Apc initiate cancer. APC forms droplets via its 20R domains and self-association domain (ASAD) and in the context of Axin. However, the mechanism involved is unknown. Here, we used peptides to study the molecular mechanism and regulation of APC droplet formation. We found that a peptide derived from the ASAD of APC-formed droplets. Peptide array screening showed that the ASAD bound other APC peptides corresponding to the 20R3 and 20R5 domains. We discovered that the 20R3/5 peptides also formed droplets by themselves and mapped specific residues within 20R3/5 that are necessary for droplet formation. When incubated together, the ASAD and 20R3/5 did not form droplets. Thus, the interaction of the ASAD with 20R3 and 20R5 may regulate the droplet formation as a means of regulating different cellular functions. Phosphorylation of 20R3 or 20R5 at specific residues prevented droplet formation of 20R3/5. Our results reveal that phosphorylation and the ability to undergo liquid-liquid phase separation, which are both important properties of intrinsically disordered proteins, are related to each other in APC. Phosphorylation inhibited the liquid-liquid phase separation of APC, acting as an ‘on-off’ switch for droplet formation. Phosphorylation may thus be a common mechanism regulating LLPS in intrinsically disordered proteins.One of the most important properties of intrinsically disordered proteins is their ability to undergo liquid-liquid phase separation and form droplets. The Adenomatous Polyposis Coli (APC) protein is an IDP that plays a key role in Wnt signaling and mutations in  Intrinsically disordered proteins (IDPs) and intrinsically disordered regions in proteins (IDRs) constitute up to 70% of the proteome . IDPs taThe tumor suppressor Adenomatous polyposis coli protein (APC) is a 2843-residue (in humans) IDP . Mutations that cause truncation of APC are responsible for 80% of sporadic colon cancers ,17,18,19APC contains several unstructured domains that bind different proteins B. For exdrosophila. It is significantly shorter than human APC with only 1067 amino acids. In APC2, the N-terminal coils preceding ASAD in human APC are lacking so that ASAD is much closer to the N-terminus of the protein and it contains a slightly different sequence. For APC2, ASAD is an essential domain that affects the size of droplets [The ability of APC to undergo LLPS involves at least two domains, ASAD and the 20R domain, and APC forms droplets together with Axin . APC2 isdroplets . Co-exprdroplets . This wadroplets . However1-L2-S3-S4-L5 of the 20Rs is essential both for binding ASAD and for regulating droplet formation. Alanine scanning of the sub-domain S1-L2-S3-S4-L5 revealed that the third serine (Ser3) was essential for regulating droplet formation. This repeat is found in all seven 20R regions. We conclude that this repeat is a ‘sticky motif’ that allows the 20R domain to undergo LLPS.Here we used peptides derived from different domains of the human APC protein to study the molecular mechanism for its LLPS. We identified short peptides representing key APC domains that undergo LLPS and used them to reveal the molecular mechanism responsible for the formation of droplets and its regulation. The ASAD adopted a helical secondary structure and formed droplets via hydrophobic interactions. Using peptide array screening we found that the ASAD binds the 20R3 and 20R5 domains of APC. Peptides derived from these two 20Rs formed droplets on their own and their droplet formation was inhibited by phosphorylation at sites known to be phosphorylated by the kinases-regulating APC activity. Phosphorylating the 20Rs at the GSK3-β kinase target residues, p20R3  and p20R5 , prevented droplet formation. Similarly, when 20R3 was phosphorylated in the phosphorylation pattern produced by the kinase CK1, p20R3 , it also did not form droplets. On the other hand, phosphorylation of 20R5 with the corresponding pattern, p20R5 (pS1863) did not inhibit its ability to form droplets. We found that the sub-domain SOur results reveal that 20R3 and 20R5 peptides, both intrinsically disordered, have a significant role in the oligomerization of APC. They undergo LLPS and bind the ASAD via the SLSXL repeat, thus participating in various oligomeric forms of APC. Phosphorylation of the 20Rs regulates their LLPS and it is known that phosphorylation of SLSXL promotes the binding of APC and E-cadherin to β-catenin ,41. We cTo determine the role of the ASAD in the LLPS of APC, we examined whether the ASAD could form droplets by itself. A peptide corresponding to the ASAD APC 396–426) formed droplets as observed by differential interference contrast (DIC) microscopy, while fusion events of the droplets were monitored to confirm LLPS B. Increa6 formed Circular dichroism (CD) studies showed that the ASAD forms an α-helical secondary structure with typical minima at 210 nm and 225 nm D. A heli396–426 (407–426 and APC401–415 (i), located at the N-terminus of the solvent-exposed Armadillo-like (ARM) repeat  is part of the 20R3 domain. The ASAD bound a peptide derived from 20R3, APC1501–1515, but did not bind the adjacent peptides on the peptide array, APC1491–1505 and APC1511–1525. This result suggests that the domain of 20R3 that binds the ASAD is 1506SLSAL1510 (1861–1875 (iii), which is derived from the 20R5 domain. The ASAD did not bind the adjacent peptides APC1851–1865 and APC1871–1885 suggesting that the ASAD binds the 20R5 domain via 1866SLSSL1870  repeat A and parC411–425 , which aLSAL1510 . The ASALSSL1870 A. In sumSince 20R3 and 20R5 bind the ASAD, we tested whether the 20R peptides also contribute to the LLPS of APC, either by forming droplets by themselves and/or together with the ASAD. Both 20R3 and 20R5 formed droplets on their own B. 20R5 fNext, we examined whether phosphorylation, a known PTM of APC in cells and an important regulation mechanism for LLPS, regulates droplet formation of the 20Rs. We examined if and how specific phosphorylation patterns corresponding to those of APC-phosphorylating kinases affected the droplet formation of the 20R peptides. We synthesized the 20R3 and 20R5 peptides with phosphorylated serine at sites known to be phosphorylated by these kinases in cells A. The peUsing peptide arrays we discovered that the ASAD bound peptides derived from 20R3 and 20R5 domains even when they were phosphorylated in the GSK3-β phosphorylation pattern A. To undWe found that the SLSXL sequence in 20R3 and 20R5 is essential for binding the ASAD. Therefore, we tested whether it is also involved in droplet formation. We selected 20R5 as a model for LLPS experiments since it contains the SLSSL repeat, which is the most common variation of the SLSXL repeat within all seven 20R domains B. We synTo identify the precise residues in SLSSL that mediate droplet formation, we performed alanine scanning of SLSSL. The peptides ALSSL, SASSL, and SLSSA all underwent LLPS at an ionic strength of 36 mM, while SLSAL formed only few droplets. Moreover, SLASL did not form droplets at all C. These LLPS of many proteins, particularly IDPs, provides versatile regulation of their interactions and functions. The tumor suppressor protein APC is an example of an IDP with multiple cellular functions that have to be coordinated for normal tissue homeostasis. APC plays a key role in Wnt signaling and it has been proposed that this is regulated by LLPS . SimilarPreviously, LLPS was reported as a unique property of disordered proteins and 50- to 100-residues-long oligopeptides ,48. It iWe found that in addition to oligomerization via previously described sites ,22,28,51Our results show that the ARM repeat can interact with different components of the 20R domain that are located more than 1000 residues removed toward the C-terminus. The binding of the ASAD to the 20Rs raises the possibility that APC can undergo oligomerization via these domains or intra-molecular interactions between them, resulting in partial folding of this largely intrinsically disordered protein. Such a folding event would bring the IDRs close to the folded N-terminal domain, the only well-structured region of APC. This in turn could regulate interactions of the N-terminal domain including cytoskeletal regulators . Mutations of APC lead to the expression of a truncated protein, lacking the 20R domain. Therefore, truncated APC cannot form ASAD-20R3/5 interactions, which would remove such potential regulation and explain why, for instance, N-terminal fragments expressed in tumor cells stimulate the ASEF protein more efficiently . FurtherIn conclusion, oligomerization of APC can be mediated by interactions between three domains: the N-terminal domain, the ASAD, and the 20R3/5 to create numerous possibilities and combinations which can produce different states of APC to initiate different pathways A.drosophila APC2 could form droplets via a hydrophobic patch, indicating that the function of ASAD in mediating and forming droplets is well-conserved between these two APC proteins.APC2 shares many of the well-conserved domains with human APC, including the ASAD, with some differences in the ASAD sequence  Table 2Table 2. The droplet formation of 20R5 occurs at a physiological and lower ionic strength, suggesting this LLPS is mediated by electrostatic interactions. On the other hand, LLPS of 20R3 was not affected by ionic strength to the same degree. These differences can be explained by differences in their sequences. First, the consensus SLSXL repeat of 20R3 has alanine on the 4th position, while 20R5 has a serine. This alone could affect the LLPS of both peptides. Furthermore, 20R5 is highly charged (33.3%) and polar (33.3%), and droplets formed by 20R5 are more likely to interact electrostatically. 20R3 is less charged (13.3%), and more polar 40%), which is consistent with the fact that its ability to form droplets was less affected by ionic strength. The 20R3 peptide we used formed droplets at identical PEG and ionic strength conditions to those reported for the longer 20R2-3 %, which ,54. ConsFinally, the SLSSL sequence is also found in the E-cadherin protein, another critical binding partner of β-catenin ,41. E-caPhosphorylation of the 20Rs increased their net charge and prevented droplet formation. This could be the result of the repulsion of the phosphate groups from each other, preventing the interactions required for LLPS, and/or increasing the solubility of the peptides in the polar solvent. 20R5 contains one CK1 phosphorylation site. We suggest that the mono-phosphopeptide does not contain sufficient charge to fully dissolve, or that the repulsion between the peptides is not strong enough to prevent droplet formation. This is supported by the fact that low ionic strength prevented droplet formation of 20R5 C. Our reIn conclusion, our results show that the 20Rs are critical regulators of the oligomerization, interactions, and activity of APC. Their location in the middle of an IDR enables them to perform this important function and to regulate it by LLPS and phosphorylation, two properties of IDRs. In the case of APC, this combination of multiple oligomerization sites and control mechanisms enables the formation of different states that direct this highly important multifunctional protein to its different functions.The peptides were synthesized using a Liberty Blue Microwave-Assisted Peptide Synthesizer (CEM) as described previously . BrieflyTM peptide micro-arrays [w/v) skimmed milk for blocking unspecific binding. Then 1 mM of the peptide dissolved in buffer A, including 4% (w/v) skimmed milk , was incubated overnight with the array at room temperature with shaking. After three washes with TBST, the array was incubated at room temperature for one hour with an anti-His HRP-conjugated antibody dissolved in buffer A , followed by three additional washes of the array with TBST. Immunodetection was performed using chemiluminescence (ECL reagents).INTAVIS Bioanalytical Instruments AG, Köln, Germany, synthesized the CelluSpotso-arrays . The pep30 µM peptides were incubated and vortexed for 3–5 min in 25% PEG400  and 10 mM phosphate buffer in various ionic strength conditions, adjusted by 1M NaCl solution (LLPS buffer) . Then 10The CD spectra of all peptides were recorded in the same manner as previously  with a JThe contact angle between ~1 µL of the ASAD in LLPS buffer solution and a glass slip covered with parafilm  was measured at the interface between the glass and line tangent to the droplet using a Theta Lite optical tensiometer  and Attension Theta software. Each result is composed of an average of 3 repeats with 100 measurements.


