SbFn) through the use of a pull-down assay. Our findings revealed the presence of an insulin-like growth factor type 1 receptor (IGF-1R) in ark clams, which was capable of binding to SbFn and was named SbIGF-1R. SbIGF-1R was found to be composed of two leucine-rich repeat domains (L domain), a cysteine-rich domain, three fibronectin type III domains, a transmembrane domain, and a tyrosine kinase domain. The ectodomain of SbIGF-1R was observed to form a symmetrical antiparallel homodimer in the shape of the letter ‘A’, with the fibronectin type III domains serving as its ‘legs’. The mRNA expression of SbIGF-1R gene was detected ubiquitously in various tissues of the ark clam, with the highest expression levels found in hemocytes, as determined by qRT-PCR. Using a confocal microscopic and yeast two-hybrid assays, the interaction between SbIGF-1R and SbFn was further verified. The results showed that SbFn co-localized with SbIGF-1R on the cell membrane, and their interaction was expected to occur on the FNIII domains of the SbIGF-1R. In conclusion, our findings highlight the identification of a putative receptor, SbIGF-1R, for SbFn, demonstrating the versatility of IGF-1R in ark clams.The ferritin secreted by mammals has been well documented, with the protein capable of localizing to cell membranes and facilitating the delivery of iron to cells through endocytosis. However, the presence of ferritin in the circulatory fluid of mollusks and its functions remain largely unknown. In this study, we aimed to investigate the potential interacting proteins of ferritin in the ark clam ( Iron is an essential catalytic cofactor of numerous enzymatic reactions that participate in fundamental cellular metabolic processes, such as energy generation, protein synthesis, and nucleic acid synthesis ,2. NeverFerritin features a nanocage that can store roughly 4500 iron atoms in a bioavailable state that serves as a buffer for reactive oxygen species inside cells . In vertDrosophila melanogaster’s secretory ferritin transports iron across the basolateral membrane of enterocytes, contributing to dietary iron delivery [Scapharca broughtonii, was discovered to mirror iron distribution [As a highly conserved iron storage molecule in nature, ferritin exists in a variety of species, from mammals to archaea . The levdelivery . Accumulribution ,18. Consribution ,20.Mactra heinesis, surf clam Spisula sachalinensis, yesso scallop Patinopecten yessoensis, pacific oyster Crassostrea gigas, and mussel Mytilus galloprovincialis [The insulin-like growth factor (IGF) system plays a crucial role in the growth, reproduction, and development of aquatic animals, and has been identified as a useful biomarker for evaluating intra-species growth differences in aquaculture ,22. Reseincialis ,25,26,27incialis . It is cincialis ,30,31.SbFn) by pulldown assay, named SbIGF-1R, and its mRNA expression pattern was detected. The interaction between SbIGF-1R and SbFn was further verified by confocal microscopic and yeast two-hybrid assays. Finally, a putative membrane anchor, an insulin-like growth factor type 1 receptor, for ferritin in mollusks was revealed, implying an important role of ferritin in iron delivery among cells.In the current study, a tyrosine kinase receptor member gene was captured as a potential interacting protein of ferritin in ark clams . The sequence is available in GenBank with the accession number OM055760. The precursor protein includes a conserved furin recognition motif RXXR located at residues 767–773, flanked by α chain and a β chain (SbIGF-1R α chain was found to be composed of two receptor L domains (L1 and L2), a cysteine-rich domain (CR), a fibronectin type III domain-1 (F1), and a portion of fibronectin type III domain-2 (F2). A portion of F2, fibronectin type III domain-3 (F3), the transmembrane domain (TM), and the tyrosine kinase domain (KD) were found in the β chain  of the full-length  β chain A. Accord β chain A.SbIGF-1R ECD was calculated using the I-TASSER online server, which was selected from five predicted models based on the C-score (−0.63) with a TM-score of 0.63 ± 0.13 and an RMSD of 10.4 ± 4.6 Å  at positions 1052–1057. A cluster of three tyrosine residues located within the activation loop of 1197–1222, which activates the kinase by allowing ATP and substrate to enter the active site, is essential for receptor autophosphorylation. In addition, the intracellular juxta-membrane domain contains a conserved NPXY motif, which may be critical for the internalization of the receptor , 52.97% to Azumapecten farreri (QFR39799.1), 42.19% to Homo sapiens (P08069.1), and 39.58% to Danio rerio (NP_694501.1) (SbIGF-1R is more closely related to IGF-1R proteins found in invertebrates than to those found in higher organisms.94501.1) . The IGF94501.1) . As a reSbIGF-1R gene was shown to be constitutively expressed with varying degrees throughout a wide range of tissues by qRT-PCR assay, including the hepatopancreas, hemocytes, adductor muscle, foot, gills, and mantle. The hemocytes had the greatest mRNA expression level , followed by the adductor muscle , the foot , and the gill  (The mRNA of  < 0.01) .SbFn and SbIGF-1R was further explored by confocal microscopic and yeast two-hybrid assays. Polyclonal antibodies against the SbFn and SbIGF-1R-FNIII were prepared, and their specificity was identified by Western blot (SbFns (red) were co-localized with SbIGF-1Rs (green) on the surface of the ark clam hemocytes  were obtained from a local farm in Yantai, China. Before treatment, these ark clams were acclimatized in aerated seawater at 18 °C for 1 week. The hemocytes from the ark clams were collected and centrifuged at 800× S.broughtonii ferritin  was amplified by PCR with the primers listed in BamHI and XhoI and ligated into the pET-30 (a) + vector . The His-fusion protein was overproduced in the Escherichia coli Transetta (DE3)  under the induction of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 6 h, then was purified using Ni-Charged Resin  according to the manufacturer’s protocol. To remove excess salt ions, the purified protein was dialyzed three times in PBS and concentrated using Millipore ultrafiltration tubes.The sequence encoding TM His Protein Interaction Kit  to identify the SbFn-interacting proteins. The input hemocyte proteins were prepared with a cell lysis buffer supplemented with EDTA-free protease inhibitors . HisPur Cobalt Resin was incubated with recombinant SbFn (rSbFn) and an equal amount of Hexa-His (GenScript), respectively, for 1 h at 4 °C with gentle rocking. After 5 washes with a wash solution containing 10 mM imidazole (GenScript), the prepared rSbFn-conjugated beads were incubated with the hemocyte proteins at 4 °C overnight with gentle rocking. Meanwhile, Hexa-His-conjugated beads were incubated with an equal volume of cell lysate as a negative control. These beads were collected by centrifugation after 5 subsequent washes. The bound proteins were finally eluted with a wash solution containing 300 mM imidazole (GenScript) and separated by gradient SDS-PAGE gels at concentrations of 8–16% (GenScript). After staining with Coomassie blue, the visual intensities of the differential protein bands between the experimental and control groups were analyzed using Image J software, and bands with ≥2-fold higher abundance (p < 0.05) relative to the control group were excised for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The LC-MS/MS analysis was performed at Guangzhou Gene Denovo Biotechnology Co., Ltd. . Briefly, the excised bands were subjected to protease degradation. The peptide samples were then separated by nanoliter liquid chromatography and detected by mass spectrometer LTQ Orbitrap Velos .A pull-down assay was performed using the PierceSbIGF-1R was obtained from the S. broughtonii genome. The specific primers , ligated into the pMD 19-T vector , and transformed into E.coli for sequencing confirmation at Shanghai Biotech . The Expasy server  was used to infer the amino acid sequence. The protein-conserved domains were identified using the SMART program  and the NCBI Conserved Domain Database . The BLASTP program  was used to analyze the homolog. The Clustal 1.81 software was used to perform the sequence alignment of IGF-1Rs, and the results were generated in the online platform . A phylogenetic tree of IGF-1Rs was constructed based on the full-length amino acid sequences by the neighbor-joining algorithm using Mega 6.0. The tertiary structures of SbIGF-1R ectodomain (ECD) were calculated using I-TASSER  and further refined by using the GalaxyRefine online server . In addition, the geometric quality of the SbIGF-1R ECD tertiary structure was validated based on Ramachandran plots and ERRAT quality factors from the Structural Analysis Validation Server , and Z-scores from the ProSA server . The validated SbIGF-1R ECD monomers were submitted to the template-free docking server ZDOCK  to predict a homodimer model for SbIGF-1R ECD. Finally, the predicted homodimer model of SbIGF-1R ECD was analyzed using PISA  to obtain the energy of the interaction.The total RNA was extracted from the hemocytes with a TRIzol reagent , and the first-strand cDNAs were reverse-transcribed by M-MLV reverse transcriptase  according to the manufacturer’s instructions. The sequence encoding the complete open reading frame of  primers  were desSbIGF-1R transcripts, with a 60S ribosomal protein subunit (RL15) as the reference gene [-ΔΔCt method was adopted to calculate the mRNA relative expression levels [The total RNA samples were extracted from the hemocytes, gills, mantle, hepatopancreas, adductor muscle, and foot of three ark clam individuals. A cDNA library was synthesized by ReverTra Ace qPCR RT Master Mix with gDNA Remover . A real-time PCR experiment was performed with a Bio-Rad CFX Connect RealTime system to detect the distribution of nce gene . The prin levels .SbIGF-1R fibronectin type III domains was amplified using gene-specific primers (E. coli Transetta (DE3) (TransGen). The recombinant fibronectin type III domains of SbIGF-1R protein (rSbIGF-1R-FNIII) were induced by 0.2 mM IPTG for 6 h at 37 °C and purified using Ni-Charged Resin (GenScript). A mixture containing 100 μg rSbIGF-1R-FNIII was used to immunize 4-week-old mice for the polyclonal antibody preparation, while the SbFn antiserum was prepared by immunizing a 6-month-old rabbit.The fragment encoding the  primers  and was SbIGF-1R-FNIII and SbFn polyclonal antibodies were identified using Western blot. The purified protein samples were separated using 12% SDS-PAGE and transferred onto nitrocellulose (NC) membranes. After blocking with 5% skimmed milk powder, the membranes were incubated overnight at 4 °C with SbFn polyclonal antibody (1:500) or SbIGF-1R-FNIII (1:500) polyclonal antibody. The membranes were then incubated with HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies  for 1 h at room temperature with shaking. Between each step, the NC membranes were washed thrice for 5 min with TBST. Protein bands were visualized in an automated chemiluminescent gel imaging system  using Western lighting ECL substrate .The specificity of the SbFn (100 μg/mL) was added and incubated for 2 h at 18 °C. Following two washes with sterile seawater, the hemocytes were fixed with 4 % paraformaldehyde for 30 min and blocked with 5% bovine serum albumin for 30 min at room temperature. Then the hemocytes were incubated with rabbit anti-SbFn polyclonal antibodies and mouse anti-SbIGF-1R-FNIII polyclonal antibodies at 4 °C overnight. After thoroughly washing, the hemocytes went through another 2 h incubation with Cy3-coupled goat anti-rabbit IgG (H + L) (AS007) and FITC-coupled goat anti-mouse IgG (H + L) (AS001) antibodies (ABclone). Finally, fluorescence was observed with DAPI staining to visualize the nuclei under a Nikon Eclipse Ti confocal microscope .Hemocytes were sampled