In recent years, thyrotropin-releasing hormone (TRH) and its analogs, including taltirelin (TAL), have demonstrated a range of effects on the central nervous system that represent potential therapeutic agents for the treatment of various neurological disorders, including neurodegenerative diseases. However, the molecular mechanisms of their actions remain poorly understood. In this study, we investigated phosphosignaling dynamics in pituitary GH1 cells affected by TRH and TAL and the putative role of β-arrestin2 in mediating these effects. Our results revealed widespread alterations in many phosphosignaling pathways involving signal transduction via small GTPases, MAP kinases, Ser/Thr- and Tyr-protein kinases, Wnt/β-catenin, and members of the Hippo pathway. The differential TRH- or TAL-induced phosphorylation of numerous proteins suggests that these ligands exhibit some degree of biased agonism at the TRH receptor. The different phosphorylation patterns induced by TRH or TAL in β-arrestin2-deficient cells suggest that the β-arrestin2 scaffold is a key factor determining phosphorylation events after TRH receptor activation. Our results suggest that compounds that modulate kinase and phosphatase activity can be considered as additional adjuvants to enhance the potential therapeutic value of TRH or TAL. Thyrotropin-releasing hormone (TRH) is a tripeptide consisting of pyroglutamyl-histidyl-proline amide. It is produced by neurons in the hypothalamus and circulates through the hypophyseal portal system to the anterior pituitary, where it binds to its receptors and stimulates the release of other hormones ,2. TRH i2+ into the cytosol, where it modulates various signaling pathways. DAG remains near the cell membrane and activates protein kinase C [The effects of TRH and TAL are mediated by the thyrotropin-releasing hormone receptor (TRH-R), which belongs to family A of G protein-coupled receptors (GPCRs). Two subtypes of TRH-R (TRH-R1 and TRH-R2) have been detected in rodents. TRH-R1 is highly expressed in the pituitary gland, whereas TRH-R2 is not detected or is detected at low levels in the anterior pituitary and was absent in the neurointermediate lobe of the pituitary ,15. TRH-kinase C . Other skinase C , phosphokinase C  and Rho kinase C ,20.It has been well-established that TRH-R is phosphorylated and binds to β-arrestin after agonist exposure . β-ArresCurrently, it is clear that the role of β-arrestins is much more diverse. These molecules can have a number of signaling functions, for example, they link Src kinases to receptors  or serveIt is known that β-arrestin2 regulates many signaling pathways in a positive or negative manner . GPCR-deThere is some evidence that certain small GTPases may also be involved in TRH-R-mediated signaling . Small GIn the present study, we investigated the phosphosignaling dynamics in rat pituitary GH1 cells treated with TRH or TAL. Moreover, our studies revealed a specific role of β-arrestin2 in determining phosphorylation events induced by TRH or TAL.The GH1 cell line (CCL 82) was purchased from the American Tissue Type Collection . Horse and fetal bovine sera were from Gibco , LipofectamineTM RNAiMAX and β-arrestin2 antibody were from Invitrogen . All others chemicals were purchased from Sigma-Aldrich  and were of the highest purity available.2 humidified atmosphere. Before starting the experiment, growth medium was replaced with medium supplemented with 1% FBS.GH1 cells (ATCC CCL-82), the tumor cells from pituitary gland of Rattus norvegicus, were routinely grown in Ham’s F-12 Nutrient Mixture supplemented with 15% horse serum, 2.5% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution at 37 °C in 5% COTM RNAiMAX reagent according to the manufacturer´s instructions. Briefly, control or β-arrestin2 siRNA were mixed with an appropriate amount of Lipofectamine RNAiMAX in serum-free Opti-MEM medium and incubated for 5 min at room temperature to obtain siRNA-lipid complexes which were then added to each flask.For transfection with siRNA, cells were grown in 75 cm, 2 tissue culture flasks, each sample in triplicate. At 50% confluence, cells were transfected with siRNA against β-arrestin2 or control siRNA using LipofectamineThen, 2 days after the transfection, cells were treated with or without 1 μM TRH or 1 μM taltirelin (TAL) to ensure that a maximal response to stimulation was elicited. After 30 min of incubation in the absence (control) or presence of ligands, cells were washed with phosphate-buffered saline (PBS) and subsequently harvested by centrifugation using Hettich Universal 320 centrifuge . Cell pellets were snap-frozen in liquid nitrogen and stored at −80 °C until use.2; pH 7.0) and briefly sonicated. Homogenates were solubilized in Laemmli buffer and separated in 10% polyacrylamide gels using the Mini-Protean II apparatus . The resolved proteins were electrotransferred from the gel to a nitrocellulose membrane and stained with Ponceau S (0.1% (w/v) in 5% acetic acid (v/v)) to verify equivalent protein loading and normalize the data. After blocking with 5% reduced-fat milk in TBS-T buffer ; pH 8.0), membranes were incubated with primary antibodies against β-arrestin1 or β-arrestin2 at 4 °C overnight. The next day, membranes were washed three times with TBS-T buffer, probed with secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature and washed three times with TBS-T buffer. Blots were visualized with the enhanced chemiluminescence technique using Thermo Scientific SuperSignal West Dura Extended Duration Substrate according to the manufacturer´s instructions. The generated images were scanned, and the signal intensities of the bands corresponding to β-arrestin1 or β-arrestin2 were quantified using ImageJ software . Data were analyzed using GraphPad Prism 8.0 . Results are presented as the mean ± standard error of the mean (SEM) from three independent experiments. Significant differences between groups were determined using Student´s t-test. Statistical significance was set as p < 0.05.Harvested cells were resuspended in TMES buffer  were homogenized by sonication using a micro probe sonicator (Bandelin Sonoplus) and lysed by boiling at 95 °C for 10 min in 100 mM Tris pH 8.5 containing 2% sodium deoxycholate. Chloroacetamide and Tris(2-carboxyethyl)phosphine to 40 mM and 10 mM final concentration, respectively, were added. The protein concentration was determined using the BCA protein assay kit (Thermo) and 250 µg of protein per sample was used for MS sample preparation. Proteins were digested by 5 µg of trypsin per sample at 37 °C overnight. Phosphopeptides were enriched using TiOy et al. .m/z) with an ion count of 1 × 106 to perform survey scans of peptide precursors from 350 to 1400 m/z. Tandem MS was performed by isolation at 1.5 Th with the quadrupole, higher energy collisional dissociation fragmentation with a normalized collision energy of 35, and rapid scan MS analysis in the ion trap. The maximum injection time was 150 ms, while the MS2 ion count target was set to 104. For MS2, only the precursors with charge states 2–6 were scanned. The dynamic exclusion time was set to 30 s with a tolerance of 10 ppm for the selected precursor and its isotopes. The cycle time was set to 2 s and the selection of the monoisotopic precursor was activated during the measurement.Nano reversed-phase PepMap C18 chromatography columns  were used for LC/MS analysis. The buffer of mobile phase A consisted of 0.1% formic acid dissolved in water, and mobile phase B consisted of 0.1% formic acid dissolved in acetonitrile. The loading buffer consisting of 2% acetonitrile, 0.1% trifluoroacetic acid, and water was used to load the samples onto the C18 PepMap100 trap column ), and sample loading proceeded at 18 μL/min for 4 min. The gradient of mobile phase B from 2% to 35% B was used to elute the peptides from the column. The eluted peptide cations were converted to gas-phase ions by electrospray ionization and then analyzed using a Thermo Orbitrap Fusion . The Orbitrap was set to a resolution of 120 K (at 200 MaxQuant software (version 1.6.3.4) was used to analyze and quantify the raw data . The falp ≤ 0.05. Phosphoproteins that met our above requirements were further analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). This bioinformatics database allowed the assignment of proteins to specific biological processes. From this database, the processes involved in signaling transduction associated with small GTPases and MAP kinases were selected and mapped using the String database.Only phosphosites of phosphoproteins whose fold change values were greater than or equal to ±2 and whose intensities were simultaneously detected in at least two of three independent samples were considered for analysis. The differences between the mean values of the intensities of the relevant groups were analyzed using GraphPad Prism 8. The significance level was set at The aim of this study was to determine how TRH or its analog TAL might affect the phosphosignalosome in the context of biological processes and protein interactions in the pituitary gland, which TRH acts on directly to stimulate thyroid-stimulating hormone production. To this end, we used a rat pituitary tumor cell line (GH1) as a model. The first specific objective was to determine which phosphosignaling pathways are affected by TRH or TAL and what kind of differential changes in phosphosignaling dynamics underlying biased agonism for TRH-R are induced by the two ligands. The next objective was to determine the role of β-arrestin2 in regulating TRH- and TAL-induced phosphosignaling pathways. In the Results section, we focus on the detailed description of the changes induced by TRH or TAL in wild-type or β-arrestin2-deficient cells for each pathway found, including the analysis of the localization and function of some phosphosites. Some descriptive results seen in the figures are mentioned in the ncifcrf.gov, assessed on 5 November 2021). The GO terms enriched in the experimental groups are related to activation and regulation of GTPase activity , protein and peptidyl-serine phosphorylation, and the MAPK cascade .To confirm the efficacy of siRNA targeting β-arrestin2, a Western blot analysis was performed with specific antibodies against β-arrestin2 . This anThe data sets of differentially phosphorylated proteins associated with small GTPase activity and protein phosphorylation consist of 571 phosphosites from 237 phosphoproteins in GH1 cells after siRNA knockdown of β-arrestin2 compared with control GH1 cells (pairwise comparison marked Arr/C) , 216 phoBioRender.com (assessed on 1 September 2021).The altered phosphoproteins were sorted according to their association with classes of small GTPases. The classes of small GTPases most affected by β-arrestin2 knockdown and/or TRH/TAL were Ras and Rho/Rac/Cdc42. For clarity, the changes in phosphorylation and phosphoprotein associations for each class of small GTPases are shown in The changes in phosphorylation of phosphoproteins involved in Ras GTPase signal transduction are shown in Knockdown of β-arrestin2 markedly affected phosphorylation of proteins involved in the Src/EGFR/Ras/PI3K/Akt pathway and downstream effectors of Akt kinase . The SrcPhosphosite Ser181 in PAK4 is located in the region responsible for PAK4 binding to 14-3-3 proteins and its phosphorylation is promoted by PI3K signaling . In the The hyperphosphorylation pattern at phosphosites Ser124, Ser126, and Ser129 in Akt1 with its subsequent activation may correlate with changes in phosphorylation of downstream effectors such as MAP3K5, MAP3K9, Sik3, Tsc1/2, or proteins that interact with mTOR  and downstream effectors of the mTOR complex  . In β-arAMPK, which affects downstream and upstream effectors and associated proteins of mTOR, was differentially phosphorylated at its catalytic and regulatory subunits Prkaa1 and Prkab1, respectively. Knockdown of β-arrestin2 resulted in hyperphosphorylation at Thr488 in Prkaa1 and at Ser108 in Prkab1 . The secFor the phosphoprotein Lats1, the change at Ser1111 occurred in all pairwise comparisons. This protein was