CD74 is a type II cell surface receptor found to be highly expressed in several hematological and solid cancers, due to its ability to activate pathways associated with tumor cell survival and proliferation. Over the past 16 years, CD74 has emerged as a commonly detected fusion partner in multiple oncogenic fusion proteins. Studies have found CD74 fusion proteins in a range of cancers, including lung adenocarcinoma, inflammatory breast cancer, and pediatric acute lymphoblastic leukemia. To date, there are five known CD74 fusion proteins, CD74-ROS1, CD74-NTRK1, CD74-NRG1, CD74-NRG2α, and CD74-PDGFRB, with a total of 16 different variants, each with unique genetic signatures. Importantly, the occurrence of CD74 in the formation of fusion proteins has not been well explored despite the fact that ROS1 and NRG1 families utilize CD74 as the primary partner for the formation of oncogenic fusions. Fusion proteins known to be oncogenic drivers, including those of CD74, are typically detected and targeted after standard chemotherapeutic plans fail and the disease relapses. The analysis reported herein provides insights into the early intervention of CD74 fusions and highlights the need for improved routine assessment methods so that targeted therapies can be applied while they are most effective. A fusion protein is the outcome of a multistep process occurring at either the DNA or RNA level and involves the cleavage of two independent genes, followed by their joining into a hybrid one. Upon translation, the newly formed fusion protein may or may not have a biological activity depending on multiple factors, such as the protein’s localization, structural architecture, and functional domains . Fusion Cluster of Differentiation 74 (CD74) is a membrane-bound protein expressed on the surface of human antigen-presenting cells (APCs). Also known as the invariant chain (Ii), the soluble form of CD74, which is found in the endoplasmic reticulum (ER), associates with major histocompatibility class II (MHC II) molecules to ensure their proper folding. CD74 can act as a receptor for the tumorigenic and proinflammatory cytokine macrophage migration inhibitor factor (MIF) . After tBesides its expression on immune system cells, CD74 has also been expressed in a variety of cancer cells, including thyroid carcinoma , bladderTranscriptional splicing can produce four different human CD74 isoforms, p33, p35, p41, and p43, which are 216 , 280 3737, and 2Drugs that target CD74 are limited despite its great clinical interest. As of 2008, the anti-CD74 monoclonal antibody (mAb), milatuzumab, received the orphan drug designation in the United States (U.S.) for the treatment of MM. Phase 1 clinical trials in MM and B-cell lymphoma patients found that Milatuzumab did not achieve an objective response rate (ORR), but patients were able to achieve stable disease with treatment ,53. Ten This review is focused on the CD74 oncogenic fusions  identified in various cancers. The structural features and expression patterns of all known CD74 fusion variants are described. Clinical patient characteristics, treatment plans, detection methods, as well as retrospective analyses of tumor samples are also included to provide insights into CD74 related oncogenic fusion proteins. While the CD74 protein alone is an attractive molecular target in multiple cancers, the study of cancer-specific proteins, such as the ones described here, offer a great opportunity for refining currently utilized cancer treatment plans and developing new methods for early detection.To date, five in-frame CD74 fusion proteins have been reported in cancer. These include CD74-NRG2α, CD74-NTRK1, CD74-ROS1, CD74-PDGFRB, and CD74-NRG1 . The disNoteworthily, the majority of CD74 fusion proteins found in patients were first reported in lung cancers, and the same trend continues to date . Of the CD74-ROS1 has seven variants reported in the literature, while CD74-NTRK1 and CD74-NRG1 have three and four variants each, respectively . CD74-PDROS1 was further characterized to encode a 259 kDa (2347 aa) protein with a glycosylated extracellular domain, a single transmembrane domain, and an intracellular tyrosine kinase domain [ROS1 gene is encoded by 43 exons according to the NCBI Reference Sequence NM_002944.2. Exons 1–32 produce the extracellular domain of ROS1, while exons 33 and exons 36–43 encode the transmembrane portion and C-terminal domain, respectively. The intracellular kinase domain or ROS1 is encoded by exons 36–41 [In an expression study of human cancer cell lines, ROS1 was found highly expressed in many glioblastoma cell lines but was not seen in normal healthy brain tissue . ROS1 hae domain . Despitee domain . The ROSns 36–41 . Tumors ns 36–41  based onns 36–41 . In 2019ns 36–41 .NTRK1 gene encodes the tropomyosin kinase receptor A (TRKA) protein, which is involved in neural development [NTRK1 gene, contains 17 exons; exons 1–9 encode a unique extracellular domain; exons 10–11 yield the transmembrane domain; and exons 11–17 produce for the intracellular domain, which includes the tyrosine kinase domain [The elopment . This prelopment : one is elopment ,80 and telopment ,81. TRKAelopment ,83. The elopment ,85. Humae domain . In respe domain . TRKA wae domain . The fine domain , thyroide domain , breast e domain , and cere domain . Oncogene domain . Larotree domain ,93 with e domain . Entrecte domain .NRG1 gene is known to produce many isoforms, which are expressed across various tissues through different promoters and alternative splicing [The splicing ,97. Furtsplicing ,99. ERBBsplicing . This lisplicing ,102,103.splicing , the strsplicing . While tsplicing , has beesplicing ,108. Liksplicing .PDGFRB gene and is known to express in mesenchymal [The platelet-derived growth factor receptor β (PDGFRB) protein is encoded by the enchymal  and smooenchymal . This reenchymal . PDGFRB enchymal . Once boenchymal , the Rasenchymal  signalinenchymal . PDGFRB enchymal , colon [enchymal , melanomenchymal , and breenchymal . Studiesenchymal ,121. Besenchymal  cancer aenchymal  cancer cCD74 and ROS1 genes. Of the CD74 isoforms, the p35 variant is involved in these fusions, and in patients has been found spliced at different exon breakpoints. Likewise, the ROS1 rearrangement occurs at different exons, resulting in the formation of unique CD74-ROS1 fusion proteins. CD74-ROS1 fusions typically have breakpoints located on ROS1 exon 32 or 34, retaining the ROS1 kinase domain [The first CD74 fusion protein, CD74-ROS1, was originally identified in NSCLC  and 14 ye domain  allowingIn 2007, the identification of CD74-ROS1 originally occurred in a 50-year-old female non-smoker with a stage IB lung adenocarcinoma . The patIn 2021, the most recently identified CD74-ROS1 variant was reported with a breakpoint fusing CD74 exon 3 to ROS1 exon 34 . This noThe expression of CD74-ROS1 has confirmed oncogenic transformation capacity towards fibroblast cells through the activation of MAPK, SHP-2, and STAT-3 pathways . AdditioRetrospective studies of tumor samples containing the CD74-ROS1 fusion were recorded to provide information about fusion variant, specimen source, detection method, age, gender, smoking status, and cancer stage . Many spClinical and case studies over the years outline additional details of CD74-ROS1 patients, such as the treatment sequence patients received, whether TKI resistance developed, and their overall survival (OS) . Based oWhile the clinical and case studies  show theCD74-NTRK1 was oncogenic through the pERK pathway; the gene fusion was also found to induce tumorigenesis in nude mice [CD74-NTRK1 was first detected by NGS in a lung adenocarcinoma patient tumor sample and confirmed using RT-PCR, followed by FISH . This fuude mice . The expude mice .Much later, in 2020, next-generation sequencing revealed a unique fusion of CD74 exon 7 with NTRK1 exon 8 (C7-N8) in a 41-year-old female stage IIIB adenocarcinoma patient  Table 1Table 1. CD74-NRG1 was first identified in a 64-year-old female never-smoker stage IB invasive mucinous adenocarcinoma (IMA) lung cancer patient through transcriptome sequencing . This fuH322 and H1568 lung cancer cell lines were virally transduced with the full CD74-NRG1 fusion, as well as with a truncated version of the fusion that lacked the EGF-like domain [An alternative translocation produced a novel C8-N6 fusion pairing that incorporated the first eight exons of CD74 and the EGF-coding NRG1 exons, resulting in a 303 aa CD74-NRG1 fusion . This fuThe same study treated EFM-19 reporter cells with media from H1299 lung cancer that expressed CD74-NRG1 and found that the fusion protein caused the phosphorylation of ERBB2 and ERBB3, which suggested the activation of HER2:HER3 autocrine receptor signaling; The phosphorylation of ERK and AKT was also observed . LapatinMultiple retrospective studies have been conducted on CD74-NRG1 patient tumor samples . Many paRNA seq first identified a CD74-PDGFRB fusion in a 2.4-year-old male with B-cell acute lymphoblastic leukemia. The novel protein was found to be a fusion between CD74 exon 6 and PDGFRB exon 11  Table S. Followiintr::PDGFRB. This fusion lacked an open reading frame to drive the translation of a functional fusion protein [Interestingly, in another case, Sanger sequencing showed a retained CD74 intron sequence that coded for a stop codon before the fusion breakpoint . Referre protein . Additio protein . Althoug protein .While this fusion has been documented in the literature several times, its oncogenic variant remains to be further studied and characterized as the oncogenic signaling pathway that is activated is still unknown. But, it is likely that the presence of CD74-PDGFRB leads to the activation of PDGFRB .The most recently identified CD74 fusion protein was CD74-NRG2α in a 70-year-old female, never-smoker, stage IIIA acinar adenocarcinoma lung cancer patient  Table S. This fuMethods that are utilized to identify CD74 fusions include DNA or RNA NGS, FISH, RT-PCR, and RT-qPCR. In clinical case studies, these methods typically identify the fusion after cancer recurrence. Both male and females are affected by CD74 fusions, with many female non-smokers reported as carriers.CD74-ROS1 has the highest number of fusion variants (seven) as well as patient data availability. Upon analysis of the published findings, it becomes clear that CD74-ROS1 is formed from very early to advanced-stage cancers. This fusion is mostly detected in NSCLC and likely occurs equally in both smokers and non-smokers. CD74-NRG1 has the second greatest number of variants (four), followed by CD74-NTRK1 (three), while CD74-NR2α