from healthy ark clams and then deposited on an attached slide for 1 h. After washing with sterile seawater to remove unadhered hemocytes, SbFn into the pGADT7 vector. Subsequently, we used the Matchmaker® Gold Yeast Two-Hybrid System  to execute a Y2H screening. In brief, the bait plasmid (FnⅢ-α/β-BD) and prey plasmid (Fn-AD) were transferred into yeast strains Y2HGold and Y187, respectively. Meanwhile, the blank pGBKT7 (CK-BD) and pGADT7 (CK-AD) vectors were treated identically as negative controls to exclude false positives. The experimental groups consisted of the Y2Hgold yeast strain transfected with FnIII-α/β-BD hybridized with the Y187 yeast strain transfected with Fn-AD, respectively. The Y2Hgold yeast strain transfected with CK-BD was hybridized with the Y187 yeast strain transfected with CK-AD to serve as a negative control. For the self-activation assay, the Y2Hgold yeast strain transfected with FnIII-α/β-BD was hybridized with the Y187 yeast strain transfected with CK-AD and the Y187 yeast strain transfected with Fn-AD was hybridized with the Y2Hgold yeast strain transfected with CK-BD. After hybridization, the cultured yeasts were diluted 10, 100, and 1000-fold with sterile water and inoculated on duplex dropout medium (SD-Leu-Trp) and quadruple dropout medium supplemented with X-a-Gal and Aba . Successful interactions were dependent on the presence of blue cells on the medium.To perform the yeast two-hybridization (Y2H) assay, we constructed the bait plasmid pGBKT7-α chain FnⅢ and pGBKT7-β chain FnⅢ as baits, respectively. For the prey plasmid construction, we cloned the full-length cDNA of p < 0.05, ** p < 0.01.All the experiments were repeated independently three times, and the data are presented as mean ± SD. Statistical analysis of the data was carried out using the Statistical Package for Social Sciences (SPSS) 21.0. The differences between the groups were assessed using one-way ANOVA. The differences are considered significant at *


Brain sections of the anterior cingulate  and corpus callosum  from cognitively normal control  and AD (n = 6) subjects were imaged using autoradiography and immunostaining. Using competition with clorgyline and (R)-deprenyl, the binding of [18F]FAZIN3 was confirmed to be selective to MAO-A levels in the AD brain sections. Increases in MAO-A, Aβ plaque, tau, and TSPO activity were found in the AD brains compared to the control brains. The [18F]FAZIN3 ratio in AD GM versus CN GM was 2.80, suggesting a 180% increase in MAO-A activity. Using GM-to-WM ratios of AD versus CN, a >50% increase in MAO-A activity was observed (AD/CN = 1.58). Linear positive correlations of [18F]FAZIN3 with [18F]flotaza, [125I]IBETA, and [125I]IPPI were measured and suggested an increase in MAO-A activity with increases in Aβ plaques and tau activity. Our results support the finding that MAO-A activity is elevated in the anterior cingulate cortex in AD and thus may provide a new biomarker for AD in this brain region.Increased monoamine oxidase-A (MAO-A) activity in Alzheimer’s disease (AD) may be detrimental to the point of neurodegeneration. To assess MAO-A activity in AD, we compared four biomarkers, Aβ plaques, tau, translocator protein (TSPO), and MAO-A in postmortem AD. Radiotracers were [ Our ong]flotaza , tau ima25I]IPPI , and TSP8F]FEPPA  with [1818F]FEH; ,29) for 18F]FEH; , and thi18F]FEH;  and tau 18F]FEH;  in AD pa18F]FEH; . Because18F]FEH; , this br18F]FAZIN3 is a polyethyleneglycol (PEG) fluoroalkyl azaindole derivative and binds reversibly and selectively to MAO-A [18F]FAZIN3 was distinctly seen in the anterior cingulate in both AD subjects . When comparing the GM/WM ratios of AD (3.43) to those of CN (1.58), a 58% increase in [18F]FAZIN3 binding was observed, suggesting an increase in MAO-A activity.The average ratio of GM/WM in the six AD subjects was 3.43. Two CN subjects shown in n the WM F,H. Thisn the WM E,F exhibn the WM , and thi5I]IBETA . The bin18F]FAZIN3 binding compared to other CN subjects, which may be due to the presence of abundant Aβ plaques -deprenyl on [18F]FAZIN3 binding, suggesting [18F]FAZIN3 as a selective MAO-A radiotracer. 18F]FAZIN3 binding. All CN subjects exhibited similar drug effects, further confirming that the observed binding of [18F]FAZIN3 is to MAO-A. This MAO-A selectivity of [18F]FAZIN3 was also reported previously Flotaza is a new fluorinated PEG derivative for Aβ plaque PET imaging and provides high-contrast imaging of postmortem AD brains [18F]flotaza binding in the anterior cingulate regions. 18F]flotaza corresponding to anti-Aβ  of the GM binding of [125I]IPPI and [18F]FAZIN3 was found in all AD subjects but was not significant . Becausn the GM G compareCN 12-21 H.18F]flotaza . A stronger MAO-A-to-tau linear regression was seen with the GM/WM ratios of [125I]IPPI , whereas [18F]FAZIN3 versus [125I]IPPI and [18F]FEPPA were not significant.The correlation of the GM/WM binding ratios of the four individual biomarkers in the 6 AD subjects and one CN subject is shown in ]flotaza A and [125I]IBETA B and was25I]IPPI C, compar25I]IPPI D. Howevenegative D. When A18F]flotaza and [125I]IBETA, with GM/WM ratios >15 at Braak stage VI. Similarly, increases in tau were seen in Braak stages V and VI with [125I]IPPI GM/WM ratios >5 in Braak stage VI. There was greater variability in the presence of tau compared to that of the presence Aβ within each of these advanced stages. In the case of [18F]FAZIN3, there was a gradual increase in the GM/WM ratios, with the highest being in Braak stage VI. [18F]FEPPA exhibited the least change in its GM/WM ratio at the different stages.Shown in 11C]harmine in the anterior cingulate and temporal cortex [18F]FAZIN3 found significant increases in MAO-A binding [11C]harmine confirmed significant levels of MAO-A in the anterior cingulate of healthy human subjects [11C]harmine binding to MAO-A were found in the anterior cingulate in major depression FAZIN3 in AD subjects was observed when comparing ratios of GM/WM in AD and CN subjects. This increase in [18F]FAZIN3 binding was very significant and greater than that reported in depressed patients [18F]FAZIN3 in AD subjects  of [125I]IPPI with [18F]FAZIN3, but it was not significant. For extended PET imaging, iodine-124 IPPI may serve as a PET imaging for tau (iodine 124 T1/2 = 4.2 days), similar to our efforts on extended imaging with iodine-124-labeled epidepride and Aβ plaques with [124I]IBETA and [124I]IAZA [The presence and spread of NFT in AD is currently being actively pursued as a more accurate biomarker for the staging of the disease . All AD 24I]IAZA ,38,46,4718F]FEPPA, which has a high affinity for TSPO, has shown promise in PET studies of AD [18F]FEPPA binding compared to the CN subjects, there was a low correlation with [18F]FAZIN3 binding. In contrast, for MAO-B, there appeared to be a better correlation between deprenyl and PK11195 [18F]FAZIN3 binding to MAO-A with [18F]FEPPA binding to TSPO. However, these findings are preliminary, and a larger study will be needed to ascertain this. Additional biomarkers for neuroinflammation will have to be explored for potential correlations with changes in MAO-A in AD FAZIN3 may be compared.This is an initial imaging study demonstrating that levels of MAO-A in the AD brain are altered. Limitations of the study include the small number of subjects in advanced stages of AD. A larger study with more subjects at different disease stages is needed to ascertain the correlation of MAO-A changes with the progression of disease. Our study here reports only one brain region; other brain regions, such as the temporal cortex and hippocampus, need to be studied to assess MAO-A changes. It must also be noted that depressive behaviors are often present in AD , althougR)-deprenyl were purchased from Research Biochemicals . FEPPA tosylate precursor and PIB (Pittsburgh compound B) were purchased from ABX Inc., Radeberg, Germany. Iodine-125 sodium iodide was purchased from American Radiolabeled Chemicals, Inc., St. Louis, MO, USA; iodine-124 sodium iodide was purchased from 3D Imaging, Little Rock, AR; and fluorine-18 was purchased from PETNET, Inc. Specialty chemicals MK-6240 and PK 11195 were purchased from AbaChemScene, New Jersey; and clorgyline and , Sun City, AZ, USA, brain tissue repository for in vitro experiments. Well-characterized frozen brain samples were obtained from BHRI, Sun City Arizona (2 (DLU/mm2) were used to quantify the extent of binding. Slides were scanned on a Hewlett Packard scanner to help delineate regions of the brain sections more clearly. At least n = 3 to 6 tissue sections per subject, per radiotracer were used for the study.Brain slices were placed in separate incubation chambers and were allowed to thaw from −80 °C to ambient temperature for 10–15 min. Subsequently, they were preincubated in PBS (pH 7.4) or Tris buffer (pH 7.4) at ambient temperature for 10 min. Fresh PBS buffer (pH 7.4) or Tris buffer (pH 7.4) containing the respective radiotracer was added to all the chambers and incubated for 60–90 min. The brain sections were air-dried, exposed  on phosphor screens, and then placed on the Phosphor Autoradiographic Imaging System . Using the Optiquant acquisition and analysis program , regions of interest were drawn in the gray matter regions of the anterior cingulate and white matter regions of the corpus callosum. Digital light units/mmThe azaindole derivative [18F]FAZIN3 was prepared in-house, as described previously . Human b[18F]Fluoroethyl harmol ([18F]FEH) was produced in-house, as reported ,29. [18FPurified [18F]flotaza and 125I]IBETA were used for autoradiographic studies IIBETA we.For tau imaging, [125I]IPPI was used for autoradiographic studies . Human bThe TSPO PET probe [18F]FEPPA, with >95% radiochemical purity and a specific activity of >70 GBq/μmol (>2 Ci/μmol), was used for autoradiographic studies . Human bImmunostaining of all brain sections was carried out by University of California-Irvine, Pathology services using Ventana BenchMark Ultra protocols. Neighboring slices were immunostained with DAKO polyclonal total tau antibody which detects all 6 six isoforms of tau, dilution 1: 3000, A0024  using reported protocols . For Aβ 18F]FAZIN3, [18F]flotaza, [125I]IPPI, [18F]FEPPA, and [18F]FEH were quantified using measurements (DLU/mm2). Immunostained sections were analyzed using QuPath. Gray matter (GM) and WM binding of each radiotracer in the AD and CN subjects were measured, and the GM/WM ratios of the AD and CN subjects were compared for each radiotracer. Using the ratio method is akin to in vivo PET methods, where the standard uptake value (SUV) between the target region is compared to a nonspecific binding reference region as a ratio and expressed as SUVR FAZIN3 was correlated to each specific binding of [18F]flotaza, [125I]IPPI, and [18F]FEPPA in order to assess any relationship between the different biomarkers.All regions of interest (ROI) in the anterior cingulate (GM) and corpus callosum (WM) autoradiographic images of [ as SUVR ,59. Grout-test, and p values of <0.05 were considered to indicate statistical significance. Spearman’s correlation was carried out in certain cases. The linear correlations and ANOVA analysis of the bindings between the different radiotracers were used to evaluate potential relationships between the different biomarkers.Group differences between AD and CN subjects were assessed using the average GM/WM ratios and were determined using Microsoft Excel 16 and GraphPad Prism 9. The statistical power was determined with Student’s Four different biomarkers, which included Aβ plaques, tau, the translocator protein for microglia, and MAO-A, were used to study the anterior cingulate cortex of well-characterized AD subjects. Our results showed that significantly greater MAO-A activity was present in the anterior cingulate of AD subjects compared to the cognitively normal controls. This increased MAO-A in AD subjects was positively correlated to Aβ plaque and tau levels. Early assessment of increased MAO-A levels using PET imaging in AD patients may provide opportunities for therapeutic interventions by MAO drugs . This ma