hypophosphorylated in β-arrestin2-deficient cells, TRH-treated cells, or TAL-treated cells. Subsequent TRH or TAL treatments of β-arrestin2-deficient cells induced hyperphosphorylation at this phosphosite . AlthougThe MEK1/2 and ERK1/2 kinases were not directly affected in their phosphorylation patterns, but their interacting partners and downstream effectors were differentially phosphorylated. Two pseudokinases, the kinase suppressors of Ras 1 and 2 (KSR1 and KSR2), serve as scaffolds linking Raf isoforms and their substrates . The KSRThe activated ERK phosphorylates many substrates, including Rps6ka3 (Rsk2) , Rreb1 , and CdkDoublecortin-like kinase protein 1 (Dclk1) was found to be differentially phosphorylated on ten phosphosites with different phosphorylation patterns under different experimental conditions . All phoThe second class of small GTPases affected by β-arrestin2 knockdown and/or TRH/TAL treatment is a Rho/Rac/Cdc42 class. The changes in phosphorylation occurred in guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and downstream effectors. For clarity, proteins with changes in phosphorylation were sorted according to their association with Rho GTPase , Rac GTPWhen proteins with GEF or GAP activities were compared, the larger set of GEFs that activate Rho GTPases were found to be differentially phosphorylated. β-Arrestin2 knockdown resulted in hyperphosphorylation of GEFs and GAPs, whereas treatment of wild-type cells with TRH and TAL resulted in hypophosphorylation of GEFs and GAPs. Treatment with TAL had a more pronounced effect on differential phosphorylation of GEFs than treatment with TRH .Of the phosphosites with changes in phosphorylation in GEFs, only one was in functional domains. Phosphosite Ser48 in dishevelled segment polarity protein 3 (Dvl3) was hyperphosphorylated in β-arrestin2-deficient cells  and was All GAPs were also differentially phosphorylated at phosphosites located in disordered regions by determining their position using the UniProt database. The two GAPs, Myo9b and Arhgap35, were hypophosphorylated at several phosphosites after TRH or TAL treatments , but knoWhereas TRH and TAL treatments caused hypophosphorylation of Rho downstream effectors in wild-type cells, they affected phosphorylation of effectors in both directions in β-arrestin2-deficient cells. The phosphosite Ser1124 in Rock2 was hyperphosphorylated in β-arrestin2-deficient cells and in wild-type cells treated with TRH or TAL, but hypophosphorylated in β-arrestin2-deficient cells treated with TRH or TAL . Three dOverall, treatment with TRH and TAL induced hypophosphorylation of GEFs, GAPs, and downstream effectors of Rac GTPases . The numThe most important Rac GEF appears to be Dock7, which was differentially phosphorylated in all five pairwise experimental groups , resultiSeveral upstream and downstream Rac GTPase effectors were found to be differentially phosphorylated . The ErbAnother interacting partner of PI3K with the stimulating effect on Rac activity is the kinase Ptk2β which was found to be hypophosphorylated at Ser389, Ser392, Ser394, Ser396, and Ser399 in wild-type cells after treatment with TRH or TAL and in β-arrestin2-deficient cells after treatment with TAL . Two SRS340SGFIYQ [Most Cdc42-GEFs were differentially phosphorylated compared with Cdc42-GAPs . Some Cd40SGFIYQ  with the40SGFIYQ . Phospho40SGFIYQ .Arhgef7 also interacts with several binding partners, such as Git1, Scribble, Pak kinases, Rac1, and Cblb ,147. GitCblb belongs to ubiquitin E3 ligases of the Cbl family, which bind to Arhgef7 after Cdc42 activation, leading to an increase in GEF activity of Arhgef7 in a positive feedback loop . Cblb waOnly five Cdc42-GAPs were differentially phosphorylated . All CdcAs mentioned above, Arhgef7 is a binding partner for Scrib and Git1 and a scaffold for members of the Hippo pathway . Git1/2 are proteins with GAP activity for Arf GTPases . The GitFive proteins with GEF activity for Arf proteins were found to have differential phosphorylation. Gbf1, an Arf1 GEF, was hyperphosphorylated after β-arrestin2 knockdown and after TRH treatment of wild-type cells and hypophosphorylated in wild-type and in β-arrestin2-deficient cells treated with TAL . Arf1 inThree Arfgef proteins were found to be differentially phosphorylated, Arfgef1 and Arfgef2 in three phosphorylation clusters. More Arf-GAPs were differentially phosphorylated than Arf-GEFs . Git2 waRab GTPases are important regulators of membrane transport and are involved in membrane fission, transport, tethering, docking, and fusion . In our Rabenosyn (Rbsn) serves as a molecular link between Rab5 and a specific phosphatidylinositol 3-kinase called hVPS45 and is recruited to early endosomes in a PI3K-dependent manner . It alsoThe other interacting Rab protein, nischarin (Nisch), interacts with Rab4, Rab9, and Rab14 localized to endosomes and can be localized to endosomes by binding PI3P through its PX domain . In our The early endosomes can be recycled to the plasma membrane via recycling endosomes . Dennd6aRabgap1 and Tbc1d9b have GAP activity to Rab11 ,178. PhoRab7 regulates cargo transport from early endosomes to late endosomes and subsequently for degradation in lysosomes . An inteAutophagy is responsible for the degradation of intracellular components by transporting them to lysosomes to maintain cellular homeostasis . Rab12 iSeveral Rab proteins are associated with the Golgi network ,183. Den321LLPXTPP329SP with PXXP motif  which is required for Grb2 binding and regulation in Ralgps1 [Although only a few proteins associated with Ral GTPases were found to be differentially phosphorylated, some alterations might be associated with a variety of signaling pathways and biological processes. Ralgps2, a GEF protein for RalA , was dif Ralgps1 , suggestRal-binding protein 1  was found to be differentially phosphorylated in its N-terminal domain in three phosphorylation clusters . All phohttps://scansite4.mit.edu, accessed on 9 December 2021). The cyclin G-associated kinase (Gak) has a region between 752 and 979 amino acids with interaction sites for clathrin, AP2, and dynamin [The μ2-subunit of AP2 can be phosphorylated by the adaptor-associated kinase AAK1, which is colocalized with many components of the endocytic machinery such as clathrin, AP2, and dynamin 1 . In our  dynamin . Gak wasRanGAP1 with the GAP activity for Ran was hyperphosphorylated at Ser427 after treatment of wild-type cells with TRH and after treatment of β-arrestin2-deficient cells with TRH or TAL . This phRanGAP1 physically interacts with a RanGEF Mycbp2, inhibits its E3 ubiquitin ligase activity and transports it to the nucleus. At the same time, Mycbp2 was a weak inhibitor of the GAP activity of RanGAP1 . Mycbp2 Ran-binding proteins coordinate with Ran to regulate nuclear import and export . In addiRapgef1 (C3G) was hyperphosphorylated at Ser375 after knockdown of β-arrestin2 and hypophosphorylated at Ser239 after treatment with TAL . This prStimulation of TRH-R also resulted in altered phosphorylation of phosphoproteins involved in the β-catenin signaling pathway. The β-arrestin complex with GPCR interacts with Src and EGFR or axin and GSK3β to affect phosphorylation or stabilization of β-catenin . While bβ-Catenin was differentially phosphorylated at Ser191 and Ser552, which was hypophosphorylated after β-arrestin2 knockdown or in wild-type cells treated with TAL and hyperphosphorylated in β-arrestin2-deficient cells treated with TAL . Both phActivation of β-catenin signaling is mediated by PI3K/Akt or Wnt/Frizzled signaling pathways . FrizzleIn this study, we investigated the changes in the phosphoproteome induced by the activation of the TRH receptor in pituitary GH1 cells as well as in cells lacking β-arrestin2. There were hundreds of alterations in the phosphoproteome in each experimental group. Detailed analysis revealed that only some altered phosphosites were located in functional domains with enzyme activity or interaction sites with other proteins. According to the UniProt database, most phosphosites were located in disordered regions of phosphoproteins. The intrinsically disordered proteins, which lack stable tertiary structure, undergo stabilization or destabilization of transient secondary structure and more global disorder-to-order or order-to-disorder transitions upon ligand binding or post-translational modifications, including phosphorylation ,228. OurAlthough GPCRs are known to initiate signaling cascades through β-arrestin , this imβ-Arrestin interacts directly with some small GTPases and their regulatory or binding proteins . It affeSome proteins were found to be associated with multiple small GTPases, so that there are multiple cross-talks between the signaling pathways of the members of the small GTPases. The crucial cross-talk is represented by a complex consisting of Arhgef7, Git1, and Scrib associated with Arf and Cdc42 signaling pathways that overlap with Rac and Ras signaling pathways via Lats1 . Akt1 kiNot only MEK and ERK, but also other MAP kinases  were not affected by TRH or TAL in wild-type GH1 cells. On the other hand, Map3k7 (TAK1) was slightly hypophosphorylated after TAL treatment and Map3k4 (MEKK4) and Map4k1 were markedly hypophosphorylated after TRH or TAL treatments of wild-type cells . All theMap4k kinases were found to phosphorylate and activate Lats1/2 kinase, a component of the Hippo signaling pathway. Activated Lats1/2 in turn promotes inhibitory phosphorylation of the transcriptional co-activators YAP/TAZ . WhereasTRH has been reported to induce hyperphosphorylation of EGFR at tyrosine phosphosites and its transactivation via PLCβ/PKC or Gβγ/Src signaling pathways ,242. In The changes in phosphorylation of proteins involved in the Rab, Arf, and Ral signaling pathways suggest the effect of TRH, TAL, and β-arrestin2 deficiency on clathrin-dependent and -independent endocytosis and cargo transport between membrane-enclosed organelles ,180,196.There are two limitations in this study. The first limitation is the use of a cancer-derived cell line. It is known that cancer cell lines may alter signaling pathways. Therefore, the results of this study may correspond more to phosphosignaling processes in pituitary tumors than in normal pituitary cells. The second limitation is that β-arrestin2 silencing was not complete under our conditions. It is likely that different levels of β-arrestin2 may affect cell signaling and biological processes to different extent. Nonetheless, it is clear that deficiency of β-arrestin2 can have significant effects on biological processes, including TRH receptor-mediated signaling and it can trigger adaptive cellular processes via phosphosignaling to compensate for the changes in β-arrestin2 scaffolding and function.Activation of the TRH receptor by TRH or TAL resulted in different phosphorylation patterns in the phosphoproteome observed in both wild-type GH1 cells and β-arrestin2-deficient cells . This suIn the present study, TRH and TAL were found to have a strong effect on the phosphorylation of GEFs, GAPs, and other members of the small GTPase signaling pathways. They affect the phosphorylation patterns of all small GTPase classes. Simultaneously, changes in the phosphorylation of MAP kinases, Ser/Thr protein kinases, Tyr protein kinases, members of the Wnt/β-catenin and Hippo signaling pathways were detected. The phosphorylation patterns triggered by TRH and TAL were found to be different, suggesting that these ligands exhibit biased agonism at the TRH receptor. It was also shown that β-arrestin2 is a key mediator that determines TRH- and TAL-induced phosphoregulation of several phosphosignaling pathways. Downregulation of β-arrestin2 resulted in alterations in the phosphorylation of EGFR and Src kinase with potential implications for their activities. These findings may be useful in the treatment of neurodegenerative disorders and neurological pathologies with TRH and TAL. Modulators of kinase and phosphatase activities and cellular levels of β-arrestin2 can be used as additives along with TRH and TAL to support their beneficial effects.