and CD74-PDGFRB have a single fusion protein variant each. CD74-NRG1 is detected often at stages I and II in retrospective studies of patient tumor tissue, while clinical studies report this fusion both in early and late-stage IMA and NSCLC. Based on patient data available, the CD74-NRG1 fusion occurs mostly in non-smokers, with a few cases in smokers.About half of the CD74 fusion proteins have been studied extensively and their signaling pathways elucidated, mostly in respect to their fusion partner. Likewise, inhibitors target the fusion partner rather than CD74. Throughout the five fusions expressed in cancer patients, all retain the transmembrane domain of CD74 that many times makes up for the transmembrane domain that biologically occurs in the fusion partner before splicing occurs. CD74 fusion proteins mainly manifest in lung cancer, apart from a single documented case in an aggressive case of inflammatory breast cancer and two cases of pediatric leukemia. Of note, the rare fusions CD74-PDGFRB and CD74-NRG2α require further studies to elucidate their exact mechanism of disease.As many studies focus on the targetable fusion partners, the oncogenic role of CD74 in these CD74 fusion proteins remains to be fully understood. In ROS1 and NRG1 fusion families, CD74 is the most common partner, emphasizing the potential significance of CD74 occurrence in fusion proteins. The participation of CD74 as a partner in five different fusion proteins, with a total of 16 different variants, highlights its involvement in the formation of various types of fusions. The data on CD74 fusions offers insight for improved therapy plans for cancer patients and the development of additional methods for early detection to improve treatment success and patient survival.


Intestinal ischemia/reperfusion (I/R) injury is a common clinical event occurring during multiple clinical pathological processes. Here, we designed this paper to discuss the role of G protein‐coupled receptor 30 (GPR30) playing in intestinal I/R injury.An oxygen‐glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate the pathological process of I/R injury. With the application of enzyme‐linked immunosorbent assay, TUNEL, and transepithelial electrical resistance (TEER) assays, the levels of inflammatory cytokines, cell apoptosis, and intestinal integrity were estimated. The corresponding proteins were estimated by applying western blot. Immunofluorescence was conducted to examine N‐terminal Gasdermin D (GSDMD‐N) expression. The interplay between KLF4 and GPR30 was demonstrated by dual‐luciferase reporter assay and chromatin immunoprecipitation.The results showed that GPR30 was downregulated in Caco‐2 cells exposed to OGD/R. GPR30 overexpression reduced the production of TNF‐α, IL‐6, IL‐1β, and IL‐18, the TUNEL‐positive cells, as well as the contents of p‐p65, Cox‐2, Inos, Bax, and cleaved‐PARP, but elevated the expression of Bcl‐2 in OGD/R‐induced Caco‐2 cells. In addition, OGD/R‐induced the reduction of TEER value and reduced expression of tight junction proteins in Caco‐2 cells, which was partially restored by GPR30 overexpression. Furthermore, GPR30 suppressed nod‐like receptor pyrin 3 inflammasome and GSDMD‐N expression. It was evidenced that Krüppel‐like factor 4 (KLF4) could directly bind to GPR30 promoter and positively regulate GPR30 expression. The regulation of GPR30 overexpression above was weakened by KLF4 knockdown.Collectively, our findings suggested that KLF4 could transcriptionally upregulate GPR30, and GPR30 prevented intestine I/R injury by inhibiting inflammation and apoptosis, and maintaining intestinal integrity that provides potential targets for mitigating the I/R injury. G protein‐coupled receptor 30 (GPR30) protects against oxygen‐glucose deprivation/reoxygenation‐induced inflammatory response, apoptosis, and intestinal integrity disruption.Transcription factor Krüppel‐like factor 4 (KLF4) directly binds to GPR30 promoter and positively regulates GPR30 overexpression.KLF4/GPR30 axis exerts function in intestinal ischemia/reperfusion injury partly by inhibiting the nod‐like receptor pyrin 3‐mediated pyroptosis. Therefore, we speculate that transcription factor KLF4 can potentially affect GPR30 expression levels by binding to the GPR30 promoter region, and thus participate in regulating the pathological process of intestinal I/R injury. Hence, this study aims to elucidate the role of GRP30 and its potential regulatory mechanism in intestinal I/R injury, which will provide a novel angle and target for intestinal I/R injury treatment.Of note, using the Human TFDB website (22.12 at 37°C.Caco‐2 cells provided by American Type Culture Collection (HTB‐37) were cultivated in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum and 1% penicillin‐streptomycin in a humidity incubator containing 5% CO2.22, 5% CO2, and 94% N2 for 8 h and further incubated with glucose‐high EBSS in the conventional incubator  for 20 h for reoxygenation.An oxygen‐glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate the pathological process of I/R as previously described.2.35 Caco‐2 cells were inoculated into six‐well plates. When cells reached 70%–80% confluence, the transfection of above plasmids into cells was implemented by adopting Lipofectamine 3000  in light of recommended protocols. Forty‐eight hours post‐transfection, cells were harvested for experiments.The GPR30 over‐expressing plasmid (oe‐GPR30), KLF4 over‐expressing plasmid (oe‐KLF4) as well as their corresponding negative control (oe‐NC) were provided by GenePharma. Short hairpin RNA (sh) targeting KLF4 (sh‐KLF4‐1 and sh‐KLF4‐2) and its negative control (sh‐NC) were also synthesized by GenePharma. 1 × 102.4Total RNA was separated by applying TRIzol kit  in light of standard specifications. The RNA purity as well as concentration was decided on adopting NanoDrop 3000 . Then, 1 μg of total RNA was reverse‐transcribed into cDNA in light of the protocol of the reverse transcription kit (Takara), followed by the quantification of mRNA level using ChamQ Universal SYBR qPCR Master Mix  in light of the recommended specification. The sequences of the primers were as follows: GRP30, forward, 5′‐AGTCGGATGTGAGGTTCAG‐3′ and reverse, 5′‐TCTGTGTGAGGAGTGCAAG‐3′; KLF4, forward, 5′‐ACCTACACAAAGAGTTCCCATC‐3′ and reverse, 5′‐TGTGTTTACGGTAGTGCCTG‐3′; GAPDH, forward, 5′‐CCATGGGGAAGGTGAAGGTC‐3′ and reverse, 5′‐AGTGATGGCATGGACTGTGG‐3′. A comparative Ct approach was employed to estimate relative gene expression using GAPDH as an internal reference.2.5Total protein was extracted adopting lysis buffer  and was quantified applying BCA Protein Assay Kit . Proteins (30 μg/lane) were separated by 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with skimmed milk, the membranes were probed with primary antibodies against GPR30 , p‐p65 , p65 , Cox2 , iNOS , Bcl‐2 , Bax , cleaved‐PARP , PARP , Claudin‐1 , Occludin‐1 , ZO‐1 , KLF4 , nod‐like receptor pyrin 3 , Cleaved‐caspase1 , caspase1 , N‐terminal Gasdermin D , and GAPDH  at 4°C overnight, after which was the cultivation with horse radish peroxidase (HRP)‐conjugated secondary antibody  at room temperature for 2 h. The visualization and analysis of protein blots were operated employing enhanced chemiluminescence and Image Lab™ software 5.2.1 , respectively.2.6The levels of tumor necrosis factor (TNF)‐α, interleukin (IL‐6), IL‐1β, and IL‐18 were assessed employing enzyme‐linked immunosorbent assay (ELISA). The detection process was performed in line with the guidelines of the manufacturer (Wuhan Huamei Bioengineering Institute).2.7Caco‐2 cells were seeded on six‐well plates and exposed to experimental procedures. After washing, the cells were exposed to 4% paraformaldehyde for fixation as well as 0.2% Triton X‐100 for permeation. Then, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Kit (Roche Diagnostics) was applied for measuring the apoptotic cells strictly in line with the manufacturer's instructions. Finally, DAPI was utilized for the staining of cell nuclei and a confocal laser microscope  was adopted for the capture of images.2.8Caco‐2 cells were seeded on six‐well plates and exposed to experimental procedures. After washing, the cells were exposed to 4% paraformaldehyde for fixation as well as 0.2% Triton X‐100 for permeation. After that, cells were blocked with 5% bovine serum albumin solution for 1 h, and then cells were incubated with primary antibody against GSDMD‐N  overnight at 4°C, following which was the probe with secondary anti‐rabbit IgG‐Alexa Fluor 488 antibody  for 1 h at 37°C in the dark. Finally, DAPI solution was added to the cells away from light. Immunofluorescence photographs were acquired employing a confocal laser microscope .2.96 cells/mL and exposed to experimental procedures. With the application of Millicell‐ERS device , the resistance was estimated. The calculation of transepithelial electrical resistance (TEER) was decided in light of the formula: TEER =  × single cell layer surface area (cm2).The cells in the logarithmic growth phase were inoculated into a 0.4‐μm 24‐well transwell chamber (Costar Incorporated) at a density of 1 × 102.10http://bioinfo.life.hust.edu.cn/HumanTFDB/#!/), a potential binding relationship between GPR30 promoter and KLF4 was predicted. To verify it, dual‐luciferase reporter assay was conducted. Wild‐type (WT) and mutant (MUT) GPR30 promoter dual‐luciferase reporter vectors were constructed at the KLF4 binding site of the 3′‐UTR region of GPR30 promoter. The well‐grown Caco‐2 cells were inoculated into 96‐well plates and cultured overnight. The transfection of above vectors and oe‐KLF4 or oe‐NC into Caco‐2 cells was operated by adopting Lipofectamine 3000 . Forty‐eight hours post‐transfection, the Dual‐Luciferase Reporter Assay system (Promega Corporation) was applied to evaluate luciferase activity. Renilla activity was used as an internal reference.Through referring to the human TFDB website  got analyzed employing GraphPad Prism 8.0 (GraphPad Software). One‐way ANOVA followed by the Tukey's multiple comparison test was adopted for the comparisons among groups, while comparisons between the two groups were conducted utilizing unpaired Student's 33.1To figure out the role of GPR30 playing in intestinal I/R injury, an OGD/R cell model was established to mimic the pathological process of I/R, and GPR30 level was firstly estimated. As Figure 3.2Subsequently, to investigate the regulatory role of GPR30 playing in intestinal I/R, untransfected and transfected Caco‐2 cells were exposed to OGD/R, and the release of pro‐inflammatory cytokines was evaluated. As Figure 3.3Then, the regulatory role of GRP30 on intestinal integrity was explored. The integrity of the Caco‐2 cell monolayer was evaluated via detecting TEER. After exposure to OGD/R, an obvious drop in TEER was observed in Caco‐2 cells, indicating that OGD/R increased the permeability of the cell membrane. Meanwhile, this elevated permeability exposed to OGD/R was reduced by GPR30 overexpression, evidenced by the elevated TEER value in the OGD/R+oe‐GPR30 group Figure . To veri3.4http://bioinfo.life.hust.edu.cn/HumanTFDB/#!/) that transcription factor KLF4 is able to bind to the GPR30 promoter region ; methodology ; formal analysis ; writing—original draft (lead). Xiaoli Xie: Data curation ; methodology ; formal analysis ; writing—original draft (supporting). Jinfeng Yao: Data curation ; methodology ; formal analysis (supporting); software (lead). Xiaoxu Jin: Data curation ; methodology ; formal analysis (supporting). Huiqing Jiang: Data curation ; methodology (supporting); software (supporting). Chenguang Ji: Conceptualization (lead); writing—review and editing (lead).The authors declare no conflict of interest.


MRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase  (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (LipMRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation.Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or Lip Datasets were collected with a Synergy HT Multi-Mode Microplate reader , with values obtained from the best linear fit using Excel 2019. In all cases, the activity was calculated by determining the absorbance per minute from the generated slopes [The activity towards TriC®) assay ,38,39. IAll reactions were performed in triplicate (n = 3) with control reactions (no enzyme added) and background signals considered, and the activity was calculated by determining the absorbance per minute from the generated slopes, as previously reported . The thrp-NP butyrate  was assessed by monitoring the continuous production of 4-nitrophenol at 348 nm  using 0.2 µg of total protein, as previously reported [3BO3, 0.04 M H3PO4, and 0.04 M CH3COOH that was titrated to the desired pH with 0.2 M NaOH. Similar assay conditions were used to assay the effects of temperature on p-NP butyrate hydrolysis, but in this case, the reactions were performed at 40 mM HEPES buffer pH 7.0. The activity was calculated by determining the absorbance per minute from the generated slopes, as previously reported [The hydrolysis of the model ester reported . In all reported , with alTd) values were determined at 220 nm between 10 and 85 °C at a rate of 30 °C per hour in HEPES buffer 40 mM, pH 7.0. A protein concentration of 1.0 mg ml−1 was used. The Td (and standard deviation of the linear fit) was calculated by fitting the ellipticity (mdeg) at 220 nm at each of the different temperatures using a 5-parameter sigmoid fit with SigmaPlot 14.0 software.CD spectroscopy was used to determine the thermal denaturation profile. CD spectra were acquired between 190 and 270 nm with a Jasco J-720 spectropolarimeter  equipped with a Peltier temperature controller employing a 0.1-mm cell at 25 °C. Spectra were analysed and processed with a Spectra Manager software , and the denaturation temperature  was used to model the opening of the lid domain in the studied lipase, as well as the binding of triolein to the lipase catalytic site. PELE is a Monte Carlo (MC)-based algorithm coupled with protein structure prediction methods ,43. This10:0) and showed slight activity towards coconut oil. There are examples of lipase engineering by lid swapping, which results in altered substrate specificity [This work provides a rational-based protein engineering approach to improve the capacity of lipases to hydrolyse large water-insoluble triglycerides. This was achieved by investigating a lipase isolated from the Marine Metagenomics MarRef Database, which contains a lid domain but was only capable of hydrolysing triglycerides up to tridecanoate (TriCcificity ,28,29, acificity  and speccificity . The rescificity ,28,29. W


IRE1 belongs to a type I transmembrane protein family harboring two functional domains, cytoplasmic domain with kinase and RNAse catalytic activity, and the luminal domain, which is involved in the sensing of unfolded proteins. IRE1 molecule undergoes dimerization in the lumenal domain, which functionally activates the catalytic C-terminal domain. IRE1 activation is directly related to transition between monomeric and dimeric forms. We have deduced two quaternary structures from the published crystal structure of IRE1. One structure with a large stable interface that requires large activation and deactivation energy to active IRE1. The other quaternary structure has low dissociation energy and is more suitable for IRE1 oligomeric transition. IRE1 structurally constitutes the sensory N-terminal domain towards the ER lumen, the C-terminal catalytic domain towards the cytosol and a linker region connecting the two domains. IRE1 undergoes dimerization/oligomerization in the lumenal domain, which functionally activates the catalytic C-terminal domain . A quaternary structure of IRE1 lumenal dimer was deduced from the crystal structure published earlier  . The proposed quaternary structure with a large stable interface is formed through a beta-sheet spreading across the dimer subunits. Several mutation studies for residues 105 , 109 , 123 , 125  corroborate the effect on dimer formation. There are, however, two problems with this form of the quaternary structure: (i), presence of a strong interface, which implies that a large activation and deactivation energy may be needed to alter the monomer-dimer equilibrium, which may not be adequate for a sensor protein. (ii), the end-to-end distance of the C-termini of lumenal domain between two monomers is 62.8 Å. For the lumenal domain to tether to the cytoplasmic domain across the membrane, a similar N-terminal end-to-end separation for cytoplasmic dimeric form of the protein is desirable. This can be found in the head-to-tail form of the dimeric proteins as shown in , where a 65.7 Å separates the dimer, which matches closely with the separation of C-terminal distance in the lumenal domain . Also, there is a ~193 residue linker region that attach the lumenal domain to the cytoplasmic domain with a 20 residue transmembrane segment located at positions 444-464. This means that there are roughly 76 and 98 residues on the lumenal and the cytoplasmic side that tethers the C-terminal domains, which must also dimerize to perform its biological functions. However, unless the 98-residue segment can form a stable structure, the lumenal---C-terminal domain tether is likely to remain floppy because of the large spatial gap between the two tethers.IRE1 is a sensor protein found in the endoplasmic reticulum (ER) of eukaryotic cells that is involved in the Unfolded Protein Response (UPR), a cellular stress response pathway. It is activated in response to the accumulation of unfolded proteins in the ER, and its activation leads to a cascade of events that help cells cope with the stress .The structural perspective from the crystal structure data somehow does not fit well with the fragmented experimental data available, and therefore better strategies need to be developed to synchronize the data. In order to solve this structural discrepancy, here we are presenting an alternative quaternary structural model for dimerization of IRE1 lumenal domain . The side-by-side orientation is obtained from the crystal lattice of the published structure and has been inferred as the potential dimer by our program. In this model, the C-termini of two monomers are separated by a shorter distance of 42.8 Å. A corresponding C-terminal domain that matches, has an N-terminal separation of 41.7 Å . In our case the lumenal domain dimer  is less stable form with 1130 Å of interface area, and -12.3 and 0.3 kcal/mol of theoretical interaction and dissociation free energy, respectively. In contrast, the other model depicts a more stable form of the quaternary structure of IRE1  with 1730 Å of interface area, and -15.2 and 7.2 kcal/mol of theoretical interaction and dissociation free energy, respectively. It means that in our model, the dimer has a reasonably large interface area and a low dissociation free energy indicating that it can easily undergo monomer-dimer transition. Also, an approximate 40 Å separation between the luminal---C-terminal domain tethers are more likely to have stable structure that stabilizes the full IRE1 protein in its functional form.The C-terminal domain here is organized in a side-by-side orientation. Interestingly, this orientation has been suggested to be the correct dimerization state of the IRE1 C-terminal domain  was done using IPAC . This program detects quaternary structures from crystal lattice by utilizing a scheme where point group symmetry works in combination with naïve Bayes classifier under Boolean framework . In the first step, by utilizing the asymmetric unit information available from the PDB file, we generate all symmetry- related molecules in the lattice. Non-protein atoms are also included. In this step, we also check presence of disulfide bonds at the interface, which are grouped together as functional units (FUs). In the next step, biological interfaces are detected by applying Bayes classifier. Smaller interfaces are connected into a large single interface. A depth-first search is used in a cyclic manner until no new interfaces are detected. In the last step, point group symmetry (PGS) is used to screen merged FUs. Cyclic PGS is applied to identify FUs of size n (>2). Symmetry with a threshold of 10.0 Aº can be identified by using backbone-trace Ca atoms. The largest FU satisfying PGS is inferred as the quaternary structure of the protein. In this particular study we have used an updated version of this program that emphasizes the contribution of symmetry in determining the final quaternary structure (yet to publish).The quaternary structures analysis of IRE1 protein (


