 10 μg/kg LPS  to construct the acute liver injury model.The ALT and AST expression was detected using corresponding kits. In brief, after LPS/D-GalN intervention for 48 h, the tail venous blood was collected from each mouse, and centrifuged to collect the supernatant to detect ALT and AST levels by ultraviolet colorimetry  according to the kit instructions. The AST and ALT results were expressed as U/L.At 48 h after LPS/D-GalN injection, mice were sacrificed through carbon dioxide suffocation. Then, the liver tissues were dissected, embedded in paraffin and prepared into the 4 μm consecutive sections. The detailed steps were shown below, xylene deparaffinage, gradient dehydration with 100%, 95% and 80% ethanol, washing with tap water for 2 min, staining with hematoxylin for 3 min, washing with tap water for 2 min, treatment with 1% hydrochloric acid alcohol for 2 s, washing by tap water for 2 min, and treatment with 1% ammonia water for 20 s, followed by 10 s staining with 0.5% eosin ethanol, gradient dehydration of ethanol, xylene transparentizing, and neutral balsam mounting. Finally, the liver tissue pathological changes were observed under the light microscope.To detect NLRP3 expression in tissues, the paraffin-embedded liver tissue block was sliced into 4 μm serial sections, deparaffinized in xylene, and treated with gradient ethanol, followed by antigen retrieval. The endogenous peroxidase was eliminated by 3% hydrogen peroxide. Later, sections were blocked with 2% bovine serum albumin (BSA) at 37° C. After non-specific antigen-antibody binding, sections were incubated with anti-NLRP3 monoclonal antibody  and subsequently with peroxidase-labeled streptomycin  for 15 min. Afterwards, each section was added with drops of freshly prepared DAB color developing solution , sufficiently washed with tap water, countered-stained with hematoxylin, and mounted.Statistical analysis was completed using SPSS19.0 software. The measurement data were expressed as mean± standard deviation The data and material were availability.LPS induced KC injury, and the cell apoptosis rate significantly increased, higher than that of DMSO group. While ACP suppressed cell apoptosis in a dose-dependent manner, and the cell apoptosis rate in ACP group was significantly lower than that in LPS group , 1B. CelIF staining results also indicated that NLRP3 was up-regulated in LPS group, and the difference was significant compared with DMSO group, while NLRP3 expression was down-regulated in ACP in a dose-dependent manner . In inflAutophagy was suppressed by the autophagy inhibitor 3-MA. The results indicated that the effect of high-dose ACP was antagonized. FCM results indicated that the cell apoptosis rate in 3-MA+ACP group significantly increased, higher than that of ACP group, which suggested that suppressing autophagy antagonized the effect of ACP , 3B. MorAccording to the results of NLRP3 staining, 3-MA antagonized the effect of ACP, NLRP3 expression was significantly up-regulated in 3-MA+ACP group, higher than that of ACP group . InflammD-GalN and LPS induced acute liver injury in mice. After H&E staining, mice in D/L group exhibited prominent inflammatory response and tissue bubble-like lesions, with obvious liver injury. ACP alleviated tissue inflammatory response and injury, and the difference was significant compared with D/L group . NLRP3 eAccording to the detection results of serum inflammatory factors, the serum expression levels of inflammatory factors increased in D/L group, which were significantly higher than those in Control group. While ACP reduced serum levels of inflammatory factors , 6B. Thein-vitro experiment, after LPS stimulation, NLRP3 expression significantly increases in HSCs and hepatocytes, along with higher levels of IL-1β, IL-18 and TNF-α [Inflammasome is a kind of multi-protein complex located in cells, which is involved in the inflammation and immune responses in human body . NLRP3 ind TNF-α . MoreoveAutophagy also exerts an important role in regulating the inflammatory response process. Numerous experimental studies have discovered that there is negative regulatory relation between autophagy and inflammasome . As founCollectively, results in this work suggest that ACP activates autophagy and induces NLRP3 degradation in KCs to alleviate inflammatory response and suppress inflammatory factor release. ACP activates autophagy and reduces NLRP3 expression in liver injury to exert its liver protective effect, which is one of the mechanisms of action of ACP.