S75E and the complex of UCHL3S75E with Ub-PA and preliminarily explained the structural mechanism of phosphorylation-enhanced UCHL3 deubiquitinating activity. Additionally, the cleavage activity of UCHL3 toward different types of synthesized poly-ubiquitin chains in vitro was tested. The results showed that purified UCHL3S75E enhanced the cleavage activity toward Ub-AMC compared to UCHL3WT. Meanwhile, UCHL3S75E and UCHL3WT did not show any cleavage activity for different types of di-ubiquitin and tri-ubiquitin chains. However, UCHL3 could hydrolyze the K48 tetra-ubiquitin chain, providing compelling in vitro evidence confirming previous in vivo results. Thus, this study shows that UCHL3 can hydrolyze and has a cleavage preference for polyubiquitin chains, which expands our understanding of the phosphorylation regulation of UCHL3 and lays a foundation for further elucidation of its physiological role.Ubiquitin C-terminal hydrolase-L3 (UCHL3), an important member of the ubiquitin C-terminal hydrolase family, is involved in DNA repair and cancer development. UCHL3 can cleave only complexes of monoubiquitin and its conjugates, such as Ub-AMC, His, or small ubiquitin-like modifier, but not polyubiquitin chains. Phosphorylation of Ser75 promotes the cleavage activity of UCHL3 toward poly-ubiquitin chains in vivo, but biochemical evidence in vitro is still lacking. Here, we first analyzed the structure of simulated phosphorylated UCHL3 Ubiquitination is a ubiquitous post-translational modification that plays important roles in various life processes, including protein degradation, signal transduction, and DNA damage repair ,2,3,4,5.Deubiquitination is the inverse process of ubiquitination and is performed by deubiquitinases (DUBs). DUBs can trim Ub molecules from target proteins and regulate the dynamic balance of protein ubiquitination ,20. The DUBs are strictly regulated to ensure precise biological functions. Post-translational modifications (PTMs), such as phosphorylation, are important regulatory mechanisms of DUBs ,33,34. CUCHL3, an important member of the UCH family, plays an important role in the occurrence and development of cancer, DNA damage repair, osteoblast differentiation, and other diseases and life processes ,50,51,52In 2016, Luo et al. first found that phosphorylated UCHL3 at Ser75 can cleave single or polyubiquitin chains on the substrate RAD51 and play an important role in DNA damage repair and tumor occurrence. Phosphorylated UCHL3 showed stronger ubiquitin chain cleavage activity against RAD51 in vivo . HoweverWT) and simulated phosphorylated UCHL3 (UCHL3S75E) were recombinantly expressed and purified using affinity chromatography, ion exchange chromatography, and gel filtration chromatography. We first analyzed the structure of UCHL3S75E and the complex of UCHL3S75E with its substrate, Ubiquitin Propargylamide (Ub-PA), and explained the mechanism of phosphorylation-promoted activity of UCHL3. Finally, the cleavage activity of UCHL3S75E and UCHL3WT on different types of synthesized ubiquitin chains was studied in vitro, and it was found that UCHL3 preferentially cleaved activity toward K48 tetramer-Ub (K48 tetra-Ub) chains. These studies are expected to lay a foundation for understanding the molecular mechanism of phosphorylation modification and the ubiquitin chain cleavage preference of UCHL3.In this study, Ser75 on UCHL3 was mutated to Glu to achieve a simulated phosphorylation modification. Wild-type UCHL3  and ligated into the pET28b vector, yielding an expression construct with an N-terminal His6 tag.S75E, and then UCHL3S75E was overexpressed and purified. The plasmid and protein purification results for UCHL3S75E are shown in The QuikChange Site-Directed Mutagenesis Kit was used to construct the plasmid UCHL3S75E was constructed by site-directed mutagenesis, overexpressed in Escherichia coli (E. coli) strain BL21 (DE3) using the vector pET-28a , and purified to homogeneity. To explore whether phosphorylation changes the deubiquitinating activity of UCHL3 by directly causing its conformational changes, crystallization trials for UCHL3S75E were carried out. The structure of UCHL3S75E was successfully determined by molecular replacement using an atomic model of human UCHL3WT . The X-solution A; the da UCHL3WT B. In add UCHL3WT C.S75E bound to Ub-PA (UCHL3S75E-Ub-PA) at high resolution  . Data cS75E-Ub-PA with UCHL3WT-Ub-VME and found that the simulated phosphorylation modification still resulted in a 3.5 Å outward shift of the α3 helix in which the amino acids at positions 74, 75, and 76 of UCHL3S75E-Ub-PA displaced relative to that in UCHL3WT-Ub-VME   and hum ID:1XD3 ). Data cS75E and the complex of UCHL3S75E-Ub-PA and attempted to confirm whether phosphorylation modification can improve the activity of UCHL3 in vitro. A ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) hydrolysis assay was performed as previously described [WT, phosphorylation increased Ub-AMC hydrolytic activity by approximately10 times , which was synthesized based on K27 di-Ub . However, UCHL3 of 5 uM C–E. Furtas 15 uM F; the fuWT or UCHL3S75E could hydrolyze longer ubiquitin chains  as a substrate to test whether UCHL3n chains A. Sodiumertra-Ub B.S75E had a slight structural change in the α3 helix and a loop near the active center compared to UCHL3WT. Therefore, the mechanism of phosphorylation-induced UCHL3 activity changes belong to the conformational changes caused by phosphorylation of the noncatalytic center, and phosphorylated UCHL3 has a greater degree of structural changes when bound to the Ub substrate. However, we did not observe obvious changes in the structures of UCHL3S75E and UCHL3S75E-Ub-PA, so as not to provide a clear illustration of the mechanism of phosphorylation-induced activity changes in UCHL3.PTMs of DUBs play important roles in various cellular activities, and phosphorylation is one of the most common modes. According to existing reports, the molecular mechanism of phosphorylation-regulated deubiquitination activity changes in DUBs is mainly divided into three ways. First, phosphorylation modification directly causes conformational changes in DUBs such as USP14; phosphorylation at Ser432 may open the BL2 loop of USP14 to release self-inhibition and promote hydrolysis of Ub substrates . AnotherS75E did not show enhanced cleavage activity compared to UCHL3WT. These results may be due to the difference between the simulated and true phosphorylation by ATM. Similar to DUBA, it is inactive by mimic phosphorylation, whereas it can be activated when it is phosphorylated by CKII kinase; there are structural changes after binding to the Ub substrate instead of apo DUBA [We found that UCHL3 had a cleavage preference toward K27/48 tri-Ub and cyclinB1-K48 tetra-Ub chains, but UCHL3apo DUBA . In addiS75E can cleave polyubiquitin chains, then there should also be an S1′ site. The Ub moiety that binds to the enzymatic S1′ site contributes to Lys, which binds to the carboxyl group contributed by the Ub bound at the S1 site to form an isopeptide bond, penetrating the active center. Therefore, we propose that UCHL3 may use di-Ub as a recognition unit to cleave polyubiquitin chains. That is, ubiquitin occupies the S1 and S2 sites of UCHL3 and then cleaves the isopeptide bond between S1 and S1′ , and the protein was overexpressed and purified using the same method.His-tagged full-length UCHL3escribed . The phoS75E and Ub-PA were removed on a Superdex 75 size-exclusion column run with 10 mM Tris pH 7.5 and 150 mM NaCl, and the final complex was concentrated to 20 mg. mL−1. The results for UCHL3S75E bound to Ub-PA are shown in Ub-PA was a gift from the Lei Liu laboratory ; UCHL3S75E and Ub-PA were incubated in a molar ratio of 2:1 in a 100 uL reaction system containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl at 4, and 18 °C, respectively; samples were taken at 1, 2, and 4 h, respectively. The complex was analyzed via SDS-PAGE, and excess UCHL3S75E- Ub-PA were grown for a week at 18 °C by the hanging drop method by mixing the UCHL3S75E-Ub-PA with an equal volume of reservoir solution containing 25% PEGMME 2000 and 0.1 M Bis-Tris (pH 6.9). Crystals were equilibrated in a cryoprotectant buffer containing reservoir buffer and 20% ethylene glycol (v/v) and were flash frozen in a cold nitrogen stream at –170 °C. Crystals of UCHL3S75E were grown for 4–7 days by mixing UCHL3S75E with an equal volume of reservoir solution containing 0.1 M Bis-Tris (pH 8.5), 0.3M MgCl2, and 30% PEG 4000 using the hanging drop method; the crystals were equilibrated using the same method as above.Crystals of UCHL3S75E-Ub-PA and UCHL3S75E were collected on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility and processed using HKL2000. Subsequent processing was performed using programs from the CCP4 suite. The UCHL3S75E-Ub-PA and UCHL3S75E structures were solved by molecular replacement using PHASER [WT-Ub-VME (PDB ID:1XD3 [WT (PDB ID:1UCH) were selected as research models for molecular replacement. The structure was refined using PHENIX software [Native diffraction datasets for UCHL3g PHASER . Human U ID:1XD3 ) and UCHsoftware . The staWT and UCHL3S75E, which were gifts from the Lei Liu laboratory . The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mg/mL ovalbumin, 5 mM ATP/MgCl2 (freshly prepared), 1 mM DTT (freshly prepared), 0.05% Tween, and 5 nM UCHL3S75E, UCHL3WT, or 50nM UCHL3WT. To start the reaction, 1 μM of Ub-AMC was added to the system. Ub-AMC hydrolysis was measured at Ex355/Em460 on an EnVision plate reader (PerkinElmer). Fluorescence intensity was recorded every 250 s for 50 min or 500 s for 150 min. The experiment was repeated thrice, and the average value was calculated. The result was calculated based on the rate from the start time to the time of starting equilibrium point.Ub-AMC was used to measure the deubiquitinating activity of UCHL3WT and UCHL3S75E