The international community has been paying close attention to the issue of food safety as a matter of public health. The presence of a wide range of contaminants in food poses a significant threat to human health, making it vital to develop detection methods for monitoring these chemical contaminants. Electrochemical sensors using emerging materials have been widely employed to detect food-derived contaminants. Covalent organic frameworks (COFs) have the potential for extensive applications due to their unique structure, high surface area, and tunable pore sizes. The review summarizes and explores recent advances in electrochemical sensors modified with COFs for detecting pesticides, antibiotics, heavy metal ions, and other food contaminants. Furthermore, future challenges and possible solutions will be discussed regarding food safety analysis using COFs. The safety of food is of vital importance to the health of people and to the long-term stability of society in general . Food foGlobally, the top 100 food and beverage companies generated revenues of USD 1.3 trillion in 2019, equivalent to approximately CNY 9.2 trillion  and heavHaving a rational and effective approach for food testing is an essential component of food safety management. Conducting well-informed research on testing techniques can provide powerful assurances regarding food safety being maintained continuously.The foundation of any food safety program is improved food safety testing techniques, which are key in addressing food safety. Food safety testing methods can be classified as traditional or rapid detection. Usually, conventional methods consist of using techniques such as gas chromatography–mass spectrometry , high-peThe primary objective of electrochemical detection techniques is to qualitatively or quantitatively analyze and measure target substances based on their electrical and electrochemical properties through the use of electrochemical sensors . There aAs a general rule, nanomaterials are materials with at least one dimension out of the three dimensions within the nanometer size range (1–100 nm), or they are formed from basic constituents with such dimensions. Nanomaterials have unique physicochemical properties in optics, electronics, magnetism, heat, mechanics, and other fields as a result of their unique surface effects, small size effects, quantum effects, and macroscopic quantum tunneling effects . The use2 g−1, respectively), high thermal stability, and permanent porosity were compared 2+. As a result, a low-potential pesticide detection can be achieved. Due to the reduction in the amount of thiol choline catalyzed by AChE as the concentration of pesticides increases, choline thiocholine is less repelled by [Ru(bpy)3]2+, resulting in the generation of redox current signals at the electrode surface. Providing enzymes with a microenvironment of superior chemical stability ensures that they will maintain a higher level of activity regardless of adverse external conditions. The research group of Lu -thiadiazol-4,7-diyl)bisbenzaldehyde (BTDD), they synthesized a COF with multiple metal ion adsorption sites . MI. MI94]. 3O4@COFs@MIPs) for the sensitive and rapid determination of TC has been proposed. TC is detectable at concentrations from 1 × 10−10 to 1 × 10−4 g/mL, with a limit of detection (LOD) of 2.4 × 10−11 g/mL. Milk and chicken samples have also been successfully tested using the prepared sensor.It is commonly known that tetracycline (TC) is a type of antibiotic that can lead to drug resistance and other side effects, such as allergic reactions, kidney toxicity, and liver damage. The detection of tetracycline antibiotics is currently performed using electrochemical sensors based on COFs in order to further improve their stability and portability. A portable on-site electrochemical sensor similar to that proposed by Yukun Yang et al.  based onOther contaminants, such as illegal additives, are also threats to the safety of food, in addition to the previously mentioned pollutants. It is possible to selectively detect these targets through the design of COF-based sensing approaches in order to address these challenges.3O4@TAPBDMTP-COF as the sensor substrate, Chen et al. were able to more easily and rapidly detect TBHQ, owing to the excellent electrocatalytic property and the large surface area of COFs [There is no doubt that tertiary butylhydroquinone (TBHQ), a good antioxidant, plays a critical role in the prevention of lipid oxidation, but the high doses of TBHQ may cause carcinogenesis . Using C of COFs . Compare of COFs . In thei of COFs . There iThe purpose of this review has been to highlight the outstanding potential of COFs as emerging porous materials for electrochemical sensing, in particular to ensure food safety. It is becoming increasingly evident that COFs  are emerging porous materials with a high crystallinity, a good degree of stability, and a controllable pore size and topology. In the field of electrochemical sensing, COFs have exhibited great potential because of their flexible design. Analytes can undergo adsorption and electrochemical reactions facilitated by COFs due to their high surface area and porosity. It is possible to enhance the selectivity and sensitivity of COFs by introducing functional groups. Furthermore, the solid-state nature of COFs makes them easy to integrate with sensing platforms, enhancing their repeatability and stability.Food safety can be improved through the use of electrochemical sensors, as they offer a high level of accuracy, simplicity, cost-effectiveness, and rapid response. COF-based electrochemical sensors are providing a platform for ensuring food safety and quality. Various designs and types of COFs have been reported, including B-O structures, imine bonds, C=C structures, triazine structures, and other types of connection. Further, the reports discuss methods for improving the performance of COF-based electrochemical sensors and discuss their application for the detection of food pollutants such as pesticides, heavy metal ions, antibiotics, etc.In spite of the excellent performance and promising prospects of COF-based electrochemical sensors, they exhibit a few challenges, pertaining to enhanced stability and repeatability, miniaturization, and on-site operation. In spite of these challenges, COF-based electrochemical sensors continue to hold great promise in the field of food analysis. It is anticipated that future research will emphasize the combination of novel COFs and advanced electrochemical technologies in order to provide electrochemical sensors with excellent analytical performance.As a part of the design of COFs, the appropriate pore size and the specific functional groups will be considered based on the physical and chemical properties of the target analytes, which will enhance the selectivity of the modified electrode. It has been proposed that post-modification methods could be employed in order to modify the prepared COF materials and achieve improved properties. Cui et al. modified the COFs with cyclodextrin in order to enhance their ability to transport amino acids selectively . It was Electrochemical sensors based on COFs are typically constructed by coating or modifying conventional electrodes, but the direct integration of COFs into the device structure can further enhance their sensing capabilities. The advantage of this approach is that it minimizes the signal-to-noise ratio and increases the surface area for the optimum binding and reaction of analytes. It is also advantageous to integrate multiple COF-based sensors on a single substrate, which allows for multiplexed detection, thus increasing the overall efficiency of the system.COF-based electrodes have recently been shown to have potential applications in the analysis of biomedical and environmental pollutants. It has been demonstrated, for example, that COF-modified electrodes exhibit superior sensitivity, selectivity, stability, and reproducibility when compared with classical electrodes. In particular, COF-modified electrodes are remarkably recyclable even after multiple cycles, demonstrating their durability. While COFs have made significant advancements in electroanalytical chemistry, their application is still relatively new, and challenges must be overcome in order to develop COF-based electrochemical sensors in the future.Looking ahead, it is vital to develop the reasonable design and controllable synthesis of multifunctional electrode materials based on COFs in order to improve their selectivity towards food pollutants and to enhance their stability and repeatability while achieving the miniaturization of the sensors. Moreover, the biocompatibility study of COF-based electrochemical sensors should be strengthened in order to expand their applications in the detection of biological food contaminants. Additionally, it would be beneficial to address the issue of electrode surface fouling and extend the lifetime of sensors by designing and synthesizing COF electrode materials with anti-fouling properties.Electrochemical sensors for COFs will be developed in part by the advancement of synthetic techniques and the development of analytical chemistry, among other factors. With the development of new synthetic techniques, COFs with more stable and controllable structures can be synthesized, enabling further investigation into the potential applications of COFs in areas such as food safety and biology. Analytical chemistry advancements have contributed to improving the performance and functionality of sensors. Since the development of synthetic techniques for COFs, researchers have been able to synthesize COFs with more stable and controlled structures. Recently, a number of new synthetic strategies and methods have been developed, including solvent evaporation, cosolvent synthesis, and interfacial synthesis. Electrochemical sensors for COFs can now be developed using these new techniques, providing more options and possibilities. Electrochemical sensors for COFs must also take into account a number of analytical chemistry considerations. It may be necessary, for example, to select the correct sensor materials and synthesis methods in order to achieve a higher level of sensitivity and selectivity. It is also important to pay attention to performance indicators, including stability, repeatability, and practicality. A further enhancement of the performance and functionality of the sensors can be achieved through the interaction of COFs with other functionalized materials. The development of electrochemical sensors with COFs in the future will be driven by these trends, which will lead to wider applications and an increased market potential.In summary, COF-based electrochemical sensors are still in the early stages of application in the field of food analysis, but this innovative research field holds great promise. It has been possible to detect antimicrobials, pesticides, and heavy metals using some of these techniques. It is anticipated that continued research into the design and synthesis of COFs, in conjunction with advanced electrochemical technologies, will contribute to the enhancement of the analytical performance of electrochemical sensors and the development of food safety testing systems.