HLA-G gene presents several functional polymorphisms distributed across the coding and regulatory regions (5′URR: 5′ upstream regulatory region and 3′UTR: 3′ untranslated region) and some of them may impact HLA-G expression and human malignancy. To understand the contribution of the HLA-G genetic background in PTC, we studied the HLA-G gene variability in PTC patients in association with tumor morbidity, HLA-G tissue expression, and plasma soluble (sHLA-G) levels. We evaluated 185 PTC patients and 154 healthy controls. Polymorphic sites defining coding, regulatory and extended haplotypes were characterized by sequencing analyses. HLA-G tissue expression and plasma soluble HLA-G levels were evaluated by immunohistochemistry and ELISA, respectively. Compared to the controls, the G0104a(5′URR)G*01:04:04(coding)UTR-03(3’UTR) extended haplotype was underrepresented in the PTC patients, while G0104a(5′URR)G*01:04:01(coding)UTR-03(3′UTR) was less frequent in patients with metastatic and multifocal tumors. Decreased HLA-G tissue expression and undetectable plasma sHLA-G were associated with the G010102a(5′URR)G*01:01:02:01(coding)UTR-02(3′UTR) extended haplotype. We concluded that the HLA-G variability was associated with PTC development and morbidity, as well as the magnitude of the encoded protein expression at local and systemic levels.Human leukocyte antigen (HLA)-G is an immune checkpoint molecule that is highly expressed in papillary thyroid carcinoma (PTC). The  Thyroid cancer (TC) is the most common endocrine malignancy, and its incidence has markedly increased during the past few decades . DiffereOver the past two decades, considerable progress has been made in the understanding of the molecular mechanisms associated with DTC development. The elucidation of genetic and epigenetic events affecting key signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway, and the role of these mediators in thyroid carcinogenesis has brought beneficial clinical management to DTC patients . More re+ T and natural killer (NK) cells and inhibit antigen presentation, lymphocyte proliferation, and antibody formation [Human leucocyte antigen-G (HLA-G) is a unique non-classical immunomodulatory major histocompatibility complex (MHC) class Ib molecule  that is ormation ,16. Alteormation .HLA-G from classical class I genes is the limited variability at the coding region. The overall structure of the HLA-G alleles has been maintained during the evolution process, in which most of its variable sites are synonymous mutations or are observed in introns [HLA-G regulatory regions (5′ upstream regulatory region: 5′URR and 3′ untranslated region: 3′UTR) may potentially influence the targeting of transcriptional and post-transcriptional elements, affecting the HLA-G gene and protein expression [HLA-G gene have been reported in pre-eclampsia [HLA-G gene variability potentially affected the magnitude of HLA-G expression [The main feature that distinguishes  introns . Current introns ,20,21. N introns . On the pression ,23. Accoclampsia , cancer clampsia ,26, tranclampsia , and autclampsia ,29, chroclampsia , and infclampsia ,32. In spression ,34.HLA-G gene has several nucleotide variation sites at the regulatory regions that have been associated with the magnitude of molecule production [HLA-G variation sites in PTC has mostly focused at the 3′UTR of the gene [HLA-G gene variability  in PTC patients in association with the clinical, histopathological, and tumor features and with the HLA-G protein expression profile. We identified HLA-G-specific regions and/or extended haplotypes associated with: (i) PTC histological and prognosis features, (ii) HLA-G expression in tumor specimens, and (iii) soluble plasma HLA-G detection before thyroidectomy.Considering that (i) HLA-G is a well-recognized immune checkpoint molecule , (ii) HLoduction , and  (p < 0.05), indicating that the alleles do not segregate randomly, justifying the haplotype inferences. Association analyses were conducted considering the three major HLA-G gene segments, taken individually  (For d 3’UTR) , and as d 3’UTR) .HLA-G 5′URR, 19 different haplotypes were found, 14 in PTC patients and 17 in the controls. The G010102a haplotype was the most frequent (32.95%), followed by G010101a (21.15%) in the total population. Compared to the control group, the frequency of the G0104a haplotype was underrepresented in PTC patients (odds ratio (OR) = 0.5273, 95% confidence interval (95% CI) = 0.3221–0.8632, p = 0.0109).Considering the HLA-G coding region haplotypes were observed for the whole group of individuals, of which 17 were observed among the PTC patients and 20 were observed among the controls. In total, the G*01:01:01:01 haplotype was the most frequent (20.66%), followed by G*01:01:02:01 (17.21%). PTC patients presented a lower frequency of the G*01:04:04 haplotype when compared to the controls .Twenty-three different HLA-G 3′UTR haplotypes were identified, 10 in the PTC group and 13 in the control group, of which UTR-02 and UTR-01 haplotypes were the most frequent in the whole group . In the PTC group, the UTR-01 haplotype was overrepresented (19.48%) , whereas the UTR-03 haplotype was underrepresented  when compared to the control group.A total of 14 different HLA-G extended haplotypes, 32 among the PTC patients and 40 among the controls. The G010101a(5′URR)G*01:01:01:01(coding)UTR-01(3’UTR) haplotype was the most frequent (19.51%), followed by G010102a(5′URR)G*01:01:02:01(coding)UTR-02(3’UTR) (16.23%), considering the total studied group. Compared to the controls, the G0104a(5′URR)G*01:04:04(coding)UTR-03(3’UTR) haplotype was underrepresented in the PTC patients .We identified 50 different HLA-G gene variability is associated with histopathological features related to PTC severity and prognosis, we analyzed the whole group of 185 PTC patients from two different geographical regions of Brazil .To assess whether the HLA-G 5′URR, the G0104a haplotype was underrepresented among patients presenting tumor multicentricity  compared to patients without this manifestation. The G*01:01:02:01 and G*01:04:01 coding region haplotypes were more frequently associated with poor incomplete response to therapy . At the HLA-G 3′UTR segment, the frequency of UTR-02 was increased among patients presenting multicentricity , whereas the haplotype UTR-03 was less frequent  in these patients. Finally, the G0104a(5′URR)G*01:04:01(coding)UTR-03(3′UTR) extended haplotype was associated with the absence of multifocal  and metastatic tumors ; i.e., this extended haplotype protected against these features.Considering the HLA-G 3′UTR segment in the same cohort of patients with different thyroidal disorders [V600E and TERT promoter mutations in PTC outcomes [HLA-G gene variability  on the magnitude of the encoded protein expression (n = 170) (We performed three previous studies concerning HLA-G in PTC: (i) a retrospective study evaluating only the HLA-G tissue expression in a different cohort of patients exhibiting several thyroidal disorders, including PTC n = 72) , (ii) a isorders , and finoutcomes , markers2 , (ii) outcomes . In the n = 170) .HLA-G 5′URR G010101c haplotype was overrepresented in tumors presenting high HLA-G expression (>50% of positive tumor cells in PTC specimens) , while the coding region G*01:01:02:01 haplotype was associated with low HLA-G expression (≤50%) . Considering the extended haplotypes, increased HLA-G tumor expression was associated with the G010101c(5′URR)G*01:01:01:05(coding)UTR-04(3′UTR) haplotype , while the G010102a(5′URR)G*01:01:02:01(coding)UTR-02(3′UTR) haplotype was associated with decreased expression . However, we should emphasize that the associations with high HLA-G tissue expression (the G010101c haplotype and the extended G010101c(5′URR)G*01:01:01:05(coding)UTR-04(3′UTR) haplotype) exhibited a very broad CI, including the number 1.The HLA-G gene variability on the sHLA-G plasma detection of PTC patients at the pre-thyroidectomy time (n = 84)  levels in PTC patients before and after thyroidectomy and the reciprocal influence of HLA-G and cytokines  by study(n = 84) .p = 0.0295); however, it is worth mentioning the wide CI of this association, including the number 1. In contrast, the coding region G*01:01:02:01 haplotype , UTR-02  and the G010102a(5′URR)G*01:01:02:01(coding)UTR-02(3′UTR) extended haplotype  were associated with PTC exhibiting no detectable plasma sHLA-G at the pre-thyroidectomy time.The 3′UTR UTR-18 haplotype was overrepresented in PTC patients exhibiting detectable plasma sHLA-G  [HLA-G gene are in LD, a single polymorphism or a specific coding region haplotype may be associated with distinct regulatory region haplotypes [HLA-G gene’s association with human tumors [HLA-G gene regions separately and as extended haplotypes in terms of their association with HLA-G tissue expression, sHLA-G plasma detection, and PTC outcomes.r 3′UTR) . Althougplotypes , indicatn tumors . Indeed,HLA-G gene, respectively [HLA-G coding variability may affect the differential pattern of alternative splicing, leukocyte–receptor interaction, and peptide coupling [The neo-expression of HLA-G in human tissues that constitutively do not express the molecule, as is the case of the thyroid, depends on the combination of factors present in the tumor milieu, such as environmental and hormonal factors, as well as genetic factors, including transcription and post-transcriptional factors . These tectively , yieldinectively . The HLAcoupling ,42. We acoupling ,12, whilcoupling ,43. The HLA-G 3′UTR gene segment is the most studied one in terms of disease susceptibility and morbidity, particularly with respect to the presence (insertion or “Ins”) or absence (deletion or “Del”) of a 14-base-pair (14-bp) fragment, in which the 14-bp Del has been associated with increased HLA-G expression, whereas the 14-bp Ins has been associated with decreased HLA-G expression [HLA-G UTR-01 haplotype was overrepresented in PTC patients compared to controls. Since UTR-01 contains the 14-bp Del and is associated with increased sHLA-G levels even in healthy individuals [(5′URR)G*01:04:04(coding)UTR-03(3’UTR) extended haplotype. Considering that the HLA-G 3′UTR and the 5′URR segments present several elements that have been functionally studied, exhibiting potential roles in HLA-G production [Undoubtedly, the pression . In thisividuals ,46, patioduction ,45, the HLA-G variability with clinical features, we observed that PTC patients carrying the (i) 5′URR G0104a haplotype, (ii) 3′UTR UTR-03 haplotype, and (iii) G0104a(5′URR)G*01:04:01(coding)UTR-03(3′UTR) extended haplotype exhibited clinical benefit with respect to PTC development, since their frequencies were underrepresented in patients exhibiting metastatic and multifocal tumors. Cervical lymph node metastases (LNMs) are the main way through which PTC spread cancer cells [With respect to the association of er cells  and preser cells . Tumor rer cells . Althouger cells . Neverther cells . Additioer cells . In conter cells .HLA-G gene variability with HLA-G tissue expression, we observed that the coding G*01:01:02:01 and the 3′UTR UTR-02 haplotypes individually or assembled as the G010102a(5′URR)G*01:01:02:01(coding)UTR-02(3′UTR) extended haplotype exhibited an association with low HLA-G expression in PTC specimens. Individuals carrying UTR-02 have already been described as low sHLA-G producers [G010101c haplotype and the G010101c(5′URR)G*01:01:01:05(coding)UTR-04(3′UTR) extended haplotype were associated with greater HLA-G expression in the PTC specimens. While UTR-04 has been associated with intermediate plasma sHLA-G