in vitro. Di-Ub cleavage reactions containing 5 μM substrate and 3 μM enzyme were conducted in a reaction buffer including 25 mM Tris-HCl (pH 8.0) and 150 mM NaCl at room temperature for different time points. Finally, the hydrolysis activity was analyzed using SDS-PAGE gels. For the tri-Ub and tetra-Ub chain hydrolysis assays, the reaction conditions were the same as above.Different types of ubiquitin chains were obtained from Yiming Li Laboratory  (Hefei US75E enhanced the cleavage activity toward Ub-AMC compared to UCHL3WT. We first analyzed the structures of the simulated phosphorylation of UCHL3S75E and the complex of UCHL3S75E with Ub-PA. We found that UCHL3S75E has slight structural changes in the α3 helix and the loop near the active center compared to UCHL3WT, and it is preliminarily speculated that the exposure of the active center of UCHL3 and the enhanced recognition of substrates are the reasons for the phosphorylation modification to enhance the activity of UCHL3 deubiquitinase. We proved that UCHL3S75E also cannot cleave any type of di-Ub chains; however, UCHL3 tends to cleave longer Ub chains and has a preference for K48-linked Ub chains. These findings expand our understanding of the phosphorylation regulation of UCHL3 and clarify the multicomplex functions of UCHL3.UCHL3 has been designated as an important DUB in various cancers and for DNA repair. A study also found that phosphorylation of Ser75 promotes the cleavage activity of UCHL3 toward poly-ubiquitin chains in vivo. Therefore, it is necessary to describe the mechanism of phosphorylation modification and the preference of Ub chains in detail. In this study, in vitro assay showed that purified UCHL3


Hypoplastic left heart syndrome (HLHS) is a rare but genetically complex and clinically and anatomically severe form of congenital heart disease (CHD).MYH6 gene inherited from the  parents. MYH6 is known to be highly polymorphic; a large number of rare and common variants have variable effects on protein levels. We postulated that two hypomorphic variants led to severe CHD when associated in trans; this was consistent with the autosomal recessive pattern of inheritance. In the literature, dominant transmission of MYH6-related CHD is more frequent and is probably linked to synergistic heterozygosity or the specific combination of a single, pathogenic variant with common MYH6 variants.Here, we report on the use of rapid prenatal whole-exome sequencing for the prenatal diagnosis of a severe case of neonatal recurrent HLHS caused by heterozygous compound variants in the The present report illustrates the major contribution of whole-exome sequencing (WES) in the characterization of an unusually recurrent fetal disorder and considered the role of WES in the prenatal diagnosis of disorders that do not usually have a genetic etiology. The first-trimester prenatal diagnosis of congenital heart disease (CHD) using transvaginal ultrasound was first reported in 1990s . In the Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of CHD . It accoNKX2.5 [NOTCH1 [MYH6 [.Although the cause of HLHS is unknown, severe cardiomyopathy is thought to be a complex genetic disorder ; the genNKX2.5 , NOTCH1  [NOTCH1 –11 and MH1 [MYH6 , 13).MYH6 variants) early in pregnancy. Written informed consent was obtained from the patient for publication of this case report and any accompanying images.The present report highlights the use of rapid prenatal whole-exome sequencing (WES) to diagnose a severe case of recurrent, neonatal HLHS (cause by compound heterozygous 2) with an unremarkable medical history other than idiopathic hypertension . After spontaneous conception, the woman was referred to our center in April 2020 for an undefined fetal cardiopathy. The results of the first-trimester ultrasound scan were normal , with a low risk of trisomy 21  and a negative, non-invasive prenatal test requested by the woman. The diagnosis of severe HLHS with mitral and aortic stenosis was made at 23 weeks of gestation (w.g.) , the women was referred for an early morphological ultrasound check-up on her next pregnancy, which revealed cardiac asymmetry and aortic stenosis  classification [MYH6 (NM_002471.4) chr14: g.23853649A > T c.5565 + 2 T > A, predicted to be a loss-of-function (splice donor) variant that was not present in population databases [MYH6 (NM_002471.4) chr14:g.23857395G > T c.4328C > A p.(Ala1443Asp) – a very rare missense variant that is predicted to be pathogenic, according to the ACMG classification and the Varsome database (https://varsome.com/). Furthermore, the wild-type and mutant amino acids’ respective physicochemical properties are very different , and the alanine at position 1443 is highly conserved from Tetraodon nigroviridis to Homo sapiens. Before receiving the WES results, the severe prognosis for HLHS prompted the woman to request termination of pregnancy at 16 w.g.In view of the recurrence of HLHS, we suggested WES. The couple agreed, and a quartet analysis was performed on fetal DNA stored from the previous pregnancy, DNA from the ongoing pregnancy, and the parents’ DNA. WES revealed that both fetuses were compound heterozygous for probably pathogenic fication ) inheritatabases . The fatMYH6 codes for myosin heavy chain α (MHC-α), the major myosin isoform in the fetal heart [MYH7, located 4 kb downstream of MYH6 and which codes for MHC-β. Relative to MHC-β, MHC-α has 1.5 to 3 times more contractile velocity and a 0.6-fold shorter actin attachment duration. In the adult heart, MHC-β expression is predominates in the ventricles and MHC-α expression predominates in the atria.Cardiac muscle myosin consist of two heavy chain subunits, two light chain subunits, and two regulatory subunits. al heart . After bMYH6 variants have been described in several cardiac diseases. For example, it has been shown that heterozygous variants are responsible for moderately severe types of CHD, such as atrial septal defects and AVSD [MYH6 is also thought to be involved in adult familial hypertrophic cardiomyopathy. The association between MYH6 variants and HLHS has already been reported; in a study of 190 unrelated HLHS subjects, rare MYH6 variants were observed in around 10% of cases [p = 0.000068), abnormally high proportion of pathogenic MYH6 variants observed in 197 patients with HLHS (vs. 813 controls) [MYH6 variants was also 10% in this population of patients with HLHS. Despite the presence of autosomal dominant transmission of left heart disease/cardiomyopathy in some families, a single pathogenic MYH6 variant alone might not be enough to cause disease: a second hit might be necessary. Interestingly, the second hit might be either a second rare MYH6 pathogenic variant (i.e. compound heterozygosity), a “common” MYH6 variant  or a variant in a modifier gene like FLNC (i.e. synergistic heterozygosity). These various possibilities might explain the broad phenotypic spectrum associated with MYH6 variants (Fig. MYH6 and CFTR are probably associated with a broad spectrum of effects and marked phenotypic variability.Pathogenic and AVSD  whereas and AVSD . MYH6 isof cases . More reLHS vs. 8 controlsnts Fig. , which rDiscordant inheritance patterns have been reported in the literature. The versatile nature of MYH6-related inheritance prompts us to think that MYH6 is at the interface between Mendelian autosomal-dominant inheritance and autosomal-recessive inheritance and thus has an unconventional pattern of inheritance.The “common variant hypothesis” has been further underpinned by several recent reports of an association between rare variants and common variants in MYH6-related conditions  and for MYH6 mutations are rare; to the best of our knowledge, the only literature report concerned cases in four families [MYH6 variant. It should be noted that MYH6 is one of the most variable genes, and so there are probably many common alleles. In the case described here, neither the fetus nor the parents carried a “common” MYH6 or FLNC variant; this is consistent with the absence of a family history of CHD and hypertrophic cardiomyopathy and the classical autosomal recessive transmission of HLHS.Cases of compound heterozygosity or homozygosity for families . Most ofMYH6 variants is associated with greater expression of MYH7, which results in hypocontractility [MYH6 mRNA and higher levels of MYH7 mRNA [Moreover, the presence of actility . AccordiYH7 mRNA .MYH6 variants. Firstly, severe valvular stenosis might led to ventricular dysmorphology and reduced preload [MYH6 variants is strongly associated with MYH7 overexpression in both atrial and ventricular tissues [There are two possible explanations for a disease-causing effect of  preload . Secondl preload . In the  tissues , leadingMYH6 variants related to fetal HLHS during an ongoing pregnancy. Our case also emphasizes the value of WES for understanding the unusual recurrence of fetal anomalies during pregnancy. Fetal anomalies are detected in 3% of all pregnancies and are responsible for 20% of prenatal deaths [The present report is the first to have described the detection of l deaths . Thus, sl deaths . WES impl deaths . Furtherl deaths . The pre