Background: Salvage procedures for distal radioulnar joint (DRUJ) arthritis, like the Darrach or Sauvé-Kapandji (S-K) procedures, often result in extensor tendon ruptures at the ulnar stump. Radioulnar instability is considered the underlying cause and stump stabilization techniques are employed. This study investigated radioulnar instability, extensor tendon irritation, and the effectiveness of stump stabilization techniques following salvage procedures.Methods: Six upper limbs from three cadavers were used. Forearm rotation was measured using magnetic position sensors to assess radial movement. The Darrach procedure was performed on two limbs, comparing radial motion ranges for different ulnar osteotomy positions. The risk of tendon rupture was assessed with applied weight. The S-K procedure was performed on four limbs, evaluating stump stabilization techniques and radial movement distance underweight.Results: Proximal osteotomy positions increased radial motion range. Extensor tendon irritation occurred when the load was applied to the volar and ulnar sides, particularly with a pronated forearm. Stump stabilization techniques did not significantly contribute to ulnar stump stabilization.Conclusions: Proximal ulnar osteotomy positions in DRUJ salvage procedures led to increased radioulnar instability and potential complications. Load application on the volar and ulnar sides, especially in a pronated forearm, increased the risk of tendon rupture. Stump stabilization techniques showed limited utility in stabilizing the ulnar stump or reducing complications. These findings can inform strategies for minimizing complications in DRUJ salvage procedures. Patients suffering from distal radioulnar joint (DRUJ) disorders related to osteoarthritis and rheumatoid arthritis commonly experience ulnar side wrist pain and subcutaneous extensor tendon rupture . SalvageSubcutaneous extensor tendon rupture can be caused by radioulnar instability and the absence of soft tissue interposition between the tendons and the ulnar stump . PreviouPrevious studies, such as those conducted by Douglas et al., who examined radioulnar instability after hemi-resection of the ulnar head, Darrach procedure, and DRUJ arthroplasty , and KitTenodesis stabilization has been proposed to improve stump instability. Minami et al., Chu et al., and Leslie et al. reported favorable outcomes using stump stabilization with the extensor carpi ulnaris (ECU) tendon -14, whilBased on our hypothesis that radioulnar instability following the Darrach or S-K procedure is influenced by the position of the ulnar osteotomy, postoperative limb position, and applied load and that stabilization procedures can improve stability, this study aimed to evaluate the instability of the ulnar stump and extensor irritation after the Darrach procedure considering the osteotomy site and forearm rotation, as well as assess the impact of stabilization techniques.SpecimensEthical approval was obtained from the Institutional Review Board of Chiba University's Graduate School of Medicine, Chiba, Japan, to conduct this study with approval number- 3224. Upper limbs from fresh frozen cadavers donated to the clinical anatomical laboratory were utilized (six upper limbs from three cadavers). The right and left upper limbs of an 84-year-old woman was used for investigating radioulnar instability and extensor tendon irritation (Experiments 1 and 2). To assess the effectiveness of stump stabilization procedures (Experiment 3), four upper limbs from two men (aged 103 and 99) were employed. After removing the dorsal skin, the extensor tendons were dissected while preserving the extensor retinaculum.Jig and fixationAn original jig consisting of a cylinder was utilized for the experiment. The ulna was fixed to the jig with K-wires, ensuring that the elbow flexion was set at 90 degrees and the fovea of the ulna, serving as the axis of forearm rotation, was positioned at the center of the circle. The wrist was immobilized using an external fixator  in a neutral position Fig .Biomechanical evaluation A magnetic position sensor  was employed for the study. Sensors were placed on the radial styloid process and the ulnar shaft while the transmitter was fixed onto the jig. The coordinates of the ulna were adjusted by measuring the distance from the ulna sensor to the center of rotation, thereby plotting the position of the radius in XY coordinates, depicting the movement of the radius around the ulna. The coordinates of the radius were measured using a sensor through manual pronation and supination, repeated approximately 10 times to record and plot approximately 10,000 coordinates on a coordinate plate, enabling the illustration of the movement of the semicircular radius centered on the ulna Fig .Experiment 1: Range of Radial Motion and Ulnar Osteotomy Position We examined how the position of the ulnar osteotomy influences the range of radial motion. Following the osteotomy and excision of the ulna head, a passive movement was used to measure the range of radial motion, observing any changes at the osteotomy site. The osteotomy positions were set at 10, 25, and 40 mm from the joint surface Fig , and theExperiment 2: Risk of Extensor Tendon Rupture We investigated the risk of extensor tendon rupture caused by stump irritation. "Irritation" was defined as the contact between the tendon and stump. The area where the stump came into contact with the extensor digitorum communis (EDC) tendon, known to be susceptible to tendon rupture from repetitive contact, was defined as the irritation area. Conversely, the non-irritation area was defined as the region where the stump did not make contact, determined by gross observation. These areas were predetermined through manual manipulation of the radius. The forearm was positioned in neutral, pronated at 30° and 60°, and supinated at 30° and 60°. A load of 1 kg was applied to the dorsal, volar, radial, and ulnar sides using an external fixator based on a previous study Fig  10]. Th. Th10]. Experiment 3: Evaluation of Stump Stabilization Techniques We evaluated the effectiveness of various stump stabilization techniques. The S-K procedure was performed following previous reports , with thStabilization with PQ suturing: Following the approach described by Johnson et al., the PQ attachment portion was separated from the ulna and used to cover the stump after osteotomy . It was Stabilization with ECU half-slip tendon: In accordance with the method outlined by Minami et al., a 3.5-mm hole was drilled from the dorsoulnar aspect of the ulnar shaft into the intramedullary cavity . The ECUStabilization with ECU and FCU half-slip tendon: Based on the technique described by Breen et al., a distally-based tendon strip of the FCU and a proximally-based tendon strip of the ECU were created . The FCUStabilization with FCU half-slip tendon: This procedure was identical to procedure #3, except that only the FCU half-slip tendon was used to stabilize the stump.For the ECU or FCU stabilization procedures, the muscles and tendons employed for stabilization were tensioned using a 500-g weight Fig . A 1-kg Data analysisTo account for variations among specimens, the movement in the no-stabilization group was standardized to 1, and the movement in the other groups was expressed as a ratio relative to the no-stabilization group. The movement in each group was compared for the four specimens. Statistical analysis was performed using Wilcoxon T-tests, with a significance level of p < 0.05.Experiments 1 and 2The range of radial motion is illustrated for each ulnar osteotomy position Fig . The uppThe results of the loading tests are presented in Table 1. In the right limb, when the ulnar osteotomy was performed at 25 mm, the stump was in the irritation area when the limb was loaded on the volar or ulnar side with a pronation angle of 60° or loaded on the ulnar side with a pronation angle of 30°. Otherwise, the stump remained in the non-irritation area. If the ulnar osteotomy was performed at 40 mm, the stump was in the irritation area when the limb was loaded on the volar or ulnar side with pronation angles of 30° or 60° or loaded on the ulnar side with the limb in a neutral position. In the left limb, when the osteotomy was performed at 25 mm, the stump was in the irritation area when the limb was loaded on the volar and ulnar sides with pronation angles of 30° and 60°. Similar findings were observed when the osteotomy was performed at 40 mm, and when the limb was loaded on the volar side in a neutral position, the stump was in the irritation area. In both upper limbs, the stump was consistently in the irritation area when the limb was loaded on the volar and ulnar sides in a pronated position.Experiment 3Based on the findings of Experiment 2, which indicated a risk of extensor tendon rupture when loads were applied to the upper limb on the volar and ulnar sides in neutral and pronation positions, the effectiveness of the stabilization technique was examined in those scenarios. Figure This study observed that the range of motion between the radius and ulna increased as the ulnar osteotomy was performed further from the articular plane. At osteotomy positions of 25 mm and 40 mm, there was an area of irritation where the stump came into contact with the extensor tendon. This suggests that as the ulnar osteotomy position becomes more proximal, the increased range of motion may elevate the risk of stump-related pain, instability, and extensor tendon rupture. Unlike a previous study , we did However, suppose there was initially a dorsal dislocation of the ulnar head in DRUJ. In that case, the stump should be positioned so that it does not protrude dorsally and does not irritate the radius. During the performance of the S-K procedure, creating a gap between the ulnar head and the stump is necessary to prevent fusion of the distal and proximal ulnar ends.Furthermore, we observed that the stump moved into a position that contacted the EDC tendon when a load was applied to the volar or ulnar side with the forearm in a pronated position. This corresponds to daily activities such as holding an object with the forearm horizontally in a neutral or pronated position. Our findings suggest that even holding a 1 kg load in such forearm positions could lead to tendon irritation. Therefore, postoperative rehabilitation and guidance in daily activities