Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), β1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-β) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell–cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells. Colorectal cancer (CRC) has been categorized worldwide as the third most common diagnosed cancer and the fourth most usual cause of cancer death . The etiAmong the post-translational modifications differentially identified in CRC tissues, mechanisms of glycosylation have been extensively studied. Alterations of glycan biosynthesis associated with abnormal glycosylation patterns of several molecules have been related with oncogenesis, cell growth, apoptosis inhibition, increased motility, cell migration, cell adhesion, tumor cell invasiveness and metastasis. Glycans act as interface molecules between cells and microenvironment and in several cases glycan structures confer functions to the molecules. Thus, tumorigenesis and metastasis could be narrowly associated with glycan profile changes in cancer cells ,6,7,8.In cancer tissues, glycan expression has been found upregulated, downregulated and, interestingly, glycan structures have been shown to be truncated or modified exhibiting new structures ,6. Thus,Glycosylation of molecules, such as proteins or lipids, takes place in the endoplasmic reticulum and Golgi apparatus in a process catalyzed in a non-templated manner by glycosyltransferases, such as fucosyltransferases, sialyltransferases, and N-acetylglucosaminyltransferases in association with glycosidases. Proteins can be N-glycosylated and O-glycosylated and these variations determine, in part, their physical and functional properties. The considerable weight of lipid glycosylation in biology has also been demonstrated ,10,11. ADownregulation or upregulation of glycosyltransferase expression, unavailability of glycosylation cofactors or substrates, changes in glycosyltransferase subcellular localization and mutations in genes encoding glycosyltransferases have been evidenced in cancer samples. These features generate aberrant glycosylation signatures that are related with tumorigenesis, metastasis and even with chemoresistance ,17.Accordingly, mutations in glycosyltransferases and glycosylation pathways associated genes, as well as altered gene expression coding glycosyltransferases have been described for different CRC cell lines and tumor tissues from several stages . AdditioIn this review, we intend to summarize the role of glycosyltransferases and glycosylation profile in CRC pathogenesis and discuss their therapeutic implications.The expression pattern of glycosyltransferase genes plays an important role in the biology of CRC cells .Among the genes coding for glycosyltransferases, observed in CRC, 16 have been found upregulated  and 16 have been found downregulated   22,23,2,30,31,32The upregulation of genes coding for beta 3 and beta 4 glycosyltransferases , fucosyltransferases , mannosyl-glycoprotein N-acetylglucosaminyltransferases (MGAT4B and MGAT5A), O-linked N-acetylglucosaminyltransferases (OGT) and sialyltransferases (ST6GAL1 and ST6GALNAC1) plays a crucial role in tumor cell proliferation, survival, induction of stem-like cell properties, epithelial–mesenchymal transition (EMT), metastasis and resistance to chemotherapy and radiotherapy .On the other hand, the downregulation of glycosyltransferases expression in CRC cells, such as beta 3-glycosyltransferases (B3GNT1 and B3GNT6), polypeptide N-acetylgalactosaminyltransferase (GALNT6), glucosaminyl (N-acetyl) transferases/xylosyltransferases (GCNT3), fucosyltransferases (FUT9), mannosyl-glycoprotein N-acetylglucosaminyltransferases  and sialyltransferases  has been also associated with tumor progression, tumor growth, cell proliferation, adhesion, migration, invasion and chemoresistance to 5-Fluorouracil (5-FU) .Regarding beta 3-glycosyltransferases, the role of β3GnT8  in CRC cell metastasis could be explained by decorating CD147 with β1,6-branched polylactosamine structures ,27,33. CThe upregulation of mannosyl-glycoprotein N-acetylglucosaminyltransferases, observed in CRC cells has implications in tumor progression and metastasis . The oveAlterations in the addition of fucose to precursor glycan structures promote cell proliferation, migration, adhesion to extracellular matrix, invasiveness, metastasis, maintenance of CSCs, TGF-β-induced EMT and multidrug-resistance (MDR) ,43,44,45Sialylation is also a process related to CRC. Modifications in the expression of sialyltransferases are associated with cancer cell survival, proliferation, migration, invasion, metastasis, induction of stem-like cell properties, chemotherapy resistance, increased sialyl-Tn antigen expression, inflammation-driven carcinogenesis and resistance to 5-FU. In this respect, overexpression of ST6GAL1 and ST6GALNAC1 has a role in cancer cell stemness, cell survival, metastasis and chemoresistance ,49,50,51Accordingly, changes in glycosyltransferase gene expression evidenced in carcinogenesis show consequences in glycosyltransferase joint activity and, therefore, in the cell glyco-code. Some of these alterations affect several molecular functions related with tumorigenesis, metastasis and cancer progression.Glycosylation is associated with several biological processes. Alterations in glycosyltransferase levels and glycosylation patterns have been evidenced in inflammatory conditions, tumorigenesis and metastasis ,8,65,66.2+ signaling and the regulation of other ion channels among other cellular processes . HS. HS20]. Glycosylation of mucins has been extensively studied in cancer tissues. In this regard, MUC1 is highly glycosylated in CRC tissues and the O-glycosylation pattern of MUC2 is altered  19,81].,81.19,81Adhesion molecules, such as selectin, integrin and their respective ligands, have an essential role in tumor cell endothelial adhesion, migration and organ invasion . ChangesRegarding the immune response, alterations of the glycosylation profile of mucins or other molecules affect tumor cells interactions with human lectin receptors on antigen presenting cells (APCs), such as MGL , DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and galectin-3. In CRC tissues, aberrant glycosylation of MUC1 and other CRC antigens, such as carcinoembryonic antigen (CEA), can alter the mammalian lectin receptor interactions with tumor cells and could participate in immune modulation and immune evasion  81]. In. In81]. Additionally, in CRC, CEA shows high levels of Lewis antigens such as Lewis X and Lewis Y. These carbohydrates mediate the binding of CEA with DC-SIGN which is expressed mainly on immature dendritic cells. The interaction of tumor cells with immature dendritic cells through DC-SIGN could be related with immune tolerance due to the fact that immature dendritic cells are not effective in priming naive T cells. Thus, recruitment of immature dendritic cells by DC-SIGN through Lewis antigens decorating CEA can arrest dendritic cell differentiation towards a mature phenotype, thereby contributing to tumor cell immune evasion  81,91].,91.81,91Aberrant glycosylation of the epidermal growth factor receptor (EGFR) has been widely studied in CRC cells . N-linkeFurthermore, insulin-like growth factor-binding protein 3 (IGFBP3) has shown an altered glycosylation pattern in serum and tumor cell membrane samples isolated from CRC patients. In CRC, IGFBP3 has been found decorated with high amounts of α2,6 Sialic acid and low levels of Fuc and GlcNAc moieties . The maiIn CRC tissues, altered glycosylation patterns of glycosphingolipids have been described  10]. Gl. Gl10]. As previously mentioned, CRC is considered one of the most lethal cancer with a high global prevalence . CurrentChemotherapy agents are varied and there are regimens including only one agent or a combination of effective drugs that affect one or several targets . Indeed,Modulation of glycosyltransferase expression by drugs has been studied for CRC. The expression of GCNT3 gene, which encodes the enzyme mucin-type core 2 1,6-N-acetylglucosaminyltransferase (C2GnT-M), has been found downregulated in bad prognosis CRC tissues . In thisRegarding tumor cell epitopes, it has been described that the altered glycosylation generates neo-antigens that become targets for tumor-specific T cells . TherebyAccording to CRC, O-GlcNAcylation has been shown to be elevated in CRC due to the Warburg effect and it has been recognized as an axis for the development and progression of metastasis . CRC celAs previously stated, alterations in glycosylation patterns, mediated by the modified glycosyltransferase expression, are involved in CRC malignant transformation ,140. TheIt has been shown that BGJ398, an OGT inhibitor, can significantly inhibit the activity of core 1 O-Glycan T-Synthase (C1GALT1) that catalyzes the transfer of Gal from UDP-Gal to GalNAc-alpha-1-Ser/Thr of cell surface proteins, such as the fibroblast growth factor receptor 2 (FGFR2), resulting in a significant decrease in the invasive capacity of CRC cells . These dRegarding tumor immune evasion, it has been described that KYA1797K, a β-catenin inhibitor, stimulates the immune response through the regulation of glycosyltransferase expression. The β-catenin and dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3 (STT3) are overexpressed in CRC. The expression of STT3 is upregulated by β-catenin. Furthermore, upregulation of both proteins correlates with CRC’s worsened prognosis. In this regard, KYA1797K mediates suppression of β-catenin expression with the subsequent downregulation of STT3. STT3 downregulation decreases the stability and the immunosuppressive activity of programmed death receptor 1 (PD-L1) through the inhibition of PD-L1 glycosylation and consequently increases the population of CD8+ T cells and the expression of granzyme B. Therefore, the inhibition of PD-L1 glycosylation reduces the immune evasion through blocking Wnt/β-catenin/STT3 signaling pathway in CRC cells  106]..106].The use of glycans as target molecules for immunotherapy has been extensively studied in cancer cells. Glycan-based cancer vaccines are used to decorate tumors with glycan structures, such as Lewis antigens, in order to enhance the presentation of tumor antigens by APCs, including dendritic cells internalization and antigen presentation, thereby improving tumor-specific T cell activation  90]. Ad. Ad90]. Drug resistance has been associated with aberrant glycosylation profiles. Hamaguchi et al. used glycosylation inhibitors in order to study the relationship between N-glycosylation and drug resistance. Specifically, they observed an effect of swainsonine, an α-mannosidase 2 inhibitor which blocks Golgi oligosaccharide processing, generating altered N-glycan structures that affect 5-FU mechanisms of resistance in CRC cell lines . In the All these considerations highlight aberrant glycosylation as a crucial issue in CRC treatment and point out some potential targets to improve and design specific therapies.Glycosylation is a regulatory mechanism implicated in a number of physiological and pathological processes. The mechanisms of glycosylation are related with several biological events such as membrane integrity, cellular interactions and cell proliferation. In cancer cells, glycosylation has been related to cell migration, chemoresistance, epithelial-to-mesenchymal transition, metastasis, reduced apoptosis, tumor growth and poor cell differentiation. In CRC, alterations in glycoprotein profiles, such as increased sialylation or branched glycan structures and overexpression of fucosylation, have been extensively found. Thereby, characterization and analysis of glycoconjugates have great potential to improve early diagnosis and therapy designs based on new therapeutic targets.