levels in healthy Brazilian and French individuals [HLA-G 5′URR yielded differential responses in the presence of HLA-G inducers or repressors. For instance, an HLA-G+ cell line (choriocarcinoma JEG-3) transfected with the G010101c haplotype significantly increased the luciferase activity cultured in the presence of interferon (IFN)-β when compared to non-treated transfectants [HLA-G 5′URR, under the influence of diverse transcription factors may differentially induce gene expression. The balance between transcriptional (primarily acting at the 5′URR segment) and post-transcriptional (primarily acting at the 3′UTR segment) factors present in the PTC microenvironment may determine the magnitude of HLA-G tumor expression, corroborating the need to evaluate the entire gene variability.Regarding the association between roducers . In contividuals , functiofectants . Therefo(5′URR)G*01:01:02:01(coding)UTR-02(3′UTR) extended haplotype was associated with undetectable sHLA-G expression. The HLA-G UTR-02 haplotype is composed of the 14-bp Ins and +3142G; both variations were previously associated with low mRNA availability and decreased HLA-G expression, whereas the UTR-18 haplotype is composed of the 14-bp Del and +3142C associated with high HLA-G expression [+ cell line (melanoma FON+) transfected with the G010102a haplotype exhibited the lowest promoter activity among all other 5′URR haplotypes when compared to the empty vector [Concerning sHLA-G detection, we observed that UTR-18 was associated with detectable sHLA-G, whereas the G010102apression ,52. Addiy vector .HLA-G genetic variability in 4640 individuals from 88 different population samples across the globe from four datasets [HLA-G locus. These variants are arranged into 514 different coding haplotypes or full-length sequences. According to the study, in terms of global diversity, there are 14 HLA-G alleles with global frequencies higher than 1%: G*01:01:01:01, G*01:01:01:04, G*01:01:01:05, G*01:01:01:06, G*01:01:01:08, G*01:01:02:01, G*01:01:03:03, and G*01:01:22:01 , G*01:03:01:02 (encoding the G*01:03 protein), G*01:04:01:01, G*01:04:01:02, and G*01:04:04 (encoding the G*01:04 protein), G*01:05N , and G*01:06:01:01 (encoding the G*01:06 protein). These alleles represent 91% of all HLA-G sequences detected worldwide, and their frequencies differ in each biogeographic region [HLA-G alleles were found in the present study and represent 96.9% of all detected haplotypes among the Brazilian subjects from the same geographical region.A recent paper published by our research group characterized the entire datasets . The stuc region . All of G*01:04:04 haplotype encodes the HLA-G*01:04 molecule and was associated with protection against disease development, (ii) the G*01:01:02:01 (HLA-G*01:01 molecule) and the G*01:04:01 (HLA-G*01:04 molecule) haplotypes were associated with poor biochemical or structural evidence of persistent disease, and (iii) the G*01:01:02:01 (HLA-G*01:01 molecule) haplotype was associated with undetectable sHLA-G. Although the HLA-G molecule may couple peptides in its groove, its major role is the modulation of immune responses through its binding to leukocyte receptors located outside the peptide-binding groove, whose interactive residues are conserved among these molecules [HLA-G coding and regulatory regions, (ii) different coding region haplotypes may generate differential alternative splicing mechanisms, yielding distinct HLA-G isoforms, and (iii) different coding region haplotypes  encoding the same molecule (HLA-G*01:04) may present distinct regulatory region haplotypes [In our study, three isolated coding region haplotypes were also associated with PTC: (i) the olecules . The preolecules . Thus, tplotypes .HLA-G haplotypes. In human genetic studies, sample size plays a critical role in conducting reliable analyses of polymorphisms [One limitation of the present study is that a few associations exhibited a very broad 95% CI and/or exhibited a 95% CI that includes the number 1, particularly involving less frequent orphisms . Thus, tIn contrast, using a Brazilian population is an important strength of our study for its remarkable and unique genetic diversity, resulting from miscegenation among Brazilian Native Amerindian, African, and European populations . GeneticThe study was conducted on 185 patients with PTC  submitted to thyroidectomy due to or the suspicion of PTC following the ultrasound-guided fine-needle aspiration (FNA) malignant cytology of the thyroid nodule  . A totalThe cytological and histopathological analyses were performed by experienced pathologists, as well as the tumor node metastasis (TNM) staging classification according to the eighth edition of the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC), ranging from stages I to IV to predict the risk of mortality from DTC. Overall, the lower the TNM stage, the lesser probability of cancer spread and the lower the risk of mortality .Clinical and histopathological data of all patients  as well as treatment details, including surgery approach and RAI therapy), are summarized in The individual response to initial conventional therapy of each patient at the last follow-up was assessed according to the criteria proposed by Momesso and Tuttle 2014 , adoptedA group of 154 unrelated healthy blood donors  with no personal or family history of thyroid diseases and exhibiting negative anti-peroxidase antibodies was recruited from “Hospital das Clínicas de Ribeirão Preto”, Ribeirão Preto-SP, Brazil.All subjects gave written informed consent to participate in the study, which was approved by local human research ethics committees .HLA-G 5′URR region was amplified by polymerase chain reaction (PCR) using the forward GPROMO.S 5′-ACATTCTAGAAGCTTCACAAGAATG and reverse GPROMO.R 5′-GCCTTGGTGTTCCGTGTCT primers, as previously described [Genomic DNA was extracted from peripheral blood leucocytes using a standard salting out procedure . The HLAescribed . The seqHLA-G coding region was PCR-amplified using the forward 5′-TAAAGTCCTCGCTCACCCAC and reverse 5′-CACCACCGACCCTGTTAAAG primers, as previously described [The escribed ,64, encoThe end of the coding region, which covers exons 5 and 6, was amplified along with the 3′UTR segment. DNA amplification was performed using the forward 5′-CCATGAGAGATGCAAAGTGCT and reverse 5′- TCTTCTGATAACACAGGAACTTC primers . The seqhttp://www.ebi.ac.uk/ipd/imgt/hla/> URL (accessed on 10 November 2020). All polymorphisms detected were individually annotated, and haplotypes of each HLA-G region were initially defined. These haplotypes were further used to construct HLA-G extended haplotypes, as previously described [The sequences were analyzed and compared to the reference sequence recognized by the IMGT, <escribed ,53.HLA-G gene variability in the present study, the HLA-G expression in the tumor was available for 170. According to the percentage of stained tumor cells, HLA-G expression was stratified into two categories: low expression (≤50% of positive tumor cells in the PTC specimens) and high expression (>50% of positive tumor cells in the PTC specimens).The HLA-G expression in the tumor microenvironment of the PTC specimens was evaluated in the same series of patients using an immunohistochemical assay as previously described . In shorPlasma sHLA-G levels were previously evaluated in the same series of PTC patients before thyroidectomy to evaluate its relationship with several cytokines . We deteHLA-G gene was evaluated by Haploview® 4.2 the program [The LD among polymorphic sites at the  program . Given t program  was used program  using AR program . A two-tHLA-G gene variability on PTC morbidity and/or the regulation of HLA-G expression in PTC was not fully understood until now. Therefore, by evaluating the entire HLA-G gene in PTC patients together with HLA-G expression in tumor specimens and sHLA-G plasma detection, we uncovered important associations between the HLA-G gene, taken as gene segments and/or as extended haplotypes, and: (i) the susceptibility to PTC development, (ii) tumor morbidity, particularly metastatic or multifocal tumors and poor response to conventional therapy, (iii) the magnitude of HLA-G expression in tumor specimens, and (iv) the presence or absence of detectable sHLA-G in pre-thyroidectomy plasma. Although several local and systemic  factors contribute to both PTC development and HLA-G expression modulation, the associations reported in the present study can have significant implications in the context of PTC outcomes, particularly the identification of specific genetic markers that can serve as potential targets for personalized therapies in the future.The immune checkpoint HLA-G molecule is highly expressed in PTC specimens, although the impact of the


To repair ionizing radiation (IR)-induced double strand breaks (DSBs), mammalian cells primarily use canonical non-homologous end-joining (cNHEJ), the homologous recombination (HR) pathway, and the alternative non-homologous end-joining (aEJ) as a backup. These pathways function either compensatively or competitively. High linear energy transfer (LET) compared to low-LET IR kills more cells at the same doses by inhibiting only cNHEJ, but not HR or aEJ. The mechanism remains unclear. The activation of each repair pathway requires the binding of different proteins to DNA fragments of varying lengths. We previously observed an increased generation of small DNA fragments (≤40 bp) in cells following high-LET IR compared to low-LET IR, suggesting that short DNA fragments were one of the major factors interfering with cNHEJ. To provide direct evidence, here we compare the efficiencies of cNHEJ, HR, or aEJ in repairing DSBs containing 30- or 60-bp fragments in vitro and in cells. We show that only cNHEJ but not HR or a-EJ was inefficient for repairing DSBs with 30-bp fragments compared to 60-bp ones, which strongly supports our hypothesis. These results not only enhance our understanding of the DSB repair pathway choice but also hold potential benefits for protection against high-LET IR-induced damage or improving high-LET radiotherapy. DNA double-strand breaks (DSBs) are a severe threat to cell survival and genomic integrity. In mammalian cells, DNA DSBs are repaired by two major pathways: canonical non-homologous end joining (cNHEJ) and homologous recombination (HR)  (one typIonizing radiation (IR)-induced DSBs (~80%) in mammalian cells are repaired by cNHEJ within the first hour after irradiation . High liWe previously found that high-LET IR generated more small DNA fragments (≤40 bp) compared to low-LET IR in cells . AnotherTo test whether the length of small dsDNA fragments affects DSB repair pathways differently, we chose 30- and 60-bp dsDNA as the comparison targets. Such a design was based on the following fact: In cNHEJ, Ku binding to DNA ends was in a sequence-independent manner, which shaped an asymmetric ring, encircling the DNA, but also left parts of the helix exposed. The exposed helix is then bound by DNA-PKcs, which is required for cNHEJ . The effBased on the hypothesis A, we desHR and aEJ are relatively complicated repair processes; they are difficult to reconstitute biochemically (particular HR) using an in vitro system compared to cNHEJ. Therefore, we used engineered reporter systems to compare these pathways in cells. Although we previously studied HR efficiency in human and mouse cells using the I-SceI-based reporter from Dr. Jasin’s lab ,33,34, tTo test the Cas9/sgRNA digestion efficiency for the inserted sequence, we used PCR to amplify the fragment with an adjacent sequence from the cNHEJ/HR vector and generated a 335-bp DNA substrate C. We theWe generated new HEK293T cell lines by stably expressing a single copy of the cNHEJ/HR or aEJ lentiviral vector, respectively. We named the cell line that was integrated with the cNHEJ/HR vector HEK293T-1, and the cell line with the