should be considered, including instructing patients to hold objects in a supinated forearm position whenever possible.We found that stump stabilization did not significantly improve radioulnar instability. Previous clinical studies have reported the usefulness of various stabilization techniques -15, but In conclusion, when performing the Darrach or S-K procedure, it is advisable to position the stump distally, considering the risk of extensor tendon rupture and other factors, and there may be no need for a stump stabilization procedure. However, in clinical practice, the stump position during the S-K procedure often ends up approximately 20-25 mm from the joint surface due to the creation of the ulnar gap, making it challenging to position it more distally and avoid an irritation area that poses a risk of extensor tendon rupture. To minimize direct stimulation of the stump measures such as bluntly scraping the bone may be considered .The first limitation of this study is the small sample size. However, experiments 1 and 2 yielded similar results for both limbs, indicating that the range of motion between the radius and ulna and the irritation of the stump on the extensor tendon are influenced by the osteotomy and forearm position. The second limitation is that the assessment of forearm muscles was conducted in a non-contracted state. Several previous studies have also evaluated the instability of salvage procedures in fresh frozen cadavers -11, and Nevertheless, we believe it was still possible to assess the effects of stump position, load direction, and the usefulness of stabilization techniques. Fourth, the cadaver specimens, being elderly, may have exhibited degeneration of the interosseous membrane, triangular fibrocartilage complex, and extensor tendons. Fifth, the measurement of the range of radial motion relied on passive movement, which could introduce variation in the observed range.In conclusion, this study utilized fresh frozen cadavers to investigate radioulnar instability following salvage procedures for DRUJ arthritis, the risk of extensor tendon rupture at the stump, and the effectiveness of ulnar stump stabilization techniques. The results suggest that the range of motion between the radius and ulna increases as the ulnar osteotomy position becomes more proximal, heightening the risk of complications. Furthermore, when the stump is loaded on the volar or ulnar side during pronation, it irritates the extensor tendon, increasing the risk of tendon rupture. This study did not observe the previously reported benefits of stump stabilization techniques. We propose that when performing the Darrah or S-K procedure on the distal radioulnar joint, reducing stump instability and associated complications can be achieved by positioning the ulnar osteotomy as distally as possible and by avoiding pronated forearm positions, particularly on the volar and ulnar sides.


The constitutive equation of thermal deformation characteristics based on strain was established, and the microstructure  under different deformation conditions was analyzed. It is shown that the steady-state flow stress can be described using the hyperbolic sinusoidal constitutive equation with a deformation activation energy of 160.03 kJ/mol. Two kinds of second phases exist in the deformed alloy; one is the η phase, whose size and quantity changes according to the deformation parameters, and the other is spherical Al3 particles with good thermal stability. Both kinds of particles pin the dislocation. However, with a decrease in strain rate or increase in temperature, η phases coarsen and their density decreases, and their dislocation locking ability is weakened. However, the size of Al3 particles does not change with the variation in deformation conditions. So, at higher deformation temperatures, Al3 particles still pin dislocations and thus refine the subgrain and enhance the strength. Compared with the η phase, Al3 particles are superior for dislocation locking during hot deformation. A strain rate ranging from 0.1 to 1 s−1 and a deformation temperature ranging from 450 to 500 °C form the safest hot working domain in the processing map.The hot deformation behavior of Al-Zn-Mg-Er-Zr alloy was investigated through an isothermal compression experiment at a strain rate ranging from 0.01 to 10 s Howependent . In recem alloys ,17,18. HDuring hot deformation, the microstructure is highly correlated with the deformation parameters, so it is very important to establish the relationship between the hot deformation parameters and the microstructure evolution. During the hot deformation process of Al-Zn-Mg alloy, dynamic recovery (DRV), dynamic recrystallization (DRX) and dynamic precipitation (DPN) influence each other and lead to changes in the microstructure . Xu obse−1 to 10 s−1 and temperatures ranging from 350 to 500 °C was studied through isothermal compression experiments. The hot deformation constitutive model was established, and the steady-state plastic working window was obtained by processing maps. The precipitation of the η phase and Al3 phase and its effect on the grain and subgrain microstructure of Al-Zn-Mg-Er-Zr alloy under different hot deformation conditions were analyzed.Er-Zr microalloying has gradually become an effective way to improve the comprehensive performance of aluminum alloys. However, there has not been sufficient focus dedicated to analyzing the hot deformation behavior of Al-Zn-Mg-Er-Zr alloys. In this study, the hot deformation behavior of Al-Zn-Mg-Er-Zr alloy at strain rates ranging from 0.01 s−1, respectively, and at the true strain rate of 0.60 using the Gleeble-3500 experimental machine. Finally, the compressed specimens were immediately water-quenched to preserve the microstructure formed during thermal compression.The chemical composition of the experimental Al-Zn-Mg-Er-Zr alloy is shown in 3 and 70% CH3OH at the voltage of ~15 V DC. The deformation microstructure, grain orientation and recrystallization degree of the alloy was performed using the Gemini SEM 300 FEG scanning electron microscope EBSD probe. Samples for TEM analysis were prepared by mechanically polishing the samples to the thickness of approximately 100 μm and subsequently punching them into 3 mm discs, and finally, twin-jet polishing with a solution of 30% nitric acid and 70% methanol electrolyte at a ~15 V DC voltage and a temperature below −25 °C was performed.The samples for microscopic characterization were sectioned parallel to the compression axis of the deformed cylindrical specimens. Then, the sections were mechanically polished and electropolished for 15 s in the mixed acid solution of 30% HNO8Cu4Er phase and AlZnMg phases containing Er. There were a large number of precipitated phases with a size of about 25 nm in the grain, as shown in 3 particles with an L12 lattice structure.The microstructures of the Al-Zn-Mg-Er-Zr alloy before deformation are shown in σ is the flow stress; Q is the deformation activation energy; R is the universal gas constant (8.314 J mol−1K−1); A, A1, A2, n, n1, α and β are the calculated material constants. The Z parameter, which can be used to express the linear relationship between flow stress and peak stress, is shown as follows [Constitutive modeling was used to estimate the thermal deformation behavior of the alloy and construct the relationship between flow stress, deformation temperature T, strain rate and deformation quantity. The Arrhenius-type equation, which includes the Zener-Hollomon parameter, is generally used to describe the hot deformation behavior of Al-Zn-Mg alloy. There are three different functions that can express the dependence of the flow stress on the deformation temperature and strain rate in high-temperature deformation. The power law equation, as shown in Equation (1), is suitable for low-stress deformation, while the exponential law equation, as shown in Equation (2), is suitable for high-stress deformation, and finally, the hyperbolic-sine-type equation, as shown in Equation (3), is suitable for all stress levels ,27.(1)ε follows :(4)Z=ε˙eσ, 1, was 6.78936. The correlation coefficient was 94.63%, as shown in β (MPa−1), was 0.1057325, and the correlation coefficient was 92.72%. The parameter α can be defined as α = β/n1, α = 0.0154260 MPa−1. The average value of the slopes in Q/nR, was 3.89649, and the correlation coefficient was 96.00%. The calculated activation energy Q was 160.03 kJ·mol−1. Deformation activation energy Q is a critical physical parameter for measuring the degree of deformation difficulty in hot deformation. Under the same deformation condition, flow stress σ decreased with the decrease in deformation activation energy Q, so the alloy with a lower Q value was deformed more easily with a lower force, as shown in Equation (3). The self-diffusion activation energy of pure aluminum is 142 kJ·mol−1. In the medium-strength weldable Al-Zn-Mg alloys with a low copper content, the deformation activation energy Q of the studied alloy was higher than that of the 7N01 alloy (152 kJ·mol−1), as reported by Liu et al. [−1), as alloy reported by Li et al. [Q of the studied alloy was lower than that of the 7075 (269.04 kJ·mol−1) [−1) alloy [Taking the natural logarithm on both sides of Equations (1)–(3), the linear relationship between u et al. , but lowi et al.  ComparedJ·mol−1)  alloy anTaking the logarithm of both sides of Equation (4), the following equation can be obtained:Z and Z parameter decreased with the increase in deformation temperature and the decrease in strain rate.The relationship between ln A and n is the intercept and the slope in the A and n could be obtained as 25.53028 and 4.79425. Thus, the relationship between flow stress and the Z parameter is as follows:The value of ln G) and some for structural deformation (J), as follows:G to J is determined by the strain rate sensitivity factor m:Prassad ,34 propoη:The power dissipative efficiency and instability coefficient are defined by the matrix that formed according to the deformation temperature T and strain rate The power dissipation efficiency represents the ratio of the energy consumed by the microstructure change to the energy consumed by the ideal linear dissipation state. Generally, the higher the power dissipative efficiency is, the better the deformation performance will be. In addition, when the instability coefficient formance .