Mitragyna speciosa (MSE) against diabetes-induced anxiety and depression-like behaviors were examined. In addition, UPLC/ESI/TOF-MS analysis was performed to identify the phytochemical nature of MSE. DM was induced using a combination of high fructose/streptozotocin, and the diabetic rats were treated with MSE (50 and 200 mg/kg) for 5 weeks. After treatment, the animals were subjected to a forced swim test, open field test and elevated plus-maze tests. Additionally, proinflammatory cytokines and oxidative stress parameters were evaluated in the brain tissues of the rats. UPLC/ESI/TOF-MS analysis revealed that MSE is abundantly rich in polyphenolic constituents, notably flavonoid and phenolic glycosides. Behavioral tests and biochemical analyses indicated that diabetic rats showed significantly increased anxiety and depressive-like behavioral deficits, brain oxidative stress and pro-inflammatory cytokines levels . Treatment with MSE (50 and 200 mg/kg) significantly attenuated increased blood glucose level, depressive and anxiety-like behaviors in diabetic rats. Additionally, the antioxidant enzymes activities were markedly increased in MSE-treated animals, while TNF-α, IL-1β and IL-6 cytokines were notably suppressed. Taken together, these results suggested that MSE has potentials as antidepressant and anxiolytic-like effects and improves the brain oxido-inflammatory status in diabetic rats.In this study, the attenuative effects of the hydro-alcoholic extract from  Diabetes mellitus is a chronic metabolic disease which has evolved over the years as a major global public health issue. Typically, diabetes mellitus is principally depicted by excessive blood glucose concentration (hyperglycemia), together with alterations in lipid, carbohydrate and protein metabolism ,2. Out oOne of the most prevailing diabetes associated comorbidity is brain disorders and psychological problems including depression, anxiety, cognitive dysfunctions and Alzheimer’s disease. In fact, it has been reported that up to 30% of DM patients are suffering from depression ,6,7. In While significant progress has been made in the development of synthetic antidiabetic drugs, unfortunately, adverse reactions as well as the inability of these drugs to impede or slow down the development of DM associated comorbidity have necessitated the search for effective antidiabetic alternatives. Medicinal plants have been enormously explored for their health promoting roles and low toxicity especially in the treatment of diabetes and its associated complications ,8,12. NuMitragyna speciosa Korth. (kratom) is an indigenous Southeast Asian plant belonging to the family Rubiaceae [M. speciosa is used in folk medicine for treating cough, fever, diabetes, pain, diarrhea, cancer, wound, fatigue, hypertension and as a substitute for opium withdrawal [M. speciosa, accumulating evidences have illustrated the antioxidant, anti-inflammatory, antiobesity, antinociceptive and cytotoxic activities of M. speciosa [M. speciosa extracts have been shown to show excellent anxiolytic and anti-depressant effects in mice using two behavioral experiments (forced swim and the tail suspension tests) [M. speciosa, several other polyphenolic constituents, such as isoquercitrin, kaempferol, caffeic acid, chlorogenic acid, rutin, apigenin, quercetin, apigenin-7-glycosides, hyperoside, quercetin-3-galactoside-7-rhamnoside, kaempferol-3-glucoside and epicatecin, have also been reported [M. speciosa, there is lack of scientific evidence relating to its antidiabetic therapeutic value as well as its efficacy against diabetes-induced comorbidity. Hence, this study investigated the effects of M. speciosa on anxiety and depression-like behaviors, oxidative and inflammatory status in the brain of fructose/streptozotocin-induced diabetic rats.ubiaceae ,15. Histthdrawal ,15,16,17speciosa ,19,20. Fn tests) ,21. Asidreported ,18,22,23p-coumaroylglucoside), cynaroside A, Kaempferol 3,7,4′-triglucoside, 6-methoxykaempferol 3,7-bis(3-acetylrhamnoside), tiliroside, neoliquiritin 2″-apioside, among several others; phenolic and phenolic glycosides, viz., isoferuloyl C1-glucuronide, protocatechuic acid-3-glucoside, leonuriside A, dihydroferulic acid 4-O-glucuronide, glucocaffeic acid and 5-caffeoylquinic acid; iridoid glucosides, viz., mussaenosidic acid, secoxyloganin, shanzhiside methyl ester, theveside, geniposide and swertiapunimarins; triterpene saponins, viz., cynarasaponin F, licoricesaponin B2, tarasaponin, mabioside C and trachelosperoside A1. In addition, alkaloids including mitragynine, lycoricidinol, 10-hydroxystrictosamide, isomitraphyllic acid (16→1)-β-d-glucopyranosyl ester, reserpiline and Echitovenine; stilbenes (Z)-resveratrol 3,4′-diglucoside and (Z)-resveratrol 3-(3″-sulfoglucoside); anthocyanins, viz., malvidin 3-(6″-acetylglucoside)-5-glucoside, cyanidin 3-(2″-glucuronosylglucoside), malvidin 3-glucoside-5- and cyanidin 3-(2-glucosyl-6-caffeoylglucoside) were also tentatively identified in MSE. Other compounds, such as acridones, purine nucleosides, hydroxycinnamic acid, catechin and coumarin acids, were also revealed in MSE  was applied for secondary metabolites profiling of MSE. As portrayed in d in MSE .p < 0.05; p < 0.05; The results showed that STZ injection leads to hyperglycemia as demonstrated by significant increase in blood glucose concentration of the DMG, MSE-L and DMSE groups compared to the CG (p < 0.05). The changes in the initial to the final body weight were evaluated. As shown in p < 0.05). However, this effect was markedly decreased in the MSE treated groups compared to the DMG group (p < 0.05). Additionally, the results also revealed significant differences in the swimming time of the DMG compared to the CG group. DMG rats spent less time swimming compared to CG rats . MSE induced a significant increase in the time spent in open arms as well as entries into the open arms . The activities of brain CAT and SOD in the DMG group were obviously lower than the CG group . MSE significantly alleviated these proinflammatory cytokine levels when compared to the DMG group. Administration of MSE markedly reduced IL-6, IL-1β and TNF-α compared to the DMG group (p < 0.05). The levels of IL-6, IL-1β and TNF-α were increased significantly in the brain of the DMG group compared with the CG group , decrease in entries and time spent in the open arms (EPMT) as well as reduction in the number of crossing and rearing (OFT), which were in corroboration with previous studies on DM-induced anxiety and depression ,26,31,32Indeed, hyperglycemia has been widely reported to promote oxidative stress, which initiates the onset, progression and deterioration of depression ,35. BraiIn addition to oxidative stress, numerous studies have reported that neuroinflammation plays a vital role in diabetes-induced depressive and anxiety owing to increased levels of proinflammatory cytokines. DM is associated with neuroinflammation, which may lead to damages or even death of brain neurons. Jawale et al. reported that DM increased the levels of TNF-α in the brain, which mediated anxiety and depressive-like behavior ,41. InflM. speciosa, indole alkaloids have been extensively identified as one of the major taxonomic markers of the plant and these compounds have been widely believed to be responsible for the effect of the plant on the brain, pain perception and other activities [M. speciosa, which strengthens the notion that several other bioactive constituents besides indole alkaloids seems to contribute to bioactive properties of the plant [The bioactivity of any natural product especially medicinal plant is largely dependent on the nature and composition of the compounds present in such plant. The existence of polyphenolic compounds in several plant extracts have been reported to be largely responsible for numerous biological activities, notably antioxidant, antidiabetic, antidepressant and anti-inflammatory properties ,42,43. Itivities . Howeverhe plant ,23. The he plant ,46,47. She plant . In addihe plant ,50. In ahe plant ,52. Takehe plant .w/v:1:10) in a shaker for 24 h. The extract was filtered, and the leaves mass was subjected to two other rounds of extraction using the same condition as earlier stated. The combined extract was evaporated using a rotary evaporator to approximately 30% of the initial volume. The remaining extract solution was refrigerated at 4 °C overnight. Thereafter, the solution was decanted, centrifuged and lyophilized to obtain a hydroscopic brown colored powder (MSE), which was stored at 4 °C until use.Fresh kratom leaves were purchased from a local garden at Nakhon Si Thammarat Province, Thailand. The plant specimen collected was authenticated at the Faculty of Traditional Thai Medicine, Prince of Songkla University, Hat Yai, Thailand and the specimen was preserved at the herbarium of the faculty . The dried and powdered kratom leaves were extracted using 80% methanol  rats (140 ± 20 g) were raised under standard conditions . The experimental protocol adhered to the instructions stipulated by Guide for the Care and Use of Laboratory Animals, National Institutes of Health, and approved by the Animal Ethics Committee of The Second Wuhu Second Peoples Hospital . All the rats were acclimatized for one week on normal rat chow and water ad libitum. After a week of adaptation, the rats were randomly divided into four treatment groups (6 rats/group) as follows: control group (CG): treated with normal saline; diabetic model group (DMG): treated with normal saline; diabetic + MSE-L (DMSE-L): diabetic rats treated with 50 mg/kg MSE; diabetic + MSE-H (DMSE-H): diabetic rats treated with 200 mg/kg MSE. Firstly, the diabetic groups  were given 30% fructose solution ad libitum in lieu of normal tap water for four weeks to induce insulin resistance, while the CG group received normal tap water during the same period. Thereafter, DM was induced in overnight-fasted fructose-fed rat groups by intraperitoneal injection of streptozotocin  solubilized in sodium citrate buffer solution (pH 4.5), while the CG rats were injected with the same volume of sodium citrate buffer. The STZ injected rats were give 5% glucose solution for 24 h in other to avoid possible hypoglycemic state due to STZ injection. The fasting blood glucose (FBG) concentration of all the animals was measured after 3 days of STZ administration with a portable Accu check guide glucometer  and rats with blood glucose concentration >13.88 mmol/L (250 mg/dL) were adjudged as diabetic. MSE was