aEJ vector HEK293T-2. To compare the repair efficiencies of 30- and 60-bp DSBs in cells using cNHEJ/HR or aEJ assays, we generated 6 groups of transient transfection samples  for HEK2The results show that group 1 of HEK293T-1 or HEK293T-2 cells did not generate clear DSB repair signals  compared to the longer one (60 bp) interfered with cNHEJ but did not affect HR or aEJ efficiency. These results provide direct evidence to support the idea that increased small DSB fragments in high-LET irradiated cells are one of the major reasons for increased cell killing, which well explains previous findings ,28,38,39Different from IR, which simultaneously generates DSBs in exposed cells, DSBs induced by external Cas9 in transduced cells through digesting the integrated two adjacent target sites (close to each other) are impossible to generate simultaneously. In vitro Cas9 digestion should be more likely to be simultaneous than cell Cas9 digestion. As shown in It is worth mentioning that the mCherry signals (cNHEJ) in Cas9/sgRNA-2 transduced HEK293T-1 cells were much higher than those in Cas9/sgRNA-1 or Cas9/sgRNA-3 transduced cells , indicatIn human cells, Ku molecules are in high abundance, and with an extremely high affinity for DNA ends, Ku can immediately bind to DSB properly (≥20 bp) and improperly (≤16 bp). However, since Ku dissociating from bound DNA required DNA-PKcs activation , the impWithout cNHEJ, high-LET IR-induced increased short DNA fragments can still be repaired by HR and/or aEJ. Such a statement is strongly supported by the results derived from synchronized S-phase Ku-deficient cells that showed the same sensitivities to high-LET and low-LET IR-induced killing , despiteAlthough cNHEJ-deficient cells showed equal sensitivities to the same doses of high-LET and low-LET IR, cNHEJ proficient cells still exhibit greater resistance than cNHEJ deficient cells following high-LET IR exposure ,28, indiDNA ladder (marker) preparation: 15-, 30-, 60-, and 108-bp dsDNA oligoes with blunt ends were synthesized by Integrated DNA Technologies (IDT) Inc.  Longer dsDNAs  were PCR amplified from pRRL sEF1a HA.NLS.Sce(opt).T2A.IFP . The dsDNA sequences are as follows: 5′-TAGAG ACGGGATGAG-3′ for 15 bp; 5′-TAGAGACGGGATGAGTGGAATTAGGACTGA-3′ for 30 bp; 5′-TAGAGACGGGATGAGTGGAATTAGGACTGAGA CTATGGTTGCTGACTAATCGAGACCCAT-3′ for 60 bp; and 5′-GCGTAGGGATAACAGGGTAATTAGATGATAGAGACGGGATGAGTGGAATTAGGACTGACGCG GGCAATCACTCGAGTGAGTAACTAGCAAGCCCGGCGTGGATCCGC-3′ for 108 bp. The PCR products were obtained using one shared primer, “CGGAATTCAAGACCTACTGCATGCAGT”, and the other primers “CGGAATTCTGAAGGTCTGGGCGCC” for 170 bp; “CGGAATTCGGTCTTCTTGTTGTTCACGAT” for 210 bp; and “CGGAATTCCCGCCG TCGTCCATGA” for 280-bp. These dsDNA fragments were labeled with ATP, [γ-32P] (PerkinElmer), using T4 Polynucleotide Kinase (T4 PNK) , and then purified with a G-25 spin column .32P] labeled dsDNA fragments , with 5 mM triethanolamine-HCl, 10 mM Tris-HCl, 50 mM Mg(OAc)2, 0.1 mM DTT, 50 ng/µL BSA, and 10 nM ATP at 37 °C for 30 min. T4 ligase was used as a positive control. The reaction products were resolved in a 12.5% (1.5 mm thick) denaturing polyacrylamide gel. The image of the gel with the radio-activated signals was obtained using a Typhoon laser scanner.For the end-joining reaction, DNA-PKcs were purchased from Thermo Fisher Scientific Inc. (PV5866). The other purified proteins, including Ku dimers Lig4/XRCC4 and XLF, were obtained from Dr. William Dynan’s lab ,44,45. TStandard molecular biology techniques were used for plasmid construction. A dsDNA oligo that had minimal homology to the human genome was synthesized, containing targeting sites for sgRNA-1 (GTCCTGTGGATCCTCTACGC), sgRNA-2 (GGACAGCATGGCCTGCAACG), and sgRNA-3 (GTCCCAGACAAGGTATAGGG), so that co-expression of Cas9 with sgRNA-1 and 2 resulted in a 30-bp fragment, and co-expression of Cas9 with sgRNA-1 and 3 resulted in a 60-bp fragment. The HR/cNHEJ reporter psf.CVL Traffic Light Reporter Ef1a Puro was modified from the lentiviral vector pCVL Traffic Light Reporter 1.1 (Sce target) Ef1a Puro (Addgene #31482) by inserting the above synthesized dsDNA fragment into the original I-SceI recognition site. Briefly, a promoter was linked to a GFP cDNA at transcriptional reading frame +1 that was interrupted by an intervening sequence . At 3 days post-transduction, cells with integrated reporters were selected using 1 μg/mL puromycin for 5 days. Puro-resistant cells were selected at the minimal multiplicity of infection (MOI) (surviving fraction < 0.1), and a monoclonal population of cells was isolated and used for further analysis. The cNHEJ/HR reporter stably-expressing cell line was named HEK293T-1, and the aEJ reporter stably-expressing cell line was named HEK293T-2.HEK293T cells were maintained in DMEM supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum. Reporters expressing lentiviral particles for the cNHEJ/HR or aEJ cassette were generated, and HEK293FT cells were transduced. Briefly, 2 × 10To generate 30- or 60-bp small dsDNA fragments, sgRNA-1, sgRNA-2, or sgRNA-3 were cloned into the pX330-U6-Chimeric_BB-Cbh-hSpCas9 vector (Addgene #42230) individually or in pairs, sgRNA-1 plus 2 (sgRNA-1/2) or sgRNA-1 plus 3 (sgRNA-1/3). For the cNHEJ/HR assay, the transduction was paired with a donor vector, pRRL SFFV d20GFP.T2A.mTagBFP Donor (Addgene #31483). Cells were harvested 72 h post-transduction and analyzed on a Flow Cytometer (BD FACS LSR II). >30,000 cells were sorted for each sample. GFP was measured using a 488 nm laser for excitation and a 530/30 filter. mCherry was excited using a 561 nm laser and was acquired with a 610/20 filter. The data was quantified using FlowJo software v10.t-test.Error bars represent the standard error, as calculated by dividing the standard deviation of replicates by the square root of the number of total replicates. For normalized data shown in In conclusion, DSBs pose a significant threat to cell survival and genomic stability. Unrepaired DSBs can lead to cell death, while misrepaired DSBs can result in chromosomal translocations, which are implicated in the development of cancer . The cur


Aim: Ferroptosis is a non-apoptotic form of cell death caused by lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein  and ABCG2. We thus sought to characterize the interactions of FINs with P-gp and ABCG2, which may provide information regarding oral bioavailability and brain penetration and predict drug-drug interactions.Methods: Cytotoxicity assays with ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2 were used to determine the ability of the transporters to confer resistance to FINs; confirmatory studies were performed in OVCAR8 and NCI/ADR-RES cells. The ability of FINs to inhibit P-gp or ABCG2 was determined using the fluorescent substrates rhodamine 123 or purpuin-18, respectively.Results: P-gp overexpression conferred resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. The FINs ML-162, GPX inhibitor 26a, and PACMA31 at 10 µM were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells. GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells.Conclusion: Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions. One of the first reported inducers of ferroptosis, erastin, was described before the concept of ferroptosis was completely understood. Erastin was found to selectively kill transformed human foreskin fibroblasts expressing mutant HRAS compared to isogenic cells expressing wild-type HRAS. The mechanism of cell death induced by erastin was not apoptosis, as the hallmarks of apoptotic cell death such as annexin V staining and caspase 3 cleavage were not observed, although death was accompanied by cell membrane permeabilization. The term “ferroptosis” was later coined for this novel form of cell death as iron chelators and antioxidants were found to potentiate erastin-mediated toxicity, suggesting an iron-dependent increase in ROS was responsible. Additionally, ferroptosis could not be inhibited by caspase inhibitors and was found to occur independently of the apoptosis effector proteins Bak and Bax.Ferroptosis is an iron-dependent form of non-apoptotic cell death arising from direct or indirect inhibition of glutathione peroxidase 4 (GPX4), leading to lipid peroxidation and unsustainable levels of reactive oxygen species (ROS)-c. Inhibition of the antiporter leads to depletion of glutathione and inactivation of GPX4. Subsequent to the discovery of erastin, other small molecules have been developed to induce ferroptosis by direct or indirect inhibition of GPX4. These ferroptosis inducers (FINs) include modified forms of erastin such as erastin2, imidazole ketone erastin and piperazine erastin, as well as the inhibitors FIN56, RSL3, FINO2, PACMA31, GPX4 inhibitor 26a, and ML-162 and ML-210.The target of erastin was identified to be the cystine/glutamate antiporter, system xet al. tested erastin toxicity in 117 cancer cell lines and identified renal cell carcinomas as being particularly sensitive to ferroptosis. This intrigued us, as renal cell carcinomas are often positive for expression of P-glycoprotein , an ATP-binding cassette (ABC) multidrug efflux pump that confers drug resistance,14. Some reports have also suggested that expression levels of another ABC transporter, ABCG2 (encoded by the ABCG2 gene), also expressed in kidney cancers, can predict overall survival in patients with clear cell renal carcinoma. Additionally, both P-gp and ABCG2 localize to the gastrointestinal tract as well as to other barrier sites, such as the blood-brain barrier (BBB). Expression at these sites is linked to their role in limiting the oral bioavailability of chemotherapy drugs and brain penetration of several targeted therapies,17. We thus sought to characterize the interactions between small-molecule ferroptosis inducers and the transporters P-gp and ABCG2.In an attempt to identify cancers that might be effectively treated by ferroptosis induction, Yang 2, GPX4 inhibitor 26a, JKE-1674, and JKE-1716 were from Cayman Chemical . Valspodar was obtained from MedChemExpress . Romidepsin was from Selleck Chemicals . Purpurin-18 was purchased from Frontier Scientific . Piperazine erastin was from TargetMol . SN-38 was obtained from LKT Laboratories . RSL3 was purchased from Tocris . Fumitremorgin C  was synthesized in-house by the Developmental Therapeutics Program at the National Institutes of Health .Erastin, doxorubicin, rhodamine 123, and FIN56 were purchased from Sigma-Aldrich . Erastin2, imidazole ketone erastin, RSL3, ML-162, FINOABCB1 or ABCG2 using Lipofectamine 2000 . Cells were selected with hygromycin, and clones were isolated by limiting dilution. Selected clones were grown in DMEM with 10% fetal calf serum, glutamine and Pen/Strep, as well as 300 µg/mL hygromycin to maintain expression of the transporters. P-gp-overexpressing MDR-19 cells and ABCG2-overexpressing R-5 cells were derived from HEK293 cells and have been previously characterized and described. Transfected HEK293 cells were grown in MEM with 10% fetal calf serum, glutamine and Pen/Strep along with 2 mg/mL G418 to maintain expression of the transporters. All cell lines were routinely tested for mycoplasma using the MycoAlert PLUS Kit  test kit and their identities were confirmed by STR analysis .OVCAR8, NCI/ADR-RES and UO-31 cells were obtained from the Division of Cancer Treatment and Diagnosis Tumor Repository, National Cancer Institute  and are grown in RPMI-1640 with 10% FBS, glutamine and Pen/Strep. A673 cells  were seeded and transfected with empty vector (EV) or vector containing full-length, human The following oligonucleotides  were used in this study:ABCB1-START: 5’- GGGGACAACTTTGTACAAAAAAGTTGGCACCATGGATCTTGAAGGGGACCGCAATGGABCB1-END: 5’- GGGGACAACTTTGTACAAGAAAGTTGATTATGCTAGCTGGCGCTTTGTTCCAGCCTGGABCG2-START: 5’- GGGGACAACTTTGTACAAAAAAGTTGGCACCATGTCTTCCAGTAATGTCGAAGTTTTTATCCCABCG2-END: 