O-(6-O-p-coumaroyl) glucosides accumulated in fruit flesh treated with glucose, sucrose, sorbitol, and fructose in vitro. Two key structural genes of the anthocyanin biosynthesis pathway, namely, PpDFR and PpUFGT, were upregulated in the flesh of sugar-treated peach fruit. By contrast, the two main transcription factors (TFs) PpMYB10.1 and PpBL regulating anthocyanin biosynthetic genes in peach fruit were not upregulated accordingly. Interestingly, two MYB family genes (PpMYB6 and PpMYB44-like) and three bHLH family genes  were upregulated. A dual luciferase assay revealed that PpMYB6 strongly activated the PpUFGT promoter when it was co-infiltrated with PpbHLH35, PpbHLH51, and PpbHLH36-like. When PpMYB44-like was co-infiltrated with PpbHLH35, it also potently activated the PpUFGT promoter. The results of this study help clarify the molecular mechanisms by which glucose, sucrose, sorbitol, and fructose regulate anthocyanin accumulation in peach fruit.Anthocyanins are important pigments in peach fruit and are beneficial to human health. Sugars are both energy-storing and signaling molecules and their roles in inducing anthocyanin biosynthesis have received a great deal of research attention. However, the mechanism by which sugars induce anthocyanin biosynthesis in peach fruit is unknown. In order to understand this induction mechanism, comprehensive transcriptome and metabolome were performed in fruit flesh treated with four different sugars for 12 and 24 h, respectively. Here, we found that cyanidin-3- Anthocyanins are flavonoid end-products and are abundant in leaves, flowers, fruits, and seeds. Their colors range from red to blue-purple in these plant organs ,2. The aS-transferase (GST) [Anthocyanins are synthesized in the cytoplasm and then vacuolated via glutathione S-transferase (GST) . Anthocyse (GST) .Anthocyanin biosynthesis is regulated at the transcriptional level by R2R3-myeloblastosis (MYB), basic helix–loop–helix (bHLH), and WD40 that form the MBW complex . In applClematis pitcheri cultured in the presence of high sucrose concentrations and nitrogen accumulated high anthocyanin levels [Sugars are both energy-storing and signaling molecules and their roles in promoting anthocyanin biosynthesis have received extensive research attention. The shoots of n levels . Exogenon levels . Glucosen levels . Strawben levels . In Arabn levels .PpPAL, PpUFGT, PpCHI, PpF3H, PpDFR, PpANS, and PpCHS reached their highest transcription levels during the early-to-middle stages of fruit development [PpBL mapped atop linkage group five. PpBL and MYB10.1 promote the transcription of the anthocyanin biosynthesis-regulating genes which leads to anthocyanin accumulation in blood-flesh fruit [bf) mapped to linkage group four [Anthocyanins accumulate in two ways during peach fruit development. First, peak anthocyanin content occurred at the late stages of fruit development in the blood-flesh peach cultivars ‘Beijingyixianhong’, ‘Wuyuexian’, and ‘Tianjin Shui Mi’. The associated anthocyanin biosynthesis-encoding genes elopment ,23. Secoelopment . Both ansh fruit ,26. The Glucose, sucrose, sorbitol and fructose are the major sugar compositions in peach fruit . Sugar iPpMYB6 (Prupe.5G065500), PpMYB44-like (Prupe.8G134900), and three bHLH family genes: Pp bHLH35 (Prupe.1G074400), Pp bHLH51 (Prupe.2G252600) and Pp bHLH36-like (Prupe.3G131500). ). Functional analysis indicates that these TFs have the potential in promoting anthocyanin biosynthesis in four different sugar treated fruits. This will help clarify the molecular mechanisms by which glucose, sucrose, sorbitol, and fructose regulate anthocyanin accumulation in peach fruit. PpMYB6PpMYB44-likePpbHLH35PpbHLH51PpbHLH36-likeIn summary, the anthocyanins biosynthesis and regulation have been studied very thoroughly in peach and other species. Moreover, sugar as signaling molecules also have been received extensive attention in promoting anthocyanin biosynthesis in many horticultural crops. However, few studies have been focused on peach anthocyanins induction by exogenous sugars and the corresponding induction mechanism. In the present study, we mainly focused on how different sugars induce anthocyanins accumulation in peach fruit flesh. We subjected the blood-flesh peach fruit cultivar ‘Tianjin ShuiMi’ to various sugar treatments. We found two MYB family genes: There were 24,228 metabolites in the flesh of peach fruit treated with sugars. Of these, 3923, 3091, 4038, and 3938 were upregulated, while 7537, 7699, 8067, and 7807 were downregulated in the flesh of peach fruit treated with glucose, sucrose, sorbitol, and fructose, respectively, for 12 h. Moreover, 5023, 5062, 4952, and 5490 were upregulated while 7602, 7444, 7734, and 7870 were downregulated in the flesh of peach fruit treated with glucose, sucrose, sorbitol, and fructose, respectively, for 24 h . The topO-(6-O-p-coumaroyl) glucoside were upregulated markedly in flesh samples of peach fruit treated with glucose, sucrose, sorbitol, and fructose for 12 h and 24 h. The p-coumaroyl CoA, chalcone, and cyanidin 3-O-glucoside levels were lower in the peach fruit treated with the sugars for 12 h and 24 h than they were in the control. All other metabolites had similar levels in both the sugar-treated and control peach fruit. These results indicated that glucose, sucrose, sorbitol, and fructose all had similar effects on metabolite accumulation in vitro. Moreover, after four sugars treatment for 12 and 24 h, anthocyanins were not accumulated as cyanidin 3-O-glucoside, the main components in blood-flesh peach fruits, but as cyanidin-3-O-(6-O-p-coumaroyl) glucoside.As the sugar-treated fruit was picked from the ‘Tianjin Shui Mi’ blood-fleshed peach landrace, we focused mainly on flavonoids in the anthocyanin biosynthetic pathway. PpDFR and PpUFGT were reported to be upregulated by the transcription factors PpMYB10.1 and PpBL, and their expression levels were also significantly higher in the flesh of the sugar-treated peach fruit compared with those of the control  glucoside rather than cyanidin 3-O-glucoside. After analyzing the transcriptome data, we found that there were three acyltransferase genes which might be associated with the acylation of ayanidin- 3-O-glucoside . In Arabidopsis, AtPAP1, AtPAP2, AtMYB113, AtGL3, AtEGL3, and AtTT8 positively influenced anthocyanin biosynthesis ,17. In ad PpUFGT ,26. In tynthesis ,32,33. TCAB1 (chlorophyll a/b-binding protein), PC (plastocyanin), and rbcS  [ATB2/AtbZIP11 (leucine zipper (bZIP)-type transcription factor) translation via an open reading frame (ORF) encoding 42 amino acids. By contrast, glucose and fructose are relatively less effective in this process [Arabidopsis uses different signal transduction pathways for sucrose and neutral sugars such as glucose and fructose. Glucose signaling molecules are sensed by the hexokinase HXK1 in Arabidopsis. HXK1 regulates the expression of sugar-related genes such as subunit) . Sucrose process . Sucrose process . HoweverPpUFGT. Hence, the foregoing TFs have positive influences on anthocyanin accumulation. However, the key regulatory genes PpMYB10.1 and PpBL [PpDFR and PpUFGT. Thus, we propose that the regulatory mechanism of anthocyanin accumulation differs between fruits subjected to exogenous sugar treatment in vitro and those that naturally ripen on a tree.As signaling molecules, sugars control regulatory genes associated with anthocyanin biosynthesis . In Araband PpBL ,26 were Based on the foregoing findings, we propose a model for anthocyanin accumulation in the flesh of peach fruit treated with glucose, sucrose, sorbitol, and fructose in vitro .The 14-year-old blood-fleshed peach cultivar ‘Tianjin Shui Mi’ was used in this study. It is an ancient Chinese landrace and contains abundant of anthocyanin compared with other cultivars. The anthocyanin content of this landrace is about 50 times that of other main cultivars. The trees were normally cultivated and managed. Ten fruits distributed along the outer crown of each tree were promptly picked at 80 d post-anthesis to measure their anthocyanin content in response to sugar treatment.The fruit flesh was cut into small pieces (2 mm × 2 mm × 2 mm) and incubated at room temperature (25 °C) in 2-(4-morpholino)ethanesulfonic acid (MES) culture medium (pH 6.5) containing various sugars  (Solarbio Beijing China). The control was sugar-free MES medium. The MES medium consisted of 100 mM MES (pH 5.5), 5 mM CaCl2, 1 mM EDTA, 10 mM vitamin C (ascorbic acid), and 100 mM glucose, sucrose, fructose, or sorbitol. After 12 h and 24 h, peach fruit flesh was immersed in liquid nitrogen and stored at −80 °C.g for 20 min and the supernatant was transferred to a new EP tube. The EP tube was placed in the LC-MS (liquid chromatograph-mass spectrometer) system  for reversed-phase separation. The column oven temperature was kept constant at 50 °C, the flow rate was 0.4 mL/min, and the mobile phase consisted of solvent A (water + 0.1% (v/v) formic acid) and solvent B (acetonitrile + 0.1% (v/v) formic acid). The gradient elution conditions were 100% phase A, 0–2 min; 0–100% phase B, 2–11 min; 100% phase B, 11–13 min; and 0–100% phase A, 13–15 min. The sample injection volume was 10 μL [Three replicates of the flesh from sugar-treated and the control peach fruit were used in the metabolomics analysis. Twenty-five milligrams of peach fruit flesh were placed in a 1.5 mL centrifuge tube  containing 800 μL of an aqueous methanol solution and pulverized in TissueLyser (QIAGEN Shanghai China) with a steel ball at 55 Hz for 4 min. The powder was then centrifuged at 30,000× as 10 μL .OF; Waters Corp.). The Q-TOF (quadrupole time-of-flight) was used in both positive and negative ion modes. The cone voltages were set to 3 kV–40 V and 1 kV–40 V for the positive and negative ion modes, respectively. The TOF mass arrangement was 50–1200 Da and the scan time was 0.2 s. For MS/MS detection, all precursors were fragmented using 20–40 eV and the scan time was set to 0.2 s. The MS data were acquired in centroid MSE mode and identified according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Metabolites differentially expressed between the sugar treatments and the control were selected based on the parameters fold-change (sugar treatment/control) > 1.2 or < 0.8333 [The metabolites eluted from the column were detected by mass spectrometry , treated with RNase-free DNAase , and reverse-transcribed with a Supremo III RT kit . Total RNA concentration and purity were assessed with an Agilent Bioanalyzer 2100 Bioanalyzer  and NanoDrop 2000 , respectively.Oligo (dT)s were used to enrich mRNA from 5 mg total RNA. The mRNA was randomly fragmented with fragmentation buffer and first-strand cDNA was synthesized with random hexamers. Double-stranded cDNA was then synthesized with dNTPs, RNase H, and DNA polymerase I. The double-stranded cDNA was enriched by adding poly-(A)s and PCR amplification. The enriched cDNA was linked to a vector, which was used to construct sequencing library, and analyzed in an Agilent Bioanalyzer 2100 system (Agilent Technologies). The cDNA library sequencing was performed in a HiSeq 2500 system . All peach fruit flesh samples were sequenced in three biological replicates. The low-quality reads were removed and the high-quality data were aligned to the peach reference genome (Lovell 2.0) with TopHat2 using its default parameters [O) annotations were analyzed according to Blast2GO [O [Magnetic beads with rameters . The genBlast2GO  and WEGOst2GO [O . DifferePp.UFGT from ‘Tianjin Shui Mi’ was synthesized at Beijing Liuhe Bgi Co. Ltd.  and infused into pGreenII 0800LUC vector [PpUFGT was taken from GDR database (Available online: https://www.rosaceae.org (accessed on 12 July 2021)). The coding sequences (CDS) of PpMYB6, PpMYB44-like, PpbHLH35, PpbHLH51, and PpbHLH36-like selected from the transcriptome analysis were also synthesized at Beijing Liuhe Bgi Co. Ltd. and inserted into the pBI121 vector under the control of the 35 s promoter. The GUS gene was infused into the pBI121 vector as a negative control [OD to 0.4–0.6 with infiltration buffer comprising 0.5 M MES, 1.0 mM MgCl2, and 1.0 μM acetosyringone. The suspension was injected into three young leaves per tobacco (Nicotiana benthamiana) plant. The leaves injected with agrobacteria were excised after 2.5 d, immersed in D-luciferin sodium salt solution for 10 min, and placed in a multifunctional imaging analysis system  to measure luminosity.The promoter sequence of C vector ,46. The  control . All recPpUFGT) in the flesh of peach fruit treated with exogenous glucose, sucrose, sorbitol, and fructose in vitro.‘PpMYB6 and PpbHLH35′, ‘PpMYB6 and PpbHLH51′, ‘PpMYB6 and PpbHLH36-like’, and ‘PpMYB44-like and PpbHLH35′ might participate in anthocyanin accumulation via up-regulating key structural gene (


Currently, there are limited treatment options for patients who developed resistance to osimertinib, a third-generation epidermal growth factor receptor (EGFR) inhibitor. Resistance to EGFR inhibitors is frequently associated with enhanced vascular endothelial growth factor (VEGF) levels. This multicenter, retrospective study aimed to evaluate the efficacy of the combination treatment with apatinib and osimertinib in 39 patients with EGFR-mutant non-small cell lung carcinoma (NSCLC) who developed osimertinib resistance. The patients received the combination of oral apatinib 250 mg qd and osimertinib 80 mg qd. The efficacy was evaluated after the first month then every 2 months thereafter. The primary endpoint was progression-free survival (PFS). The overall response rate (ORR) and the disease control rate (DCR) of the combination of apatinib and osimertinib was 12.8% (5/39) and 79.5% (31/39), respectively. The median PFS was 4 months [95% confidence interval (CI): 3.5–4.5 months]. Fourteen patients were administered with at least 6 months of combination therapy, and 11 of them remained on treatment programs. The 6-month PFS rate was 38%. Nine patients underwent biopsies after failing osimertinib treatment, and five of six patients with TP53 mutations had PFS of less than 3 months. The spectrum of resistance to osimertinib mechanisms included c-mesenchymal-epithelial transition factor (c-Met) amplification, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gain-of-function mutation, phosphatase and tensin homolog (PTEN) loss-of-function mutation, Erb-B2 receptor tyrosine kinase 2 (ERBB2) amplification, and insulin-like growth factor 1 receptor (IGF1R) mutation. The most common adverse events were hypertension , diarrhea , and proteinuria . The combination of apatinib and osimertinib improved the ORR and the DCR of patients with osimertinib-refractory EGFR-positive NSCLC, thus making it a reasonable treatment choice after the development of osimertinib resistance. Osimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), can selectively block mutations that are sensitive to EGFR-TKI. It is resistant to EGFR T790M mutation. In patients with non-small cell lung carcinoma (NSCLC) who had EGFR T790M mutations that are resistant to both first- and second-generation EGFR-TKIs, it can help achieve a median progression-free survival (PFS) of approximately 10 months . HoweverResistance to EGFR inhibitors is frequently associated with enhanced vascular endothelial growth factor (VEGF) levels. Dual inhibition of the VEGF receptor (VEGFR) and EGFR signaling pathways shows the potential to overcome osimertinib resistance . Apatinin = 24), Second Affiliated Hospital of Zhejiang University School of Medicine (n = 9), Beijing Chest Hospital (n = 4), and Peking University Third Hospital (n = 2), between March 1, 2018 and November 1, 2019  4.0 classification.n = 9) enrolled in the study was subjected to several biopsies (liquid or tumor) once osimertinib resistance progressed and prior to the administration of apatinib. We obtained a biopsy of the tumor of a patient from a lesion, after which we performed targeted next-generation sequencing (NGS) according to previously described methods (n = 6), pleural effusion (n = 1), and cerebrospinal fluid (n = 1).A subgroup of the patients  was used to calculate and produce the results of all statistical analyses performed in this study. Of the 39 patients, 18 were male and 21 were female. Thirty patients were never smokers, while nine of them were current or former smokers. Every patient was a carrier of EGFR mutations; and according to their TKI-naïve samples, there were 16 exon 19 deletion, 17 exon 21 L858R mutation, and 6 exon 18 G719X mutation cases. In total, 4, 25, and 10 patients received osimertinib as first-, second-, and later-line treatment, respectively. The median PFS for osimertinib was 8 months (95% CI: 7.1–8.9). NGS data from 9 patients who developed osimertinib resistance were available for analysis. The clinical characteristics of the patients are listed in n = 1), PIK3CA gain-of-function mutation (n = 1), PTEN loss-of-function mutation (n = 1), ERBB2 amplification (n = 1), and IGF1R mutation (n = 1).Rebiopsy for NGS was performed in nine patients during osimertinib treatment and prior to the combination treatment. We found that of all somatic variations in nine patients, mutations related to EGFR sensitivity were the most common. TP53 mutation was found in seven of nine patients (77.8%), whereas T790M mutation was identified in four of nine patients (44.4%). The spectrum of resistance to osimertinib mechanisms included c-Met amplification , and the remaining 14 patients (35.9%) were still being treated with combination therapy . The medn = 3), hand–foot syndrome (n = 3), hoarseness (n = 2), thrombocytopenia (n = 2), and liver dysfunction (n = 1). Notably, a patient achieved partial response; however, the combination therapy was terminated because of tachycardia and markedly decreased left ventricular ejection fraction , diarrhea , and proteinuria . Other less common adverse events included rash , MET amplification, aberrations in downstream signaling pathways (mutations in the RAS and RAF genes), and epithelial–mesenchymal transition . For patin vitro and in vivo conditions . XY, JZo, HC, BJ, and JW: analysis and interpretation of data . XY, YX, LX, LL, and JZa: writing, review, and/or revision of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JD declared a past co-authorship with several of the authors XY, JZo, HC, JZa, TA, and YW to the handling editor.


TNF, MMP9, and TLR4. Drug–gene network revealed that chrysin targeted more genes, such as TNF and MMP9. JMJD1C-AS-hsa-miR-204-HDAC4/SIRT1 and LINC01144-hsa-miR-128-ADRB2/TGFBR3 regulatory axes were found from ceRNA network. From these networks, 125 candidate RNAs were obtained. Of which, an optimal 38 RNAs signatures  were identified to construct a Support Vector Machine classifier. The predictive value of 38 biomarkers had an AUC of 0.990. Similar predictive performance was found in external validation dataset (AUC of 0.845). Our findings provided the potential for 38 RNAs to serve as biomarkers for predicting the risk of SAH. However, their application values should be further validated in clinical.This study aimed to investigate the molecular mechanism under the pathophysiology of subarachnoid hemorrhage (SAH) and identify the potential biomarkers for predicting the risk of SAH. Differentially expressed mRNAs (DEGs), microRNAs, and lncRNAs were screened. Protein–protein interaction (PPI), drug–gene, and competing endogenous RNA (ceRNA) networks were constructed to determine candidate RNAs. The optimized RNAs signature was established using least absolute shrinkage and selection operator and recursive feature elimination algorithms. A total of 124 SAH-related DEGs were identified, and were enriched in inflammatory response, TNF signaling pathway, and others. PPI network revealed 118 hub genes such as  IA is cBASP1, CEBPB, ECHDC2, GZMK, KLHL3, and SLC2A3, were determined as biomarkers to assess the progression and rupture of IAs [MALAT1 expression was independently associated with the poor overall survival for IAs, and the overexpression of MALAT1 predicted an higher risk of death in IA patients [miR-16 and miR-25) may be novel biological markers that are useful in assessing the likelihood of IA occurrence [With the development of bioinformatics, gene expression profiling has been widely used to identify the biomarkers for the diagnosis and treatment of SAH . Wang ete of IAs . Interese of IAs . Besidese of IAs . For inspatients . Circulacurrence . Unfortucurrence ,15. EspeIn the present research, we aimed to screen the SAH-related RNAs as biomarkers to provide new insights for the early screening, diagnosis, and treatment of SAH. For this aim, GSE36791  and GSE722.1Homo sapiens” were acquired in the GEO database as of 2 January 2021. For the purpose of this research, the dataset screening criteria were as follows: (1) blood samples; (2) samples of SAH patients and controls; and (3) the total number of samples >50. There were two datasets meeting the screening criteria, GSE73378 [The microarray datasets searched by terms of “subarachnoid hemorrhage” and “GSE73378  and GSE3GSE73378  datasets2.22 FC| > 0.263 (1.2 fold changes). Euclidean distance-based two-way hierarchical clustering analysis [In GSE73378 dataset, the differentially expressed mRNAs (DEGs), miRNAs (DEMs), and lncRNAs (DELs) from SAH samples and normal samples were analyzed using the limma package (Version 3.34.7)  in R 3.6analysis ,19 was panalysis  in R 3.62.3http://software.broadinstitute.org/gsea/index.jsp) [SAH-associated genes were downloaded from DisGeNET database  by term dex.jsp)  to furthdex.jsp) ,24. FDR 2.4Interactions among the protein-coding genes in SAH-related DEGs were retrieved from the STRING database (Version 11.0)  with PPI2.5Connectivity Map (CMap) resource was created to connect human diseases with the genes that underlie them and drugs that treat them. CMap is the first installment of a reference collection of gene-expression profiles from cultured human cells treated with small bioactive molecules, for uncovering the functional connections among diseases, genetic perturbation, and drug action ,28. The 2.6The connection relationship between DELs and DEMs was constructed by the DIANA-LncBase v2 database , and the2.7https://cran.r-project.org/web/packages/lars/index.html) [https://cran.r-project.org/web/packages/caret) [All RNAs  contained in these three networks were used to screen characteristic RNAs by two different algorithms: least absolute shrinkage and selection operator (LASSO) and recursive feature elimination (RFE). In brief, R 3.6.1 lars package   was useds/caret)  was also2.8https://cran.r-project.org/web/packages/e1071) [https://cran.r-project.org/web/packages/pROC/index.html) [We first extracted the expression of the optimal feature RNAs from GSE73378 dataset and GSE36791 dataset. Their expression levels in SAH and normal samples were displayed. Afterward, the Support Vector Machine (SVM) from R 3.6.1 e1071   was usedex.html)  to calcu33.12 FC| > 0.263 and FDR < 0.05. Of which 228 RNAs were upregulated, and 435 were downregulated  were identified based on the cutoff value of |logegulated . The pheegulated .3.2From the DisGeNET database, a total of 470 genes associated with SAH were obtained. Then, GSEA was performed for all genes in SAH with the reference gene sets of SAH-associated genes from DisGeNET, and a total of 354 SAH-related genes were obtained . Next th3.3TNF, AKT1, TP53, MMP9, TLR4, STAT3, IL1B, TLR2, MYC, and CXCR4, were screened with the highest degree.The SAH-related DEGs were entered into the STRING database, and a total of 830 PPI networks were generated. The PPI network, including 118 gene nodes, was constructed as shown in 3.4R| > 0.75 and P < 0.05. The drug–gene pairs related to these 23 chemicals were further selected from the CTD database, and a total of 22 drug–gene pairs were obtained to establish a drug–gene network , indicating that LINC01287 – hsa-miR-204 – STAT3 was a potential important ceRNA regulatory axis.Using the DIANA-LncBase v2 database, seven lncRNA–miRNA interactions involving three miRNAs and five lncRNAs with the negative correlation of their expression levels were obtained. Then, the target genes were predicted for 3 miRNAs in lncRNA–miRNA interactions, and then the target genes were filtered by SAH-related DEGs, and a total of 21 pairs of miRNA–mRNA connections were found. The ceRNA network was established via integration with lncRNA–miRNA and miRNA–mRNA interactions . The ceR network , and wea3.6JMJD1C-AS1 and LINC01144), 1 miRNA (hsa-miR-510), and 35 genes  (JMJD1C-AS1 and LINC01144), hsa-miR-510, and mRNAs (KLF4 and TRPM4) showed no statistical difference on their expression levels between SAH and normal samples , a total of 90 RNAs and 52 RNAs were obtained using LASSO and RFE, respectively . Further others) . The exp others) . While,  samples .To validate the diagnostic ability of the optimal characteristic RNAs signature, the SVM classifier were constructed in GSE73378 dataset, which showed well predictive value for SAH patients with an AUC of 0.990 . The pre4This study aimed to discover effective diagnosis biomarkers for SAH by the analysis of sequencing data, which have the potential to guide future clinical and basic medical studies. In the present research, we first identified 621 DEGs, of which 124 SAH-related DEGs were obtained using DisGeNET and GSEA. These genes were enriched in the inflammatory response, cellular response to mechanical stimulus, TNF signaling pathway, and cancer-related pathways. Increasing studies have revealed that IA is closely related to the inflammatory response ,41. MoreTNF, MMP9, and TLR4 were hub genes. It has been reported that venous levels of TNF-R1 were associated with poor outcomes at 6 months for SAH [TNF-α can inhibit the formation of IAs in vivo [TNF expression may have the potential to inhibit SAH. MMP9 was found to be associated with TLR4 signaling activation, and downregulating MMP9 induced by LPS has a neuroprotective effect on brain injury caused by SAH [TLR4 is a key player in the regulation of inflammation, and it has been found to be correlated with poor prognosis in SAH [TLR4 was up-expressed in SAH. Subsequently, by constructing a ceRNA network, it was suggested that the downregulated lncRNA MEG3 may be particularly important for SAH, as it may function as a ceRNA for upregulating hsa-miR-128 expression, thus leading to the downregulation of ADRB2 and TGFBR3. Previous studies have shown that MEG3 is highly expressed in SAH, and MEG3 may promote SAH-induced neuronal cell injury by inhibiting the PI3K/AKT signaling pathway [MEG3 has also been found to promote platelet phagocytosis by decreasing miR-128 expression to protect VECs from senescence [MEG3 in SAH need further experimental confirmation. Furthermore, TGFBR3 is involved in the activation of the TGF-β signaling pathway, and TGFBR3 is downregulated in pancreatic ductal adenocarcinoma cells [TGFBR3 was downregulated in SAH, and overexpression of TGRBR3 may be an important therapeutic target in SAH treatment.PPI network for SAH-related DEGs showed that  for SAH , and dow in vivo . Thus, dd by SAH . In addis in SAH . Our pre pathway . Howevernescence . To the ma cells . In presJMJD1C-AS1, LINC01144, hsa-miR-510, TLR4, ADRB2, TGFBR3, and so on) were potential biomarkers for predicting SAH. MiR-510 has been reported to be significantly downregulated in ovarian serous carcinoma (OSC), and it is a novel candidate biomarker for predicting the symptoms of OSC [miR-510 and lncRNAs, JMJD1C-AS1 and LINC01144, in SAH has not been reported. LINC01144-hsa-miR-128-ADRB2/TGFBR3 regulatory axis was found from our ceRNA network, LINC01144 may play a role in SAH by regulating ADRB2 and TGFBR3 expression. ADRB2 encodes adrenoceptor beta 2. Adrenoceptor polymorphisms are associated with an increased risk of cardiac abnormalities after SAH [IL-6 after SAH in rats [TGFBR3 is a transforming growth factor (TGF) beta receptor. TGF-β1/Smad/CTGF pathway was inhibited by rhDecorin to prevent development of hydrocephalus after SAH [TGF-β1 in human umbilical cord-derived mesenchymal stem cells could attenuate SAH-induced chronic hydrocephalus, upregulation of inflammatory cytokines, and other behavioral changes [ADRB2 and TGFBR3 in SAH, we speculated that LINC01144 was involved in the development of SAH. However, elucidation of the roles of these lncRNAs associated with the screening and prevention of patients with SAH requires further investigation.We identified 38 optimal characteristic RNAs signatures from the RNAs in these networks, which were used to construct the SVM classifier. The results of ROC curves investigated that these RNAs , which further indicated the stability and reliability of feature RNAs in predicting risk of SAH. Additionally, though the expression and predictive value of these feature RNAs have been validated using another external independent dataset (GSE36791), experimental verification is still indispensable in the future. In addition, the clinical value of these biomarkers should be further confirmed.5JMJD1C-AS1 and LINC01144), 1 miRNA (hsa-miR-510), and 35 genes  as potential blood biomarkers for screening patients with SAH. This 38 RNAs signature had a better predictive performance for SAH risk. LINC01144 might regulate ADRB2/TGFBR3 expression by sponging hsa-miR-128. These findings of the present study contributed to understanding the molecular mechanism of SAH deeply and also provided the potential biomarkers for the screening and prevention of SAH. However, their application values should be further validated in clinical.In summary, gene expression profile analysis revealed a large scale of expression pattern changes in RNAs under the pathophysiology of SAH, and they were mainly implicated in the inflammatory response, TNF signaling pathway. We further identified 38 RNAs, including 2 lncRNAs (


Lung cancer is one of the most common cancers and has a high mortality rate. Due to its high incidence, the clinical management of the disease remains a major challenge. Several reports have documented a relationship between the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway and lung cancer. The recognition of this pathway as a notable therapeutic target in lung cancer is mainly due to its central involvement in the initiation and progression of the disease. Interest in using natural and synthetic medications to target these signaling pathways has increased in recent years, with promising results in vitro, in vivo, and in clinical trials. In this review, we focus on the current understanding of PI3K/AKT/mTOR signaling in tumor development. In addition to the signaling pathway, we highlighted the therapeutic potential of recently developed PI3K/AKT/mTOR inhibitors based on preclinical and clinical trials. Lung cancer is the most common cancer worldwide and the leading cause of cancer-related death, affecting millions of people every year . Among tThe main strategy in the treatment of lung cancer is chemotherapy, which may improve the quality of life of patients . ChemothAs lung cancer treatment increases interest in targeted therapy, systemic chemotherapy is still the standard treatment for lung cancer . UnfortuAt present, the treatment paradigm for lung cancer has shifted to targeted therapy. This type of treatment attacks specific features of cancer cells known as molecular targets that are either cell surface receptors or intracellular molecules. Many types of targeted therapies for lung cancer are currently available, such as epidermal growth factor receptor (EFGR) inhibitors (erlotinib and gefitinib) or anaplastic lymphoma kinase (ALK) inhibitors . The development of specific targeted therapy such as EGFR inhibitors has been shown to increase overall survival (OS) at around 42.6% (from 4.7 to 6.7 months), which shows the benefit of targeting an oncogenic driver compared to the standard first-line chemotherapy . TargeteCurrently, PI3K/AKT/mTOR signaling has been reported as an emerging source of lung cancer aggressiveness . The devThe protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is initiated by the activation of phosphoinositide 3-kinases (PI3K). PI3K, an enzyme consisting of a large family of lipid and serine/threonine kinases, is normally involved in lipid synthesis . PI3K, aC-terminal tail domain that binds to mTOR complex 2 (mTORC2). Several studies have reported that phosphorylated AKT (p-AKT) promotes aggressive cancer behaviors such as cell proliferation, invasion, metastasis, and angiogenesis, and prevents programmed cell death through the regulation of several downstream effectors [AKT is a serine/threonine kinase that contains a core kinase domain with a threonine residue (T308) and a ffectors ,38,39,40ffectors ,42,43.Overexpression of mTOR is commonly observed in various types of cancers . mTOR foThe PI3K/AKT/mTOR signaling pathway is engaged in a wide spectrum of metabolic processes in which it monitors energy, nutrient and stress levels. Transcription of genes associated with the cancer promotor stimulated by mTORC1 occurs through the phosphorylation of its downstream effectors eukaryotic translation factor 4E-binding protein 1 (4EBP1) and p70S6 kinase 1 (S6K1) . They rePIK3CA, the gene encoding the catalytic subunit of the PI3Kα isoform, is frequently mutated in various human cancers, including breast, ovarian and lung cancers [Deregulation of the PI3K/AKT/mTOR signaling pathway by either the mutation or amplification of genes involved in the PI3K pathway, loss of the tumor suppressor PTEN, or overactivation of RTKs, has been observed in various cancer cells, contributing to tumor progression and metastasis ,49,50,51 cancers ,53,54. T cancers ,56. The  cancers .Mutation or loss of PTEN, a negative regulator of the PI3K signaling pathway, has also been observed in many cancers ,59. PTENCell survival and chemotherapeutic resistance are inseparable from the PI3K/AKT/mTOR signaling pathway. Several factors related to this signaling pathway include the Bcl-2 family, X-linked inhibitor of apoptosis protein (XIAP), mouse double minute 2 homolog (MDM-2) and Forkhead box O3 (FOXO3a) transcription factor  63,64,6,66,67. BN-terminal transactivation domain of p53, which mediates p53 degradation by the ubiquitination process [Several studies have shown that XIAP is regulated by AKT signaling ,64,65. A process ,75. The  process ,77. In cThe FOXO3 protein is a FOXO transcription factor and a member of a subgroup of the Forkhead family. FOXO3 promotes apoptosis signaling by either inducing the expression of multiple proapoptotic members of the Bcl-2 family proteins or stimulating the expression of death receptor ligands such as Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) . FOXO3 iCIP1/WAF1) and cyclin-dependent kinase inhibitor 1B (p27Kip1), which inhibit these cyclin kinase inhibitors [Activated AKT/mTOR are considered important key elements that regulate cell proliferation . Severalhibitors ,83. mTORhibitors .Cancer metastasis is the process by which cancer cells dissociate, migrate, and invade other sites. The PI3K/AKT/mTOR pathway participates in cancer metastasis in which cancer cells are stimulated by the activation of RTKs, cytokines, or hormones. Active AKT induces phosphorylation of mTORC1 and its downstream targets, such as 4EBP1 (Thr 37/46) and p70S6K (Thr 389). Activation of 4EBP1 induces the translation of several transcription factors, such as Snail, Slug, and Twist ,85,86. AAccording to previous findings, the PI3K/AKT/mTOR pathway governs cancer cell migration and invasion by regulating F-actin reorganization . AKT actAngiogenesis or the development of new blood vessels is one of the common hallmarks of cancer and is crucial for cancer progression, development and metastasis. This process is necessary to supply nutrients and oxygen to compensate for rapid tumor growth ,99. TumoHIF-1α was reported to be regulated by the PI3K/AKT/mTOR signaling pathway. AKT/mTOR induces downstream signaling, such as 4EBP1, which is essential for inhibiting cap-dependent mRNA translation and increasing the translation of the HIF-1α transcription factor . In addiDue to the substantial increase in the number of new therapeutic agents that target specific molecular pathways, a higher degree of biochemical precision in therapeutic drugs can now be achieved than were previously available with conventional chemotherapeutic drugs. Since the PI3K/AKT/mTOR pathway is considered a potential target for anticancer drug research and development, numerous potent molecules or combinations are undergoing clinical trials. This review summarizes several drugs targeting PI3K/AKT/mTOR signaling pathways that are being investigated in various phases of clinical trials .N-terminal SH3 domain, a Rho-GAP domain, an nSH2 domain, an iSH2 domain and a cSH2 domain [N-terminal p85-binding motif that controls its activity [The PI3K family is divided into four groups  according to their structure, regulation and substrate specificity . Class I2 domain ,111. In 2 domain . Class Iactivity .Class IA PI3Ks are activated in response to activation of RTKs and Ras proteins. The SH2 domain of p85 is released from the complex and binds to the phosphorylated tyrosine motif (pyxxm) in RTKs, and the RBD domain also directly interacts with the active Ras protein (Ras-GTP) to promote membrane localization of PI3K . ATP occ50 values of 52 nM, 166 nM, 116 nM and 262 nM, respectively, in an ATP-competitive manner, thereby inhibiting the activation of the secondary messenger phosphatidylinositol–3,4,5-trisphosphate [Numerous studies indicate that hyperactivity of PI3K signaling is strongly associated with tumor growth, tumor microvessel density and the increased invasive and chemotactic abilities of cancer cells . Currenthosphate ,118. It hosphate ,120,121.hosphate .50 of 3 nM [Pictilisib or GDC-0941 is a potent inhibitor of PI3Kα/δ with an IC of 3 nM . In a cl of 3 nM . Pictili of 3 nM . Based o of 3 nM .50 of 2.5 nM [Idelalisib is an oral competitive inhibitor of the ATP binding site, specifically at the PI3Kδ catalytic domain, with an ICf 2.5 nM . Idelalif 2.5 nM . Currentf 2.5 nM .50 of 5 nM [Alpelisib (BYL719) is a selective PI3Kα inhibitor derived from 2-aminothiazole with an IC of 5 nM ,131. Alp of 5 nM ,133. Alp of 5 nM , using t of 5 nM .50 of 21 nM). A phase I study on dose escalation reported that TAK-117 showed an acceptable safety profile at the intermittent MTD (900 mg). Observations of grade ≥3 drug-related ALT/AST elevations were lower after the intermittent treatment than after daily treatment with TAK-117 [Serabelisib (TAK-117) is a potent and selective oral PI3Kα inhibitor  is a potent and selective inhibitor of class I PI3Ks  with ICt models . Taselist models . For lunt models .50 values of 0.4 and 5.4 nM, respectively [Gedatolisib (PF05212384) is another type of PI3K inhibitor that also functions as an mTOR inhibitor. Gedatolisib is potent and selective for PI3Kα and PI3Kγ with ICectively . In an iectively . Due to ectively . Anotherectively .50 of 9 nM for PI3Kγ [Voxtalisib (SAR245409/XL765) is a dual-targeting drug that acts by inhibiting the kinase activities of PI3K and mTOR with an ICor PI3Kγ . An in vor PI3Kγ ; howeveror PI3Kγ .N-terminal pleckstrin homology (PH) domain, a central kinase catalytic (CAT) domain and a C-terminal extension (EXT) containing a regulatory hydrophobic motif (HM)  . The PH The development of small-molecule AKT inhibitors is mainly focused on the inhibition of AKT-mediated phosphorylation. To date, ATP-competitive inhibitors are some of the most common AKT inhibitors and have been shown to strongly suppress AKT activity. These inhibitors directly bind to the ATP binding pocket of active AKT in a PH-out conformation that leads to paradoxical increases in AKT phosphorylation at threonine and serine residues . Hyperph50 values of 8, 12, and 65 nM, respectively [MK2206 is an AKT inhibitor that targets all three isoforms of AKT  with ICectively . Accordiectively . In a phectively . A combiectively ,161. MK2ectively . The addectively . A similectively .50 of 3 nM for AKT1 and 8 nM for AKT2 and AKT3) [Capivasertib (AZD5363) is similar to MEK2206, as it inhibits all isoforms of AKT (ICnd AKT3) . A phasend AKT3) . Currentnd AKT3) .50 values of 180, 328, and 38 nM for AKT1, AKT2, and AKT3, respectively [Uprosertib (GSK2141795) is an oral ATP-competitive AKT inhibitor with ICectively ,167. An ectively . An addiectively .Perifosine is an alkyl-phospholipid that functions as a dual PI3K/AKT inhibitor, which is being investigated in a phase I trial in patients with NSCLC (NCT00399789). This clinical trial aimed to determine the MTD of perifosine that is tolerated in the gastrointestinal tract. Patients with NSCLC will receive daily or weekly doses of perifosine (150 mg/900 mg) ,170. ApaAspirin is a nonsteroidal anti-inflammatory drug (NSAID). In vitro and in vivo experiments showed that additional treatment with aspirin sensitizes NSCLC cells to osimertinib through a Bim-mediated apoptosis induction . In indi2-terminat HEAT (N-HEAT), middle HEAT (M-HEAT)), FAT, FRB, and kinase domains  and ATP-competitive mTOR kinase inhibitors . RapalogAs the first-generation mTOR inhibitor, rapamycin is currently being investigated in a clinical trial in patients with NSCLC in several combinations of sunitinib (NCT00555256) and afatinib (NCT00993499) ,185. Its2/day) and temsirolimus (15 mg i.v. weekly) for stable disease were observed in 37.5% of the total patients [Another drug derived from rapamycin, temsirolimus, has been approved by the FDA for the treatment of advanced renal cell carcinoma. Preclinical treatment with temsirolimus inhibits the proliferation of several NSCLC lines as well as the antitumor activity of NSCLC xenografts . In anotpatients . The Waspatients ,193.Metformin is the first-line medication for the treatment of type 2 diabetes mellitus, but recently, several studies have also shown that metformin has anticancer properties by inhibiting mTOR. Metformin is effective at reducing the cancer incidence, and it improves the prognosis of patients whose cancer is diagnosed . Previou50 values of 0.208, 0.2 and 1.039 µM, respectively. An evaluation of onatasertib in a patient-derived lung adenocarcinoma xenograft showed a 47% reduction in tumor growth after treatment with 10 mg/kg/day [Onatasertib (CC223) is an mTOR inhibitor that binds to the ATP-binding region in the catalytic site of mTOR. A preclinical experiment showed that onatasertib inhibits the proliferation of several NSCLC cell lines, such as A549, H460, and H23 cells, with ICg/kg/day . A phaseg/kg/day . In addig/kg/day ,202.Approximately two-thirds of the anticancer drugs currently available are synthetic products that are derived from natural sources . The incOrchidaceae family, particularly Dendrobium species, including 4,5,4′-trihydroxy-3,3′-dimethoxybibenzyl (TDB) and gigantol [Dendrobium genus of the orchid family, specifically Dendrobium ellipsophyllum [50 values ranging from 100–190 µM. TBD upregulated the expression of the tumor suppressor p53 and proapoptotic protein Bax and downregulated the expression of several antiapoptotic proteins, such as Bcl-2 and Mcl-1. Further experiments reported that TDB also significantly diminished the level of p-AKT [Bibenzyl compounds have been mainly detected in the gigantol ,207. Sevgigantol ,209,210.ophyllum . TBD belof p-AKT . As an aDendrobium genus (Dendrobium draconis), exhibits potent anticancer activity against NSCLC cell lines (H460 and H292 cells) [Gigantol, a bibenzyl compound isolated from the 2 cells) ,214,215.2 cells) . The exp2 cells) . TherefoOrchidaceae family, especially Dendrobium and Juncaceae [Dendrobium infundibulum. As a promising natural compound, ephemeranthol A exerts anticancer activity by inducing apoptosis through the activation of several apoptosis-related enzymes, such as caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP). In addition to apoptosis induction, ephemeranthol A inhibits the migration of lung cancer cells by inactivating AKT and subsequently suppressing the expression of EMT markers such as N-cadherin, vimentin and slug [Phenanthrene, a group of polycyclic aromatic hydrocarbon-containing compounds composed of three connected benzene rings, was identified in the uncaceae . Phenantuncaceae . Each phand slug .Dendrobium densiflorum, also induces apoptosis and inhibits migration [Cypripedin, a phenanthrene quinone isolated from igration ,220. CypDendrobium formosum that was recently reported to have potent antimetastatic and cytotoxic effects on lung cancer [Likewise, erianthridin is a phenanthrene derived from g cancer ,221. EriDendrobium venustum and is abundant in northern, central and western Thailand. A previous study suggested that phoyunnanin E induces apoptosis in NSCLC cell lines via a p53-dependent pathway [Phenolic compounds are characterized by molecules containing at least one hydroxyl group attached to aromatic hydrocarbons. Phenolic compounds exhibit significant benefits in several diseases, mostly through their antioxidant activity . Phoyunn pathway . An in v pathway .Curcuma longa. As a well-known natural product, curcumin possesses several pharmacological activities, such as antioxidant, anti-inflammatory, antidiabetic, antihypertension, and anticholesterol activities [Curcumin, a bright yellow polyphenol-containing compound, is abundant in the species tivities . In lungtivities . In additivities .Cycas revoluta. Sotetsuflavone is classified as a bioflavonoid, a polyphenol compound comprising two identical or nonidentical flavones connected to each other by an alkyl or an alkoxy-based linker of varying lengths [In addition, sotetsuflavone, an active constituent of many traditional Chinese medicines, is present in several medicinal plants, especially in the  lengths . This co lengths . Its ant lengths , and it  lengths .Chrysanthemum morifolium. Zhou and coworkers revealed that luteoloside inhibits lung cancer cell proliferation by inducing G0/G1 cell cycle arrest. A significant downregulation of p-AKT/p-mTOR/p-p70S6K was observed in response to luteoloside treatment. Moreover, luteoloside induces intracellular ROS formation associated with the suppression of the AKT/mTOR signaling pathway [Luteoloside is also one of the active flavonoids detected in several medicinal herbs, especially in  pathway .Boesenbergia rotunda, one species in the Zingiberaceae family [Cardamonin is a chalcone flavonoid that is abundant in e family . Its ante family . As a grXestospongia sp.). Jorunnamycin A shares a similar structure to the famous ecteinascidin 743 (trabectedin), which was approved by the FDA in 2015 as an anticancer drug targeting unresectable or metastatic liposarcoma [50 values of 220 and 160 nM, respectively [Quinoline is a group of molecules composed of heterocyclic aromatic rings that exhibit various pharmacological activities, such as antibacterial, antioxidant, and anticancer activities . Jorunnaosarcoma . Accordiectively . Jorunnaectively .Xestospongia sp.) [Likewise, renieramycin M is a novel tetrahydroisoquinoline natural marine compound that is isolated from a Thai blue sponge (gia sp.) . Renieragia sp.) . Additiogia sp.) ,240. TheIn this review, we summarized several drugs and natural compounds that target the PI3K/AKT/mTOR pathway in lung cancer. Thousands of species from plants, animals, and marine organisms have been investigated for their pharmacological activities. Both natural products and semisynthetic drugs are currently being investigated in preclinical and clinical trials. The majority of natural products that target this pathway and their downstream signaling intermediates, such as p70S6K, 4EBP1, and HIF1α, are associated with apoptosis and/or autophagy induction, suppression of the EMT, inhibition of migration and invasion and sensitization to chemotherapy. PI3K/AKT/mTOR inhibitors are designed to directly interact and inhibit these molecules.According to accumulated studies of the PI3K/AKT/mTOR signaling pathway in lung cancer, this active signaling pathway clearly offers tremendous possibilities for therapies and challenges for drug research and discovery. However, targeted therapy acting on the PI3K/AKT/mTOR pathway may cause numerous side effects and defects due to the resistance acquired. The study of the specificity in cancer should be a serious concern, and the identification of novel dosing regimens that result in greater tolerance and overall efficiency of PI3K/AKT/mTOR inhibitors is required. Additional research should strive to overcome resistance to PI3K/AKT/mTOR inhibitors and suggest additional rational drug combinations.


The scaffolding protein family Fe65, composed of Fe65, Fe65L1, and Fe65L2, was identified as an interaction partner of the amyloid precursor protein (APP), which plays a key function in Alzheimer’s disease. All three Fe65 family members possess three highly conserved interaction domains, forming complexes with diverse binding partners that can be assigned to different cellular functions, such as transactivation of genes in the nucleus, modulation of calcium homeostasis and lipid metabolism, and regulation of the actin cytoskeleton. In this article, we rule out putative new intracellular signaling mechanisms of the APP-interacting protein Fe65 in the regulation of actin cytoskeleton dynamics in the context of various neuronal functions, such as cell migration, neurite outgrowth, and synaptic plasticity. APBB1, APBB2, and APBB3, respectively [APBB stands for APP-binding family B and refers to the observation that all three Fe65 proteins bind to the amyloid precursor protein , whereas human Fe65 and Fe65L1 have a long N-terminal domain with a length of 258 and 290 amino acids, respectively. The corresponding region is missing in Fe65L2. In addition, there are several splice variants of all members of the Fe65 family. At least six different isoforms have been reported for Fe65: Isoform 1, also called p97Fe65, is the longest isoform with 710 amino acids; isoforms 2 and 3 lack two amino acids, E462 and R463, resulting from a deletion of mini-exon 9 ; isoformFe65 homologs have been reported for many different vertebrates, including humans, mice, and fish. Furthermore, in non-vertebrates, a homolog protein of Fe65, Feh-1 from Caenorhabditis elegans, was reported that also has a conserved domain structure ,20. HoweRecent X-ray crystallography and NMR measurements suggest a homotypic dimerization of Fe65 via the PTB2 domain, involving unwinding of a C-terminal α-helix at the end of one PTB2 domain, binding to the PTB2 domain of a second Fe65 molecule . This inBox 1The amyloid precursor protein (APP) is a type I transmembrane protein with a large extracellular and a short intracellular domain. It undergoes a complex proteolytic processing by sequential cleavage of different sheddases that cleave extracellular portions of transmembrane proteins, releasing the soluble ectodomains, followed by γ-secretase cleavage of the residual membrane tethered stub. Best investigated sheddases are the α- and β-secretase, releasing the soluble fragment sAPPα or sAPPβ, respectively, with only 13 amino acids difference in length but severe differences in function. While sAPPα has clear neuroprotective properties, these were mostly observed in lower activity or not at all in comparative studies for sAPPβ. Depending on the sheddase cleavage, also the fragments released after intramembranous cleavage by γ-secretase differ dramatically. In the so-called amyloidogenic pathway after sAPPβ was cleaved off, two fragments are generated. The APP intracellular domain that gets released in the cytoplasm and the extracellular released Aβ peptide that forms oligomers and large aggregates that accumulate in form of β-amyloid plaques in Alzheimer’s disease. In contrast, in the non-amyloidogenic pathway after sAPPα generation, a non-toxic instable P3 fragment and AICD get released. Notably, the AICD is proposed to be involved in regulation of transcriptional activity together with Fe65. However, extensive research revealed more complex APP processing, involving a large variety of different secreted factors (>10) with diverse neuroprotective or pathogenic properties. Supplementary to the function of the secreted fragments, additional functions of full-length membrane-bound APP have been proposed as co-receptors for very different signaling pathways, involved in neurite outgrowth and synaptic plasticity. Interestingly, a function of APP forming transcellular dimers as a synaptic adhesion molecule has also been suggested in this context. However, the molecular signaling of APP is not yet understood, genetic studies, particularly considering the overlapping function of the two APP homologous proteins, APLP1 and APLP2, clearly showed an essential contribution in diverse cellular functions such as neuronal outgrowth, synaptic plasticity, and vesicular trafficking ,24,25,26Important insights into the putative function of the Fe65 family come from the analysis of interaction partners. Most attention has been paid to the Fe65-PTB2 domain as it directly interacts with the APP C-terminus that links Fe65 to a central protein of AD .In addition to APP, more than 20 different Fe65 interaction partners have been reported  that canA second group of interaction partners links Fe65 function to lipid metabolism. For instance, the family of low-density lipoprotein receptors (LDLR), including the low-density lipoprotein receptor-related protein 1 (LRP1), very low-density lipoprotein receptor (VLDLR), Megalin/LRP2, and ApoEr2 were shown to bind to the Fe65 PTB1 domain ,46,47,48A third group of interaction partners was identified in a mass spectrometry-based analysis, reporting more than a hundred Fe65 interaction partners, including some involved in calcium regulation . In thisAnother major group of Fe65 interacting proteins is involved in actin regulation, encompassing Mena , cortactin, and ELMO1/DOCK1  52,53,5,55,56. IMena, the mammalian homolog of Drosophila enabled, is a member of the Mena/VASP protein family, consisting of vasodilator-stimulated phosphoprotein (VASP), Ena-VASP-like protein (EVL), and Mena. All members are composed of two Ena/VASP homology domains (EVH1 + EVH2) and a proline-rich core region ,61,62,63Mena/VASP members are highly abundant in the brain and concentrated in regions of high actin dynamics such as focal adhesions, stress fibers, lamellipodia, filopodia, ruffles, and growth cones ,71,72,73Mena and its homologs act as anti-capping proteins and support filamentous actin polymerization ,81,82,83Initial evidence that Fe65 may be involved in actin cytoskeleton regulation comes from a biochemical study showing that Mena preferentially interacts with the WW domain of Fe65 via two central PPxPP motifs, analyzed in detail by crystallography ,57. ConsCortactin, a class II nucleation promoting factor, recruits N-WASP , which in turn activates the Arp 2/3 complex by changing the position of Arp2 forming an Arp2–Arp3 short-pitch dimer and generating a new daughter actin filament ,96. AfteCortactin undergoes different posttranslational modifications, regulating its activity. Phosphorylation by distinct kinases, including Src, Abl, Arg, Erk, PAK, and PKD ,114,115,As Fe65 binds to the histone acetylase Tip60 that is capable of increasing cortactin acetylation, it inhibits the association of cortactin with actin ,116. In In addition to the regulation of actin dynamics, the interaction of cortactin and Fe65 might also play a role in the nucleus, as acetylated cortactin gets translocated to the nucleus, similarly as shown for the Fe65-APP-Tip60 tripartite complex ,33,124. Rac (Ras-related C3 botulinum toxin substrate 1) is a member of the Rho family of small GTPases that regulates axon and dendrite differentiation, prolongation, and arborization while it is antagonized by RhoA activity ,126,127.Rac proteins get activated by guanine exchange factors (GEFs) that promote nucleotide exchange from GDP to GTP . The GEFELMO proteins were first discovered in a genetic screen to identify components required for engulfment of dead cells and cell motility in C. elegans . ELMO anThe interaction of Fe65 and ELMO1 was identified by the group of Dr. Lau ,145,146,The basic idea of scaffolding protein functions is that they bring components of a signaling cascade together into spatial proximity. This can increase the efficiency of signal transduction as well as signal specificity. This function appears crucial as modeling has shown that kinases in a cascade without scaffold proteins have a higher probability of being dephosphorylated by phosphatases before they are even able to phosphorylate downstream targets . In thisOne well-studied example of a scaffolding protein involved in actin cytoskeleton regulation is β-catenin, which binds to the cytoplasmic end of E-cadherin and to α-catenin, which interacts with the underlying actin cytoskeleton . CadheriAlthough not yet shown experimentally, it appears well feasible that Fe65 also functions as a highly dynamic adaptor protein. First, Fe65 was shown to form dimers via the PTB2 domain using the interaction site of the APP-C-terminus , indicatSince Fe65 is a phosphoprotein, it seems quite conceivable that phosphorylation is involved in regulating spatial and temporal Fe65 dynamics. Fe65 can be phosphorylated by various protein kinases, including extracellular signal-regulated kinase (ERK1/2), serum- and glucocorticoid-regulated kinase (SGK1), Abelson tyrosine kinase (c-Abl), ataxia telangiectasia mutated/ataxia-telangiectasia- and Rad3-related protein (ATM/ATR) kinase, and glycogen synthase kinase 3β (GSK3β) ,156,157.Important insights into the Fe65 function were gained by analyses of genetically modified mice ,168,169.Additionally, Fe65 and APP family KO mice show severe learning and memory deficits, resulting likely from impairments of synaptic plasticity . Some ofFe65 and APP can regulate cell motility. Therefore, overexpression of APP or Fe65 in MDCK cells increased the cell migration velocity in a wound-healing assay and co-expression of APP and Fe65 further accelerates cell movement . This suIn line with changes of the motility and of the plasma membrane traction, Fe65 was also shown to suppress invasion by binding to Tip60 and cortactin . Actin-dTo unravel the functional interdependence of Fe65 and its interaction partners in cell motility, additional genetic studies using the same cell lines and comparable mouse models will be necessary.Overall, there is clear evidence for a central function of the APP-binding protein family Fe65 in actin regulation. Fe65 likely supports the formation of branched and unbranched actin polymerization, affecting multiple cellular functions, including axonal/dendritic outgrowth, cell migration, structural synaptic plasticity, and intracellular vesicular trafficking. Those diverse functions could be mainly explained by Fe65 interaction with central regulators of actin dynamics. First, Fe65 binds to Mena, which promotes actin elongation by enhancing profilin function. Increased actin polymerization then favors the growth of unbranched actin filaments, leading to filopodia formation, important for environment scanning and positioning as well as for dendritic spine initiation. However, depending on the availability of Arp2/3 complexes, Mena/profilin may also contribute to increasing actin meshwork formation . FurtherIn addition to the question of how the scaffolding protein Fe65 helps to orchestrate the reorganization of the actin cytoskeleton, it will be central to understand what the upstream regulators of Fe65 are. Based on the high-affinity binding of the Fe65 family members to the APP family, and due to widely overlapping phenotypes in APP/APLP1/APLP2 TKO and Fe65/Fe65L1 DKO mice, it appears very likely that APP/APLPs are important upstream regulators of Fe65 family members  1,4,5,6,9,23,234The pathological role of the Fe65 family in AD is extensively investigated, but so far, only minor changes in APP processing and amyloid pathology were reported ,256,257.In summary, we found convincing evidence for an important role of Fe65 in the regulation of the actin cytoskeleton in neuronal development and synaptic plasticity. To better understand the pathophysiological role of the Fe65 family in the different actin-dependent processes and the transitions between the different types of actin networks, it will be important to study the control and dynamics of the different Fe65 complexes in more detail.


Orthohepevirus C detected in rats and in human patients with persistent hepatitis have been detected in ferrets, minks, and foxes, providing a rationale for investigating the presence of HEV or HEV-like viruses in wild carnivores. Herewith, we report the identification and the nearly full-genome (7212 nucleotides) characterization of an HEV Gt3 strain detected in a faecal specimen collected from a wolf found dead in Northwestern Italy. This finding poses questions concerning the potential role of wolves as HEV mechanical, passive carriers, favouring the introduction and spread of HEV in the environment.Data on hepatitis E virus (HEV) strains circulating in carnivores are still limited. However, serological evidence suggests that their exposure to HEV infection is more common than expected. In addition, close relatives of Hepatitis E virus (HEV) infection is a major health problem worldwide. In developed countries, zoonotic transmission of HEV genotypes (Gt) 3 and 4 is caused by the ingestion of raw or undercooked meat of infected pigs and wild boars, the main reservoirs of HEV. However, additional animals may harbour HEV or HEV-related strains, including carnivores. In this study, we investigated the molecular epidemiology of orthohepeviruses in wild canids by screening a total of 136 archival faecal samples, collected from wolves (42) and red foxes (94) in Northwestern Italy. Orthohepevirus RNA was identified in a faecal specimen, collected from a wolf carcass in the province of La Spezia . The nearly full-length (7212 nucleotides) genome of the strain HEV/81236/Wolf/2019/ITA (GenBank accession no. MZ463196) was determined by combining a sequence-independent single-primer amplification (SISPA) approach with the Oxford Nanopore Technologies sequencing platform. Upon phylogenetic analysis, the HEV detected in wolf was segregated into clade HEV-3.1, displaying the highest nucleotide (nt) identity (89.0–93.3%) to Gt3 strains belonging to subtype c. Interestingly, the wolf faecal sample also contained porcine astrovirus sequences, endorsing the hypothesis of a dietary origin of the HEV strain due to preying habits.  Hepeviridae [Hepeviridae into two genera, namely the genus Piscihepevirus (cutthroat trout virus) and Orthohepevirus [Orthohepevirus is further subdivided into four distinct species, Orthohepevirus A–D, including hepeviruses of mammalian and avian origin [Orthohepevirus A have been assigned to eight distinct genotypes (Gt1-Gt8) [Orthohepevirus A [Orthohepevirus A, hepeviruses closely related to rat orthohepeviruses C (HEV-C1) [Hepatitis E virus (HEV) is a common cause of human acute viral hepatitis . HEV is eviridae . In the epevirus . The genn origin . Based oGt1-Gt8) , with foGt1-Gt8) , althougevirus A . HEV infevirus A ,11,12,13evirus A . Additioevirus A . In addi(HEV-C1) , but clu(HEV-C1) ,16.Canis lupus italicus) and red foxes (Vulpes vulpes) were analysed molecularly, using primer sets targeting highly conserved regions among the four major Gts (Gt1-Gt4) of Orthohepevirus A and a primer set designed to amplify all members of the genus Orthohepevirus. The nearly full genome sequence of an HEV Gt3 strain detected in a wolf faecal sample was characterized.In this study, faecal samples from wolves , Liguria (6), and Valle D’Aosta (3) between February 2017 and September 2020. Additionally, in the analysis were included 94 red foxes found dead or shot during the regular hunting season in the time frame September 2013–November 2017.Orthohepevirus A-specific real-time reverse transcription PCR (qRT-PCR) [7 to 102 copies) of a plasmid containing the 68-nt ORF3 fragment of a Gt3 HEV wild boar strain  [Orthohepevirus was done by a pan-orthohepevirus heminested RT-PCR using broadly reactive primers designed to amplify all members of the genus Orthohepevirus [http://www.ncbi.nlm.nih.gov, accessed on 10 October 2021) and FASTA  with default values were used to find homologous nucleotide sequences. Total RNA was extracted from each faecal specimen (0.5 mL) by using the TRIzol LS  procedure and analysed by qRT-PCR) . TenfoldU508285)  were useepevirus  and targepevirus . The posA sequence-independent single-primer amplification (SISPA) approach  combined3 RNA copies/gram faeces  in one specimen  collected from an adult male wolf  found dead in La Spezia province, (Liguria Region). The cause of death was trauma with organ rupture and exsanguination likely due to car accident. The same specimen resulted positive when re-screened with a pan-orthohepevirus broadly reactive RT-PCR [Out of 136 faecal samples collected from wild canids, qRT-PCR detected HEV RNA with a viral load of 2.1 × 10e RT-PCR  and withe RT-PCR . BLAST ae RT-PCR . The stre RT-PCR . Upon phe RT-PCR ,27,28, dOrthohepevirus A [Orthohepevirus A. The SISPA technique in combination with ONT, a third-generation sequencer, which recently emerged as complement to or replacement for second-generation sequencers, was applied to target viral RNA sequences contained in the wolf faecal sample. This method allowed for successful sequence assembly of the nearly full genome of the strain HEV/81236/Wolf/2019/ITA. Upon visual inspection of the complete genome based-tree, consistent with previous analyses [Since the initial discovery of animal HEV and the understanding of its zoonotic potential , the knoevirus A . Besidesevirus A , naturalevirus A ,11,12,13evirus A  that hasevirus A . In our analyses ,27,28, tanalyses ,28, the analyses . In addianalyses ,32,33, aanalyses . AccordiOrthohepevirus A susceptibility of this wild canid. Meanwhile, we cannot exclude its potential role as an HEV mechanical, passive carrier, likely due to preying on actively infected hosts. The wolf HEV-positive stool was collected in a geographical area  characterized by a high density of wild boar populations in which HEV Gt3 subtypes 3a, 3c, 3e, and 3f have been found to circulate with prevalence rates ranging from 2.3% to 4.4% [Sus scrofa [To date, there is no clear evidence that zoonotic HEVs may infect or cause disease in canids. Experimental inoculation of two dogs with a swine Gt4 strain was able to elicit specific IgG antibodies, but HEV RNA was not found in infected animals . Overall to 4.4% ,33. In os scrofa , but it s scrofa ,40,41,42In conclusion, the findings of this study pose questions concerning the role of wolves as potential HEV carriers, thus sustaining viral circulation in the ecosystem. Large-scale serological and molecular investigations in wolves and red foxes could be useful for carefully assessing their role in the HEV epidemiological cycle, chiefly in geographical settings in which animal reservoirs and predators live in sympatry. Furthermore, upon nearly complete genome characterization by using a third-generation sequencer, it was clear that HEV Gt3 strains of subtype c are circulating in the surveyed geographical area , confirming previous studies ,32,33. A


Recent advances in imaging techniques have identified localized Ca2+ transients within the fine processes of the astrocytic structure, which we term microdomain Ca2+ events. These Ca2+ transients are very diverse and occur under different conditions, including in the presence or absence of surrounding circuit activity. This complexity suggests that different signalling mechanisms mediate microdomain events which may then encode specific astrocyte functions from the modulation of synapses up to brain circuits and behaviour. Several recent studies have shown that a subset of astrocyte microdomain Ca2+ events occur rapidly following local neuronal circuit activity. In this review, we consider the physiological relevance of microdomain astrocyte Ca2+ signalling within brain circuits and outline possible pathways of extracellular Ca2+ influx through ionotropic receptors and other Ca2+ ion channels, which may contribute to astrocyte microdomain events with potentially fast dynamics. Astrocytes are complex glial cells that play many essential roles in the brain, including the fine-tuning of synaptic activity and blood flow. These roles are linked to fluctuations in intracellular Ca Physiologically, astrocytes are critical for brain homeostasis . They bu2+  that are2+ ,13,14,15 GCaMP6f ,23,24,252+ transients are dictated by the resting, basal intracellular Ca2+ concentration [2+ events [2+ signal onset timescales (a few milliseconds after the start of stimulation), this astrocytic Ca2+ signalling was deemed too slow to modulate rapid processes such as synaptic activity or blood flow [2+ dynamics have recently been described within fine astrocyte structures in response to physiological stimuli in vivo [2+ signalling to play a rapid role in fine-tuning circuits as discussed below.Astrocyte MCEs are heterogenous; they vary in amplitude and duration, and occur within astrocytes at rest  ,18. The ntration , which intration . The numntration ,28,29 fontration ,18,30,31+ events ,33,34 an+ events ,18 activood flow ,33,34. H in vivo . In part in vivo . This su2+ dependent, suggesting that astrocyte Ca2+ events are a key component of bidirectional astrocyte-neuron interactions [2+ domains or more global astrocyte Ca2+ signals may modulate neuronal networks and dictate animal behaviour [Astrocytes are active contributors to brain processes through the release of gliotransmitters or vasoactive molecules that modulate the nearby neuronal activity or blood flow ,11,12. Tractions . Specifiractions ,48,49,50ehaviour ,53,54,552+ signalling and gliotransmitter release influences basal synaptic activity, excitatory and inhibitory neurotransmission, and synaptic plasticity  have repetitive [2+ events that locally regulate circuit activity, modulate the processing of information in large networks and impact animal behaviour. Fast onset MCEs evoked by neuronal activity could be of critical importance for rapidly tuning changes at single synapses that amount to alterations in activity over larger circuits. Again, future studies specifically targeting pathways that contribute directly to astrocyte MCEs will help to link MCEs to the modulation of single synapses, but will also help determine how the scaling of astrocyte Ca2+ signalling and the recruitment of MCEs influence larger neuronal networks and behaviour.During periods of neuronal activity, astrocyte Caial area ,29,30,31response . For exaresponse ,70, sincllations . Additiooeruleus ,33,71,72ormation , while mpetitive , depresspetitive , or autipetitive . Thus, a2+-dependent release of vasoactive molecules, such as arachidonic acid metabolites  [2+ influx in astrocytes [A number of mechanisms are known to contribute to localized astrocyte MCEs ,15,20,25 release  via the trocytes ,86,87,882+ events is the release of Ca2+ from the endoplasmic reticulum following inositol-1,4,5-trisphosphate receptor (IP3R) and upstream Gq-G-protein coupled receptor (GPCR) activation  when activated by synaptic glutamate (N-methyl-D-aspartate (NMDA) receptors [2+, deactivation kinetics, and Ca2+ permeability [2+ and therefore do not require membrane depolarization to be activated. These subunits are also less permeable to Ca2+ than GluN2A/2B receptors, and they have slower deactivation kinetics [Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that conduct cations . There is evidence of astrocyte kainate receptor subunit expression at the mRNA and protein levels [Astrocytes express the genes of iGluRs, albeit at lower levels than neurons. All four AMPA receptor subunits GluA1-GluA4) have been detected in astrocytes -GluA4 ha, althougtrocytes ,109,110.n levels ,112; hown levels ,116,117.2+ permeability and their contribution to Ca2+ signalling, has been controversial. Early Ca2+ imaging studies were conducted in primary astrocyte cultures  by combining Ca2+ imaging dyes with GFAP-eGFP transgenic mice to better label astrocytes [2+ events in fine structures. Using GCaMP3 and GCaMP6f, Haustein et al. [2+ events [2+ responses happened on the same time scale as neurons and preceded local vasodilation. Blockers for AMPA or NMDA receptors were applied directly to the brain and both drugs reduced fast Ca2+ events in astrocyte processes, but only CNQX reduced fast Ca2+ events in endfeet [Although iGluR agonists evoke Catrocytes ,128,129.n et al.  showed t+ events . In simi+ events ,31, whic endfeet . This su endfeet , making 2+ in the soma, limited studies have found a functional role for astrocyte AMPAR. In the cerebellum, Bergmann glia astrocytes express the GluA1 and GluA4 subunits [2+ impermeable [While it is clear that AMPA receptor activation can cause an elevation in astrocyte Casubunits . When Beermeable  or (b) termeable , structuermeable  and defiermeable . Clearly2+ stores [2+ in astrocytes through elevation of store uptake [2+ concentrations, which has implications for Ca2+ microdomain activity and their dynamics [2+ transients in cultured cortical astrocytes upregulate the Cdk5/Nrf2 pathway, a key regulator of genes for cell antioxidant machinery [Astrocyte NMDA receptors have functional roles in maintaining astrocyte Ca+ stores , antioxi+ stores , gliotra+ stores , and the+ stores . First, e uptake . Thus, Ndynamics ,27. Secoachinery . This inachinery , and conachinery . 2+ events in astrocytes, then gliotransmitter release may happen in a temporal realm that can rapidly tune synaptic activity. Finally, astrocyte NMDA receptors may also regulate synaptic strength in the hippocampus by maintaining paired-pulse ratio heterogeneity, which is seen between two presynaptic neurons that target the same postsynaptic cell [2+ transients and astrocytic NMDA receptor signalling in diversifying the presynaptic strengths and potentially elevating the network dynamics of dendrites [2+ signalling mediated by these receptors and their physiological roles.As discussed in tic cell . Paired-endrites . This tyendrites . While t1/5 [7 subunits [2+, K+, and Na+ into the cell. The subunit composition confers ATP binding affinity and Ca2+ permeability [7 receptors are only activated by high extracellular ATP levels and have been linked to pathology and astrocyte reactivity [1/5, with its higher affinity for ATP and good Ca2+ permeability, is more likely to be involved in astrocyte MCEs, particularly with a fast onset during local circuit activity. So far, the contribution of P2X1/5 activity to astrocyte MCEs has not been explored with GECIs, but P2X activation causes astrocyte Ca2+ transients (primarily somatic) in brain slices and acutely isolated astrocytes, as measured with Ca2+ dyes [Astrocytes express ionotropic P2X purinergic receptors , likely 1/5  or homotsubunits . These leability ,143. P2Xactivity ,145. Thea2+ dyes ,146.1 receptors have been linked to endfoot Ca2+ transients and capillary dilation during neurovascular coupling, suggesting that these ionotropic receptors induce the release of vasoactive molecules that specifically act on capillaries and not arterioles [Coincidently, astrocyte P2X receptor activation enhances purinergic signalling in different brain regions. In the cortex, astrocyte P2X receptors increase ATP release , which mterioles . Astrocyterioles ,147, whiterioles . These e2+, Na+ and K+ and are made up of 16 possible subunits. Astrocytes express homomeric alpha-7 nicotinic acetylcholine receptors , which has an important role in buffering intracellular Ca2+ in exchange for Na+ influx  glutamate activation of Na+-permeable ionotropic kainate or NMDA receptors [+ gradient to drive synaptic glutamate uptake [+ into the cell during GABA uptake [2+ events due to NCX reversal may also trigger Ca2+-induced Ca2+ release from intracellular Ca2+ stores, suggesting NCX reverse function amplifies agonist-induced Ca2+ events in astrocytes [Several possible mechanisms increase intracellular astrocyte Naeceptors ,162,163,e uptake ,164,165,A uptake ,166. Ca22+ may evoke gliotransmitter release, such as glutamate [2+ transients and regulate excitatory transmission. While a number of studies have attempted to model the contribution of NCX to astrocyte MCEs in fine processes [Astrocyte NCX reversal and increased cellular Calutamate ,168, ATPlutamate , and homlutamate . An incrlutamate . This isrocesses ,170,171,2+ channels (VGCCs) [2+. VGCCs do not contribute to spontaneous MCEs [+ uptake [+ and glutamate accumulate. Functionally, astrocyte VGCCs induce the release of glutamate [Astrocytes also express voltage-gated Ca (VGCCs) ,173,174, (VGCCs) . These cous MCEs , but the+ uptake . Thus, alutamate . However2+ that grant them the potential to quickly regulate local processes such as synaptic activity and blood flow. By aiding the integration of information at multiple synapses and among different neuronal types in brain circuits, astrocytes may impact neuronal activity at the population level, thereby contributing to information processing and animal behaviour. Future experiments should focus on GECI tools to better identify MCEs, particularly with a fast onset during nearby synaptic activity. Additionally, pathways of extracellular calcium influx outlined here have important implications for astrocyte calcium physiology and are gaining interest in the field. Approaches to selectively target these pathways will help to better understand their contribution to rapid onset astrocyte MCEs and their functional relevance regarding neuronal network activity.Astrocytes have localized, rapid fluctuations in intracellular Ca