Using UPLC-MS/MS, ten new DPP-IV inhibitory peptides were identified from the oat protein hydrolysate by neutrase. Among these peptides, IPQHY, VPQHY, VAVVPF, and VPLGGF exhibited the strongest DPP-IV inhibitory activity with IC50 values below 50 μM, and all of them acted as mixed-type inhibitors. Molecular docking indicated that the above four oat-derived peptides were predicted to form hydrogen bonds, attractive charge, and hydrophobic interactions with the residues of the active site of DPP-IV. Therefore, our results suggest that oat is an excellent protein source for food-derived DPP-IV inhibitory peptides and it has the prospect of becoming a dietary supplement for T2DM.In this study, flavourzyme, papain, neutrase, and alcalase, as well as gastrointestinal digestion simulated with pepsin and pancreatin, were used to hydrolyze oat protein, and the dipeptidyl peptidase-IV (DPP-IV) inhibitory activities of the oat protein hydrolysates were investigated. The results indicated that the oat protein hydrolysate by neutrase showed the most potent DPP-IV inhibitory property with an IC Diabetes mellitus (DM), a metabolic syndrome, is one of the most prominent health problems in the world. Most diabetes can be divided into two main etiological mechanisms. Type I diabetes mellitus T1DM) is an autoimmune disease in which absolute insulin deficiency due to the immune-mediated destruction of beta cells in the pancreas leads to hyperglycemia. However, insulin resistance is the primary feature of type 2 diabetes mellitus (T2DM) and can be prevented by interventions of lifestyle and diet DM is an . This imIt is known that synthetic DPP-IV inhibitors used in clinics are often reported to have various side effects, such as headache, hypoglycemia, pancreatitis, arthralgia, and infections . As a coAs one of the most widely cultivated cereal grains, oat contains 12% to 20% protein, higher than many other kinds of cereal . Oat-proIn this study, the oat proteins were hydrolyzed using different enzymes and the DPP-IV inhibitory activities of the oat protein hydrolysates were determined using glycyl-prolyl-7-amino-4-methyl coumarin (Gly-Pro-AMC) as the substrate. The peptide sequences of the oat protein hydrolysate were further analyzed using an ultra-performance liquid chromatography−tandem mass spectrometer (UPLC-MS/MS). Additionally, the type of inhibition of oat-derived DPP-IV inhibitory peptides and their interactions with the active site of DPP-IV were further explored.The oats were provided by Lvba Grain and Oil Group Co., Ltd. . Flavourzyme, papain, neutrase, and alcalase were purchased from Pangbo Biological Engineering Co., Ltd. . The 2,4,6-trinitrobenzene sulfonic acid (TNBS) was purchased from Huaxia Reagent Co., Ltd. . Pepsin , pancreatic , Tris-HCl, human DPP-IV, acetonitrile (ACN), and formic acid (FA) were purchased from Sigma-Aldrich . Acetic acid was purchased from Acro Biosystems Co., Ltd. . L-Leu and HEPES were purchased from Solarbio Technology Co., Ltd. . Gly-Pro-AMC was purchased from AAT Bioquest . All of the other chemicals were of analytical grade.w/v) ratio, and the pH was adjusted to 10.5 with 1 M NaOH. After stirring for 2 h, the supernatant was collected after centrifugation at 4000× g for 20 min at room temperature. Next, 1 M HCl was used to bring the pH of the supernatant down to 4.3, and the mixture was then agitated for 10 min. After centrifugation at 7000× g for 20 min at room temperature, the protein precipitate was recovered, vacuum freeze-dried, and stored at −20 °C.The oat powder was mixed with water in a 1:10 (w/v), and heated to 90 °C for 10 min to denature the protein. After cooling down, the pH of the oat protein solution was adjusted with 1 M NaOH or HCl. Flavourzyme, papain, neutrase, or alcalase at an enzyme/substrate ratio of 4% (w/w) was added to hydrolyze the oat protein for 180 min at the proper temperature  and pH . In addition, in vitro simulated gastrointestinal digestion (SGID) of the oat protein by pepsin  and pancreatin  was also performed. Pepsin and pancreatin were added at a ratio of 2%. Boiling for 5 min was used to inactivate the enzymes. The hydrolysate was centrifuged at 1300 g for 10 min. Then, the supernatant was fractionated by an ultrafiltration membrane with a molecular weight (MW) cut-off of 1000 Da . The fraction with MW less than 1000 Da was finally collected, freeze-dried, and stored at −20 °C.The oat protein was dissolved in distilled water at a concentration of 5%  of the sample and blank groups, respectively. V (L) and m (g) are the volume and mass of the sample used, respectively.The TNBS method was used to measure the amino nitrogen content of the sample, with minor modifications . In bries, Ac, and Ab are the intensity of the fluorescence of the sample, control, and blank groups, respectively.The DPP-IV inhibitory activity assay of the sample was performed using Gly-Pro-AMC as the substrate, as previously described . Briefly50) of the sample was calculated according to the DPP-IV inhibition activity against the concentrations of the sample. In addition, the type of inhibition of the sample was obtained from the Lineweaver–Burk equation and double reciprocal plots.The half-maximal inhibitory concentration  and an analytical nano-column of Acclaim PepMap RPLC C18 column . Gradient elution was used with a mobile A of 0.1% FA in 2% ACN and a mobile B of 0.1% FA in 80% ACN. Mobile B increased from 6% to 9% in 5 min, 9% to 14% in 15 min, 14% to 30% in 30 min, 30% to 40% in 8 min, and 40% to 95% in 2 min. The flow rate was set at 600 nL/min. The sample was then analyzed using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometry (Thermo Scientific). The spray voltage and the capillary temperature were set at +2.2 kV and 270 °C. The m/z for the full scan was set from 100 to 1200. The scan resolutions for MS and MS/MS were set to 70,000 and 17,500, respectively. The normalized collision energy setting was set at 40. The raw MS data file was analyzed and searched against the database of UniProt Avena sativa L. (Oat) using Byonic. The precursor ion mass tolerance and MS/MS tolerance were set at 20 ppm and 0.02 Da, respectively. Only highly confidently recognized peptides were used.The sample dissolved in 0.1% FA was analyzed in an Ultimate 3000 UPLC system  using a pre-column of Acclaim PepMap RPLC CThe interactions between the oat-derived peptides and human DPP-IV were investigated by the molecular docking technique using Discovery Studio 2017 client software. The X-ray diffraction crystal structure of human DPP-IV was obtained from the RCSB Protein Database (PDB code: 2BGR). After removing water and adding hydrogen atoms, the structure of human DPP-IV was defined as the receptor. The structures of oat-derived DPP-IV inhibitory peptides were defined as the ligands after the energy was minimized using a CHARMm force field. The CDOCKER protocol was used for molecular docking with a binding site of coordinates x = 39.964044, y = 51.623473, and z = 38.513685, and a radius of 9 Å. The best binding conformation was output according to CDOCKER energy. The receptor–ligand interaction analysis was further used to explore the interactions between the peptides and DPP-IV.n = 3). The difference between two groups was analyzed using Student’s t-test, and the difference between three and more groups was analyzed using one-way analysis of variance (ANOVA) with the least significant difference (LSD) test. The statistical analysis was performed using SPSS 19.0 software. The significance level was p < 0.05.All data were presented in the form of mean ± standard deviation (p < 0.05), both of which were higher than the other enzymes and SGID (p < 0.05), as shown in In this study, the amino nitrogen contents of the oat protein hydrolysates by different enzymes were determined using the TNBS method. A higher amino nitrogen content represented a higher degree of hydrolysis. The results revealed that the oat hydrolysate by neutrase had the highest amino nitrogen content, followed by papain (p < 0.05). However, the hydrolysate by neutrase exhibited the highest DPP-IV inhibitory activity with an IC50 of 2.55 ± 0.38 mg/mL compared with that of other enzymes (p < 0.05). At a concentration of 20 mg/mL, the DPP-IV inhibitory activity of the oat hydrolysate by neutrase was determined to be 81.70% ± 1.18%. In addition, the oat hydrolysates by SGID and flavourzyme also showed high activities, with an IC50 of 10.55 ± 1.24 mg/mL and 14.20 ± 5.64 mg/mL, respectively, followed by alcalase, with an IC50 of 20.34 ± 5.44 mg/mL (p < 0.05) (The DPP-IV inhibitory activities of the oat protein hydrolysates by different enzymes with Gly-Pro-AMC as the substrate are shown in  < 0.05) . In cont50 of 2.04 mg/mL [50 of 0.99 mg/mL [The DPP-IV inhibitory property of the oat hydrolysate has also been reported elsewhere. For example, the tryptic hydrolysate of oat globulin was found to inhibit DPP-IV activity, with an IC04 mg/mL , which w04 mg/mL . In addi99 mg/mL . What shThe peptide sequences of the oat hydrolysate by neutrase were identified using UPLC-MS/MS. In total, 710 peptides with 5 to 10 amino acids were successfully identified. It is well known that the peptide containing a Pro or Ala at the second position of the N-terminus and a hydrophobic amino acid residue at the N-terminus usually shows a DPP-IV inhibitory activity . In the p < 0.05, 50 of 25.72 ± 1.06 μM, 30.78 ± 1.93 μM, 33.91 ± 4.83 μM, and 40.22 ± 0.95 μM, respectively; followed by LPQHY, VAEVPF, and PPHCP, with an IC50 of 62.66 ± 71.25 μM, 65.33 ± 13.08 μM, and 77.14 ± 2.75 μM, respectively; and MAGQVF of 223.31 ± 6.16 μM and AAVPF of 282.31 ± 27.18 μM, respectively (p < 0.05).Among the 10 novel peptides, only LPQYQA showed a relatively low activity, whereas the other nine peptides  all exhibited superior DPP-IV inhibitory activities with a significant dose−effect at concentrations ranging from 25 μM to 400 μM  and an S2 pocket  [ractions . The curThe oat protein hydrolysate by neutrase had the highest degree of hydrolysis and the highest DPP-IV inhibitory capacity. From the oat protein hydrolysate by neutrase, 10 peptides were identified with DPP-IV inhibitory activities. Among them, IPQHY, VPQHY, VAVVPF, and VPLGGF showed the strongest inhibition activities and all behaved as mixed-type inhibitors. In addition, IPQHY, VPQHY, VAVVPF, and VPLGGF were predicted to form multiple interactions, including hydrogen bonds, attractive charge, and hydrophobic interactions with the active site of DPP-IV. This work demonstrated that oat protein is a good source for food-derived DPP-IV inhibitory peptides and has the potential for the management of T2DM.


We investigated the effects of the intake of low amounts of ice slurry at −2°C on the tympanic temperature and exercise performance during repeated high-intensity intermittent exercises in a hot environment. This study was a randomized, crossover study, with a 6-day washout period. Twelve university rugby union players performed two 30-min sessions of high-intensity intermittent exercises separated by a 15-min half-time break on a cycle ergometer in a hot environment . The participants ingested 450 g of −2°C-ice slurry (ICE), or a 30°C-beverage (CON) having the same composition as ICE, or 30°C-water (WAT) during the half-time break. The tympanic temperature and skin temperature were measured as the physiological data, and the peak power and mean power as the exercise performance data. The tympanic temperature at the half-time break and beginning of the 2nd session was significantly lower in the ICE group as compared with the CON and WAT groups. The skin temperature at the half-time break was significantly lower in the ICE group as compared with the WAT group. While the peak power and mean power during the 2nd session were significantly greater in the ICE group as compared with the CON and WAT groups. Our findings suggest that even the intake of lower amounts, as compared with those used in previous studies, of low-temperature ice slurry can reduce the body temperature and improve the peak power. These results suggest that intake of low-temperature ice slurry as a strategy for internal body cooling is useful for improving endurance exercise performance in hot environments.Ice slurry ingestion enhances exercise performance by lowering the core body temperature. However, an operational issue related to this ingestion is the requirement for a high intake of 7.5 g·kg Performing exercises in hot environments can lead to excessive increase of the core body temperature, resulting in heat exhaustion or heat stroke, and potentially, serious central nervous system dysfunction , 2. In gCooling strategies adopted in athletes during sports competitions can be divided into two categories, external cooling and internal cooling , 13. Ext-1 of ice slurry (−1°C) before and during exercise attenuated increase of the core temperature and improved the exercise performance [-1  of ice slurry before exercise or at every break-time during exercise resulted in a significant reduction of the core temperature by 0.4°C-0.7°C [Ice slurry refers to a homogeneous mixture of small ice particles and a carrier liquid. Intake of ice slurry before exercise, as compared with intake of cold fluids, leads to a greater reduction of the core temperature, with larger heat-sink effects , therebyformance , 27. How-1 of low-temperature ice slurry (−1.7°C) prepared using a carbohydrate-electrolyte solution, which is lower than the intake amount prescribed in previous studies, before exercises attenuated the increase in the core temperature in firefighters [Whilst several adjunctive cooling methods, such as concurrent use of an ice vest , have befighters . Howeverfighters , 31.There are currently no studies on the effects of ice slurry ingestion during the half-time break in rugby union or soccer matches, which involve performance of high-intensity intermittent exercises before and after the half-time break. Adoption of recovery strategies during the short 15-min half-time break is particularly important during, for example, a rugby union match, which is classified as a full-contact sport. Tackles and collision-based activities, which occur during both match play and training in rugby union, have a high energy cost  and can The purpose of the present study was to determine whether intake of low-temperature ice slurry in even smaller amounts than those examined in previous studies during the half-time break might lower the body temperature to improve the high-intensity intermittent exercise performance in athletes. It was hypothesized that lowering the ice slurry temperature would reduce the amount necessary to lower the core temperature sufficiently and improve high-intensity intermittent exercise performance.2 max: 49.2 ± 3.4 mL·kg-1·min-1) volunteered to participate in this study, which was conducted between February and March 2020 . All the participants provided written informed consent prior to commencement of the study, after receiving a full explanation about the risks and benefits of participating in this study. None of the participants were smokers or had any injuries. This study protocol was approved by the Ethics Committee of Keio University Graduate School of System Design and Management  and the Ethics Committee of the Keio University Sports Medicine Research Center  in Japan, and the study protocol was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN000039272). This study conducted in compliance with the latest version of the Declaration of Helsinki.Twelve male non-heat-acclimatized healthy university rugby union players , or a 30°C-beverage with the same composition as ICE (CON), or 30°C-water (WAT). The participants completed one familiarization session before the first experimental trial. For each participant, the three experimental trials were implemented at the same time of the day, on the same day of the week to standardize the influence of the circadian rhythm. All trials were conducted between February and March 2020, to limit heat acclimation, in a temperature-controlled room to simulate hot conditions , in Kanagawa, Japan. Throughout the study period, the participants maintained their normal lifestyles, including their physical activity and nutritional habits. Participants were instructed to avoid strenuous exercises, alcohol consumption, and intake of any supplements from the day before the start of the study to the end of the study.st session (HT0), the second portion 5 min after HT0 (HT5), and the remaining portion 10 min after HT0 (HT10). The compositions of ICE and CON were the same: carbohydrate 56 kcal·100 g-1, Na+ 94.0 mg·100 g-1, K+ 20.3 mg·100 g-1.The participants ingested 450 g of ICE (−2°C), CON (30°C) or WAT (30°C) during the 15-min half-time break, with the amount divided into three equal portions to be ingested every 5 min. The participants ingested the first portion immediately after the end of the 1ty) was continuously recorded using an infrared sensor, CE Thermo probe  [-2) = 0.965 (W·kg-1°C-1) × m × ΔTty·AD-1; m: body mass; ΔTty: tympanic temperature change; AD: body surface area [0.425 × height0.725 [sk) was calculated using the formula proposed by Roberts et al. [sk = 0.43 × (chest temperature) + 0.25 × (forearm temperature) + 0.32 × (thigh temperature) [-1 and 95% limits of agreement  compared with an electro-cardiographic monitoring device [nd session  were calculated relative to the values at the end of the 15th sprint of the 1st session. The total peak power and total mean power were calculated as the cumulative percent changes of the peak power and mean power, respectively. Adverse events related to the ice slurry ingestion were assessed by self-reported symptoms of the participants during the half-time break and after the exercise.The body mass (kg) of the participants was measured before and after the exercise sessions using an InBody770 body composition analyzer . The tympanic temperature (T± 0.1°C) . Tympani± 0.1°C)  and brai± 0.1°C) , 39, alt± 0.1°C) . Heat stace area . The bods et al. : Tsk = 0erature) –46. An og device . All devg device  and BorgDescriptive data are presented as means ± standard errors (SE), unless stated otherwise. All statistical analyses were performed using the SAS software package version 9.4 . Normality of the data and homogeneity of variance were tested using Shapiro-Wilk’s test and Bartlett’s test. Differences in the means of the measured values among the beverage conditions and among different time-points were analyzed by Friedman’s two-way ANOVA. When significant differences were found, post-hoc analysis was performed using Wilcoxon’s signed-rank test, with Holm’s adjustment applied for multiple comparisons. P<0.05 was set as the significance level for all the analyses.st 30-min session of the exercise protocol, there were no significant differences in the peak power or mean power among the beverage groups. The changes in the peak power and mean power during the 2nd session calculated relative to the value at the end of the 15th sprint of the 1st session are shown in Figs st (p = 0.016 vs. CON), 4th (p = 0.001 vs. CON), 6th , 9th  and 11th  sprint, and changes in the mean power at the 4th (p = 0.009 vs. CON) and 11th (p = 0.012 vs. WAT) sprint of the 2nd session were significantly higher in the ICE group as compared with the those in the CON or WAT group. The total peak power was also significantly higher in the ICE group than that in the CON (p = 0.023) and WAT (p = 0.027) groups. There were no significant differences in the total mean power among the groups. No significant differences were observed between the CON and WAT groups.During the 1ty were significantly greater in the ICE group than that in the CON or WAT group at 5 min (p = 0.012 vs. WAT), and 10 min  during the half-time break, Pre 2nd session: , 5th sprint of the 2nd session  was significantly higher in the ICE group than that in the CON or WAT group , 5th  and 10th  sprint of the 2nd session  was found in the VAS. Likewise, a main effect for time (p < 0.001) was also found in RPE. During the trials, both the subjective scale scores increased significantly over time as compared with Pre, at all time-points  related to the ice slurry ingestion were observed during the study period.ty and improved the exercise performance  during the 2nd session, lending support to the hypothesis that even intake of a small amount of low-temperature ice slurry during the half-time break of field sports activity simulating exercises in a hot environment would improve the exercise performance during the 2nd session.This study was conducted in university rugby union players to determine the effects on the body temperature and exercise performance  of intake of low-temperature ice slurry. The main findings of this study were that intake of 450 g of low-temperature (−2°C) ice slurry during the half-time break decreased the T-1), calculated on the basis of the mean body mass of the participants (86.3 kg). However, although the time of intake and exercise protocol differed from that used in previous studies, the change in Tty in the ICE group was significantly greater as compared with that in the other groups even after intake of only 450 g (5 g·kg-1) of low-temperature ice slurry [-1, 12 times) and at the half-time break (7.5 g·kg-1), while in the present study, the participants ingested 150 g of −2°C-ice slurry every 5 min during the 15-min half-time break, to avoid adverse events caused by rapid ingestion and to control the temperature of the ice slurry. This intake amount of ice slurry seemed to be effective, because the body temperature decreased to the same degree as that reported in the previous study.This is the first study to show that intake of low-temperature ice slurry during the half-time break of a rugby union match simulating exercise designed based on the physical demands of repeated-sprint activities in rugby union match play , 50, decicipants 6.3 kg. He slurry . In a pre slurry , the parThere was a significant recovery effect on the peak power during the second half of the exercise activity, after the half-time break, in the ICE group as compared with that in the CON and WAT groups, while no significant difference was observed between the CON and WAT groups. Therefore, it was considered that the peak power recovery effect may not be due to the intake of carbohydrates in the ice slurry, but to the cooling effect of the low-temperature ice slurry. Increase in the heat storage capacity and decrease in central fatigue due to lowering of the brain temperature has been speculated as the mechanism underlying the effect of the low-temperature ice slurry intake , 51. As st session significantly decreased the Tty and Tsk as compared with intake of CON or WAT; the Tty and Tsk were the lowest at the beginning of the 2nd session  and remained low until the 5th sprint of the 2nd session (25 min after the low-temperature ice slurry intake). Athletes and coaches should consider the lengths of breaks  in each sporting activity to determine the time and amount of ice slurry to be consumed. Since it is difficult to clarify the relationship between the amount of ice slurry ingestion and exercise performance from the results of this study alone, further research is necessary to explore the intake of appropriate amount of ice slurry during break times in each competitive athletic activity.Our findings have some potential implications for athletes competing in field sports activities. Intake of low-temperature ice slurry after the 1While discussing the results, some limitations of this study must be recognized. Firstly, we did not measure the core temperature , thermal sensation, or thermal comfort. The cold sensation experienced by the participants in the ice slurry condition could have affected the physical performance –55. Fututy and improved the exercise performance in a hot environment. Our data demonstrated that the strategy of internal body cooling via intake of low-temperature ice slurry might be effective for maintaining high exercise performance of athletes in sports competitions, such as rugby union matches, held under severe environmental conditions.In this study, the intake of low amounts of low-temperature ice slurry during the half-time break reduced the TS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