−1 and 10 s−1, the power dissipative efficiency increased with the increase in deformation temperature. This is because high temperatures enhance the effect of thermal activation and promote microstructure evolution such as DRV and DRX during the hot deformation of the studied alloy. When the strain was 0.1, the experimental alloy had three high dissipation efficiency regions, as shown in −1 and 450–500 °C; (2) 0.01–0.05 s−1 and 430–480 °C; (3) 0.01–0.03 s−1 and 350–375 °C. The dissipation efficiency of these three regions exceeded 30%. With the increase in strain, the DOM#2 region gradually decreased and then disappeared when the strain reached 0.3. The DOM#3 region also gradually decreased until it disappeared when the strain reached 0.5. However, the temperature range suitable for processing in the DOM#1 region expanded with the increase in strain. In general, with the increase in strain, the area of high dissipation efficiency of the investigated Al-Zn-Mg-Er-Zr alloy gradually decreased. When the strain reached 0.6, only DOM#1 had high dissipation efficiency (0.1~1 s−1 and 425~500 °C), as shown in −1 and 1 s−1.By superimposing the processing instability diagram onto the power dissipation efficiency diagram of the experimental alloy, we can obtain the thermal processing diagram of the experimental alloy, as shown in In order to explore the microstructure evolution of experimental alloy during thermal processing, microstructure analysis and discussion were carried out on the deformation samples under different processing conditions in the thermal processing diagram when the deformation was 0.6. It can be seen from −1, the deformed structure in the alloy accounted for 18.93%, so the power dissipation efficiency was low at 18.3%. When the strain rate was 0.1 s−1, the proportion of deformed structure of the alloy gradually decreased to 10.84%, the dynamic recovery of the alloy was relatively complete, and the power dissipation efficiency was 40.8%. When the strain rate was increased to 1 s−1, a large number of recrystallized grains of a smaller size appeared at the subgrain boundary, but the proportion of deformed structure increased to 19.88%, so the power dissipation efficiency decreased to 35.6%. When the strain rate rose to 10 s−1, because the deformation time was too short, the dislocation did not decrease or increase quickly enough to cut elongated deformation grains to form polymorphic subgrain boundaries. So, a large number of deformed structures were preserved inside the alloy, accounting for 47.01%, and the power dissipation efficiency was 3.0% at this time. During the thermal deformation of the alloy, the energy provided by the external environment for the deformation of the alloy structure was partly used for the dynamic recovery and recrystallization, and partly for the nucleation and growth of the precipitated phase in the alloy. When the strain rate was low, the alloy had enough deformation time to complete the dynamic recovery, but at the same time, the precipitate had enough time for nucleation growth to hinder the dynamic recovery and recrystallization of the alloy, so the power dissipation efficiency of the experimental alloy was low at the low strain rate. When the strain rate was too high, the deformation time of the alloy was too short, leading to a short dislocation migration time, and thus the dynamic recovery of the alloy was not complete. In this case, the power dissipation efficiency was also very low, so the alloy remained in the processing instability region. Therefore, when the strain rate was too high or too low, it was not suitable for thermal processing. In summary, the experimental alloy thermal processing range is between a deformation temperature of 450 and 500 °C and a strain rate of 0.1~1 s−1.η phase with a size between 50 and 200 nm, which could easily change with the change in deformation parameters. The other was spherical Al3 particles with sizes ranging from 10 nm to 20 nm, which had good thermal stability. During thermal compression, the evolution of the microstructure of these particles played an important role. The microstructures of samples deformed at 450 °C and 0.1 s−1 are shown in η phases, and the dislocation wall formed in the subgrain boundaries. In the dislocation wall, as shown in η phase with a size of 80~150 nm and the Al3 particles with a size of 10~20 nm both played a role in pinning the dislocation movement, and there were still large areas of wrought structure in the interior of the subgrain, which is consistent with the results of EBSD, as shown in 3 particles. The number density of Al3 particles in the deformed alloys was obviously higher than that in the undeformed alloys, indicating that the dynamic precipitation of Al3 particles also occurs during hot deformation.As shown in −1, as shown in 3 particles were distributed more uniformly, and the microstructure showed characteristics typical of dynamic recovery. When the deformation temperature rose, the η phase became scattered and its size increased, and the subgrain boundary became more distinct, as shown in η phase dynamically coarsened, and its density decreased. The dislocation locking of particles was weakened, and the dislocation walls gradually transformed into subgrain boundaries. When the temperature rose to 500 °C, the η phase did not precipitate any longer.TEM images of the Al-Zn-Mg-Er-Zr alloy deformed under different conditions are shown in 3 particles were distributed in two ways: one is random distribution, and the other is chain arrangement. The random distribution of Al3 particles was dominant at low deformation temperatures, as shown in 3 particles began to form chains. With the increase in strain rate, the number density of Al3 particles increased, and the chain arrangement became more dominant. This is perhaps because the driving force of precipitation of Er and Zr in α-Al increases exponentially with the increase in the diffusion rate, and these dislocations or substructures provide higher diffusion rates for Er and Zr, and so Al3 nucleates predominately on the dislocations or substructures. Thus, Al3 particles exhibit chain arrangement. It has also been mentioned in previous studies [3 precipitation distribution observed in 3-precipitation-induced chain arrangements.Al studies ,37,38,39η phase also had the effect of pinning dislocation. However, when the temperature was higher than 500 °C, a large number of η phases dissolved or coarsened, and their density decreased, and so dislocation locking of the η phase reduced significantly. The diffusivity and the solid solubility of Er and Zr in an aluminum matrix are low, and the growth and coarsening of Al3 particles are rare, which is correlated with the thermal stability of precipitated phases. Therefore, the deformation parameters had little effect on the size of Al3 particles; the dislocation locking effect of Al3 particles existed under various deformation conditions and was not reduced with the rise in temperature. So, at higher deformation temperatures, Al3 particles still pin dislocations and thus refine subgrain and enhance the strength. Conclusively, compared with the η phase, Al3 particles are superior for dislocation locking.When the deformation temperature was below 450 °C, the (1)The steady-state flow stress increases with the increase in strain rate or the decrease in deformation temperature, and can be described with the hyperbolic sinusoidal constitutive equation with a deformation activation energy of 160.03 kJ/mol.(2)−1 and a deformation temperature ranging from 450 to 500 °C form the safest hot working domain. The microstructural characteristics of the alloy in the safe working domain were predominantly dynamic recovery, accompanied by a small number of recrystallized microstructures.According to the processing maps and EBSD results, when the deformation temperature was 350 °C, the alloy was in the processing instability domain, and the wrought structure dominated in the studied alloy. With the increase in deformation temperature, sufficient driving force was provided for the studied alloy, dislocation density decreased, the subgrain structure was dominant, and the main softening mechanism of the alloy was dynamic recovery. When the strain rate was too high, the deformation time interval of the alloy was too short, leading to a short dislocation migration time, and thus the dynamic recovery of the alloy was not complete. In this case, the power dissipation efficiency was also very low, so the alloy processing was in the processing instability domain. Therefore, a strain rate that is too high or too low is not suitable for hot deformation. In summary, a strain rate ranging from 0.1 to 1 s(3)η phase, whose size and density changed significantly with the variation in deformation conditions, and the other was the spherical Al3 particles, which had good thermal stability. Both kinds of particles played a role in dislocation locking. When the strain rate was decreased or the deformation temperature was increased, the η phase coarsened dynamically and its density decreased, and so its dislocation locking ability was weakened.The investigated alloy contained two kinds of second-phase particles during hot deformation. One was the (4)3 particles are distributed in two ways: one is random distribution, and the other is chain arrangement. The random distribution of Al3 particles was dominant at low deformation temperatures, but when the deformation temperature reached 500 °C, some Al3 particles began to show chain arrangement. When the deformation temperature was increased, the number density of Al3 particles increased, and the chain arrangement structure became more dominant. However, the deformation conditions had little effect on the size of Al3 particles. So, compared with the η phase, Al3 particles are superior for dislocation locking during hot deformation.AlIn the current work, the grain microstructure and precipitate evolution in Al-Zn-Mg-Er-Zr alloy during hot deformation was investigated. Based on the experimental data, the constitutive equation and the thermal processing maps were established, and the effects of hot deformation conditions on the microstructure  of the alloy were analyzed. The microstructure characteristics of the alloys in the steady deformation zone and unstable deformation zone in the thermal processing maps were also analyzed. The conclusions are as follows: