This minireview gives an up-to-date overview of the recent advances during the past 3 years, encompassing enhanced electrosynthesis technologies, novel synthetic applications, and ideas for improving reaction sustainability.With the renaissance of chemical electrosynthesis in the last decade, the electrochemistry of hypervalent iodine compounds has picked up the pace and achieved significant improvements. By employing traceless electrons instead of stoichiometric oxidants as the alternative clean “reagents”, many hypervalent iodine compounds were efficiently electro-synthesized  Hypervalent iodine reagents (HIRs) have recently attracted considerable attention in organic synthesis because of their valuable oxidizing abilities, superior leaving group abilities, unique electrophilic properties, and environment-friendly features . To datem-chloroperbenzoic acid (m-CPBA), CH3CO3H, NaBO3, NaIO4, oxone (2KHSO5·KHSO4·K2SO4), H2O2, and Selectfluor, thus inevitably leading to an excess of waste products , Hg(II), and Tl(III) reagents . Neverthproducts . Hence, in situ, so, organic electrosynthesis is recognized as a reliable and sustainable alternative to conventional approaches  precursors, and attempted to use in situ electrogenerated HIRs as important mediators for various iodine (III)-mediated transformations  by anodic oxidation of iodobenzene in the presence of silver fluoride. Shortly after that, the first electrochemical preparation of diaryliodonium salts was disclosed by Miller and Hoffmann in 1967 benzene , [(bistrifluoroacetoxy)iodo]benzene , and Koser’s reagent and its derivatives 4 were achieved via facile ligand exchange reactions. Furthermore, this protocol allowed for the simple synthesis of electron-deficient HIRs that were previously difficult to prepare using conventional methods. In addition, the productivity of this procedure  is higher than the similar transformations under batch electrolysis conditions.In 2019, the first generic method for synthesizing bench-stable classic HIRs -workers . By usinrkers arenes, PIDA 2, IBD , and PhI[OCH(CF3)2]21b, could also be produced in good to outstanding yields with great selectivity by simply substituting H2O with AcOH, KCl, or replacing the solvent to HFIP in the absence of H2O. 2-Iodobenzoic acids precursors could be efficiently converted into benziodoxoles 7 and 8 in high yields under slightly modified electrochemical conditions. Furthermore, a variety of diaryliodonium salts 9 were obtained in satisfying yields employing TfOH as both the electrolyte and counteranion, and CH3CN and TFE as the mixed solvent. When subjected to additional ligand-exchange, the electrogenerated PIDA 2 or hydroxy benziodoxolone 7 could readily convert into many useful HIRs, including Koser’s reagent 4, Weiss’ reagent 10, [hydroxy(phosphoryloxy)iodo]benzene 11, amidoiodane 12, iodonium imide 13, alkynyliodonium salt 14, aryl iodonium ylide 15, and benziodoxole reagents 16 .Very recently (2021), He and others  have previa two-electron anodic oxidation of iodoarene in the presence of a suitable ligand. The cathodic half-reaction is the reduction of protons, which produces H2 as a by-product. In the latter case arenes, and benziodoxoles, and the other for diaryliodonium salts. In the first case, the aryl iodide(III) reagents are proposed to proceed ter case , one ele9 (9 were achieved in high to excellent yields via anodic oxidation of iodobiaryls and iodoarene/arene (9a (>95%). The large-scale experiment’s solvent mixture was easily recovered (>97%) and recycled several times without a notable drop in yield.Almost at the same time, Moran and co-workers also established an electrochemical approach to synthesis cyclic and acyclic diaryliodonium salts 9 . In a sine/arene . It was 3, using 0.1 M LiClO4 as the supporting electrolyte and 1% H2O in MeCN as the O-atom source (7) and its derivative  were obtained with Faradaic efficiency of 89 and 78%, respectively. According to isotopic (18O) labeling studies using H218O, the authors suggest that the O-atom in the hypervalent product is initially coming from the oxidized, O-atom-containing surface of the glassy carbon electrode, and then the surface oxide of the glassy carbon is regenerated by the O-atoms from water solution. These results provide evidence that H2O serves as the oxygen source for the newly formed functional groups in hypervalent iodine products in this electrosynthesis process.Also in 2021, Folkman and others have demonstrated a novel electrochemical synthetic route to HIRs using water as an abundant, green chemistry O-atom source . The bulm source . IBA (7)Whereas several methods for electrochemical synthesis of iodine (III) compounds have been developed during the past few decades, the methods for anodic electrosynthesis of iodine (V) species were less explored and have just recently been established.20), through anodic oxidation of 2-iodobenzoic acid (IBA) in a divided cell using a boron-doped diamond (BDD) electrode in 0.2 M H2SO4 aqueous solution  compounds could be electrochemically generated only in aqueous electrolyte solutions  compound, 2-iodoxybenzoic acid  derivatives were unsuccessful due to decomposition under experimental conditions.Apart from iodine (III) compounds, the aforementioned method by Folkman and others  also enain situ generated at the anode, often immediately used as in-cell or ex-cell or in-flow mediators in subsequent redox reactions, thus efficiently triggering many valuable organic transformations, including fluorination, oxidative cyclization, trifluoroethoxy-lactonization, aziridination, and enantioselective cyclizations, etc. methyl oxazoline 26, which then suffers an SN2 nucleophilic attack from fluorine, resulting in the formation of the halogenated product along with the regeneration of ArI.The proposed mechanism  showed t224, a similar electrochemical protocol  with platinum electrodes and the addition of 1.2 equiv. of mediator and Bu4NBF4 electrolyte. Fluorinated alcohol TFE was chosen as the solvent for stabilizing iodine (III) reagents generated by the anodic oxidation of iodoarenes. Under these conditions, a range of diketo carboxylic acids were smoothly converted into chiral six-membered lactones in 36–87% yield and 0–71% ee mediated by in situ generated chiral iodine (III) intermediate 27. Intermolecular α-alkoxylation of diketo ester derivatives can also be achieved using the same protocol, and the corresponding coupling products were obtained in 38–66% yield and 31–79% ee. Furthermore, this batch-type process could be adapted to an electrochemical flow microreactor to reduce or minimize the use of supporting electrolytes.The first application of electrogenerated chiral hypervalent iodine compound in enantioselective electrosynthesis  was deve3 with ruthenium-(II) catalyzed C-H activation, a wide array of aromatic amides and ketones were selectively converted to their corresponding oxygenated products (2), using a mixture consisting of TFA, TFAA, and n-Bu4PF6 served as electrolyte. Chemical oxidants such as m-CPBA or ozone, which are commonly employed to synthesize the HIRs, failed to give the desired products in adequate yields. Mechanistic studies by experiment, computation, cyclic voltammetry, and in operando NMR spectroscopy showed that an in situ formed HIR served the dual role of electron-shuttle and a transfer reagent of carboxylate anions. The proposed mechanism is shown in 28, and then the resulting complex 29 suffer indirect oxidation by anodically generated PIFA, leading to the formation of ruthenium(IV) intermediate 30. The ruthenium(IV) intermediate undergoes reductive elimination, releasing the product and closing the catalytic cycle.In 2020, Ackermann and co-workers demonstrated how iodoarene mediators can be integrated into metallic-electrocatalytic processes . By a coproducts . The rea31  intermediate 31 . An undi31 . This elvia anodically generated iodine (III) intermediate 31 was reported by Wang, Xu, and co-workers in 2021  species 31 . By usin of 7 mA . A broad32, which could trigger the oxidative cyclization of 2-acetamidobiphenyls.In 2019, Francke’s group developed an electrochemical protocol to achieve intramolecular oxidative C–N bond formation . In this1a exhibits excellent efficiency and reliability in organic electrosynthesis 2 with the second flow of thioanisoles, hydroquinone, 3-(4-hydroxyphenyl)propanoic acid, ortho-hydroxyimines, acetophenones, propiophenones, or acetamide in TFE solvent, a series of oxidative transformations including mono-oxidation, dearomatization, cyclization, α-acetoxylation, tosyloxylation, etc. were successfully achieved using simple reaction setup  ynthesis . By coupon setup . The yie1a  reagents mentioned above, He and his co-workers further developed two oxidative transformations, trifluoro-ethoxylactonization, and tosyloxylation, enabled by anodically generated iodine (III) intermediate 1a . The two solvent . The cor1b, which in situ generated from iodobenzene in the presentence of n-Bu4NOAc in HFIP, smoothly promoted the annulation process and constructed highly strained cyclopropane-fused lactam in 72% yield  intermediate . The iod2% yield .in situ generated HIRs can utilize as powerful batch or flow mediators and trigger valuable oxidative coupling reactions, including fluorocyclizations, trifluoro-ethoxylactonisation, aziridination, asymmetric spirolactonization, etc.As shown herein, the growing interest in hypervalent iodine chemistry together with the “renaissance” of organic electrosynthesis opened new possibilities for developing synthetically attractive transformations in a more sustainable way. By means of anodic oxidation of iodine (I) precursors, HIRs can be electrochemically generated using electric current as an inexpensive and traceless oxidant under environmentally benign conditions. The 3·nHF electrolytes or fluorinated alcohols is highly desirable. Finally, the exploration of more challenging electro-synthetic applications, especially asymmetric electrocatalysis, mediated by electro-generated iodine (III) or iodine (V) is also in urgent demand. Although electrochemistry of hypervalent iodine compounds is still in its infancy, the authors believe that this research front may enjoy imminent prosperity in the near future.Despite considerable progress and achievements, there are major challenges still yet to be overcome. Firstly, the number and types of electrochemically accessible HIRs, especially iodine (V) species, still need to be expanded. Moreover, the development of low-cost, non-toxic, and more sustainable electrolytes as a replacement of commonly used NEt


TMPRSS4 was positively correlated with tumor immune infiltration and the expression of gene markers of immune cells. Notably, its expression was lower in the lymphocyte-depleted subtype than in other immunosubtypes in TC. Moreover, TMPRSS4 was closely related to chemokines as well as their receptors and the immunosuppressive checkpoints CTLA-4, PD-1, and HLA-G. In conclusion, TMPRSS4 may act as a novel biomarker predicting prognosis and immune infiltration in TC.Transmembrane protease serine 4 (TMPRSS4) is a cancer-associated protease associated with prognosis in various types of cancer. Mechanistically, TMPRSS4 mainly regulates malignant phenotypes, such as tumor invasion and metastasis, by either the epithelial to mesenchymal transition (EMT) program or promoting the proliferation of cancer cells. To date, TMPRSS4 and immune infiltration in thyroid carcinoma (TC) are largely unknown. Thus, this paper evaluated the expression of TMPRSS4 in tumor tissue through the Tumor Immune Estimation Resource (TIMER) database, and Oncomine, and its correlation with clinical parameters by UALCAN databases. Furthermore, we analyzed its prognostic value from Kaplan-Meier Plotter database, and the relationship between TMPRSS4 and the abundance of tumor-infiltrating lymphocytes (TILs) in TC in TISIDB, screening potential immune targets to explore novel mechanisms for the clinical management of TC. Finally, we assessed the correlation between TMPRSS4 and some immune markers to uncover a potential immune-related biomarker in TC patients by TIMER2.0. The results revealed that TMPRSS4 was highly expressed in TC and was also associated with lymphatic metastasis, advanced stage, histological subtype, and favorable clinical outcome. The stratified analysis based on immune cell content showed that decreased TMPRSS4 had worse prognosis in CD8The online version contains supplementary material available at 10.1186/s12902-022-01203-3. Thyroid carcinoma (TC) is the most common endocrine malignancies. In the past few decades, the incidence of TC cases has sharply increased globally and seriously threatens public health . AlthougThe transmembrane protease, serine 4 (TMPRSS4), is a member of the serine protease family and is located in the long arm of chromosome 11 (11q23.3). TMPRSS4 protein harbors proteolytic, stem, transmembrane, and cytoplasmic domains. Abundant evidence revealed that TMPRSS4 played an oncogenic role in various types of cancer, including pancreatic carcinoma , gastricIn TC, upregulated TMPRSS4 accelerated cancer cell proliferation by mediating CREB phosphorylation . More imIn our present study, we performed a comprehensive assessment of the expression profiles and clinical significance of TMPRSS4 by analysis from public databases such as Oncomine, TIMER, and the Kaplan–Meier plotter, UALCAN. Finally, we explored the relationship between TMPRSS4 and tumor-infiltrated immune cells using TIMER and TISIDB. We also analyzed its correlation with chemokines, their receptors, and some immune inhibitors. Our results not only offer new insights into the function of TMPRSS4 in TC but also highlight a possible molecular mechanism of the interaction between TMPRSS4, immune cells and tumor cells. The best understanding of TMPRSS4 will provide some clues for favoring immunotherapy for TC.https://www.oncomine.org/resource/main.html).The Oncomine database is a single comprehensive database integrating 86,733 samples, 715 gene expression datasets, and 19 cancer types. It’s designed to screen differentiated expressed genes, co-expression genes by data mining. We analyzed the expression profile of TMPRSS4 in tumors by using this database (http://timer.cistrome.org/) is a comprehensive resource for systematical analysis of immune infiltrates across diverse cancer types. The abundances of six immune infiltrates ) are estimated by TIMER algorithm. We explore the differential expression between tumor and adjacent normal tissues for any gene of interest across all TCGA tumors by Gene_DE module, the correlation between TMPRSS4 expression and the abundance of immune infiltrates based on the different methods for immune-oncology  in the gene module, the association between clinical outcome and abundance of immune infiltrates or gene expression in the Outcome module, and the correlations between TMPRSS4 expression and some particular markers associated with immune cell infiltration of tumors in the Gene_Corr module.TIMER2.0 web server , which serves as an analysis tool for assessing the effect of 54 k genes  on survival in 21 cancer types based on discovery and validation of survival biomarkers. RNA sequencing (RNA-seq) data were utilized from the Cancer Genome Atlas . We explore the predicting role of TMPRSS4 expression for prognosis in multiple types of cancer patients and specific immune cell-enriched TC patients by employing the pan-cancer Kaplan–Meier plotter database with the best performing cutoff value.Kaplan–Meier plotter consists of GEO, EGA, and TCGA database (http://cis.hku.hk/TISIDB/index.php). We conduct the correlation analysis between TMPRSS4 and the abundance of 28 tumor-infiltrating lymphocytes (TILs) in TC. And its correlation with various chemokines and their related receptor, and immunoinhibitors.TISIDB is a web database integrating diverse heterogeneous data types to analyze the interaction between tumor and the immune system . We perform the further validation of the analysis in the former database, compare the expression of TMPRSS4 in TC with its corresponding normal tissue, and confirm the correlation of TMPRSS4 expression and the clinical features.UALCAN is a web portal containing abundant cancer OMICS data , and type 1 T-helper cells (Th1). Unfortunately, there was a limitation that clinical samples of enriched type 2 T-helper cells (Th2) cells and natural killer T (NKT) cells were too small to analyze further.We employed the pan-cancer Kaplan–Meier plotter database and analyzed the association between TMPRSS4 expression and clinical outcome. We found that the predictive role of TMPRSS4 for prognosis varied in different types of cancers. Among them, decreased TMPRSS4 predicted a poor prognosis for BLCA, cervical squamous cell carcinoma (CSCC), LUSC, and THCA. In contrast, patients with elevated TMPRSS4 in KIRP, LIHC, and PAAD had a shorter OS time Fig. . Given t+ T cells , neutrophils  and DCs , and a weakly negative correlation with B cell  and macrophages  in TC  and NK cells  , gamma delta T cell (Tgd) , and Treg  , activated CD4 T cells (Act CD4) , activated CD8 T cells (Act CD8) , CD56dim NK cells (CD56 dim) , CD56 bright (CD56 bright) cells , immature B cells (imm B) , memory B cells (mem B) , effector memory CD4 T cells (Tem CD4) , central memory CD8 T cells (Tcm CD8) , T follicular helper cell (Tfh) , Th1 , type 17 T-helper cells (Th17)  and other immune cells , T cell exhaustion, TAMs, Tfhs, Th1 cells, Th2 cells, Th17 cells, and Tregs. Meanwhile, considering that tumor purity is a major confounding factor in this analysis, we adjusted the analysis results by tumor purity and pinpointed out a significant correlation between TMPRSS4 expression and B cell markers (CD19 and CD79A), CD8+ T cells (CD8A and CD8B), DCs markers , M1 macrophages markers (PTGS2 and IRF5), M2 macrophages markers , monocyte markers (CSF1R and CD86), neutrophils markers , T cell general markers , T cell exhaustion markers , TAMs markers , the Tfh marker BCL6, Th1 markers , Th2 markers , Th17 markers (STAT3 and IL17A), and Treg markers . Undoubtedly, all these analysis results revealed that TMPRSS4 might be related to the onset and progression partially through regulating immune infiltration.We further evaluated the correlation of TMPRSS4 expression with the markers of each tumor infiltrating immune cells in TC from the TIMER2.0 database. The analysis illustrated that TMPRSS4 was associated with most of the immune cells Table , includir = 0.719, P < 0.001), CCL20 , CCL17 , CCL22 , CCL18  and CCL23  , PD-L1  and HLA-G , Programmed cell death 1 ligand 1 (PD-L1), and HLA-G. TMPRSS4 displayed positive correlation with CTLA-4  in TC patients from the TIMER2.0 database. TMPRSS4 expression showed strongly positive association with DCs and neutrophils, weakly positive correlation with CD8+ T cells, and negatively correlated with tumor purity, B cells, macrophages,. Furthermore, the survival analysis showed that TMPRSS4 may be as a prognostic predictor and protective factor for TC by Kaplan-Meier Plotter. Its prognostic role was arguably attributed to the immune cell infiltration in the TME. We also investigated its predictive value in a lymphocyte enriched cohort of TC and uncovered a similar phenomenon only in CD8+ T cell-enriched group by Kaplan-Meier Plotter analysis. Meanwhile, TMPRSS4 expression positively correlated with the abundance of NK cells, Tcm_CD4, Tem_CD8, Tgd, and Treg from the TISIDB database. TMPRSS4 might be involved in antitumor immunity and thus result in a favorable prognosis. In general, the immune subtype was classified into six subtypes: wound healing, IFN-gamma dominant, inflammatory, lymphocyte depleted, immunologically quiet, and TGF-β dominant. Notably, lower TMPRSS4 mRNA expression was shown in the lymphocyte-depleted subtype. It suggested that there was an intrinsic link between TMPRSS4 and tumor immune infiltration. Therefore, we speculated that TMPRSS4 might play an immune-regulatory role in TC.Based on the TCGA data, the Kaplan-Meier plotter database revealed upregulated TMPRSS4 levels were correlated with a worse outcome for patients with KIRP, LIHC, and PAAD. In contrary, increased TMPRSS4 positively correlated with the OS of BLCA, CSCC, LUSC. Notably, the survival analysis was in favour of the previous reports in LIHC , PADD 4, 26, whiOn the other hand, we performed a similar analysis between TMPRSS4 expression and gene markers of each type of immune cells. TMPRSS4 displayed a significant and positive correlation with most of the markers, except for NK cells. TMPRSS4 may be involved in the progression of TC by regulating innate and adaptive immunity. To date, increasing studies have shown evidence that T cell exhaustion leads to immune escape . SignifiGiven the diversity and complexity within the TME, innate immune cells  and adaptive immune cells (lymphocytes) communicate with cancer-associated cells such as fibroblasts, adipocytes, endothelial cells, and extracellular matrix via chemokines, adipocytokines, and cytokines , 29. MorDespite PTC patients having an excellent clinical outcome, a small portion of patients also experience recurrence or death. Currently, cancer immune escape plays a critical role in cancer development. Cancer cells exploit immune checkpoints  to break the physiologic maintenance of self-tolerance and inhibit the antitumor immune response. Na KJ et al.  found thBriefly, TMPRSS4 was a promising potential candidate biomarker for the immune treatment of TC in the future. Highly expressed TMPRSS4 was strongly associated with aggressive clinicopathological features, beneficial prognosis, and immune cell infiltration. Moreover, our study offers insights into a novel mechanism by which TMPRSS4 may affect the clinical outcome of TC through tumor immune infiltration but also paves the way for further studies on immunotherapy against TC. However, our current research may be limited by tumor heterogeneity, transcriptome-based cell-type quantification methods for immuno-oncology , and datAdditional file 1: Supplementary Table 1. The associations between TMPRSS4 and distinct immune populations using TIMER database. Supplementary Table 2. The associations between TMPRSS4 and distinct immune populations using TISIDB. Supplementary Table 3. The associations between TMPRSS4 and distinct immune populations using xCell. Supplementary Table 4. The associations between TMPRSS4 and distinct immune populations using CIBERSORT. Supplementary Figure 1. The correlation between TMPRSS4 and abundance of TILs in TC from TISIDB. Supplementary Figure 2. The correlation between TMPRSS4 and chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL14, CX3CL1, CCL19, and CCL28 in TC from TISIDB. Supplementary Figure 3. The correlation between TMPRSS4 and receptors of chemokines CCR2, CCR5, CCR7, CCR8, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CX3CR1 in TC from TISIDB. Supplementary Figure 4. The correlation diagram between TMPRSS4 and expression of immunosuppressive markers in TC by TIMER2.0. A: the correlation analysis without any adjustment, B: the correlation analysis adjusted by tumor purity.