The Lineweaver-Burk diagram showed that GSPPPSG is a non-competitively dominant mixed-type PLI, whereas KLEGDLK is a competitive inhibitory-type PLI. Moreover, molecular docking suggested that both peptides can stably bind to the key amino acid residues of the PL active site, mainly through hydrogen bonding, hydrophobic, and electrostatic interactions. In general, we not only established a method to rapidly fish out potential PLIs from protein hydrolysate, but also provided safe and efficient lead compounds for the development of novel diet foods or drugs.Obesity has become an increasingly serious public health problem. Pancreatic lipase (PL) is identified as a ideal target for the prevention and treatment of obesity. Orlistat, the only approved PL inhibitor (PLI), is a powerful weight loss drug but has many side effects. Therefore, there is an urgent need to discover powerful PLIs with high safety. Protein hydrolysate has been demonstrated to be a treasure trove of PLIs, but recognizing responsible functional peptides from them is like looking for a needle in a haystack. In this work, we synthesized and optimized a PL ligand fishing model (PLLFM) using magnetic nanoparticles (MNPs), then PLLFM was used to quickly fish out potential PLIs from the Cod meat hydrolysate (CMH). Finally, two new PLIs, GSPPPSG and KLEGDLK were identified with IC Obesity is a state of excessive fat deposition caused by adipocyte hypertrophy and proliferation . The latPancreatic lipase (PL) is a lipolytic enzyme that participates in the hydrolysis of dietary triglycerides for absorption and utilization. It can digest 50–70% of total dietary fat and has been proven to be a crucial target for the development of anti-obesity agents . OrlistaHow to capture active peptides from complex protein hydrolysates has always been a research hotspot in the field. The traditional activity-guided separation procedures involve repeated fractionation steps and bioactivity tests, which are time-consuming, labor-consuming, and inefficient –20. LigaThis study aimed to efficient screening of PLIs from protein hydrolysate through ligand fishing strategy. In particular, the pancreatic lipase ligand fishing model (PLLFM) was synthesized and optimized. Then, PLIs were targeted mining, and identification from protein hydrolysate by PLLFM and LC-Q-TOF-MS/MS. Finally, the inhibitory mechanism of active peptides on PL was investigated by Lineweaver–Burk plots and molecular docking.Pseudostellaria heterophylla, Red snapper scale, and Silver carp skin, and preserved in Dalian Polytechnic University and Fuzhou University. Carboxyl-terminated Fe3O4 magnetic nanoparticles SM3-P100  were purchased from Aorun Weina New Material Technology Co., Ltd. . Porcine pancreatic lipase , p-nitrophenyl butyrate  were purchased from Sigma Chemical Co. . N-hydroxysucci-nimide (NHS), 2-(N-morpholino) ethanesulfonic acid (MES), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Macklin Biochemical Co., Ltd. . The other chemicals and solvents were all of analytical reagent grade.Five protein hydrolysates were from Cod meat, Sea cucumber, PLLFM was prepared following a previous paper with a little modification . BrieflypNPB was dissolved in acetonitrile, and diluted with tris-HCl buffer. Add 100 μL of the sample, 200 μL of Tris-HCl buffer, 150 μL of PL (5 mg/mL) to a 1.5 mL EP tube, incubate at 37°C for 10 min, and then add 150 μL of 10 mmol/mL pNPB, shake and mix, and incubate at 37°C for 10 min. After the reaction was completed, the absorbance value at 405 nm was measured with a multifunctional microplate reader, and the PL inhibitory activity was calculated according to the following equation: Inhibition rate (%) = 1- (absorbance in the presence of inhibitor/fluorescence intensity in negative control) × 100%.Grind small pieces of cod meat into surimi, add alkaline protease, and hydrolyze for 2 h. The enzyme was then inactivated by high temperature and centrifuged. Finally, the supernatant was spray dried to obtain cod peptide (CP). According to the method of Hou et al. , the PL Pseudostellaria heterophylla, Red snapper scale, and Silver carp skin were taken to test their LP inhibitory activity. The most active sample was separated by G-15 gel, and the most active fraction was determined as the fishing target. Specifically, 20 μL (10 mg/mL) of the most active fraction (S0) was mixed with the constructed PLLFM and incubated at 37°C for 2 h. After incubation, PBS buffer was added and eluted 3 times to remove non-specific components (S1), and then 10% acetonitrile was used to elute the active ligands with a strong affinity for PL (S2). S0–S2 were analyzed by HPLC , with the mobile phase containing solvent A  and solvent B : 5–55% B at 0–30 min.Five protein Hydrolysates from Cod meat, Sea cucumber, The specific peak of S2 was collected and loaded onto Nano LC-Q-TOF-MS/MS  at the Instrumental Analysis Center of Shanghai Jiao Tong University to identify the peptide amino acid sequence.The target peptides were synthesized by Cellmano Biotech Co. Ltd.  by solid-phase method.pNPB was added for 10 min. the absorbance of the reaction system at a wavelength of 405 nm was measured to determine the reversibility of the reaction. Next, the optimal PL concentration (5 mg/mL) and peptide  were fixed, and pNPB with different concentrations  was added to react for 10 min, a value was measured every 30 s, and the wavelength was 405 nm. The Lineweaver-Burk double reciprocal curve was used to determine the maximum velocity (Vmax) and Michaelis–Menten constant (Km). The slope and y-intercept of the Lineweaver-Burk plot vs. peptide concentration give a straight line with the intercepts on the horizontal axis as Ki (competitive inhibition constant) and K'i values (uncompetitive inhibition constant), respectively.According to the method reported in the previous literature, with a slight modification, the type of inhibition of PL by active peptides was determined , 30. TheDiscovery Studio 2017 R2 software was used to further evaluate the interaction mode and binding affinity between PL and the peptide. The 3D structure of PL (PDB ID: 1LPB) was obtained from the Protein Data Bank database, dehydrated and hydrogenated, and the peptide was minimized by the CHARMM force field. LIBDOCK and CDOCKER were chosen to perform the docking program. Phe 77, Ile 78, Tyr 114, Ser 152, Ala 178, Pro 180, Phe 215, Ala259, and His 263 were active centers .P-value was below 0.05.All values in this study were reported as the mean ± standard deviation (SD) based on triplicate independent experiments. Statistical comparisons were performed using Duncan's multiple range test and the least significant difference (LSD) test in SPSS 18.0 software. It was determined to be significant when the −1, while PLLFM has a broad and strong absorption peak at 1,715.87 cm−1, which indicated that -COOH on the MNPs reacts with -NH2 on the PL. Subsequently, we examined the stability, and reproducibility of PLLFM. In order to improve the enzymatic immobilization capacity and relative enzymatic activity of the PLLFM, single-factor experiments and response surface optimization of its synthesis conditions were carried out. The results  showed tPseudostellaria heterophylla, Red snapper scale, and Silver carp skin for PL inhibitory activity tests. As displayed in 50 = 3.33 mg/mL). Therefore, the CMH was selected as the further research object. Immediately after, the CMH was separated into five fractions (F1-F5) by Sephadex G-15, and their PL inhibitory activities were tested at a concentration of 1 mg/mL. Among them, F3 showed the strongest PL inhibitory ability, and its inhibitory rate was about 1.67 times that of GMH  was incubated with PLLFM at 37°C for 2 h, the supernatant was removed by magnetic separation, and eluted with PBS and 10% acetonitrile solution in turn to obtain S1 and S2 eluates for HPLC analysis. As shown in +; −10lgP 34.05) and KLEGDLK , which were considered as potential PLIs for further activity validation due to their high confidence and relative abundance. These two peptides are non-toxic and stable, and their molecular weight, isoelectric point, hydrophilicity and other properties are within the range of the reported physicochemical properties of PL-inhibiting peptides The characteristic peak 3 was collected, and its sequence was identified by Nano LC-Q-TOF-MS/MS, and further analyzed by Peaks studio 10.0 software combined with the NCBI database. As shown in 50 values of different purities of CMH for PL. The IC50 value of GSPPPSG for PL was 0.60 mg/mL, and the IC50 value of KLEGDLK was 1.08 mg/mL, which were 5.5 and 3.1 times higher than the initial CMH activity (IC50 = 3.33 mg/mL), respectively. The activity of these two peptides was about one-third that of orlistat , which was also similar to the reported activities of natural products and synthetic compounds  was higher than the non-competitive inhibition constant K'i (1.46 mM)  of KLEGDLK was lower than that of GSPPPSG, which suggests that KLEGDLK has a stronger affinity for the active site of PL.The type of inhibition of PL by GSPPPSG and KLEGDLK was shown in decrease ; Table 11.46 mM) . InteresVm value ; Table 1Molecular docking was performed using the “CDOCKER” and “LIBDOCK” functions in Discovery Studio 2017 R2 software to understand the interaction model of the two peptides with amino acid residues in the PL active pocket . From th50 values of 0.60 and 1.08 mg/mL, respectively. Further studies showed that GSPPPSG is a non-competitively dominant mixed-type PLI, while KLEGDLK is a competitive inhibitory PLI. Molecular docking revealed that both peptides bind to amino acid residues in the active site of PL by hydrogen bonds, hydrophobic interactions, and electrostatic interactions.In summary, rapid fishing of two new PLIs  from CMH using PLLFM prepared by MNPs was reported for the first time. Peptides GSPPPSG and KLEGDLK showed strong PL inhibition with ICThe original contributions presented in the study are included in the article/YT conceived, designed research, and wrote the manuscript. CL performed the whole experiment. SW provided the modification of this manuscript. MD and BZ conceived of and proposed the idea. All authors read and approved the manuscript, and agreed to the published the manuscript.This work was supported by National Key R&D Program of China (2018YFD0901106), Natural Science Foundation of China (No. 31771922), and Central Government Subsidy Project (202006).