administered to DMSE-L and DMSE-H at 50 and 200 mg/kg, respectively, via oral gavage, while normal saline was administered to the CG and DMG groups daily for five weeks. The dose selection of MSE was based on previous studies ,54,55,56The test was performed using a rectangular wooden open field with dimensions 40 × 50 × 65 cm, and the floor of the field was divided into six concentric units. The rats were placed in the middle of the field. The rats’ ambulation including the total number of crossings with all four paws and the number of rearing was recorded for 5 min. A trained observer blinded to the treatment protocol evaluated the total number of cross and rearing of each animal.The EPMT was made up of two open and two closed arms with (50 × 10 cm) perpendicularly arranged and linked by a central sheath 50 cm high from the floor. Each rat was gradually positioned in the central square of the apparatus, facing the open arms and allowed to explore for 300 s. The time spent and number of entries into the open arms was measured during a 5 min period. In other to eliminate any olfactory cues, the apparatus was cleaned with 70% alcohol between each examination.The forced swimming test apparatus consisted of a cylindrical shaped glass container (40 × 30 cm) filled with water (23 ± 2 °C) to a depth of 30 cm. The rats were initially forced to swim for 15 min. After 24 h, the rats were subjected to the same procedure for 5 min, during the period the immobility and swimming time were manually recorded. Immobility time was taken as the period where no activity was initiated by the rats except for movements that kept the rat’s head above water. A trained observer blinded to the treatment protocol evaluated the swimming and immobility time of each animal. The experimental timeline is shown in The rats were fasted for 12 h and humanely sacrificed by euthanizing with sodium thiopental (150 mg/kg). The brain tissues were dissected from the rats, cleaned with physiological saline solution and instantly fixed in 10% neutral buffered formalin solution for further histopathological analysis. Another portion of the brain tissues were preserved at −70 °C for further analysis.g for 30 min at 4 °C), the supernatant collected was used for evaluating glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) levels using assay kits from Jiancheng Biotechnology, Nanjing, China. Likewise, proinflammatory cytokine levels; interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) were measured using ELISA kits obtained from Abcam, Cambridge, UK, following the manufacturers procedures.The isolated whole brain tissues were homogenized using potassium phosphate buffer (pH 6.5). After centrifugation  and normal distribution of the data was checked by the Kolmogorov–Smirnov test. Data displayed mean ± standard deviation and were analyzed by one way ANOVA followed by Tukey’s post-hoc test. The findings from this study demonstrated that the administration of MSE attenuated depressive and anxiety-like behaviors induced by fructose/STZ diabetic rats. The alleviative effects of MSE were mediated through the reduction of oxidative stress and neuroinflammation  in the brain of the treated diabetic rats. Furthermore, LC-ESI-MS analysis of MSE revealed the overwhelming majority polyphenolic compounds, including phenolic, flavonoids, terpenoids, iridoids and their glycosidic bound forms. Overall, the results highlighted the potential biological effects of MSE in the treatment of diabetes and its associated comorbidity.


FtNAC70, FtPUL, and FtGBSS1-3 in the lightcyan module and FtbHLH5, C3H, FtBE2, FtISA3, FtSS3-5, and FtSS1 in the brown. All the above genes were preferentially expressed in seeds, further suggesting their role in seed starch biosynthesis. These results provide crucial guidance for further research on starch biosynthesis and its regulatory network in Tartary buckwheat.Starch is a major component of crop grains, and its content affects food quality and taste. Tartary buckwheat is a traditional pseudo-cereal used in food as well as medicine. Starch content, granule morphology, and physicochemical properties have been extensively studied in Tartary buckwheat. However, the complex regulatory network related to its starch biosynthesis needs to be elucidated. Here, we performed RNA-seq analyses using seven Tartary buckwheat varieties differing in starch content and combined the RNA-seq data with starch content by weighted correlation network analysis (WGCNA). As a result, 10,873 differentially expressed genes (DEGs) were identified and were functionally clustered to six hierarchical clusters. Fifteen starch biosynthesis genes had higher expression level in seeds. Four trait-specific modules and 3131 hub genes were identified by WGCNA, with the lightcyan and brown modules positively correlated with starch-related traits. Furthermore, two potential gene regulatory networks were proposed, including the co-expression of  Fagopyrum tataricum, also called Tartary buckwheat, belongs to the Fagopyrum genus of Polygonaceae Mill and is a type of traditional pseudo-cereal for both medicine and food. Tartary buckwheat is famous for its high nutritional value, i.e., balanced amino acids, vitamins, minerals, and flavonoids  [ercetin) ,2,3. Theercetin) .F. esculentum), wheat (Triticum aestivum) and barley (Hordeum vulgare), with 19.3–20.2% iodine affinity, 1.36–1.48 blue values, and 645–657 nm maximum absorption wavelength [Starch, comprised of amylose and amylopectin, is the major component of Tartary buckwheat seeds, accounting for the highest proportion of their dry weight . Starch velength . The stavelength . Meanwhivelength .In endosperm, starch biosynthesis is a complex process controlled by the synergistic action of five starch synthetases, namely ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE) ,15. AGPaOryza sativa), maize (Zea mays), wheat, and barley [ZmNAC128 and ZmNAC130, which can bind to the prompters of Brittle 2 (BT2), Zpu1 (encoding DBE), GBSSI, Sh2 (encoding AGPL), SSV, ISA2, and SSIIa, and thus positively affect starch synthesis in the endosperm of maize [Genes encoding the starch biosynthesis enzymes have been massively characterized and functionally analyzed in cereals, such as rice , has been isolated [In Tartary buckwheat, starch-related research has focused on the starch content, granule morphology, and physicochemical properties ,11,12,13isolated . Meanwhiisolated . These wAmylose and amylopectin contents were measured in the matured seeds of 20 Tartary buckwheat varieties (lines), which presented obvious characteristics in their seed size, morphology, shell color, and shell thickness. The results showed that the amylose content ranged from 17.61% to 21.00%, with an average of 19.24%. The amylopectin content ranged from 19.67% to 28.49%, with an average of 23.63%. The ratio of amylopectin/amylose ranged from 1.05 to 1.43, with an average of 1.23 . We subsTo discover the global changes in gene expression level, high-throughput RNA-seq was carried out for late-filling-stage seeds of seven selected varieties , with three biological replicates for each variety. In each sample, 40,261,724–102,065,872 clean reads and 6.04–15.31 G clean bases were obtained. Q30 ranged from 88.86% to 89.77% and GC content ranged from 47.64% to 50.03% . The cleUsing PK1 as a control, 10,873 DEGs were identified, among which 9573, 3490, 1655, 977, 909, and 906 were identified in the comparisons of M11_vs._PK1, JQ2_vs._PK1, DMQ_vs._PK1, BK2_vs._PK1, QK2_vs._PK1, and XMQ_vs._PK1, respectively a. Of theThe metabolic pathways that the DEGs were related to were analyzed based on KEGG annotation. Among the top 20 enriched pathways in the six comparisons, four metabolic pathways, namely “starch and sucrose metabolism”, “biosynthesis of secondary metabolites”, “circadian rhythm—plant”, and “metabolic pathways” were enriched in all six comparisons . InteresMeanwhile, the identified DEGs in each comparison were annotated to a KOG category. As a result, “carbohydrate transport and metabolism”, “secondary metabolite biosynthesis”, “posttranslational modification, protein turnover, chaperones”, “transport and catabolism”, “signal transduction mechanisms”, and “general function prediction only” were listed in the top annotated categories in all comparisons . The catWe also performed GO analysis for the DEGs and the top 50 enriched items related to biological processes, cellular components, and molecular functions were identified . All comFtAGPL1~FtAGPL4 and FtAGPS1~FtAGPS2), 5 GBSS encoding genes (FtGBSS1~FtGBSS5), 12 SS encoding genes , 4 BE encoding genes (FtBE1~FtBE4), and 4 DBE encoding genes (FtISA1~FtISA3 and FtPUL) , 3 GBSS encoding genes , 5 SS encoding genes , 2 BE encoding genes (FtBE1 and FtBE2), and 2 DBE encoding genes (FtISA3 and FtPUL) had higher expression levels, suggesting they had potential functions in the starch biosynthesis of Tartary buckwheat seeds. In addition, 20 out of the 31 genes in the starch biosynthesis pathway were identified as DEGs, including 12 in the hierarchical cluster of C2, 4 in C4, and 4 in C6  were more highly expressed in the varieties with higher starch content  . Their ed FtPUL) . Among t 4 in C6 . It is w content c.p value < 0.05 level. The greenyellow and pink modules were negatively correlated to the amylose content, amylopectin content, and amylose/amylopectin, but positively correlated to amylopectin/amylose. On the other hand, the lightcyan module was positively correlated to amylose content, amylopectin content, and amylose/amylopectin, but negatively correlated to amylopectin/amylose. The brown module was positively correlated to amylose content, amylopectin content, and amylose/amylopectin. Gene expression patterns of four trait-specific modules are shown in WGCNA was performed using 22,191 identified genes whose average count number was equal to or larger than 10 in at least one variety. As a result, nine modules were identified based on the association of gene expression levels with starch-related traits a. Among The hub genes were then identified using the threshold of the top 30% in intramodule connectivity, absolute KME (module eigengene-based connectivity) value ≥0.8, and absolute GS (gene trait significance) value ≥0.2, resulting in a total of 3131 hub genes in four trait-specific modules, including 2409, 217, 415, and 90 hub genes in greenyellow, pink, brown, and lightcyan, respectively .p-value < 0.05 level, resulting in 43, 15, 2, and 5 pathways, which were enriched in the greenyellow, pink, lightcyan, and brown modules, respectively  and carbohydrate metabolism (B09101) were listed on the top two enriched pathways  [TFs in the hub genes of four trait-specific modules were screened; the resulting 60, 1, 4, and 36 TFs were enriched in the greenyellow, pink, lightcyan, and brown modules, respectively a. In theAC77/15) ,34,35, oFtNAC70 and FtNAC15, one AP2/ERF-ERF (FtPinG0002070100.01), and one RWP-RK (FtPinG0002032100.01), were clustered to C6 based on their gene expression patterns; meanwhile, all five TFs in the brown module, namely one E2F-DP (FtPinG0001244300.01), two bHLH  [FtWRKY39) [TFs are often in the upstream of a regulatory network, so we identified the TFs in the hub genes of the top 50 in intramodule connectivity, resulting in nine TFs in total, including four TFs in the lightcyan module and five TFs in the brown module . InterestbHLH91) , one WRKtWRKY39) , and oneFtNAC70, was co-expressed with two starch biosynthesis genes, FtPUL and FtGBSS1-3, in the lightcyan module  but negatively correlated to four starch biosynthesis genes, FtBE2, FtISA3, FtSS3-5, and FtSS1 (Pearson R ≤ −0.91), at the significance level of 0.01  seed kernels, genes involved in starch biosynthesis, including AGPase, GBSS, SS, SBE, and ISA encoding genes, were also enriched in the developing seed [RNA-seq is a well-established and widely used method for identifying global gene regulation at a transcriptional level. We carried out RNA-seq analyses for seven varieties that differed in their starch content, resulting in 29,978 mapped genes and 10,873 DEGs, using PK1 as a control. Through metabolic pathway enrichment analysis, the “starch and sucrose metabolism” pathway was found to be significantly enriched and was positively related to starch content. Meanwhile, “carbohydrate transport and metabolism”, which included the process of starch biosynthesis and metabolism, was listed in the top annotated KOG categories. The KOG categories result was consistent with the KEGG enrichment result, suggesting the starch biosynthesis pathway differed in the seven varieties. In recent transcriptome analyses of developing Chinese chestnut , has been isolated and involved in amylose biosynthesis [FtGBSS1-2 and was highly expressed in the seeds in our study. Another reported starch-related gene in F. esculentum, namely FeGBSS (Genbank accession: HW041459.1), was highly homologous to FtGBSS1-1; however, FtGBSS1-1 was not preferentially expressed in the seeds in our study, suggesting it might participate in amylose biosynthesis in other tissues than the seeds of Tartary buckwheat. In addition, we previously performed a transcriptome analysis of three seed developmental stages in Tartary buckwheat and identified 23 DEGs corresponding to starch biosynthesis, among which six DEGs were expressed positively with seed development and showed higher expression in the later developmental seeds [Based on our RNA-seq data, we identified 31 genes related to the starch biosynthesis pathway, including ynthesis . This geFtGBSS1-3 and FtPUL were listed in the lightcyan module and FtBE2, FtSS3-5, FtISA3, and FtSS1 were listed in the brown module.With the combination of the four starch-related traits and the gene expression data achieved through WGCNA analysis, nine modules were identified, of which four modules  were identified as trait-specific modules. Though the greenyellow and pink modules included more hub genes and more enriched pathways, the starch-related pathway was absent in them but significantly enriched in the lightcyan and brown modules, which were positively correlated to amylose content, amylopectin content, and amylose/amylopectin based on the module-trait relationship. Among the starch biosynthesis genes, ZmNAC128 and ZmNAC130 could bind to the prompters of the starch biosynthesis genes, including Brittle 2 (BT2), Zpu1 (encoding DBE in maize), GBSSI, Sh2 (encoding AGPL), SSV, ISA2, and SSIIa, and thus positively affect starch synthesis in the endosperm of maize [TaNAC019 was reported to regulate starch biosynthesis by the regulation of SSIIa and Susy1, and thus accelerate starch accumulation in wheat [FtNAC70 and FtNAC15, were identified as top-50 hub genes. Both of their expression patterns have been reported to be increasing with the seed development of Tartary buckwheat [FtNAC70 showed that it was a seed-specific NAC TF. Meanwhile, its expression was significantly positively correlated to FtGBSS1-3 (Pearson R = 0.84) and FtPUL (Pearson R = 0.79), suggesting that FtNAC70 should be a key positive regulator in regulating starch biosynthesis in Tartary buckwheat.According to the results of TF analysis, 4 and 36 TFs were identified in the lightcyan and brown modules, respectively, among which nine TFs were in hub genes of the top 50 in intramodule connectivity. Some of the above TFs, such as NAC, AP2/ERF-ERF, bHLH, and WRKY, were homologous to the functionally characterized regulators related to starch metabolism in cereal crops. For example, NAC family genes are involved in starch biosynthesis in rice and maize. of maize . TaNAC01in wheat . In our uckwheat ,34. It iFtbHLH5 and FtbHLH91, were identified as top-50 hub genes. FtbHLH5 was more highly expressed in seeds and stems, whereas FtbHLH91 was more highly expressed in leaves rather than other tissues [FtbHLH5 might be involved in regulating starch biosynthesis in seeds and stems, whereas FtbHLH91 might be involved in regulating starch biosynthesis in the stem. The expression of FtbHLH5 was significantly negatively correlated to FtBE2 (Pearson R = −0.96), FtSS3-5 (Pearson R = −0.93), FtSS1 (Pearson R = −0.93), and FtISA3 (Pearson R = −0.92), suggesting that FtbHLH5 should be a key negative regulator in regulating starch biosynthesis in Tartary buckwheat.TFs in the bHLH family could regulate starch biosynthesis in an indirect manner. A novel FLOURY ENDOSPERM2 (FLO2) could interact with the bHLH proteins in a transcriptional complex that could regulate starch storage by influencing the development of endosperm in rice . OPAQUE1 tissues . In addi tissues , suggestZmEREB156, was sucrose- and ABA-inducible and positively regulated starch synthesis by directly binding to the promoter of ZmSSIIIa in maize [Sugar Signaling in Barley2 (SUSIBA2), a plant-specific WRKY TF, could bind to the promoters of BEIIb; the heterologous expression of it in rice produced a rice variety with a high starch content [OS1, encoding an RWP-RK TF, downregulated certain genes in specific cell types, including a majority of zein- and starch-related genes in central starch endosperm cells [FtWRKY39), and an RWP-RK TF (FtPinG0002032100.01), were identified as top-50 hub genes, suggesting they might be involved in starch biosynthesis in Tartary buckwheat. The expression level of FtWRKY39 increased gradually with the development of seeds [An AP2/ERF-ERF family gene, in maize . Sugar S content . A mutanrm cells . In our of seeds , suggestFtbHLH5, significantly positively correlated to FtbHLH5 (Pearson R = 1.00) and significantly negatively correlated to FtBE2, FtSS3-5, FtSS1, and FtISA3. In addition, C3H was preferentially expressed in seeds rather than other tissues. These suggested that C3H might interact with FtbHLH5 and be another key negative regulator in regulating starch biosynthesis in Tartary buckwheat.Though no evidence has been shown at present, an E2F-DP TF (FtPinG0001244300.01) and a C3H-type Zn-finger protein (FtPinG0003310400.01) were also identified as top-50 hub genes, suggesting they might be involved in starch biosynthesis in Tartary buckwheat. Notably, the C3H-type Zn-finger protein was in the co-expression network of A total of 20 Tartary buckwheat varieties/lines were used in this study, namely Biku 2 (BK2), Damiqiao (DMQ), Jinkuqiao (JKQ), Jinqiao 2 (JQ2), Jiujiangkuqiao (JJ), Miku 11 (M11), Miku 127 (M127), Miku 13 (M13), Miku 18 (M18), Miku 5 (M5), Miku 55 (M55), Pinku 1 (PK1), Qianheiqiao 1 (QHQ1), Qianku 2 (QK2), Sanya A5 (SYA5), Sanya B60 (SYB60), Sanya B68 (SYB68), Sanya B69 (SYB69), Sanya C28 (SYC28), and Xiaomiqiao (XMQ). In the early August of 2020, their seeds were sowed in the Anshun experimental field of our laboratory and grew with normal field management. When they were nearly matured, seeds at late filling stage were taken with three replicates, immediately frozen in liquid nitrogen, shelled on ice, and stored in an ultra-low-temperature freezer for starch measurement and RNA-seq. When the plants were matured, the ripened seeds were harvested and dried for starch measurement. Then the ripened seeds of all 20 varieties/lines and the late-filling-stage seeds of seven varieties, namely PK1, BK2, QK2, XMQ, DMQ, JQ2, and M11 were used for starch measurement, with three biological replicates and two (or three) technical replicates. Meanwhile, the late-filling-stage seeds of the seven mentioned varieties were used for RNA-seq analysis, with three biological replicates.(597 nm–480 nm) and OD(541 nm–700 nm), respectively. The equation of the standard curve for amylose wasAmylose and amylopectin content were measured in accordance with a previous report , with so2O was added. Then, 200 µL solution was placed into a new 2 mL centrifuge tube and 1 mL ddH2O was added. The pH was adjusted to 3.0 using 2 mol/L HCl. Then, 20 µL iodine reagent was added and mixed. The volume was titrated to 2 mL with ddH2O. After standing at room temperature for 25 min, the mixtures were applied to a spectrophotometer for amylose measurement at 597 nm and 480 nm and amylopectin measurement at 541 nm and 700 nm. Then, the amylose and amylopectin content were calculated using Equations (1) and (2), respectively.For each variety/line of Tartary buckwheat, 0.1000 g ground powder of the seeds was accurately weighed and placed in a 50 mL centrifuge tube. Then, 10 mL absolute ethanol was added and mixed. The amylose and amylopectin were extracted in a water bath at 80 °C for 30 min. After cooling, the samples were centrifuged at 12,000 rpm at 25 °C for 5 min. The supernatant was discarded, and 10 mL KOH (1 mol/L) was added to the retained precipitate. The sample was placed into a water bath at 100 °C for 15 min, followed by being transferred to a freezer at 4 °C. Afterward, a 1 mL sample was transferred into a new 50 mL centrifuge tube and 10 mL ddHhttp://www.mbkbase.org/Pinku1/ (accessed on 8 September 2022) and the chromosomal location of the mapped genes were obtained by HISAT2 with default parameters [ftp://ftp.ncbi.nih.gov/blast/db (accessed on 8 September 2022), KEGG https://www.genome.jp/kegg (accessed on 8 September 2022), KOG https://www.ncbi.nlm.nih.gov/COG/ (accessed on 8 September 2022), and GO http://geneontology.org/ (accessed on 8 September 2022) using the BLAST program . Gene expression was quantified by FPKM (fragments per kilobase of transcript per million fragments mapped) as the following equation:RNA was extracted using the RNAprep Pure Plant Plus Kit produced by TIANGEN . Library construction was carried out by Metware Biotechnology Co., Ltd. . After that, the library was sequenced on an Illumina NovaSeq 6000 platform  and 150 bp paired-end reads were generated. Data filtering and quality control were also completed by this company using fastp, resulting in more than 6 G clean data with Q20 > 95% and Q30 > 85% for each sample ,43. Aftehttps://cran.r-project.org/web/packages/corrplot/index.html (accessed on 8 September 2022).Pearson correlation was analyzed and visualized using “corrplot” in R language 2(FoldChange) ≥1 and padj  < 0.05 [https://cran.r-project.org/web/packages/available_packages_by_name.html#available-packages-C (accessed on 8 September 2022). For the hierarchical cluster of all identified DEGs, the scale was set as “row”. For the hierarchical cluster of partial analyzed DEGs, the scale was set as “none”. KEGG pathway and GO item enrichment were performed by TBtools [https://cran.r-project.org/web/packages/ggplot2/index.html (accessed on 8 September 2022).DEGs were identified by “DESeq2” in R language with default parameters, using absolute value of Log) < 0.05 . The ups) < 0.05 . The hie TBtools . The topFor tissue-specific analysis, gene expression data of four different tissues  were obtained from the published genome data for Tartary buckwheat , whereashttp://itak.feilab.net/cgi-bin/itak/online_itak.cgi (accessed on 8 September 2022). The gene co-expression networks of the starch biosynthesis genes and TFs were visualized using Cytoscape [WGCNA was performed using “WGCNA” in R language . After fytoscape .FtAGPL2, FtAGPL4, FtAGPS1, FtGBSS1-2, FtGBSS1-3, FtGBSS1-5, FtSS1, FtSS2-1, FtSS3-2, FtSS3-5, FtSS4-3, FtBE1 FtBE2, FtISA3 and FtPUL, had a higher expression level in seeds. In addition, four trait-specific modules  and 3131 hub genes in these modules were identified by WGCNA, among which the lightcyan and brown modules were positively correlated to amylose content, amylopectin content, and amylose/amylopectin. Starch and sucrose metabolism and carbohydrate metabolism pathways were enriched in both the lightcyan and brown modules. Based on TF analysis in trait-specific modules, FtNAC70 was co-expressed with FtPUL and FtGBSS1-3 in the lightcyan module; FtbHLH5 and C3H were co-expressed with FtBE2, FtISA3, FtSS3-5, and FtSS1 in the brown module. All nine genes in the co-expression networks were preferentially expressed in seeds rather than other tissues, suggesting they might be responsible for starch biosynthesis in the seeds. These results would not only enrich the gene resources related to starch, but also provide a molecular insight into the starch biosynthesis regulatory mechanism in Tartary buckwheat. The results here appear to be an excellent starting point for further research on Tartary buckwheat. This work will be of benefit in the future for a complementary theoretical study using quantum chemical topology to identify the vdW interactions that play a role in the RNA-seq analysis. The estimation of non-covalent interaction energies present in the structures of RNA presented here could help to better guide further research on starch biosynthesis and its regulatory network in Tartary buckwheat.In this work, we performed RNA-seq analyses using seven Tartary buckwheat varieties that differed in starch content and combined their RNA-seq data with starch content using WGCNA. As a result, 10, 873 DEGs were identified and functionally clustered to six hierarchical clusters based on their expression patterns. Subsequently, starch biosynthesis genes were identified, and 15 of them, including