CPT2) gene expression during duck adipocyte differentiation.Fat deposition in poultry is an important factor in production performance and meat quality research. miRNAs also play important roles in regulating adipocyte differentiation process. This study was to investigate the expression patterns of miRNAs in duck adipocytes after differentiation and explore the role of miR-214 in regulating carnitine palmitoyltransferases 2  miRNAs and their target genes. The expression patterns of miR-214 and CPT2 showed contrasting expression patterns before and after differentiation, and they were selected for further research. The expression of miR-214 was decreased during the first 3 days of duck adipocytes differentiation, and then increased, while the expression of CPT2 increased both in the transcriptional and protein level. The luciferase assay suggested that miR-214 targets the 3′untranslated region of CPT2. Overexpression of miR-214 not only promoted the formation of lipid droplets but also decreased the protein abundance of CPT2.There were 481 miRNAs identified in duck adipocytes, included 57 DE miRNA candidates. And the 1,046 targets genes of DE miRNAs were mainly involved in p53 signaling, FoxO signaling, and fatty acid metabolism pathways. miR-214 and Current study reports the expression profile of miRNAs in duck adipocytes differentiated for 4 days. And miR-214 has been proved to have the regulator potential for fat deposition in duck. Deposited fat in poultry mainly consists of abdominal fat, subcutaneous fat, and intramuscular fat (IMF). Excessive fat deposition can reduce feed efficiency and carcass yield . While tAdipocytes, which are derived from mesenchymal stem cells, play an important role in energy balance in both poultry and mammals . AdiposeCTNNB1) to promote differentiation by interfering with the Wnt/β-Catenin signaling pathway, in 3T3-L1 preadipocytes , CCAAT/enhancer-binding protein-α [C/EBPα], adipocyte fatty-acid binding protein 4 [FABP4], carnitine palmitoyltransferases 1A [CPT1A], CPT2, and acyl-coenzyme A oxidase 1 [ACOX1]), were detected in the adipocyte cells ; on average, 10,979,173 and 10,757,684 clean reads were mapped for the CVC and CVT libraries, respectively. Further, >90% of the bases had base quality >Q20. Most of the clean reads were 21 to 24 nt; 22 nt reads were the most abundant, followed by 23 nt reads , acetyl CoA acyltransferase 2 (ACAA2), CPT2, acyl-CoA synthetase 1 (ACSL1), fatty acid synthase (FASN), hydroxyacyl-CoA dehydrogenase (HADH), hydroxyacyl-CoA dehydratase 3 (HACD3), and acyl-CoA synthetase bubblegum family member 2 (ACSBG2) were associated with fatty acid metabolism . KEGG pathway enrichment revealed that the target genes were enriched in the p53 signaling pathway, protein processing in endoplasmic reticulum, and the FoxO signaling pathway ; furthertabolism . Interessm , for both preadipocytes and differentiated adipocytes, than in the groups transfected with the miR-NC . FABP4, ol group . miR-214ol group . Besidesol group . The numol group . TogetheIn this study, we found that, after 4d of culture in differentiation medium, TG content increased, and lipid droplets gradually accumulated inside cells. The expression level of fatty acid synthesis genes increased, and that of fatty acid transport genes increased slightly. And, several of the miRNAs identified in our miRNA-seq analysis, such as miR-103, miR-21, miR-30, miR-27, miR-130 and let-7, were also detected by other researches ,24. FurtHADHB, ACAA2, CPT2, ACSL1, FASN, HADH, HACD3, and ACSBG2) were involved in this pathway. They play important roles in fatty acid synthesis, transport, and oxidative utilization. FASN and HACD3 are two key enzymes required for fatty acid synthesis [ACSL1 and ACSBG2 enable fatty acids to enter into lipid pools, and activate fatty acids [CPT1 and CPT2 mediate fatty acid transfer into the mitochondrial matrix for β-oxidation [CPT1 regulated fatty acid oxidation and thus affects intramuscular adipogenesis [CPT2 was proved to be a common up-regulated protein during differentiation of preadipocytes from bovine omental, subcutaneous and intramuscular adipose depots [HADH, HADHB, and ACAA2 are key enzymes in fatty acid oxidation [HADHB, in chickens [ACSBG2 expression [CPT2, miR-214 and CPT2 are selected to explore their regulatory mechanism during adipocyte differentiation in duck.We conducted KEGG pathway analysis to reveal the biological significance of the target genes of the DE miRNAs. The “fatty acid metabolism” pathway was enriched in KEGG analysis and 8 genes  [miR-214 is an important miRNA that has been well studied in humans. It is also identified in chicken , duck 1, and pigMFN2) , and supMFN2) ,46. We fMFN2) . FurtherMFN2) . Taken tCPT) enzyme system, which operates throughout the body, enables long-chain fatty acids to enter the mitochondrial matrix [CPT system comprises two separate proteins located in the outer and inner mitochondrial membranes  [CPT2 expression increased with differentiation time which is coincide with the result of bovine during differentiation of adipocytes [CPT2 regulated fatty acid transportation and differentiation in duck adipocytes: lipid droplets accumulated and CPT2 expression increased with differentiation time, enhancing fatty acid transport; while the later up-regulated miR-214 promoted the differentiation process, inhibited the increasing expression of CPT2, and these further increased the accumulation of lipid droplets. CPT2 is also found to be under the expression regulation of peroxisome proliferator-activated receptor γ (PPARγ) [CPT2, thereby reducing fatty acid transport and oxidation. Altogether, we suggest that miR-214 and CPT2 are involved in the complex regulatory network during the duck adipocytes differentiation.The carnitine palmitoyltransferase (l matrix . The CPTctively) . Carnitictively) . During ipocytes . Here, w (PPARγ) . Besides


CDC20/APC/CCDH1 inhibitors, namely Apcin and proTAME, are effective against lymphoma and multiple myeloma cells, resulting in mitotic arrest and apoptosis and synergizing with clinically-relevant drugs. The evidence and hypothesis presented in this review provide the input for further biological and chemical studies aiming to dissect novel potential CDC20 roles and targeting strategies in hematological malignancies.Cell division cycle 20 homologue (CDC20) is a well-known regulator of cell cycle, as it controls the correct segregation of chromosomes during mitosis. Many studies have focused on the biological role of CDC20 in cancer development, as alterations of its functionality have been linked to genomic instability and evidence demonstrated that high CDC20 expression levels are associated with poor overall survival in solid cancers. More recently, novel CDC20 functions have been demonstrated or suggested, including the regulation of apoptosis and stemness properties and a correlation with immune cell infiltration. Here, we here summarize and discuss the role of CDC20 inside and outside mitosis, starting from its network of interacting proteins. In the last years, CDC20 has also attracted more interest in the blood cancer field, being overexpressed and showing an association with prognosis both in myeloid and lymphoid malignancies. Preclinical findings showed that selective CDC20 and APC/CThe online version contains supplementary material available at 10.1186/s13046-022-02363-9. Cancer hallmarks attempt to organize the complexity of tumor biology into major features. To date, cell growth, signaling regulation, apoptosis evasion, uncontrolled replication, neo-angiogenesis, tissue invasion and metastasis are the most known and investigated mechanisms . An incrCDC20 has been already described in a variety of human solid tumors [CDC20 expression level has been reported as a putative marker of clinical outcome in many cancer types, being associated with advanced stage, high grade and poor prognosis [Many studies focused on the mechanisms underlying the control of the correct separation of twin chromatids during the final step of cell division. It has been shown that the cell division cycle 20 (CDC20) plays a crucial role in the final steps of mitosis. Recently, there is different evidence which suggest that CDC20 contributes to a number of cellular processes that have been partially explored. An overexpression and/or oncogenic role of d tumors , 10 incld tumors , 12, bred tumors , 14, lund tumors , 16, prod tumors –19, gastd tumors , hepatocd tumors , kidney d tumors , ovariand tumors , 24, ostd tumors –27 and gd tumors , 29. Morrognosis , 31.The following sections summarize CDC20 functions, including mitosis-related and unrelated ones, and the available data sustaining the oncogenic impact of CDC20 in hematological malignancies in order to evaluate its potential role as prognostic factor and therapeutic target in these neoplasms.CDC20 gene localizes on the short arm of chromosome 1 and encodes for a 499-amino acid and a 51 kDa protein (Human Protein Atlas proteinatlas.org). CDC20 is composed by two main segments: the N-terminal region characterized by low structural complexity and the C-terminal region containing the WD40 repeats [The  repeats . The N-t repeats . The C-t repeats . The KEN repeats . Mutatio repeats .Fig. 1CDCDC20 loss determined embryonic lethality due to prolonged metaphase arrest caused by securin stabilization [CDC20 was discovered in 1970 by Hartwell’s group for its role in initiating anaphase and chromosome segregation in yeast models . The keylization –41, and lization .CDC20 complex is finely regulated. In addition to CDK1 and other mitotic kinases [500) [CDC20 exerts its function during the metaphase to anaphase transition ensuring proper chromatids segregation [During metaphase, CDC20 complexes with MAD1, MAD2, BUBR1, BUB1, and BUB3, to generate the mitotic checkpoint complex (MCC), a crucial effector of the spindle assembly checkpoint (SAC) . The SAC kinases , 72, Tan kinases , CCNB3 [ kinases , Apc1 loes [500)  and Hemaes [500)  were alsregation   were positively correlated with CDC20 in cancer cells [CDC20 substrates, that are also critical cell cycle regulators, as Securin (PTTG1) [The key role exerted by CDC20 during mitosis is further supported by the analysis of the CDC20 interactome. Overall, 817 physical interactions with 171 partners have been reported in humans by proteomic analyses, according to The Biological General Repository for Interaction Datasets . The exer cells . Moreove (PTTG1) , CCNB1 [ (PTTG1) , CCNA1/2 (PTTG1) , NEK2 [8 (PTTG1) , 84, Zwi (PTTG1)  and p21  (PTTG1) , indicatPathway analysis of CDC20 interacting proteins suggested additional roles of CDC20 outside cell cycle regulation, with a significant enrichment of specific cellular processes, including protein modification, localization and degradation, telomeres regulation, transcription, and other signaling pathways, as Hippo, TGF-β, β-catenin, MAPK Table S. AccordiCDC20 is active, and that CDC20 depletion allows a significant up-regulation of BIM, activating the DNA damage-induced apoptosis of cancer cells [CDC20 contributes to its degradation during mitosis, and MCL-1 expression levels help distinguish prolonged arrest from normal mitotic events [CDC20 binding, thus affecting the rate of CCNB1 degradation and slowering mitotic slippage [CDC20 knockdown resulted in an increased phosphorylation of the anti-apoptotic BCL-2 and BCL-xL proteins and MCL-1 degradation, promoting CDK1 signaling activation and apoptosis [CDC20 downregulation. In breast cancer models CDC20-depleted cells underwent mitotic arrest and were primed to die by apoptosis, which was, at least in part, dependent of BCL-xL phosphorylation on serine 62 residue [CDC20 has been also involved in the apoptotic response, by regulating phosphatidylcholine (PC) cycle , 103 ander cells . Regardic events , 107. Slslippage . Moreoveslippage . Converspoptosis . The ove residue . Therefo+ T cells, monocytes [+ T cells, regulatory T cells, B lymphocytes, and natural killer cells [Recently, CDC20 expression has been also associated with immune infiltration in cancer. A positive correlation has been demonstrated with the infiltration of cancer-associated fibroblasts and myeloid-derived suppressor cells across several cancer types , with anonocytes , 115, exonocytes , CD4+ T In summary, CDC20 is involved in a number of biological and tumor-related functions, that deserve further investigation in the field of hematological malignancies.CDC20 is rarely mutated across cancers [G284V, E413G and A128T) have been reported . All three mutations reported above have been identified the diffuse large B cell lymphoma (DLBCL) cohort. Moreover, copy number alterations also occur rarely and have been detected in AML and DLBCL, with a frequency lower than 0.2% . They include both amplifications and deletions, with concordant consequences on the gene expression levels in most cases  and paclitaxel (PTX) that act through the induction of mitotic arrest and activation of the spindle checkpoint [Few data are available on the role of CDC20 in ALL pathogenesis and drug-induced changes of its expression. In the Jurkat T-ALL model, APC/Cansition . Likewisn of MLL , a lysinn of MLL . This aseckpoint . In part+ T-cell malignancy caused by infection from human T-cell leukemia virus type 1 (HTLV-1). The progression from infection to malignant transformation has not been fully described, but it has been linked to the activation of the viral trans-activator/oncoprotein, Tax. Tax mediates activation of viral transcription and alters cellular mechanisms in a pleiotropic manner inducing NF-κB activation, cell cycle perturbation and cell transformation [S. cerevisiae, rodent, and human cells leading impaired chromosome segregation and causing severe aneuploidy [CDC20, supporting the idea that Tax promotes aberrant activation of APC/CCDC20 to avoid the block of mitotic exit and progression of aneuploid cells, highly represented in ATL [CDC20 is a hub gene in the protein-protein interaction network of differentially expressed genes between ATL samples and normal CD4+ T cells [Adult T-cell leukemia/lymphoma (ATL) is a CD4ormation . Liu andeuploidy . The stud in ATL . Accordi T cells .CDC20 was also reported as a hub protein among tumor-associated genes in DLBCL [CDC20 expression correlates with poor overall survival (OS)  and hig= 0.0247 ) in DLBCin DLBCL . Deregulic mouse . CDC20 eic mouse . Indeed,CDC20 is highly expressed not only in DLBCL but also in mantle cell lymphoma (MCL) [CDC20 is among the top five altered genes involved in the development of MCL and it is significantly associated with shorter OS  , 125. FuCDC20 expression has been reported in the high-risk category characterized by unmutated IGHV (U-CLL) [IGHV mutated (M-CLL) cases, the more aggressive U-CLL subtype exhibited an increased expression of cell cycle genes, including ATF2, CCNB2, CDC20, CDC25A, CREB1, E2F4, ESR1, FOXM1, MKI67, MYC, POU2F2, RBL2, SP3, TYMS, UBE2C, VRK1. CDC20 was also associated with CD38 expression, another marker of disease aggressiveness [+ B-CLL samples had an increased level of APC/C subunit 5 (APC/C 5), which controls some regulatory sub-functions of the APC/C complex [+ CLL cells could represent an alternative strategy adopted to mimic the effects of CDC20 overexpression.In CLL, high  (U-CLL) . Indeed,siveness . Moreove complex . As demo complex . This evAURKC, PLK2, PLK3) compared to normal plasma cells and higher levels of others, including CDC20, and were able to bypass the SAC-mediated arrest when challenged with nocodazole [CDC20 transcript were also confirmed in primary cells from high-risk MM patients [BUB1B [CDH1 [CDC20 knockdown reduced the viability of MM cell lines, by inducing cell growth arrest and accumulation of the APC/CCDC20 substrate CCNB1 [CDC20 expression was associated with inferior OS both per se  patients is highly unstable and is characterized by chromosome translocations and aneuploidy, affecting the disease outcome . The higcodazole . High lepatients , that als [BUB1B  together1B [CDH1 , that sute CCNB1 . Moreoveectively ) and in P < 0.05 ).CDC20 and MAD2 characterized MDS patients with severe thrombocytopenia and complex karyotypes [P = 0.013) [Emerging evidence shows that MDS patients harbor deregulated expression of several components of SAC machinery. In particular, MDS patients with hypercellular and normocellular bone marrow, reflecting a more aggressive disease, had higher expression of SAC components in comparison with those characterized by hypocellular bone marrow . Moreoveryotypes . Notably= 0.013) .CDC20 and PLK1 and low RAD50 expression was able to discriminate the aneuploid from euploid cases [CDC20 [CDC20 also showed an aberrant interaction with NUP98 fusion oncoproteins [CDC20 and the physical interaction is dependent on the phosphorylation of a PEST sequence within NUP98 C-terminal domain, which occurs prior to mitotic entry [CDC20 during mitosis. Conversely, Salsi et al. demonstrated that NUP98 fusion oncoproteins bind APC/CCDC20 during mitosis, through the NUP98 GLEBS-like domain in the absence of the RAE1 partner protein. This interaction led to BUBR1 displacement and consequent attenuation of the SAC [More than 20% of AML patients have defects in chromosome segregation, also supported by an altered activity of the SAC components, caused by BUB1 deregulation, BUBR1 repression, or aberrant expression of MAD2 and CDC20 , 145, thid cases . In addis [CDC20 . CDC20 ws [CDC20 , MEIS1, s [CDC20  and NUP9s [CDC20 . CDC20  patients during the last 10 years , controlCDC20/APC/CCDH1 inhibitors [Due to the potential oncogenic role of CDC20, different chemical compounds and inhibitors have been developed and tested for their efficacy as antineoplastic agents: tosyl-L-arginine methyl ester (TAME) and its pro-drug (pro-TAME), APC inhibitor (Apcin), Withaferin A, N-alkylated amino acid-derived (NAHA), Ganodermanontriol, Genistein, CARP-1 functional mimetic 4 (CFM-4) and 6-brominated coumarin hydrazide-hydrazone derivative (BCHHD), that have been extensively revised by Wang et al . Among thibitors , respectCDC20 inhibitor) or mitotic slippage depending on the low or high SAC activity, respectively [Apcin is a small molecule that binds in a competitive manner the D-box-binding domain of CDC20 thus preventing its substrate recognition capacity and inhibiting the ubiquitination of CDC20 targets  Fig. 7AA7A. Surpectively . Regardiectively . In vivoectively . Apcin aectively .Fig. 7ScCDC20 and APC/CCDH1 substrates’ degradation and mitotic arrest [pro-TAME is a small molecule that mimics the IR motif of CDC20 and CDH1 involved in their recruitment to the APC/C. It binds the APC/C core complex in a competitive manner and prevents its association with APC/C activators  Fig. 7BB7B, leadc arrest . Pro-TAMc arrest .CDC20 substrate CCNB1, together with phosphorylation-mediated inactivation of the anti-apoptotic factors BCL-2 and BCL-xL, reduction of cell viability and activation of caspase-3 dependent apoptosis [In MCL and DLBCL cellular models proTAME induced metaphase arrest, resulting in accumulation of the APC/Cpoptosis . Pro-TAMConsistently with the high CDC20 expression in MM, treatment of cell lines and primary cells with proTAME resulted in the stabilization of CCNB1 and cell cycle arrest at G2/M phase , 132. MoIn line with evidence from solid cancer, CDC20 overexpression also plays a critical oncogenic role in hematological malignancies. The available data gathered in this review revealed that the up-regulation of CDC20 is associated with inferior OS in different hematological malignances. In agreement with its potential role as prognostic marker, higher expression of CDC20 was observed in high-risk MM, CLL, MDS and AML patients. Moreover, over-expression of CDC20 has been reported as a biomarker of resistance to TKI therapy in CML patients. In line with these observations, studies performed in DLBCL, MCL and MM cells treated with Apcin or proTAME, or their combination, demonstrated that targeting CDC20 is a promising therapeutic strategy in hemato-oncology. Indeed, it has been shown that CDC20 inhibitors significantly potentiate the efficacy of conventional therapeutic agents in different hematological malignances. In addition, novel therapeutic combinations based on the synthetic lethality mechanisms could be explored. For example, Apcin effectiveness is enhanced in cells carrying defective sister chromatid cohesion, that also characterize a subgroup of AML patients , as showCDC20 enhances the sensitivity of cancer cells to microtubule interfering agents [Further development towards clinical application of CDC20 inhibitors is hampered by the poor bioavailability of the compounds, because of the high dosage needed to achieve a therapeutic response. This evidence and the current knowledge provided the rationale for the development of other specific inhibitors. We have synthesized and tested novel tryptamine derivatives bearing aminopyrimidyl- or imidazolyl- moieties, which are also present in Apcin . In partg agents  (Fig. 7COverall, this evidence proves a growing therapeutic interest in CDC20 targeting in hematological malignancies, which will promote novel studies towards the development of better combination strategies, the identification of patients’ cohorts that will mostly benefit of them and the definition of optimal therapeutic windows.Additional file 1.


In England, the emergence the more transmissible SARS-CoV-2 variant Alpha (B.1.1.7) led to a third national lockdown from December 2020, including restricted attendance at schools. Nurseries, however, remained fully open. COVID-19 outbreaks (≥ 2 laboratory-confirmed cases within 14 days) in nurseries were investigated to assess the risk of SARS-CoV-2 infection and cumulative incidence in staff and children over a three-month period when community SARS-CoV-2 infections rates were high and the Alpha variant was spreading rapidly across England.This was a cross-sectional national investigation of COVID-19 outbreaks in nurseries across England. Nurseries reporting a COVID-19 outbreak to PHE between November 2020 and January 2021 were requested to complete a questionnaire about their outbreak.Three hundred and twenty-four nurseries, comprising 1%  of nurseries in England, reported a COVID-19 outbreak. Of the 315 (97%) nurseries contacted, 173 (55%) reported 1,657 SARS-CoV-2 cases, including 510 (31%) children and 1,147 (69%) staff. A child was the index case in 45 outbreaks (26%) and staff in 125 (72%) outbreaks. Overall, children had an incidence rate of 3.50%  and was similar irrespective of whether the index case was a child  or staff . Among staff, cumulative incidence was lower if the index case was a child  compared to a staff member , with the highest cumulative incidence when the index case was also a staff member . Compared to November 2020, outbreak sizes and cumulative incidence was higher in January 2021, when the Alpha variant predominated. Nationally, SARS-CoV-2 infection rates in < 5 year-olds remained low and followed trends in older age-groups, increasing during December 2020 and declining thereafter.In this cross-sectional study of COVID-19 outbreaks in nurseries, one in three staff were affected compared to one in thirty children. There was some evidence of increased transmissibility and higher cumulative incidence associated with the Alpha variant, highlighting the importance of maintaining a low level of community infections.The online version contains supplementary material available at 10.1186/s12889-022-14228-z. The rapid spread of SARS-CoV-2, the virus responsible for COVID-19, forced many countries to impose national lockdowns . Cases iCompared to adults, children have been reported to have a lower risk of symptomatic disease, hospitalisation or death , 5, 6. TFrom mid-August 2020 and prior to schools reopening to all students, SARS-CoV-2 infections started increasing nationally, first in adults and then in children, leading to a second national lockdown during November 2020, whilst keeping educational settings fully open. SARS-CoV-2 cases subsequently declined, first in adults and then in school-aged children . The emeCompared to primary and secondary schools, nurseries provide a different challenge from an infection control perspective. The premises are often much smaller, with a higher staff-to-child ratio, and the very young age of the children makes physical distancing from staff unfeasible , 17. PubIn England, educational settings are regularly provided with updated guidance on infection control measures, including recommendations for good ventilation, regular hand-sanitising, social distancing, and enhanced cleaning; face masks and face coverings are not recommended for staff or children in nurseries , 20. AllWe used the same methodology to investigate COVID-19 outbreaks in nurseries as for primary and secondary school outbreaks, defined as “ ≥ 2 laboratory-confirmed cases within 14 days” . “NurserData were analysed using R studio (version 1.3.1056) and Stata v.15.1 , and are mainly descriptive. The start of an outbreak was taken as the symptom onset date in the index PCR-confirmed case or, for asymptomatic cases, their date of PCR sample. Denominators for staff and children by age-group and bubble were reported by individual nurseries. Denominators for the number of nurseries and educational settings in England were estimated using the total number of possible settings for different age-groups. A setting with a combination of nurseries, primary or secondary schools was counted separately for each setting (Source: Department for Education) . The proPHE has legal permission, provided by Regulation 3 of The Health Service (Control of Patient Information) Regulations 2002, to process patient confidential information for national surveillance of communicable diseases and as such, individual patient consent and approval from an ethics committee is not required.Between 02 November 2020 (week 45) and 31 January 2021 (week 4), 324 nurseries reported a COVID-19 outbreak to PHE, comprising 1.0%  of nurseries across England  nurseries were contacted and 181 (57%) completed the survey. Eight settings provided insufficient information; therefore, 173 nurseries from all English regions were included in the analysis Fig. . There wOverall, children had an cumulative incidence of 3.50% 95%CI, 3.21–3.81%) compared to 30.92%  among staff  or a staff member . Among staff, overall, cumulative incidence was lower if the index case was a child  compared to a staff member , with the highest cumulative incidence among direct staff when the index case was also a direct staff member  (Table Nurseries experiencing a COVID-19 outbreak reported that 49% 85/172) of their staff members were able to maintain physical distancing between each other most of the time throughout the day, while 39.5% (68/172) reported social distancing “rarely”, “never” or “some of the time”. Within individual bubbles, nurseries reported that 74% (127/172) of children were “never” able to maintain physical distancing with each other, and 64% (110/172) staff were “never” able to maintain physical distancing with children  and 78% 131/169) had shared bathrooms used by staff members across different bubbles. Most settings had designated eating spaces  and bathrooms for children , assigned per bubble  nurseries reported a COVID-19 outbreak compared to 2%  primary schools and 10%  secondary schools. This compares with 1% , 4%  and 12% , respectively, between 02 November 2020 and 31 January 2021, which included a period when primary and secondary schools had restricted attendance for children of keyworkers and vulnerable children in January 2021. When outbreaks did occur in nurseries, however, cumulative incidence in staff (24.26%) and children (2.34%) during November 2020, when there was little circulation of the Alpha variant, was significantly higher than primary school teaching staff (9.81%), secondary school teaching staff (3.97%), secondary school students (1.20%) or primary school students (0.84%) during September and October 2020, although these estimates were generated during a period of different community prevalence . Table S2. Reported social distancing between and within bubbles in nurseries reporting a COVID-19 outbreak to Public Health England (PHE). Table S3. Shared facilities in nurseries reporting a COVID-19 outbreak to Public Health England (PHE). Table S4. reason for testing in the first four cases, where known, among staff and children in nurseries reporting a COVID-19 outbreak to Public Health England (PHE).


Moreover, FOX proteins were closely correlated with TP53 and KRAS mutation. (4) Conclusions: Our study confirmed previous reports about FOX box protein in the regulation of tumor growth. A remarkable observation about the unclear crosstalk with crucial genes, as TP53 and KRAS need deeper investigation.(1) Background: The present study aimed to investigate the influence of forkhead box (FOX) on endometrial cancer (EC) progression. For a better understanding, the driving mechanisms are vital to identifying correlations between genes and their regulators. (2) Methods: The study enrolled one hundred and three white female patients with confirmed EC. For the analysis, we used next-generation sequencing with the Hot Spot Cancer Panel provided by Illumina Inc., San Diego, CA, USA, and an immunohistochemical analysis of FOXA1, FOXP1, and estrogen receptors. (3) Results: FOXA1 silencing led to a worse outcome based on the correlation with FOXA1 (test log-rank  In past decades, morbidity and mortality associated with endometrial cancer (EC) increased despite diagnostic progress. Obesity is particularly associated with EC, and EC is prevalent in high-income countries owing to their associated high rate of obesity . CurrentFOXA1 and FOXP1 are members of the forkhead box (FOX) family of transcription factors, formerly known as the hepatocyte nuclear factor (HNF) family. Recent studies have shown that the FOX transcription factor family is closely associated with hormone-dependent carcinogenesis, especially with steroid receptors ,5,6.CDKN2A), a key negative regulator of MDM2 activity. Importantly, tumorigenesis in this system depends on sustained Notch activity, as attenuation of the Notch expression dramatically increases p53 levels and tumor regression through apoptosis [The role of FOXA1 in cancer remains not fully defined, as both pro- and anti-tumorigenic functions have been uncovered. Research has identified that FOXA1 and FOXA2 expression inversely correlates with disease progression and/or aggressiveness; for example, FOXA1 and FOXA2 are readily detected in normal epithelium and precancerous lesions, but the expression is commonly lost in poorly differentiated disease (and event associated with EMT-like phenotypes) . In addipoptosis . This inThe purpose of the present study was to explore the expression of FOX protein, and its influence on EC molecular basis and cancer progression.A total of one hundred and three white female patients with confirmed EC were enrolled. All patients underwent surgery and other oncological procedures between 2005 and 2017. The collective evaluation data and follow-up data were tabulated. To align tumor staging, each case was re-diagnosed according to the Eighth Edition of TNM Classification . DetaileAll participants underwent surgical treatment without previous radio-chemotherapy to conduct a credible comparative analysis of tumor characteristics, the treatment, and unchanged molecular profiling.To achieve this goal, we used the Hot Spot Cancer Panel provided by Illumina Inc., San Diego, CA, USA. The gene battery comprised BL1, EGFR, GNAS, KRAS, PTPN11, AKT1, ERBB2, GNAQ, MET, RB1, ALK, ERBB4, HNF1A, MLH1, RET, APC, EZH2, HRAS, MPL, SMAD4, ATM, FBXW7, IDH1, NOTCH1, SMARCB1, BRAF, FGFR1, JAK2, NPM1, SMO, CDH1, FGFR2, JAK3, NRAS, SRC, CDKN2A, FGFR3, IDH2, PDGFRA, STK11, CSF1R, FLT3, KDR, PIK3CA, TP53, CTNNB1, GNA11, KIT, PTEN, and VHL. The sequencing target was 2800 COSMIC mutations from 50 oncogenes and tumor suppressor genes.In addition, we performed tissue microarray  for immunohistochemistry. We performed the immunohistochemical assays using the automated IHC/ISH slide staining system BenchMark Ultra . After deparaffinization and rehydration of the samples, we performed the unmasking processes using CC1  and incubation with primary antibodies . In addition, further routine steps were performed. Moreover, we used the Ventana ultra-View Universal DAB Detection Kit and Opti View Detection Kit. The immunohistochemistry details are presented in ® Genomic DNA FFPE One-Step Kit . The quality was quantified using DeNovix DS-11 Spectrophotometer  and QuantiFluo® ONE dsDNA System .DNA isolation: cancer genomic DNA was extracted from formalin-fixed paraffin-embedded tissue using the MagCoreThe assay generated a library of 207 gene-specific amplicons and targeted ~2800 clinically relevant mutations.Sequencing: the products were analyzed by next-generation sequencing (NGS) using the Illumina platform, MiSeq Dx.https://wannovar.wglab.org, accessed on 2 May 2022). The results were visualized using the R Bioconductor package Maftools  [Data analysis: an analysis of the NGS data was performed using the GALAXY platform . Sequencing reads (FASTQ files) were aligned to the human reference genome hg19 using the Bowtie2 tool. Variant calling was performed using the Varscan2 tool. Parameters used for the analysis were minimum allele frequency—0.05, minimum quality—20, and minimum coverage ×80. All variants were annotated with ANNOVAR  was performed on variables that were statistically significant in univariate analysis.http://www.statsoft.com/, accessed on 2 May 2022.All statistical tests were two-sided, and values <0.05 were considered significant. Computations were performed using STATISTICA , StatSoft, Inc. (2014), version 12, Tulsa, OK, USA, Available online: The estimation of targeted protein frequency was conducted , revealip = 0.04220 (p = 0.033), revealing that FOXA1 silencing led to a poor outcome. The correlation was found between FOXA1 and FOXP1 (R = 0.2872 p = 0.0041). We noted the correlation of OS with FOXP1 (p > 0.05).A detailed OS analysis was conducted to investigate the impact of the clinical outcome. The Cox univariate analysis provided us with significant results concerningFOXA1  and FOXA1 with KRAS mutation . This information confirmed the impact on advanced-stage FIGO . The multivariate Cox models covering all tested genes and FOX proteins unveiled prognostic values for TP53 , KRAS , and lack of FOXA1.Interestingly, we noted the coincidence of a lack of FOXP1 with TP53 mutation + breast cancer. In our cohort study, we recorded over 90% concordance in co-expression with ER and FOXA1. Our results confirm a protective effect of ER/FOXA1 co-expression ,21,22. OThe clinical impact of forkhead protein box FOXP1 is still unclear. We did not observe the significance of FOXP1; however, another report suggested that high nuclear expression of FOXP1 would be a recent event in EC carcinogenesis. Like the FOXA1, a tight connection with ER and AR was also noted . The abop = 0.0106), revealing a correlation of FOXA1 with KRAS mutation (p = 0.0446).Our study noted a 25% frequency of KRAS mutation and 20% TP53 mutation. Moreover, a lack of FOXP1 was correlated with TP53 mutation  and activated TGFβ signaling pathway in prostate cancer . More inBecause Notch signaling is observed in many malignancies, there is hope for universal treatment for improving clinical survival.Our study confirmed previous reports about the FOX box protein in the regulation of tumor growth. A remarkable observation about the unclear crosstalk with crucial genes, as TP53 and KRAS need additional research.This study was limited by the population selected from one region in Poland, and the focused follow-up time varied among the same participants. In future research, we plan to add a control group. Moreover, our gene panel did not cover the POLE gene. The strengths of this work include the long-term observation and the use of modern methods of genetic testing.


Background. Nonalcoholic fatty liver disease (NAFLD) is commonly observed in patients with type 2 diabetes (T2D). Semaglutide, a glucagon-like peptide 1 receptor agonist, may have a therapeutic role by targeting common mechanisms involved in the pathophysiology of T2D and NAFLD. The study aimed to assess the effectiveness of Semaglutide on NAFLD in patients with T2D. Methods. Forty-eight patients were treated with subcutaneous Semaglutide in add-on to metformin for 52 weeks. After the baseline visit (T0), follow-up was scheduled quarterly  and then at 12 months of therapy (T12). During each visit, body composition was analyzed by phase-sensitive bio-impedance, and NAFLD was diagnosed and staged by Ultrasound (US) imaging. Surrogate biomarkers of NAFLD were also calculated and followed over time. Results. A significant decrease in anthropometric and glucometabolic parameters, insulin resistance, liver enzymes, and laboratory indices of hepatic steatosis was observed during treatment. Similarly, fat mass and visceral adipose tissue (VAT) decreased over time more than skeletal muscle and free-fat mass. US-assessed VAT thickness and the 12-point steatosis score also declined at T3 up to T12. Liver steatosis improved in most patients (70%), showing a reduction by at least one class in the semiquantitative US staging. Conclusion. Besides glucose control and body composition improvements, Semaglutide was effective in ameliorating the clinical appearance and severity of NAFLD in T2D patients. Nonalcoholic fatty liver disease (NAFLD) is the foremost liver disease in the world, affecting up to 30% of the general population . Its preNAFLD is the result of a metabolic disorder related to a sedentary lifestyle, excessive calorie intake, insulin resistance (IR) , as wellThe disease is characterized by macrovesicular fat storage in more than 5% of hepatocytes in non-drinkers or mild drinkers (ethanol consumption <30 g/day in men and <20 g/day in women) . A dysfuThe association between NAFLD and metabolic disorders is strong, with a bidirectional relationship. Insulin resistance induces an imbalance between lipid production and disposal, leading to lipid accumulation in hepatocytes, chronic low-grade inflammation, progression to NASH, and adverse hepatic and extrahepatic outcomes . T2D andTo reinforce the association between T2D and NAFLD, a recently recognized definition of hepatic steatosis in patients with such metabolic disorders has been devised, namely Metabolic Associated Fatty Liver Disease (MAFLD) . CompareThe assessment of body composition is a promising approach for studying the pathogenesis and prognosis of NAFLD in T2D patients . Low skeA healthy diet, lifestyle changes, and physical activity are the first-line therapy of NAFLD, demonstrated to improve IR, reduce systemic inflammation, promote weight loss, reduce fat accumulation, increase SMM, and, lastly, prevent negative outcomes . HoweverThiazolidinediones, glucagon-like peptide-1 receptor agonists (GLP-1 RAs), and sodium-glucose transporter type 2 inhibitors (SGLT2i) may have an interesting role as pharmacological treatment of MAFLD, since they promote loss of VAT, reduce systemic inflammation, and provide CV protection. In particular, GLP1-RAs and SGLT2i, but not thiazolidinediones, promote weight loss, an important therapeutic target in this setting. GLP-1 RAs appear to have the most promising therapeutic role in NAFLD ,23, althSemaglutide is one of the most potent GLP-1 RAs, as demonstrated by its beneficial effects on glucose control ,25, CV aThe aim of this study was to evaluate the effectiveness of diabetes therapeutic dosages of subcutaneous Semaglutide on fatty liver disease and surrogate markers of NAFLD in patients with T2D, treated once weekly for 52 weeks.This is a 52-week prospective, single-arm, real-life study. The study was carried out in the Metabolic Disorders Outpatients Clinic of the Department of Internal Medicine at the University of Bari, Italy, in accordance with the general ethical principles for medical research involving human subjects of the Declaration of Helsinki. The study protocol was formally approved by the Ethics Committee of the University of Bari .A total of 150 patients with T2D attending our clinic were screened for eligibility from 1 January to 30 June 2021. The patients were scheduled for intensification of antihyperglycemic therapy due to poor glycemic control, background cardiovascular and renal status, excess weight, or need for weight loss. Among them, 48 (32%) patients  who were willing to receive once-weekly Semaglutide subcutaneously were included in the study. Only 2 of 48 patients discontinued Semaglutide at T6 due to a relevant increase in pancreatic enzymes, and underwent further investigation.2, eligibility for GLP-1 RAs intensification according to current recommendations and guidelines , and the need to minimize weight gain or promote weight loss.Inclusion criteria were an established diagnosis of T2D, age > 18 years, stable estimated glomerular filtration rate (eGFR) > 15 mL/min/1.73 mThese included other forms of diabetes mellitus, pregnancy or lactation, inadequate compliance, or contraindications to GLP-1 RAs, previous or ongoing treatment with pioglitazone and/or SGLT2i and/or GLP-1 RAs, inadequate ability to comply with follow-up or to provide informed consent, assumption of oral contraceptives or corticosteroids, viral hepatitis B and C, and excessive ethanol consumption (more than 30 g/day in men and 20 g/day in women) .Eligible patients were fully informed about the study’s purposes and gave written consent to participate. Follow-up visits were carried out, during four scheduled appointments, at baseline (T0), and after 3, 6, and 12 months of treatment . A complete medical history was collected, and a physical examination was carried out during each follow-up visit. The clinical and anthropometric parameters analyzed were office arterial pressure, heart rate, body weight (BW), waist circumference (WC), and body mass index (BMI). Laboratory tests were prescribed according to the general principles of clinical practice and the recommended follow-up. In particular, the laboratory tests included a complete total blood count, fasting glycemia, glycated hemoglobin (HbA1c), serum creatinine with eGFR, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γGT), insulin, and C-peptide. Liver ultrasound (US) examination with the grading of hepatic steatosis was also performed.Once-weekly (qw) Semaglutide was administered subcutaneously in accordance with general recommendations and guidelines. As per good clinical practice, a dose of 0.25 mg qw was prescribed for 4 consecutive weeks and then raised to 0.5 mg qw. After six months of treatment, Semaglutide was further intensified to 1 mg qw to reinforce extra glycemic effects, especially for inducing additional body weight loss, when indicated. All participants were on metformin at the maximum tolerated dose, and Semaglutide was prescribed as an add-on. Glinides, sulfonylureas, dipeptidyl peptidase type IV inhibitors, were discontinued properly. No patient was on insulin therapy at baseline, nor was it prescribed to any patient before the end of the study.The Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) index was calculated as (fasting insulin × fasting glucose)/405, and used as an indirect measure of systemic insulin resistance .The AST to Platelet Ratio Index (APRI) score was calculated for estimating the risk of liver fibrosis and cirrhosis according to the following formula: . A value over 36 was indicative of NAFLD and a value lower than 30 should exclude it [0.953 × loge(triglycerides) + 0.139 × BMI + 0.718 × loge(γgt) + 0.053 × waist circumference − 15.745)/(1 + e0.953 × loge(triglycerides) + 0.139 × BMI + 0.718 × loge(γgt) + 0.053 × waist circumference − 15.745) × 100. An FLI lower than 30 (negative likelihood ratio up to 0.2) suggests that the presence of liver steatosis may be excluded, whereas an FLI equal to or more than 60 (positive likelihood ratio starting from 4.3) was highly indicative of liver steatosis [The Fatty Liver Index (FLI) was calculated according to the formula: FLI = , as described elsewhere . BrieflyThe hand grip (HG) strength test was performed using a manual hydraulic dynamometer  to assess the muscle strength in both hands . Three sVAT thickness was also estimated with a US method (US-VAT) using a General Electrics Logiq E9 ultrasound machine  according to a validated method . BrieflyLiver steatosis was graded on the basis of US features assessed with a General Electrics Logiq E9 ultrasound machine. First of all, according to a validated semiquantitative score, NAFLD was classified as absent (0), mild (1), moderate (2), or severe (3) based on standardized US characteristics of liver echogenicity . MoreoveThe primary study outcome was to estimate the mean changes in serum liver enzymes , validated clinical scores , and US liver appearance over the entire follow-up .Additional study outcomes included mean changes in glucometabolic control , and body weight and composition .Descriptive statistics were represented as mean, standard deviation, frequency, and percentage. Changes in the variables over time  were evaluated by Mixed Models for repeated measures, and means were estimated by the least-squares method. Pearson correlation for the association among quantitative variables was used, while the Kendall coefficient was employed when appropriate, and the chi-square test was used for the association between categorical variables. To evaluate significant changes in steatosis degree from the first to the last observation time, the McNemar Bowker test was used. Comparisons between Responder and Non-Responder patients were evaluated by unpaired t Student. The analyses were carried out on the entire study population and split by gender. Statistical analysis was performed using SAS software 9.4. .2) and WC (120 cm) suggested a high prevalence of central obesity among the study participants, who also exhibited clinically relevant insulin resistance . According to baseline BMI, patients were classified as normal weight (BMI < 25 kg/m2) only in 2.1% of the cases, overweight (25 < BMI < 30 kg/m2) in 6.2%, with mild obesity (30 < BMI < 35 kg/m2) in 27.1%, and moderate-to-severe obesity (BMI ≥ 35 kg/m2) in 64.6%  were in the normal range  as indicated in The APRI score was within the normal range (<0.5) in all. The HSI, FLI, and quantitative LSS suggested the presence of NAFLD in all patients . NAFLD wp < 0.001). To assess the internal consistency of measurements, some statistical correlations were calculated at baseline . In partBody weight loss was statistically and clinically relevant at each follow-up visit compared to baseline, with a mean loss in body weight of 7.4% at T3, 9.2% at T6, and 10.3% at T12 . SimilarAfter one year of therapy, a significant improvement in fasting glycemia, HbA1c, serum insulin, and HOMA-IR index , as wellNo significant changes were observed throughout the study period in systolic and diastolic blood pressure, heart rate, eGFR, and fasting serum c-peptide .As shown in p < 0.001). These patients were defined as Responders (R) to treatment. In the remaining 30%, namely Non-Responders (NR), the steatosis grading was unchanged; no patient got worse. Importantly, the improvement in liver steatosis was independent of gender (p = 0.16) and intensification of anti-hypercholesterolemic therapy at baseline (p = 0.11). No differences were found between R and NR with regard to age (p = 0.12) and duration of T2D (p = 0.19), or changes in BMI (p = 0.39), HOMA-IR (p = 0.28), and liver enzymes (p > 0.05) from baseline to the end of the study.Most patients (70%) achieved a significant improvement in liver steatosis severity from baseline by the end of the observation period, as expressed by at least one-class reduction in the 4-point semiquantitative US staging  was consistent with the concurrent effects of Semaglutide in reducing body weight, BMI, WC, and triglycerides, all influencing the calculation of NAFLD biometric markers. On the other hand, the clinical improvement of liver steatosis was also highlighted by the early decrease in US-assessed liver fat, which was significant soon after 3 months of therapy, and persisted up to 52 weeks. Moreover, the percentage of R patients, exhibiting at least a one-class improvement of liver steatosis severity according to the US-based semiquantitative score, was very high (80%) therefore suggesting that Semaglutide may have a relevant role in treating MAFLD in T2D. There is still debate as to whether GLP-1 RAs may improve liver steatosis directly, since the GLP-1 receptor is not expressed at appreciable levels in the liver . On thisA special consideration should be made about metformin therapy. All participants were on metformin before the study entry and received a stable dose over the follow-up. Although metformin has shown anti-inflammatory properties and potentially positive effects on hepatic steatosis and steatohepatitis, by suppressing de novo lipogenesis, enhancing fatty acid oxidation, and promoting degradation of intracellular lipids that accumulate in hepatocytes ,42 no siIn line with our results, two large clinical trials, in which NAFLD was an established comorbidity among the enrolled patients, have recently shown a dose-dependent reduction of serum ALT and high-sensitivity C-reactive protein values after treatment with Semaglutide . In a spOur study has some strengths and limitations. Strengths include the robust study design, a well-standardized and comprehensive assessment of participants, a long-term follow-up (52 weeks), and the real-life nature of the observations which, in turn, may provide useful information for clinical practice. The main limitations of the study are the small  sample size and the lack of more sensitive, specific, and accurate instrumental examinations providing a quantitative classification of hepatic fat accumulation, such as liver biopsy or magnetic resonance imaging. Based upon the latter point, minor steatosis or small changes over time in hepatic fat deposits would preferably be studied with the Controlled Attenuation Parameter (CAP) algorithm implemented in the transient elastography (FibroScan) system, and the Ultrasound-Guided Attenuation Parameter (UGAP). These imaging techniques are an accurate diagnostic tool for the point-of-care investigation of MAFLD , but theOur data show that Semaglutide improves liver steatosis in patients with T2D. Since NAFLD plays a crucial role in the pathophysiology of T2D and related cardiovascular and renal complications, Semaglutide should be considered as an optimal approach for the treatment of patients with T2D and fatty liver disease.


The discovery of melanocortins in 1916 has resulted in more than 100 years of research focused on these peptides. Extensive studies have elucidated well-established functions of melanocortins mediated by cell surface receptors, including MSHR (melanocyte-stimulating hormone receptor) and ACTHR (adrenocorticotropin receptor). Subsequently, three additional melanocortin receptors (MCRs) were identified. Among these five MCRs, MC3R and MC4R are expressed primarily in the central nervous system, and are therefore referred to as the neural MCRs. Since the central melanocortin system plays important roles in regulating energy homeostasis, targeting neural MCRs is emerging as a therapeutic approach for treating metabolic conditions such as obesity and cachexia. Early efforts modifying endogenous ligands resulted in the development of many potent and selective ligands. This review focuses on the ligands for neural MCRs, including classical ligands (MSH and agouti-related peptide), nonclassical ligands , and clinically approved ligands . Melanocortin peptides, primarily consisting of α-, β-, and γ-melanocyte-stimulating hormones  and adrenocorticotropin (ACTH), are elements of an ancient modulatory system . MelanocG protein-coupled receptors (GPCRs) have been tractable drug targets for decades, with over one-third of currently marketed drugs targeting GPCRs ,10,11. OMc3r or Mc4r or both revealed that these two neural MCRs served important nonredundant roles in regulating energy homeostasis, with MC3R regulating feed efficiency and MC4R regulating both food intake and energy expenditure .,29.1-MSH2 and α-MSH -OH). NDP-MSH has nanomolar to subnanomolar potencies in MC1R and MC3-5R , melanotan II . In facckleback ,148,149.Astyanax mexicanus) mc4r mutations contribute to physiological adaptations to nutrient-poor conditions by increasing appetite, growth, and starvation resistance -α-MSH (NDP-MSH) for binding to the MC4R, sharing several binding determinants with peptide agonists but also possessing unique binding determinants [THIQ A, a tetr 2.1 nM) . Structurminants . This smrminants . We showrminants .Scatophagus argus) [Monopterus albus) [Lateolabrax maculatus) [THIQ binds to human MC4R orthosterically, but binds to fish Mc4r allosterically. In teleosts, including spotted scat (s argus) , swamp es albus) , and spoculatus) , THIQ faIpsen 5i B, as theA recent study with two teleost Mc4r showed that Ipsen 5i serves as a neutral allosteric modulator at cAMP signaling pathway but allosteric agonists at the ERK1/2 signaling pathway . In thisThe peptidomimetic compound ML00253764 C was inicis-7-ring retinal for rhodopsin [Dysfunction in folding caused by genetic mutations in numerous genes causes protein conformational diseases . Pharmachodopsin , Org 425hodopsin , and IN3hodopsin .MC4R mutations have been identified from 141,456 humans and have been registered in gnomAD v2.1.1 database  [MC4R mutations showed that the most common defect is decreased or lack of cell surface expression [The MC4R is a critical regulator of energy homeostasis, regulating both food intake and energy expenditure . Numeroust 2022) ,184. Manpression ,185.MC4R mutation [ML00253764, the first pharmacoperone for MC4R, was identified by our team in 2009 ,186. Sinmutation ,186,187.mutation .50s are in the tens of micromolar range [It was suggested that studying nature-derived peptides confer advantages for developing GPCR ligands as drug discover effort . Full agar range .Animal venoms contain numerous chemicals with diverse biological activities. In a bank of 3597 toxins, 8% are found to interact with human MC4R . Two pepACTH is an old forgotten melanocortin peptide that might potentially be rescued and repurposed for new indications . ACTH re® Gel is the only treatment available for infantile spasm, a medical condition usually diagnosed within the first year of life, consisting of seizures and mental retardation with poor prognosis [ACTH was the gold standard therapy for multiple sclerosis in the 1960s, but its use decreased with the advent of glucocorticoid . H.P. Acrognosis ,196. A rrognosis ,198. Therognosis . It is wrognosis ,200,201.Interestingly, exactly 50 years after ACTH was approved by the FDA, Getting et al. envisaged a novel mechanism of action and discovered that the anti-inflammatory effects of ACTH persist in adrenalectomized rats using a model of knee gout . ACTH alIn addition, the ability of ACTH to directly modulate local CNS inflammation has been reported in several in vitro studies. Using rat brain cultures containing oligodendroglia, astrocytes, and microglia, preincubated with cytotoxic agents, ACTH was shown to protect mature oligodendroglia and oligodendroglia progenitor cell from death induced by staurosporine, kainate, quinolinic acid, or reactive oxygen species ,205. UsiObesity has reached epidemic proportions in developed countries and represents a major public health challenge. The pandemic of obesity is growing at an alarming rate, with an estimated direct and indirect cost of $150 billion per year in the US alone, and one out of five deaths is linked in one way or another to obesity . Obesity50 at MC4R is 0.032 nM and EC50 at MC3R is 0.69 nM) [POMC), leptin receptor (LEPR), or proprotein convertase subtilisin/kexin type 1 (PCSK1) [Over the past few decades, concerted efforts have been made to translate melanocortin ligands into clinical therapies ,135. Bot0.69 nM) . This sy (PCSK1) .MC4R deficiency [POMC and LEPR deficiency, with 80% of POMC-deficient subjects and 45% of LEPR-deficient subjects losing >10% of their weight within approximately one year [In a 28-day Phase 1b clinical trial, setmelanotide led to moderate weight loss in obese people with ficiency . The effone year ,217. In one year ,215,217.The safety of setmelanotide might be caused by differential signaling pathways ,221. SetIn addition to obesity therapies, the other potential use of MC4R agonists as therapeutics for erectile dysfunction and premenopausal hypoactivity sexual desire disorder has also been investigated, including melanotan II (University of Arizona) , bremela50 of 0.25 nM), making it possible to modulate brain pathways involved in sexual response [Bremelanotide (PT-141) B is a MCresponse ,233.The most common adverse events of bremelanotide are nausea, flushing and headache, with no marked dosage dependence ,234. MeaMC3R and MC4R are inextricably linked to obesity. However, research on the old drugs repurposing for new therapeutic uses for the melanocortin system is scarce. The FDA-approved drug fingolimod, a sphingosine 1-phosphate receptor agonist, is used in monotherapy for the treatment of relapsing-remitting multiple sclerosis ,237. RecAnother case is the marketed drug fenoprofen. Long known for the antiarthritic activities and efficacy in rheumatoid arthritis and osteoarthritis, fenoprofen is used as a nonsteroidal anti-inflammatory drug and inhibits cyclooxygenase . MonteroAnother recent study suggested that metformin, used as first-line treatment for type 2 diabetes mellitus, inhibits the activation of MC2R and MC3R . At the Numerous melanocortin ligands have been developed in the 70 years since the sequences of the first endogenous ligands were elucidated. While much of the early focus was on the development of compounds that alter pigmentation, the cloning and structure analysis of the MCRs led to the development of potent and selective ligands. Targeting neural MCRs (MC3R and MC4R) is emerging as a therapeutic approach for metabolic diseases and chronic inflammation. This review summarized the advances in the ligands for neural MCRs, including classical endogenous ligands, nonclassical ligands such as small molecules and pharmacoperones, and some ligands approved for human medicine. Development of novel nonclassical ligands and old drugs repurposed for targeting the neural MCRs to treat obesity and related diseases are of imminent importance to fulfill a highly unmet medical need in the future.


VvSGR, VvPPH2, and VvFtsH6-2 corresponded to the changes in Chl content among P, S, and ST. The changes in the transcription of VvNYC1, VvSGR, VvPAO1, VvPAO2, VvPAO4, VvPPH1, VvPPH3, and VvFtsH6-1 only contributed to low Chl levels in P. The cis-element analysis indicated that these SAGs possessed several light- and hormone-responsive elements in their promoters. Among them, ABA-responsive elements were found in twelve of the sixteen promoters of SAGs. Correspondingly, ABA-signaling components presented various changes in transcription among P, S, and ST. The transcription changes in VvbZIP45 and VvSnRK2.1 were similar to those in VvSGR, VvPPH2, and VvFtsH6-2. The other nine ABA-signaling components, which included VvRCAR2, VvRCAR4, VvRCAR6, VvRCAR7, VvRCAR2, VvPP2C4, VvPP2C9, VvbZIP25, and VvSnRK2.3, were highly expressed in P but there was no difference between S and ST, with similar expression patterns for VvNYC1, VvSGR, VvPAO1, VvPAO2, VvPAO4, VvPPH1, VvPPH3, and VvFtsH6-1. These results suggested that the senescence of P and ST could be regulated by different members of Chl breakdown-related SAGs and ABA-signaling components. These findings provide us with important candidate genes to further study the regulation mechanism of leaf senescence order in grapevine.Natural leaf senescence is an acclimation strategy that enables plants to reallocate nutrients. In the present study, interestingly, we found that the basal mature leaves of grapevine primary shoots (P) exhibited the earliest senescence, followed by the apical young leaves of secondary shoots (ST), and then the basal mature leaves of secondary shoots (S). The Chl level decreased with the extent of leaf senescence. According to the genome-wide identification and expression analysis, sixteen senescence-associated genes (SAGs) involved in Chl breakdown were identified in the grapevine genome. Their expression patterns showed that the transcript changes in  NON-YELLOW COLORING1 (NYC1) and NYC1-LIKE (NOL), in rice (Oryza sativa) [PPH gene has been identified to encode pheophytinase, which catalyzes hydrolysis reactions in Arabidopsis [HCAR (hcar) maintained green leaves during dark-induced and/or natural senescence, while HCAR-overexpressing plants exhibited more tolerance to reactive oxygen species than the wild type in rice (Oryza sativa) [pph-1 mutant showed a stay-green phenotype since it was unable to degrade chlorophyll during senescence in Arabidopsis [LpNOL RNA interference (NOLi) in perennial ryegrass (Lolium perenne L.) has been shown to significantly block Chl degradation in senescent leaves and also delayed the initiation and progression of leaf senescence [SGR homologs, i.e., SGR1/NYE1, SGR2, and SGRL. SGR1/NYE1 has been characterized to promote Chl breakdown during leaf senescence in many plants [SGRL contributes to Chl degradation in pre-senescent leaves [SGR2 negatively regulates the degradation of Chl under age-, dark-, and stress-induced senescence conditions [CCEs, chloroplast protein, and FtsHs protease, are thought to be a type of senescence-induced senescence executers (SIEs), whose high expression levels directly result in senescence syndromes [PAO and NYC1) to promote ABA-mediated Chl breakdown and leaf senescence [PYL9 over-expressing plants exhibited dramatically increased drought-induced leaf senescence and ABA-promoted leaf senescence via activating core ABA-signaling pathways (PP2Cs-SnRKs-ABFs). These genes belong to the senescence-induced senescence regulators (SIRs) group, in the context that they are activated during leaf senescence and regulate leaf senescence [Leaf senescence is a complex but orderly physiological process that enables plants to remobilize nutrients from old leaves to developing organs . It is i sativa)  and Arab sativa) . The nexbidopsis . The resbidopsis . Specifi sativa)  and acce sativa) . The pphbidopsis . LpNOL Rnescence . In addinescence ,15. The y plants , while St leaves . By contnditions . FtsHs anditions . The chlnditions . These gyndromes . Recentlnescence . Zhao etnescence  found thnescence .CitPPH was directly regulated by an ethylene response factor involved in chlorophyll degradation during citrus fruit ripening [Grapevine, a worldwide important crop with highly commercial products such as table berries, juice, and wines, presents a full seasonal senescent syndrome. Leaf senescence is tightly related to crop yield and biomass production and needs massive changes in the expression level of SAGs . A numberipening . HoweverGenerally, leaf senescence in shoots occurs gradually from bottom to top. In the present study, the bottom leaves on primary shoots, indeed, turned yellow first. Interestingly, we observed senescence in the top leaves of secondary lateral shoots prior to the bottom leaves of secondary lateral shoots during natural autumn leaf senescence A,B. The NOL , two NYE1/SGR1 (SGR and SGRL), one ACD2/RCCR (VvRCCR), one HCAR (VvHCAR), four PAO/ACD1 , three PPH , and two FtsH6 (VvFtsH6-1 and VvFtsH6-2). The exon-intron distributions of the grape SAGs presented variations among different groups , tobacco (Nicotiana tabacum), and sweet orange (Citrus sinensis) were resolved into three distinct groups. VvNYC and other NYC orthologs in Arabidopsis , tobacco (Nicotiana tabacum), and sweet orange (Citrus sinensis) clustered into one subgroup, VvNOL1 clustered together with AtNOL, while the only member comprising the third subgroup was VvNOL2. SGR orthologs of grapevine, Arabidopsis , and cabbage (Brassica campestris) were clustered in a subgroup. VvSGRL was clustered with AtSGRL , pepper (Capsicum annuum), sweet orange (Citrus sinensis), cabbage (Brassica rapa var. parachinensis), and Arabidopsis  (VvPPH4 was divided into a subgroup with characterized PPH orthologs of tobacco (Nicotiana tabacum), sweet orange (Citrus sinensis), cabbage (Brassica rapa var. Parachinensis), perennial ryegrass (Lolium perenne), and Arabidopsis , while VvPPH1 and VvPPH3 were divided into two subclusters  E. VvPPH4clusters F. VvFtsH AtFtsH8 G.NYC1, NOL2, SGRL, SGR, RCCR, HCAR, PAO1, PAO2, PAO4, PPH2, FTsH6-1, and FTsH6-2. One auxin-responsive element (AuxRR-core) was observed in promoters of PAO3 and PAO4; two MeJA-responsive elements (CGTCA-motif and TGACG-motif) presented in promoters of NOL2, SGRL, PAO3, PAO4, and PPH2. Gibberellin-responsive elements, i.e., P-box and GARE-motif, were found in different promoters of SAGs. The former was in the promoters of HCAR, PAO2, PPH2, PPH3, FTsH6-1, and FTsH6-2; the latter was in the promoters of NOL2, SGR, PAO1, PAO2, PPH2, PPH3, FTsH6-1, and FTsH6-2. TCA-element, a cis-acting element involved in salicylic acid responsiveness, was identified in the promoters of NOL2, SGRL, SGR, RCCR, HCAR, PAO3, PPH1, PPH2, PPH3, and FTsH6-1.The cis-acting regulatory elements of the SAG promoters were analyzed . Except VvSGRL and VvRCCR were characterized by high expression levels in basal leaves on the secondary shoots (S) and low expression levels in basal leaves on primary shoots (P); VvSGR, VvPPH, VvFtsH6-1, and VvFtsH6-2 presented the highest expression level in P, but the lowest expression level in S; and VvHCAR showed increasing expression from P to ST. The expression level of the other six SAGs, including VvNYC1, VvPAO1, VvPAO2, VvPAO4, VvPPH1, and VvPPH1, were obviously higher in P as compared with S and ST, while VvNOL2 presented the opposite expression pattern. VvPAO3 exhibited a significantly higher expression level in ST but no differences between P and S. The diverse expression profiles of these genes in different leaf positions could suggest the specific function of each gene during leaf senescence.To identify the causal relationships between the order of grapevine leaf senescence and the transcript abundance of SAGs involved in Chl degradation, the expression level of 16 Chl degradation-related SAGs were quantified in different leaf nodes on grape shoots . In totaVvRCAR2, VvRCAR4, VvRCAR5, VvRCAR6, and VvRCAR7. The ABA-signaling components, such as VvPP2C4, VvPP2C9, VvSnRK2.3, and VvZIP25, showed similar expression patterns. VvRCAR3 exhibited no significant differences among the three leaf positions, while VvRCAR1 was most abundant in S. VvSnRK2.3. VvZIP25 was significantly differentially expressed among the three leaf positions and presented the highest abundance in P but the lowest abundance in S. VvZIP08 showed a high expression level in ST but low expression levels in S and P.ABA is a well-known regulator of plant senescence. Twelve out of sixteen Chl degradation-related SAGs were found to contain ABA-responsive elements in their promoters. Therefore, 14 orthologs of ABA-signaling components were focused on the transcription changes in the different leaf positions of shoots . Five ouNYC1 homologs (NYC1 and NOL) [HCAR [PPH [PAO/ACD1 [RCCR/ACD2 [SGR homologs  [CBR, HCAR, PPH, PAO, RCCR, SGR, and AtFtsH6, respectively, and playing roles as senescence executers during leaf senescence. In the present study, twelve genes encoding CCEs , two encoding chloroplast-located proteases (VvFtsH6-1 and VvFtsH6-2), and two encoding chloroplast proteins (SGR and SGRL) were identified in the grapevine genome by using the above Arabidopsis orthologs as queries  present in the Arabidopsis genome, SGR1/NYE1 [SGRL [SGR2 [VvSGR increased with leaf senescence, while VvSGRL showed high abundance in pre-senescent leaves (S) but low abundance in senescent leaves (P). Therefore, VvSGR could be the ortholog of Arabidopsis SGR1, and VvSGRL could be the AtSGRL ortholog. Further tests of gene expressions and Chl changes at different nodes of leaves showed that Chl content decreased with the extent of leaf senescence  had significantly different expressions between S and ST; their expression patterns corresponded to a low level of Chl in ST and a high level of Chl in S. This could suggest a complex regulation of cellular Chl degradation during leaf senescence. Among the differently expressed CCEs, the PPH expression level has been found to positively correlate with the extent of leaf senescence during the natural leaf maturation process [CBR isoforms, NYC1 and NOL, have been suggested to have a similar function in Chl metabolism in Arabidopsis but act at different developmental stages, resulting from their different mutant phenotypes and expression patterns [VvNYC expression patterns differed considerably from VvNOL1 and VvNOL2 in different nodes of grapevine leaves  and confirmed that only the ACD1 gene encodes PAO. Using the ACD1 protein as the query sequence to blast the grapevine genome database, four VvPAO homologs were searched in the grapevine genome database. Their expression patterns indicated that all of them were involved in Chl breakdown at different nodes of leaves. Red Chl catabolite reductase (RCCR) catalyzes the conversion of red Chl catabolite (RCC) to red Chl catabolite (RCC), which is essential in Chl breakdown [VvRCCR in different-aged leaves seem not to be related to Chl breakdown  , one HCAL) [HCAR , one PPHCAR [PPH , one PAOPAO/ACD1 , one RCCCCR/ACD2 , three Snd SGRL) , and onend SGRL)  encoding queries . SignifiGR1/NYE1  and SGRLE1 [SGRL  contribuRL [SGR2  negativenescence , and sixnescence . The explow in S , which clow in S . However process . Two CBRpatterns ,11. Simie leaves . A signibidopsis ,11. Simibidopsis , a singldatabase . Its exp , two of the major binding partners of the ABA receptor (VvPP2C4 and VvPP2C9) [ABF3/4 orthologs  [VvSnRKs expressed highly in leaves (VvSnRK2.1 and VvSnRK2.3) [VvRCAR2, 4, 6, 7, VvPP2C4, 9, VvbZIP25, and VvSnRK2.3 were obviously high in P but showed no difference between S and ST, which presented similar expression patterns with six Chl degradation-related SAGs. The ZIP25 ortholog in Arabidopsis (ABF4) has been confirmed to mediate ABA-triggered Chl degradation and leaf senescence by directly activating the expression of AtNYC1 and AtPAO [VvbZIP45 and VvSnRK2.1 were similar to those of VvSGR, VvPPH2, and VvFtsH6-2, and their expression patterns corresponded to changes in Chl content in leaves with different extents of leaf senescence. The PPH orthologs in perennial ryegrass (LpPPH) have been shown to be positively related to the extent of leaf senescence and could be a direct downstream target gene of transcription factors in the ABA-signaling pathways [To explore potential SIRs regulating these SIEs, an analysis of the cis-element components of sixteen SIEs was performed. The results showed that some SIE members shared conserved core nucleotide sequences potentially recognized by abscisic acid (ABRE), auxin (AuxRR-core), methyl jasmonate (CGTCA-motif/TGACG-motif), gibberellin (TATC-box/GARE-motif/P-box), and salicylic acid (TCA-element) signaling . Among tnescence . A numbenescence ,11. HoweVvPP2C9) , three AvbZIP45) , and twoSnRK2.3)  were invSnRK2.3) . In thisnd AtPAO . The exppathways . It seemTwo-year-old vines of V. vinifera cv. ‘Huangjinmi’ were grown in potting soil conditions in a greenhouse at Yangzhou University, Yangzhou, Jiangsu Province of China . The vines were grafted on rootstock varieties ‘Beta’ (Vitis riparia × V. labrusca). Seven uniform trees were selected and 18 branches arising from the lateral buds on the primary shoot and developing into primary and secondary lateral shoots were marked and used for sampling. Six branches served as one biological replicate. Leaves at the same positions were pooled and frozen in liquid nitrogen and then stored at −80 °C.For each replicate, 0.1 g of frozen leaves was extracted, and Chl a/b content was determined using the method reported by Burnison et al. (2011) .−5 E-value as the threshold. The obtained grape SAG proteins were then aligned using a conserved domain search tool (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 19 April 2020)). Genes lacking conserved domains or that were less than 200 bp were discarded.Arabidopsis thaliana Chl degradation-related SAGs were downloaded from the leaf senescence database LSD3.0  and usedArabidopsis thaliana), tobacco (Nicotiana tabacum), sweet orange (Citrus sinensis), cabbage (Brassica rapa var. parachinensis), cabbage (Brassica campestris), rice (Oryza sativa), perennial ryegrass (Lolium perenne), maize (Zea mays), bamboo , and soybean (Glycine max) were downloaded from the leaf senescence database LSD3.0 [http://gsds.cbi.pku.edu.cn accessed on 1 May 2020) [http://meme-suite.org/tools/meme accessed on 1 May 2020) [The protein sequence of functionally characterized Chl degradation genes in Arabidopsis (e LSD3.0  and thenay 2020) . Putativay 2020) . The proay 2020) .−ΔΔCt method [Total RNA was extracted from frozen leaves using a CTAB-based method . First-st method .The Chl content and RT-qPCR results were subjected to a one-way analysis of variance and the means were compared using the Tukey test with the SPSS software 22.0. Figures were constructed using Excel 2010.VvNYC1, VvSGR, VvPAO1, VvPAO2, VvPAO4, VvPPH1, VvPPH2, VvPPH3, and VvFtsH6-1 in the former leaves. The greater degree of Chl degradation in the apical young leaves of secondary shoots than that in the basal mature leaves of secondary shoots was related to the higher expression levels of VvSGR, VvPAO3, VvPPH2, and VvFtsH6-2 in the former leaves. ABA-responsive elements were mostly identified in the promoters of these SAGs. Further analysis of the expression pattern of ABA-signaling components suggested that ABA signaling was involved in regulating the order of leaf senescence, but the regulation mechanism of ABA-mediated Chl degradation could be different in different nodes of leaves. Further studies are required to fully understand the mechanism of the different extents of leaf senescence in different leaf orders of grapevine leaves.In this work, we found that the grapevine leaves on primary and secondary shoots aged in a certain order in autumn. The Chl level decreased with the extent of leaf senescence and sixteen Chl breakdown-related SAGs were identified in grapevine leaves. The greater degree of Chl degradation in the basal mature leaves of grapevine primary shoots than that in the basal mature leaves of secondary shoots was associated with higher transcript levels of