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Studies have demonstrated that adults with idiopathic generalized epilepsy (IGE) have functional abnormalities; however, the neuropathological pathogenesis differs between adults and children. This study aimed to explore alterations in the cerebral blood flow (CBF) and functional connectivity (FC) to comprehensively elucidate the neuropathological mechanisms of IGE in children.We obtained arterial spin labeling (ASL) and resting state functional magnetic resonance imaging data of 28 children with IGE and 35 matched controls. We used ASL to determine differential CBF regions in children with IGE. A seed-based whole-brain FC analysis was performed for regions with significant CBF changes. The mean CBF and FC of brain areas with significant group differences was extracted, then its correlation with clinical variables in IGE group was analyzed by using Pearson correlation analysis.Compared to controls, children with IGE had CBF abnormalities that were mainly observed in the right middle temporal gyrus, right middle occipital gyrus (MOG), right superior frontal gyrus (SFG), left inferior frontal gyrus (IFG), and triangular part of the left IFG (IFGtriang). We observed that the FC between the left IFGtriang and calcarine fissure (CAL) and that between the right MOG and bilateral CAL were decreased in children with IGE. The CBF in the right SFG was correlated with the age at IGE onset. FC in the left IFGtriang and left CAL was correlated with the IGE duration.This study found that CBF and FC were altered simultaneously in the left IFGtriang and right MOG of children with IGE. The combination of CBF and FC may provide additional information and insight regarding the pathophysiology of IGE from neuronal and vascular integration perspectives. Epilepsy is a chronic disorder of the nervous system disease caused by excessive brain neuronal firing or abnormal synchronous activity that causes brain dysfunction . It affeArterial spin labeling perfusion imaging is a non-invasive MRI technique for measuring CBF without using contrast agents . BecauseWith rs-fMRI, changes in blood oxygen level-dependent (BOLD) signals are detected. These can be used to study the spontaneous neural activity of the brain in the resting state and to evaluate the FC and networks of various brain regions. Furthermore, because rs-fMRI does not involve the need to perform specific tasks, participants can cooperate well. It has been used in the field of neuropsychiatry, where it is especially suitable for studying neuropsychiatric diseases of patients with cognitive and attentional disorders, or the inability to cooperate, including bipolar disorder , psychosBased on these previous studies, CBF and FC are abnormal in IGE patients. However, the neurobiological mechanism of IGE in children is unclear, and there are no effective treatments available. Moreover, the correlation between CBF and internal brain function changes is unclear. Recently, This study involved sixty-three subjects. There were 28 children with IGE and 35 healthy controls (HCs). The study protocol was approved and was performed in accordance with the recommendations of the Medical Research Ethics Committee of Zunyi Medical University’s Affiliated Hospital. Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. Children with IGE underwent a complete neuropsychological assessment using the Chinese version of the Wechsler Intelligence Scale for Children, including verbal, performance, and full-scale intelligence quotient (IQ) assessments, on the same day as the MRI scan.Idiopathic generalized epilepsy patients were required to fulfill the following inclusion criteria: (I) diagnosis of IGE according to the 2010 International League Against Epilepsy diagnostic criteria (II) underwent a normal routine MRI examination of the brain (III) and age 6–16 years. The exclusion criteria were as follows: (I) other neuropsychiatric diseases, (II) history of drug abuse, (III) craniocerebral trauma or surgical history, (IV) intracranial organic lesions or serious artifacts; and (V) head motion exceeding ± 2 mm or ± 2°.The following were inclusion criteria for the HCs: (I) underwent a normal routine MRI examination of the brain and (II) age 6–16 years. The exclusion criteria were as follows: (I) other neurological/psychiatric conditions, (II) history of drug abuse, (III) cognitive and memory disorders, and (IV) head motion exceeding ± 2 mm or ± 2.A 3.0-T HDxt system  was used to acquire all MRI results. ASL perfusion imaging was performed using pseudocontinuous labeling with three-dimensional fast spin-echo acquisition and background suppression. The following parameters were used: repetition time, 4,599 ms; echo time, 9.8 ms; post-label delay, 1,525 ms; spiral-in readout of eight arms with 512 sample points; field of view, 240 mm × 240 mm; slice number, 36; and slice thickness, 4 mm. The rs-fMRI data were acquired using an echo planar imaging sequence with the following parameters: repetition time, 2,000 ms; echo time, 30 ms; field of view, 240 mm × 240 mm; flip angle, 90°; slice number, 33; slice thickness, 4 mm; and volume, 210. The participants were instructed to keep their eyes closed, relax, remain still, think of nothing, and not fall asleep during the scanning process.Preprocessing of the MRI data was performed with Data Processing Assistant for Resting-State Brain Imaging (DPARSF) . First, 1. The CBF images of each patient’s structural space were transformed to the Montreal Neurological Institute standard space. Subsequently, we calculated a z-score from the spatially normalized CBF images of each participant by subtracting the mean and dividing by the standard deviation of all voxels across all subjects. Finally, all CBF images were smoothed using a 6-mm × 6-mm × 6-mm Gaussian kernel filter with full-width at half-maximum. Further statistical analyses using an independent two-sample t test were performed to compare the groups using age and sex as covariates. The significance threshold was set using the Gaussian random field (GFR) correction (P < 0.05). Based on Pearson’s correlation analysis, abnormal CBF was correlated with IQ, disease course, and age at onset. The significance level was set at P < 0.05.Cerebral blood flow images were obtained from the original ASL data using an MRI scanner post-processing workstation. We acquired CBF images and processed the data using SPM12t test with age and sex as covariates, we compared the two groups (IGE patients vs. HCs). The significance threshold was set using the GFR correction . Finally, we extracted the abnormal FC of brain regions. Pearson’s correlation analysis was performed for brain regions with abnormal FC in children with IGE and IQ, disease course, and age at onset. The significance level was set at P < 0.05.A seed-based FC analysis was conducted. First, based on the CBF results, four regions with significant differences between groups were selected as masks and defined as ROIs during the FC analysis. Thereafter, using an independent two-sample P = 0.958, P > 0.05, chi-square test), age , and education  between the IGE patients and HCs.Demographic and clinical characteristics of the 28 children with IGE  and 35 HCs  are described in t test; t = 0.29; P = 0.773) in the whole brain matter. Increased blood flow was detected mainly in the left inferior frontal gyrus (IFG), middle frontal gyrus, cingulate gyrus, bilateral middle temporal gyrus (MTG), thalamus, right middle frontal gyrus (MOG), and right medial prefrontal cortex.The spatial distribution map of CBF in the HCs and IGE patients are shown in t-tests to examine the differences in FC of the regions of interest in the IGE patients and HCs. Compared with the HCs, we found there was a significant decrease in connectivity in the left IFGtriang with the CAL  and increased CBF in IGE patients was mainly present in the right MTG, MOG, and superior FG (SFG) . By iden the CAL , and tha the CAL .Cerebral blood flow of the right SFG was negatively correlated with the age at onset in the IGE group . AdditioThis study combined three-dimensional ASL perfusion with rs-fMRI to examine changes in CBF and potential FC disruptions in CBF-altered regions in children with IGE. Compared with HCs, children with IGE had significant abnormal perfusion in the right MOG and left IFGtriang. A seed-based FC analysis was used to evaluate the potential changes in these abnormal regions and found simultaneous decreases in the FC between the left IFGtriang and CAL and between the right MOG and bilateral CAL, which could help comprehensively elucidate the neuropathological mechanism of IGE in children.+ influx leads to depolarization, causing vascular neuronal damage to a certain extent. In particular, when the endothelial function of the local capillaries is damaged, local CBF is reduced (Previous studies have consistently reported significant abnormal perfusion in the frontal, temporal, and occipital lobes of IGE patients. The regions showing perfusion abnormalities in the frontotemporal lobe also showed structural and functional abnormalities similar to those reported by other MRI studies . In this reduced . TherefoThere have been reports of increased CBF during the postictal phase of typical absence seizures and the interictal phase of myoclonic epilepsy in adolescents . In anotThe MOG is the visual sensory cortex, which is mainly responsible for processing visual information . We obseDuring the seed-based FC analysis, we found a significant decrease in the FC in the left IFGtriang with the CAL, and the FC between the right MOG and bilateral CAL was decreased in children with IGE.Previous studies have reported abnormal FC of the frontal lobe with other regions, such as the thalamus, posterior cingulate gyrus, and orbitofrontal cortex in IGE patients . During The MOG and CAL are important components of the visual sensory cortex and visual network, and they have a critical role in the processing of visual information. During our study, we found that areas with perfusion abnormalities in children with IGE had alterations in FC. The BOLD signal depends on the concentration of deoxyhemoglobin in the blood and is regulated by changes in the CBF and cerebral blood volume  and abnoDuring this study, we observed that CBF in the right SFG was negatively correlated with the age at IGE onset, signifying that a younger age at onset is associated with a greater CBF increase. The frontal lobe is the advanced center of brain development and embodiment of human wisdom. It is closely related to attention, cognition, and executive control function. Therefore, it can be proposed that CBF changes in the SFG may hurt the ability of the region to recruit when responding to task demands, and thus may cause impaired cognitive functioning and attention disturbances. Furthermore, between abnormal FC of the left IFGtriang and CAL was negatively correlated with IGE duration. The frontal lobe and CAL have important roles in cognitive and visual processing. Previous studies have found that changes in the prefrontal function correlate well with disease duration. There were some limitations to this study. First, intelligence of the HCs was not evaluated; however, they attended normal primary and secondary schools. The performance of the HCs during the MRI scanning sessions and at school was to be normal. Second, there were no data regarding the IGE subtypes. Third, previous studies showed that antiepileptic drugs may affect the results of functional MRI. We observed simultaneous changes in the CBF and FC in the left IFGtriang and right MOG in children with IGE. The combination of ASL and rs-fMRI and the integration of neuronal and vascular information can add to our understanding of the pathophysiology of children with IGE.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Affiliated Hospital of Zunyi Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.GC and JH contributed to the conception, design, and radiological expertise, helped to select and assess cases, and drafted manuscript. JL, GS, and GX contributed radiological expertise and carried out the statistical analysis and designed the study. HR and LN contributed to the drafting and revision of the manuscript. WT, QL, XL, and YH offered data collection. TZ and HL reviewed and critiqued the manuscript and approved the final manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.