5’- GGGGACAACTTTGTACAAGAAAGTTGATTAAGAATACTTTTTAAGAAATAACAATTTCAGABCB1 and ABCG2 were constructed by PCR amplification of cDNA sequences flanked by Gateway Multisite recombination sites . PCR was carried out with 200 nM of each oligo listed in the table above using Phusion polymerase  under standard conditions and an extension time of 180 s. PCR products were cleaned using the QiaQuick PCR purification kit . The final PCR products were recombined into Gateway Donor vector pDonr-253 using the Gateway BP recombination reaction using the manufacturer’s protocols. The subsequent Entry clones were sequence verified throughout the entire cloned region.Entry clones for Gateway Multisite LR recombination was used to construct the final mammalian expression constructs from the Entry clones using the manufacturer’s protocols . The Gateway Destination vector used was pDest-305 , a mammalian expression vector containing a Gateway attR4-attR2 cassette based on a modified version of pcDNA3.1. This vector backbone contains a hygromycin resistance marker for antibiotic selection. A human elongation factor 1 (EF1) promoter was introduced using a Gateway att4-att1 Entry clone . Final expression clones were verified by restriction analysis and maxiprep DNA was prepared using the Qiaprep Maxiprep kit .ABCB1 in UO-31 cells was achieved by co-transfecting cells with knockout and homology-directed repair vectors for ABCB1  using Lipofectamine 2000 (Invitrogen) and subsequent selection with puromycin (3 µg/mL). Knockout clones were subsequently isolated, and loss of P-gp was verified by flow cytometry following antibody staining with phycoerythrin-labeled UIC-2 antibody as described below.CRISPR-mediated knockout of 50). Where noted, cytotoxicity assays were performed with 10 µM valspodar to inhibit P-gp.Cells were seeded in opaque white, 96-well plates at a density of 2,500 cells/well and allowed to attach overnight. Cells were then treated with increasing concentrations of the desired compound and incubated for 72 h. Cell TiterGlo (Promega) was then used to determine luminescence values for each concentration according to the manufacturer’s instructions. The data were modeled using nonlinear regression curve fitting  to determine the concentration at which 50% of cell growth was inhibited (GI. Cells were trypsinized and incubated for 30 min in complete medium (phenol red-free Richter’s medium with 10% FCS and penicillin/streptomycin) with the desired fluorescent substrate (0.5 µg/mL rhodamine 123 to detect P-gp or 15 µM purpurin-18 to detect ABCG2) in the presence or absence of 25 µM concentrations of the desired FIN or a positive control inhibitor  for 30 min at 37 °C in 5% CO2. Subsequently, cells were washed and incubated in substrate-free medium for 1 h at 37 °C continuing with or without inhibitor. Cells were subsequently analyzed with a FACSCanto flow cytometer  and data analysis was performed using FloJo v 10.4.2 .To measure cell surface expression of P-gp or ABCG2, trypsinized cells were incubated for 20 min. at room temperature in 2% bovine serum albumin/PBS with phycoerythrin-labeled UIC-2 antibody or phycoerythrin-labeled 5D3 antibody, respectively, according to the manufacturer’s instructions . Cells were also incubated with the corresponding phycoerythrin-labeled isotype control - IgG2a kappa for P-gp and IgG2b kappa for ABCG2 (both from ThermoFisher). P-gp or ABCG2 transporter activity was measured using rhodamine 123 or purpurin-18, respectively. Total membrane vesicles were prepared from High Five insect cells overexpressing P-gp. The membranes were diluted with ATPase assay buffer  to reach a final concentration of 100 µg/mL and were incubated with the compounds at the noted concentrations for 10 min in the presence or absence of 0.3 mM sodium orthovanadate. The addition of 5 mM ATP (5 mM) started the reaction (20 min at 37 °C), after which SDS  was added to terminate the reaction. The amount of inorganic phosphate released was quantified and the results were reported as a percentage of vanadate-sensitive ATPase activity with DMSO.The ATPase assay was performed as described previously and did not express P-gp or ABCG2, we transfected this cell line with either empty vector (A673 EV) or vectors containing the genes encoding human P-gp (A673 B1) or ABCG2 (A673 G2). We selected single clones with high levels of P-gp or ABCG2 based on measurement of antibody staining by flow cytometry and further characterized positive clones. As seen in As the A673 cell line was reported to be sensitive to FINsTo verify that transporter levels were adequate to confer resistance to known substrates, we performed cytotoxicity assays with the P-gp substrate doxorubicin as well as the ABCG2 substrate SN-38. As seen in A673 EV, A673 B1 and A673 G2 cells were then used in 3-day cytotoxicity assays to determine whether the transporters could confer resistance to the FINs. While resistance due to the expression of ABCG2 or P-gp was not seen with most FINs examined, this was not true for some erastin derivatives that had been modified to improve water solubility. Overexpression of P-gp conferred relatively high levels of resistance to imidazole ketone erastin and piperazine erastin , but we Supplementary Table 1], in agreement with the results from the A673 cells.Since P-gp overexpression appeared to confer resistance to some FINs, we validated the results in parental OVCAR8 ovarian cancer cells and P-gp-overexpressing NCI/ADR-RES cells that were derived from OVCAR8 cells by selection with doxorubicin. As shown in . To determine if the expression of P-gp in this cell line is high enough to confer resistance to imidazole ketone erastin or piperazine erastin, two of the best substrates for P-gp, we performed CRISPR-mediated deletion of ABCB1 and selected two clones that had lost expression. As shown in ABCB1 gene had been deleted. The knockout clones also demonstrate increased sensitivity to the P-gp substrate romidepsin [Supplementary Table 2]. Similar results were obtained when we performed cytotoxicity assays with imidazole ketone erastin and pierazine erastin in the presence of 10 µM of the P-gp inhibitor valspodar [Supplementary Tables 1 and 3]. Thus, even relatively low levels of P-gp may cause resistance to the erastin analogs.The renal carcinoma cell line UO-31 is known to have detectable levels of P-gp and displays rhodamine effluxmidepsin . When we. While P-gp does not appear to confer resistance to erastin in our studies, erastin does stimulate ATPase activity. This suggests that erastin interacts with P-gp, but likely has a slow off rate, potentially acting more as a weak inhibitor than a substrate.The effect of erastin, imidazole ketone erastin, and piperazine erastin on the ATPase activity of P-gp was subsequently examined. While several P-gp substrates and some inhibitors have been shown to stimulate the ATPase activity of P-gp, not all substrates do so. We found that all three of the compounds stimulated the ATPase activity of P-gp to a degree comparable to that of verapamil, which is used as a positive control . The ATPAs targeted therapies are known to act as inhibitors of ABC transporters, we next characterized the ability of the FINs to act as inhibitors of P-gp or ABCG2. At a concentration of 10 µM, erastin2, ML-162, GPX4 inhibitor 26a, and PACMA31 inhibited P-gp-mediated rhodamine 123 transport, resulting in a 5- to 10-fold increase in rhodamine fluorescence in MDR-19 cells . In ABCG,23. FINs are effective in xenograft mouse models and have been suggested as potential cancer treatments. Despite the recent proliferation of papers describing novel molecules that can induce ferroptosis-11,24, very few studies have addressed potential interactions with ABC transporters that might limit bioavailability or brain penetration. In our study, we found that the FINs FIN56, imidazole ketone erastin and piperazine erastin are transported by P-gp, suggesting that their oral bioavailability and/or brain penetration may be compromised. Additionally, we found that the FINs ML-162, GPX inhibitor 26a, and PACMA31 act as inhibitors of P-gp. Interestingly, the most potent P-gp inhibitor, GPX inhibitor 26a, was also the most potent ABCG2 inhibitor, suggesting that treatment with FINs may cause drug-drug interactions.Ferroptosis induction by small molecules is a novel way to induce cell death in cancer cells and several cancer cell types are sensitive to ferroptosis induction, such as renal cell carcinoma, diffuse large B-cell lymphomas, as well as many chemotherapy-resistant cancer subtypeset al. who reported erastin as a P-gp substrate. However, we note that they used a P-gp-expressing cell line that was generated by gradually increasing exposure to paclitaxel. While selection with paclitaxel can lead to P-gp overexpression, other mechanisms of resistance can arise. It cannot be ruled out that other mechanisms besides efflux by P-gp may have caused the resistance to erastin observed by Zhou et al. Unfortunately, they did not perform cytotoxicity assays in the presence of a P-gp inhibitor, which would have confirmed the role of P-gp. In contrast, we did not observe erastin resistance in cells that were transfected to express P-gp without selection with an anticancer drug, and this result was confirmed in a selected cell line. We thus conclude that erastin is not a P-gp substrate and that the cell line used by Zhou et al. may have another mechanism at work that can confer resistance to erastin.Our findings regarding erastin differ from those of Zhou . The BCR-ABL inhibitors imatinib, nilotinib, dasatinib, and bosutinib have been found to be substrates of P-gp and ABCG2 at low concentrations, while they act as inhibitors of the proteins at higher concentrations-31. Overexpression of ABCB1 has been demonstrated in tumor samples obtained from patients whose tumors have developed resistance to the ALK inhibitor ceritinib in the absence of secondary ALK mutations. Ceritinib has also been reported to inhibit P-gp- and ABCG2-mediated transport. Additionally, both P-gp and ABCG2 have been shown to confer resistance to several structurally different aurora kinase inhibitors,35.It is not surprising that the FINs, which are essentially targeted therapies, interact with drug transporters, as many targeted therapies have been shown to either be substrates or inhibitors of P-gp or ABCG2ABCB1 homologs Abcb1a and Abcb1b are knocked out, the ABCG2 homolog Abcg2 is knocked out, or all of the homologous transporters have been deleted. Brain concentrations of the Janus kinase 1/2 inhibitor momelotinib 24 h after oral administration were 6.5-fold, 3-fold and 48-fold higher in mice deficient in Abcg2, Abcb1a/b, or Abcg2;Abcb1a/b, respectively, compared to control mice. Similarly, 24 h after oral administration of the BCR-ABL inhibitor ponatinib, brain concentrations were 2.2-fold, 1.9-fold and 25.5-fold higher in mice deficient in Abcg2, Abcb1a/b, or Abcg2;Abcb1a/b, respectively, compared to wild-type controls. Thus, P-gp and ABCG2 can have a profound effect of limiting brain penetration of targeted therapies that are substrates of the transporters. Brain accumulation or oral bioavailability of FIN56, imidazole ketone erastin or piperazine erastin could similarly be affected, as they were found to be transported by P-gp.The ability of P-gp and ABCG2 to affect brain penetration of targeted therapies has most dramatically been demonstrated in mouse models in which the In conclusion, we have demonstrated that the FINs FIN56, imidazole ketone erastin, and piperazine erastin are substrates of P-gp, suggesting a potential reduction in oral bioavailability and brain penetration. ML-162, GPX inhibitor 26a and PACMA31 were found to inhibit the transport activity of P-gp and/or ABCG2, suggesting potential drug-drug interactions. Thus, our findings will be valuable as these compounds are pursued clinically.


For metabolomics results, HFD could significantly alter the levels of 15 endogenous metabolites, and genistin could reverse them. Creatine might be a beneficial biomarker for the activity of genistin against hyperlipidemia, as revealed via multivariate correlation analysis. These results, which have not been reported in the previous literature, may provide the foundation for genistin as a new lipid-lowering agent.Genistin, an isoflavone, has been reported to have multiple activities. However, its improvement of hyperlipidemia is still unclear, and the same is true with regard to its mechanism. In this study, a high-fat diet (HFD) was used to induce a hyperlipidemic rat model. The metabolites of genistin in normal and hyperlipidemic rats were first identified to cause metabolic differences with Ultra-High-Performance Liquid Chromatography Quadrupole Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS). The relevant factors were determined via ELISA, and the pathological changes of liver tissue were examined via H&E staining and Oil red O staining, which evaluated the functions of genistin. The related mechanism was elucidated through metabolomics and Spearman correlation analysis. The results showed that 13 metabolites of genistin were identified in plasma from normal and hyperlipidemic rats. Of those metabolites, seven were found in normal rat, and three existed in two models, with those metabolites being involved in the reactions of decarbonylation, arabinosylation, hydroxylation, and methylation. Three metabolites, including the product of dehydroxymethylation, decarbonylation, and carbonyl hydrogenation, were identified in hyperlipidemic rats for the first time. Accordingly, the pharmacodynamic results first revealed that genistin could significantly reduce the level of lipid factors ( In its MS/MS spectrum, the DPIs at m/z 137 ([M+H-C8H6O]+) and m/z 119 ([M+H-C7H4O3]+) were generated due to RDA fragmentation [Daidzein (M2) exhibited the theoretical [M-H]− ion at m/z 431.09837  with a retention time of 1.24 min. Meanwhile, the fragment ion at m/z 92 ([M-H-C15H15O9]−) indicated the loss of B-ring, and the DPI at m/z 162 ([M-H-C11H12O7]−) indicated that the reaction of RDA cleavage occurred at positions 1 and 4 of M2. The two fragment ions were found in genistin in the MS/MS spectrum [Genistin spectrum . (M3) was eluted at 0.98 min with the theoretical [M-H]− ion at m/z 429.08273 . It was 176 Da higher than that of daidzein. In the MS2 spectra, the DPIs at m/z 162 ([M-H-C12H14O7]−) and m/z 160 ([M-H-C12H16O7]−) indicated the occurrence of an RDA reaction. The characteristic fragment ion at m/z 92 ([M-H-C15H13O9]−) indicated that the glucose group should be not introduced into the B-ring. The fragment ion at m/z 187 ([M-H-C10H10O7]−) was produced by the rupture of the A-ring [The glucuronidation product of daidzein e A-ring . (M4) possessed the theoretical ion [M-H]− at m/z 433.11405  with a retention time of 0.91 min. It was 18 Da higher than that of daidzin. In the MS2 spectra, the DPIs at m/z 92 ([M-H-C15H17O9]−) and m/z 197 ([M-H-C6H10O5-C2H2O3]−) indicated that the hydration reaction occurred in the C-ring [The hydration product of daidzin (M5) was 30 Da higher than that of daidzin with a retention time of 9.57 min. It exhibited the theoretical ion [M-H]− at m/z 445.11395 . In the MS2 spectrum, the methoxy group might be added to the A-ring due to the DPI at m/z 407 ([M-H-C3H2]−), which was produced by the loss of the B-ring. The fragment ion at m/z 148 ([M-H-C11H16O7]−) also confirmed the above conclusion. The hydroxylation and methylation product of daidzin (M7) was eluted at 12.53 min and produced the theoretical [M+H]+ ion at m/z 215.10667  in the positive ion mode. It was 28 Da lower than that of equol. In its MS2 spectrum, the fragment ion at m/z 149 ([M+H-C5H6]+) was produced due to the removal of the B-ring, and the fragment ion at m/z 146 ([M+H-C4H5O]+) was generated by the loss of positions 5 and 8 on the A-ring. The decarbonylation product of equol (M8) was eluted at 9.04 min and possessed the theoretical [M+H]+ ion at m/z 245.08077 . It was 2 Da higher than that of equol. In its MS2 spectrum, the simultaneous removal of the A-ring and the B-ring produced the DPI at m/z 83 ([M+H-C10H10O2]+). The fragment ions at m/z 93 ([M+H-C8H7O3]+), m/z 226 ([M+H-H2O]+), and m/z 69 ([M+H-C10H8O3]+) indicated that the possible reaction could only occur at position 3 of the C-ring. The hydrogenation product of equol (M10) was eluted at 9.97 min, at which point it produced the theoretical [M-H]− ion at m/z 223.04003 . In the MS2 spectrum, the characteristic fragment ion at m/z 111 ([M-H-C5H4O3]−) was produced via RDA rearrangement, which occurred on the 3’ and 6’ positions of the B-ring. The DPIs at m/z 183 ([M-H-C2O]−) and m/z 92 ([M-H-C8H3O2]−) were observed, suggesting that the hydroxyl and methyl groups were lost in the A-ring. The cleavage behavior was similar to that of the standard. The dehydroxymethylation product of daidzein (M11), which was eluted at 9.37 min, contributed the theoretical [M+H]+ ion at m/z 257.08088 . It was 2 Da higher than that of daidzein. The DPIs at m/z 69 ([M+H-C11H8O3]+) and m/z 189 ([M+H-C4H4O]+) were produced due to the RDA reaction on the B-ring in its MS2 spectrum.The carbonyl hydrogenation product of daidzein (M12) exhibited the theoretical [M-H]− ion at m/z 225.05573 , which was 28 Da lower than that of daidzein. In its MS2 spectrum, the fragment ion at m/z 183 ([M-H-C2H2O]−) was produced by the B-ring cleavage, and the fragment ion at m/z 165 ([M-H-C2H2O-H2O]−) confirmed our conclusion.The decarbonylation product of daidzein (M1) was eluted at 9.39 min and produced the theoretical [M-H]− ion at m/z 401.08777 , which was 132 Da higher than that of genistein. In the MS2 spectra, the DPIs at m/z 225 ([M-H-C5H8O4-CO2]−), m/z 357 ([M-H-CO2]−), and m/z 313 ([M-H-CO2-CO2]−) confirmed the theory above. The arabinylation product of genistein (M6), the retention time of which was 9.87 min, was 28 Da lower than that of genistein in the positive ion mode. In the MS2 spectra, the DPIs at m/z 107 ([M+H-C7H4O3]+), m/z 226 ([M+H-O]+), m/z 69 ([M+H-C10H6O3]+), and m/z 93 ([M+H-C8H6O3]+) suggested that a decarbonylation reaction should not occur on the B-ring. However, the fragment ion at m/z 121 ([M+H-C7H6O2]+) indicated that the decarbonylation reaction could occur on the C-ring. The decarbonylation product of genistein (M9) was eluted at 10.09 min; it possessed the theoretical [M-H]− ion at m/z 461.10887 . It was 30 Da higher than that of genistin. In the MS2 spectrum, the characteristic fragment ion at m/z 137 ([M-H-C15H16O8]−) was produced due to the RDA reaction on the C-ring. Meanwhile, the DPIs at m/z 407 ([M-H-C3H2O]−), m/z 92 ([M-H-C6H10O5-C10H7O5]−), m/z 425 ([M-H-2H2O]−), and m/z 84 ([M-H-C6H10O5-C12H7O4]-) indicated the methoxyl group should occurr on the A-ring.The hydroxylation and methylation product of genistin (M2) served as a metabolic center to gradually produce secondary metabolites. For the common metabolic pathways of normal and hyperlipidemic rats, genistin was metabolized to M6, M1, and M9 mainly by decarbonylation, arabinylation, hydroxylation, and methylation. The reaction of three metabolites should be the conventional pathway of genistin metabolism in vivo. In normal rats, genistin was metabolized to daidzein (M0), and M0 was further metabolized to M3, M5, M4, M7, and M8 underwent glucuronidation, hydroxylation and methylation, hydration, decarbonylation, and hydrogenation, respectively. These metabolic reactions might be stress patterns of genistin being cleared in a normal organism. In hyperlipidemic rats, genistin was metabolized mainly to M10, M12, and M11 by dehydroxymethylation, decarbonylation, and carbonyl hydrogenation. It has been suggested that genistin may be metabolized into these three metabolites which are involved in the pathogenesis of hyperlipidemia.Thirteen metabolites (parent drug included) were found in normal and hyperlipidemic rats after oral administration of genistin. The proposed metabolic pathways of genistin are illustrated in p < 0.01), whereas HDL-C was decreased compared with that in rats in Con (p < 0.01), which indicated that HFD could indeed cause hyperlipidemia in rats. After treatment, simvastatin and genistin at different doses significantly reduced the levels of plasma TC, TG, and LDL-C (p < 0.01), and elevated the standard of HDL-C (p < 0.01). This beneficial function was also observed in the liver color of rats in all groups. All results indicated that genistin has the remarkable function of regulating blood lipids.During the experiment, the body weights of rats in all groups were measured per week. As shown in p < 0.05) while decreasing the level of SOD compared with that in Con rats (p < 0.05). The result indicated that excessive HFD could accelerate the process of lipid peroxidation in vivo. Moreover, lipid peroxidation emerged as having a negative relationship with liver function. For H&E staining, the livers of rats in Mod indeed showed increased symptoms of cavitation and fibrosis, which also corroborated the above statement. Moreover, these elevated levels of ALT in Mod rats also corroborated the abnormal liver function compared with Con (p < 0.05). Surprisingly, genistin obviously reversed the above situation; this means that genistin could reduce lipid accumulation in the liver. Concurrently, genistin also significantly reduced the level of MDA (p < 0.05) and increased the content of SOD (p < 0.05). It was shown that genistin has an inhibitory effect on lipid peroxidation. Genistin also had a beneficial effect on liver function by reducing fibrosis and vacuolation of liver cells. This result was also reflected in the decreased ALT due to genistin. In brief, genistin could reduce HFD-induced lipid factor levels and hepatic lipid accumulation in rats.For Oil red O staining, more lipids accumulated in the liver after being raised with HFD. As we know, the excessive lipids could cause a lipid peroxidation reaction with the participation of reactive oxygen species, and lipid peroxidation was associated with cell permeability, DNA damage, and protein synthesis disorders. In Mod rats, HFD significantly increased the content of MDA  and Q2  in positive and negative ion modes revealed the reliability of the model, and the 200 permutation tests also confirmed the above results.p < 0.05 could be used to screen and identify differential metabolites in the OPLS-DA model (p < 0.05), and nine metabolites were significantly decreased (p < 0.05) with HFD intervention. Nevertheless, genistin could still obviously down-regulate six metabolites (p < 0.05), including L-Tryptophan, L-Proline, Octadecanoic acid, 4-Amino-4-cyanobutanoic acid, sn-Glycero-3-phosphoethanolamine, and 5-Aminolevulinate. Genistin also up-regulated 9 of 15 metabolites, involving L-Leucine, Creatine, L-Carnitine, O-Ureido-L-serine, anserine, N-Formimino-L-aspartate, L-Ornithine, 2,3-Dihydroxycarbamazepine, and 5-Hydroxyisourate. Approximate changes in multiples of metabolites are shown in Additional OPLS-DA was utilized to identify significantly changed metabolites after genistin treatment. On the score plots of OPLS-DA in positive and negative ion modes, there were obvious separation trends between Con and Mod and between Mod and Gen. Subsequently, the S-plot with the threshold of VIP > 1.0 and DA model C,F. FinaDA model . Among tFifteen differential metabolites were imported to the MetaboAnalyst 5.0 dataset, and their results revealed the metabolic pathway of genistin against hyperlipidemia A. The rep < 0.05), illustrating that two metabolites might aggravate the damage of hyperlipidemia to the body. The insignificant relationship between two metabolites and HDL-C also proved that they focused only on the transport of total cholesterol from the liver to the plasma. In addition, 4-Amino-4-cyanobutanoic acid and octadecanoic acid were all negative correlated with SOD (p < 0.001); the result suggested that they were associated with lipid peroxidation in the liver. 5-Aminolevulinate, sn-Glycero-3-phosphoethanolamine, and L-Tryptophan all showed negative correlations with SOD and HDL-C (p < 0.05). 5-Aminolevulinate, sn-Glycero-3-phosphoethanolamine, and L-Tryptophan could not only induce hyperlipidemia but also cause adverse effects on liver function based on the positive relationship between these compounds and TG, MDA, ALT, and LDL-C (p < 0.05).Moreover, the Spearman correlation analysis was used to determine the significant correlations between metabolites and cytokines. As shown in p < 0.05), illustrating the function of liver protection. However, three of nine metabolites, namely anserine, creatine, and O-Ureido-L-serine, were negatively correlated with TG, TC, and LDL-C (p < 0.05). The relationships between nine other metabolites and cytokines were contrary to the aforementioned situation. For indicators, all metabolites were negatively correlated with ALT (p < 0.01) and was positively correlated with HDL-C (p < 0.05). The result suggested that creatine seemed to show a unique function in alleviating hyperlipidemia. Moreover, the correlations between creatine and SOD and MDA also confirmed that creatine could inhibit lipid peroxidation in the liver. Other metabolites that could demonstrate this function were anserine, 2,3-Dihydroxycarbamazepine, and N-Formimino-L-aspartate (p < 0.05). Among those metabolites, only creatine could show the negative correlation with the above three factors  was applied to determine the differential biomarkers of genistin against hyperlipidemia. 4-Amino-4-cyanobutanoic acid and octadecanoic acid were negatively associated with SOD while showing a positive correlation with TG, TC, and LDL-C. In addition, creatine was more closely associated with cytokines than other metabolites. Thus, creatine should be considered a beneficial biomarker for genistin in the treatment of hyperlipidemia.(M6), L-arabinylation (M1), and hydroxylation and methylation (M9). Meanwhile, seven metabolites were found only in rats administered normally, such as deglycosylation (M0), glucuronidation (M3), hydroxylation and methylation (M5), hydration (M4), decarbonylation (M7), and hydrogenation (M8) products of genistin. Based on the above results, we speculated that the reactions of three metabolites detected in both normal and pathological rats should be the routine pathways for genistin metabolism in vivo, and metabolic reactions of seven metabolites in normal rats might be stress patterns of genistin being cleared in a normal organism. Three other metabolites could be detected only in hyperlipidemic rats, including the reactions of dehydroxymethylation (M10), decarbonylation (M12), and carbonyl hydrogenation (M11). This fact demonstrates that genistin may be metabolized into three metabolites to participate in the pathogenesis of hyperlipidemia, or three metabolites have some advantages in the treatment of hyperlipidemia with genistin. It is worthwhile for this latent problem to be studied in the future.The metabolic behaviors of drug or natural products in vivo have always been the focus of study for their continued development. The reactions involved in their metabolic pathways may be associated with the involved targets or endogenous pathways. In addition, the metabolic differences of drugs or natural products under normal and pathological conditions have been reported in the previous literature ,23,24, wp < 0.05); this effective activity was also confirmed by pathological results. Thirdly, hyperlipidemia was characterized by abnormally elevated levels of TC, TG, and LDL-C and abnormally reduced HDL-C levels. Genistin significantly improved this pathological change. At present, the research of genistin in the treatment of hyperlipidemia has never been mentioned, such that these results can construct the foundation for genistin as a new lipid-lowering agent. Isoflavones, also known as estrogen-like substances, have been proven to have remarkable abilities in regulating hormone homeostasis, cell proliferation, and metabolic regulation in women, such as soy isoflavone and Pueraria isoflavones ,27,28. Ip < 0.05), and these metabolites were significantly reversed by genistin (p < 0.05), which belonged to the metabolic pathways of arginine and proline metabolism and arginine biosynthesis. Spearman correlation analysis and db-RDA revealed that creatine should be considered a beneficial biomarker of genistin against hyperlipidemia. Creatine in mammal body could be synthesized from arginine, methionine, and proline found in the kidney, liver, and pancreas , H [0–60], O [0–10], N [0–5], ring unsaturated double bond (RDB) [0–15], and the mass accuracy error was within 5–10−6.The UHPLC-Q-Exactive Orbitrap MS data were processed with Compound Discoverer 3.0 software  for noise cancellation, baseline correction, and normalization to obtain reliable datasets with some information, including p-value (formed from relative intensity). Subsequently, the S-plot with the threshold of VIP > 1.0 and p < 0.05 could be used to screen and identify differential metabolites in the OPLS-DA model. The structures, molecular weights, and codes of differential metabolites were assigned according to the human metabolome database ). Finally, the Spearman correlation analysis and db-RDA analysis were used to determine the relationship between lipid factors and differential metabolites (https://www.bioincloud.tech/ (accessed on 1 February 2023)). Afterward, the processed datasets were added to the SIMCA-P 14.0 software  to perform the principal component analysis (PCA) and orthogonal to partial least-squares-discriminant analysis (OPLS-DA). Among them, PCA was applied to discriminate the separation trends of all groups. OPLS-DA was used to characterize metabolic perturbation of hyperlipidemia. In addition, the S-plot scores were used to screen the differential metabolites of hyperlipidemia treated by genistin combined with other judgment methods, such as variable importance in projection (VIP) (generated in the OPLS-DA mode) and t-test was performed for a two-group comparison. For multiple comparisons, ANOVA was used. p < 0.05 was defined as statistically significant. The statistical analyses and figures were performed using GraphPad Prism 8.0 software . Fasting body weight data were analyzed using one-way ANOVA. The statistical analysis of the data was performed using SPSS 22.0 software . An unpaired Student’s In this study, the metabolic differences and similarities of genistin in normal rats and hyperlipidemic rats were compared. These results are able to provide the foundation for the metabolic mechanism of genistin in pathological and normal rats. The efficacy results revealed the explicit function of genistin against hyperlipidemia, which has been rarely reported; thus, the results demonstrate the possibility of genistin as a new lipid-lowering agent. The mechanism on genistin in the treatment of hyperlipidemia was preliminarily elucidated using metabolomics. Genistin may treat hyperlipidemia by regulating the level of creatine, which can be produced by the arginine and proline metabolic pathway in vivo. However, there are some limitations of the present study. Firstly, the certain relationship between the differences in metabolic behavior and metabolic pathways of genistin in normal and hyperlipidemic rats and the results of pharmacodynamics is still unclear. Secondly, it is also ambiguous whether there are differences in the levels of this metabolite. Finally, the character of genistin metabolites in the treatment of hyperlipidemia by mediating the level of creatine in vivo should also not be ignored. In summary, the relationship between genistin and hyperlipidemia may be further revealed in subsequent studies.