Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms. Acanthamoeba spp., facilitate their access to susceptible hosts, making Acanthamoeba an opportunistic pathogen to humans and other mammals (Acanthamoeba antigens (Acanthamoeba sp. can cause keratitis (AK), by direct exposure of the eyes to contaminated water or lack of hygiene when wearing contact lenses, posing serious risk of blindness if left untreated  causing an often fatal Acanthamoeba granulomatous encephalitis (AGE) (Free-living amoeba (FLAs) are single-cell ubiquitous protozoa, found in environments such as soil, air, fresh and saltwater, dust, as well as men-built devices such as air conditioning units and cooling towers . The ubi mammals . In factantigens . Acanthantreated . Acanthais (AGE) . Currentis (AGE) .A. castellanii and other FLAs might have imposed selective pressures on microorganisms. Those that adapted and resisted to the ameboid intracellular environment , the causative agent of the fatal meningoencephalitis in immunosuppressed individuals (Histoplasma capsulatum (Hc), extremely resistant to the host’s defense , often associated with candidemia and invasive candidiasis in immunosuppressed individuals  on the surface of eraction . Both leeraction . BesidesA. castellanii and macrophages by assessing their relative impact on the interactions with fungi and fungal survival. As part of our objective, we performed a targeted search of surface proteins present in A. castellanii and murine macrophages to identify possible high affinity ligands and receptors to mannoproteins and fungal cell wall mannans. Therefore, we were able to establish similar relationships between mannose affinity pathways in both phagocytes, which could also evidence the possibility of convergent evolution regarding their interactions with fungal pathogens and fungal adaptation in both models.In this work, we sought to evaluate the importance of MBLs from A. castellanii ATCC 30234  was cultured in Peptone- Yeast Extract- Glucose (PYG)  medium (pH 6.5) containing 20 g/L of peptone, 1 g/L yeast extract and 100 mM glucose, supplemented with 0.4 mM CaCl2, 0.4 mM MgSO4, 2.5 mM Na2HPO4, 2.5 mM KH2PO4, 1 g/L sodium citrate, 0.05 mM Fe(NH4)2(SO4)2) and 1% penicillin/streptomycin  at 28°C  was cultured in Sabouraud medium (pH 5.6) containing 10 g/L of peptone and 40 g/L dextrose (Cn H99 serotype A (ATCC 208821) in a minimum medium (pH 5.5) composed of glucose (15 mM), MgSO4 (10 mM), KH2PO4 (29.4 mM), glycine (13 mM), and thiamine-HCl (3 μM) (Hc G217B (ATCC 26032), in Ham’s F-12 medium (pH 7.2) supplemented with glucose (18.2 g/L), glutamic acid (1 g/L), HEPES (6 g/L) and cysteine (8.4 mg/L)   and Hc G.4 mg/L) . All funA. castellanii trophozoites and RAW 264.7 macrophages were grown for 48 h and the percentage of viability was determined by 0.4% Trypan Blue dye exclusion, as the number of viable (white) × 100, over total cells (white + blue), with values ~100% for both cells (2HPO4 and 1.8 mM KH2PO4) at 4°C, with centrifugation at 250xg for 10 min for amoebas and 400xg for 5 min for macrophages. The cell concentration was adjusted to 25x106cells/mL and surfaces biotinylated by incubating cells with 2 mM Sulfo-NHS-LC-Biotin (ThermoFisher) at 4°C for 30 min , with addition of protease inhibitors , and disrupted with a dounce homogenizer and zirconium beads, for 20 cycles in an ice bath, to obtain the total cell extract. The preparations were centrifuged at 18,000xg for 30 min at 4°C to obtain the clarified total extracts. The protein concentrations in both extracts were determined by the Pierce™ Bicinchonic Acid (BCA) Protein Assay Kit, ThermoFisher, following manufacturer’s instruction. To analyze the protein pattern of the extracts, 10 μg of proteins were resolved by 10% SDS-PAGE, as described elsewhere , with washes at 640xg for 5 min. Then, clarified extracts were incubated with the resins, in a proportion of 2.5 mL resin for every 5 mL of clarified extract (2 mg/mL), for 1 h at room temperature. Subsequently, resins were packed, and fractions of unbound proteins (F1) were collected. Then, the columns were washed with 10 bed volume of DPBS, at a 1 mL/min flow to remove non-specific binding proteins (F2). Thereafter, column-bound proteins were eluted with PBS/Ca+2/Mg+2, plus 2 mM EDTA and 150 mM soluble mannose, and the eluent (F3) was collected in 0.5 mL fractions. The collected fractions F1, F2, and F3 had absorbances monitored at 280 nm, along with quantification by the BCA method, and were evaluated by SDS PAGE and Western blot as described.Clarified surface protein extracts of the phagocytes were purified by affinity chromatography assay, using a mannose column, according to manufacturer’s instruction  . After wAc and RAW 264.7 was scaled up to 96-well microplates previously sensitized with 50 µL of mannose solution (10 µg/mL) for 1 h at 37°C, and subsequently overnight at 4°C. After, the plates were blocked with blocking buffer (SuperBlock™ (PBS) Blocking Buffer, ThermoFisher), for 1 h at 37°C. Thereafter, plates were incubated with 200 µg/mL (50 µL/well) of phagocytes’ clarified extracts in DPBS, for 1 h at 37°C. Posteriorly, plates were washed three times with PBS to remove mannose unbound proteins. Then, mannose-attached proteins were recovered with stripping buffer  for 1 h at 70°C. Next, the content of each well was collected and dialyzed in dialysis cassettes  against PBS/Ca+2/Mg+2 at 4°C, under 200 rpm shaking for three days, with daily buffer exchanges. Then, the mannose-purified surface proteins (MPPs) were collected, identified, and analyzed by Western blot. The protein banding pattern of the MPPs were compared between the distinct phagocytes.The mannose-purification of clarified surface protein extracts of Ac and RAW MPPs were processed for analysis by mass spectrometry. We sought to identify and compare the sequences of mannose-binding proteins from both phagocytes to establish any possible degrees of similarity regarding mannose affinity. For isolation of biotinylated surface proteins, the MPPs were purified using the Dynabeads® M-280 Streptavidin Kit with 100 μg of magnetic beads. The beads were washed with PBS + Tween 20 (0.01%) at pH 7.4, separated using a magnet for 5 min and incubated with biotinylated proteins for 30 min at room temperature under orbital shaking. Beads were washed five times with PBS and dehydrated in a speed vac. Beads were resuspended in Urea (7 M) and Thiourea (2 M), added of 1 M HEPES , followed by 100 mM dithiothreitol (to 10 mM), and incubated for 1 h at 30°C. Iodoacetamide (400 mM) was added to a final concentration 40 mM, with subsequent incubation for 30 min in the dark. Then, 175 μL of ultrapure H2O TEDIA and trypsin solution in 0.1 M acetic acid   were added. Then, peptides were purified in an UltraMicroSpin™columns (Havard Apparatus) and dried in a speed vac. At the end of the process, tryptic peptides were solubilized in 10 μL of 0.1% TFA and about 4 μL of the mixture wwere loaded onto an in‐house packed column (15 cm × 75 μm) filled with 3 μm ReproSil C18 resin (Dr. Maisch GmbH) using the Nano LC-Ultra nano liquid chromatography system (Eksigent Technologies) (ologies) .® analytical column (75 μm × 20 cm) packed with Reprosil-pur C18-AQ 3 μm resin (Dr. Maisch). For each sample, peptides elution used a linear gradient of solvent solution ACN 95%, 0.1% TFA from 5% to 20% for 85 min, 20%-40% for 22 min, 40%-95% for 5 min and 95% for 8 min, using a flow rate of 250 nL/min. Mass spectra were acquired in a positive mode top 10 DDA (Data-Dependent Acquisition) method. MS1 scan was acquired in an Orbitrap analyzer set for a 350-1800 m/z range, 60000 resolution (at m/z 400) with a minimal signal required of 10000, isolation width of 2.0. The 10 most intense ions were fragmented by collision-induced dissociation (CID) at 30 NCE  with a dynamic exclusion of 30s. The acquired spectra were processed using the peptides search engine BSI PEAKS® X+ 10.5 (Bioinformatics Solution Inc.) for peptide identification. The considered parent mass error tolerance and fragment mass error tolerance were both set to 0.6 Da and 0.5 Da, with precursor mass search type monoisotopic. The maximum missed tryptic cleavages was set to two, and one non‐specific cleavage was allowed. Carbamidomethylating was set as fixed modification (MW = 57.02 Da) and as variable modifications, methionine oxidation (MW = 15.99 Da) and lysine Sulfo-NHS-LC-Biotin conjugation (MW = 339.16 Da) were allowed. The A. castellanii trophozoites  and Mus musculus  databases were downloaded at www.uniprot.org as for April 2021. The considered false discovery rate was set at 1%. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE , for LC-based separation, coupled to a nano-ESI LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher). Peptide separation was performed in NanoAccquity system equipped with C-18 ReproSil 5 μm resin (Dr. Maisch) and a New Objective PicoFrithe PRIDE  partner de novo peptides was obtained from the Peaks®X+ software and analyzed in the DeNovo Sequencing (PepExplorer) of the PatternLab for Proteomics (http://www.patternlabforproteomics.org). The PepExplorer was set up against the same target-decoy sequence database of Ac and RAW as described above, according to instructions informed elsewhere (Ac), minimal identity of 0.8, a minimum peptide size of 5 amino acids (for M. musculus), and a denovo cut off score of 90, for both. After analysis, the dynamic report showed a list of identified proteins and the corresponding peptides based on sequence alignments that were compared between samples for the determination of proteins with affinity to mannosylated residues. Proteins abundances were calculated according to the number of spectra of all peptides identified and validatedby de novo sequencing a specific protein divided by the sum of all counted spectra for each of the candidate proteins (https://david.ncifcrf.gov). To detect similarities between A. castellanii and M. musculus MPPs sequences identified, we performed a BLASTp search  for 1 h at 37°C. After three washes, the biotinylated clarified extracts were serially diluted (200 to 0.09 µg/mL) in blocking solution and added to wells for 1 h at 37°C. Non-biotinylated clarified extracts of both phagocytes were used as negative controls. Thereafter, the plates were washed three times and incubated with an alkaline phosphatase-streptavidin conjugate (SouthernBiotech) diluted at 0.5 µg/mL in blocking solution, for 1 h at 37°C. The wells were washed three times with TBS-T and reactions developed with p-nitrophenyl phosphate , at 37°C for 15 to 30 min. The absorbances were read at 405 nm on a microplate reader  .Likewise, MPPs binding performances were evaluated under similar indirect ELISA, as previously described, and reactions compared between both phagocytes and to clarified total extracts.6 yeasts/well) and blocked with blocking solution for 1 h at 37°C. Subsequently, plates were incubated with the serially diluted MPPs of both phagocytes, alkaline phosphatase-streptavidin conjugate, and upon incubation with substrate, absorbances were measured as previously described. Non-biotinylated clarified extracts were used as negative controls. For the flow cytometry and fluorescence microscopy assays, yeasts were washed three times and blocked with 2% BSA in PBS for 2 h at room temperature with shaking. Upon washes, 107 yeasts were incubated with clarified or MPPs (100 µg) diluted in 1 mL PBS/Ca+2/Mg+2 plus 2% BSA and incubated for 2 h at room temperature. After three washes with PBS/Ca+2/Mg+2, yeasts were incubated with the streptavidin-Alexa 488 conjugate (ThermoFisher) at 5 μg/mL blocking solution for 1 h at room temperature, in the dark, and subsequently washed three times. As a positive control for the binding to mannosylated components on the fungal cell wall, yeasts were incubated with ConA-TRITC  for 1 h at room temperature, in the dark. After washing, yeasts were incubated with 0.5 µg/mL of Uvitex 2B  for 30 min at room temperature in the dark, with three further washes with PBS/Ca+2/Mg+2. The yeasts were examined using an Axio Imager microscope , using a 495 nm filter, appropriate for the fluorochrome used, at 100x. In addition, the preparations were analyzed in a FACSCalibur  and the intensity of FL1+ cells (for binding to streptavidin-Alexa 488) and FL2+ cells (for binding to ConA-TRITC) were recorded, Yeasts were also incubated with either only non-biotinylated clarified extracts or fluorophore conjugates, as negative controls  diluted in PBS, for 1 h at room temperature. After five washes, the yeasts were pre-incubated with 50 µg/mL of MPPs from either phagocyte for 1 h at room temperature. Following three washes with PBS, yeast concentrations were adjusted and added to phagocytes at a multiplicity of infection (MOI) of 5 fungi: 1 phagocytic cell in a crossed-way: (i) yeasts pre-incubated with MPPs of RAW 264.7 interacted with amoeba and (ii) yeasts pre-incubated with MPPs of Ac interacted with macrophages, for 30 min and 2 h, under appropriate conditions for each phagocytic cell. The stipulated interaction times were based on information from live-microscopy experiments, for early adhesion and late interaction  was calculated:To confirm the similarity of recognition to fungal mannosylated residues by phagocytic cells, their crossed-inhibiting effect on fungal interaction was evaluated. eraction . After, 5 cells/well) in 24-well plates under the appropriate growth conditions. Yeasts were washed, NHS-Rhodamine stained as described, and diluted in the required media for each phagocyte (PYG or DMEM) with or without 100 mM soluble mannose, at a MOI of 5 fungi: 1 phagocytic cell. Plates were incubated for 30 min and 2 h  and internalization (Φintratio) ratios describe respectively the proportion of adhered and internalized fungi relative to the total number of interacting fungi. Symmetrized adhesion (Φadhsym) and internalization (Φintsym) indices reveal the overall probability of a fungus to be respectively adhered or internalized by a host cell. All parameters were compared between the mannose treated versus untreated and between both phagocytes for early adhesion and late interaction events.The Interaction ratio (Φh) describes the ratio of host cells that has at least one interacting pathogen. The adhesion (Φadh5 cells/well), incubated overnight, and yeast added at a MOI of five fungi: one phagocyte in the presence or absence of 100 mM soluble mannose. Initially, interactions occurred for 2 h to allow phagocytosis of the majority of yeasts, as described in previous procedures  upon interaction inhibition with mannose. Phagocytic cells were plated , diluted in 1 mL of glucose buffer , for 30 min at 37°C, in the dark . After wwww.graphpad.com). Comparisons between three or more groups were made by two-way ANOVA, with subsequent Tukey’s or Dunnett’s multiple comparisons test. Comparisons between two groups were made using the Student t-test. Values of p ≤ 0.05 were considered statistically significant. Each experiment was repeated at least three times.Statistical analyses were performed using GraphPad Prism version 8.00 for Windows . The maximum binding capacity was higher for macrophages (Bmaxmacrophage= 1.55) versus Ac MPPs (Bmaxamoeba= 0.88), confirming the higher affinity.Upon purification, the evaluation of MPPs’ affinity to mannose  and late (2 h) events  events Figure 6Ac was observed for Ca . Regarding macrophages, within the first 30 min, soluble mannose affected RAW-fungi association, to Ca and Hc . At 2 h, mannose inhibition was less impacting, but it still impaired the phagocytosis of Ca and Hc , at 24 h upon internalization. The absolute CFU numbers obtained were normalized by the symmetrized internalization (phagocytic) index, to estimate relatively the number of viable pathogens that were internalized by phagocytes. For Ac and RAW, the samples were processed for identification by mass spectrometry. In the Ac MPPs pool, 43 proteins were identified and relatively quantified  and glururonoxylomanogalactan  polymers, along with some mannoproteins that modulates the host’s immune response system, whereas the external α-1-3-glucan plays a major role in the immune escape and fungal virulence  and MBL- Pathogen Recognition Receptors (PRRs) of the C-tyle lectin receptors (CLRs) family- are active participants in the interaction between fungi and macrophages . In the Acanthamoeba is a possible common ancestor of macrophages, as these phagocytes, besides sharing similarities on cell morphology and ultrastructure, biology, and phagocytosis, also display conserved pathogen´s recognition mechanisms. The MBLs identified in fungal recognition by both amoeboid and macrophage surfaces, belong to a ConA-like super family proteins, suggesting the possibility of convergent evolution  and MBP1 (Q6J288), as well as the lectins Mrc1- Macrophage mannose receptor 1 (Q61830), and Mrc2- C-type mannose receptor 2 (Q64449), in addition to the participation of other proteins with transmembrane domains in the recognition of mannose in both models. The alignment between these proteins displayed conserved regions in the carbohydrate binding domains, mainly between the transmembrane proteins MBP1, Mrc1 and Mrc2. It is possible to justify the complexity of RAW macrophage receptors, as already observed in results throughout this work, by the presence of eight Lectin C-type domains in each of the structures of Mrc1 and Mrc2 (Q61830 and Q64449), compared to a short Con-A like domain in MBP and MBP-1 (L8GXW7 and Q6J288). However, the abundant presence of highly conserved residues among these proteins leads to the proposal of evolution of this interaction pathway through the recognition of mannosylated residues. Probably, in simpler organisms, such as amoeba, the interaction Acanthamoeba can harbor and supply new coronaviruses to the environment, contributing to the infection of susceptible hosts  in humans, currently considered by the World Health Organization (WHO) as a global public health emergency . We knowle hosts . Likewishis host . Acanthapidemics .Due to the fundamental role of MBLs in the specificity and the high affinity recognition of universal antigens by phagocytic cells, there is a need to expand the knowledge, with comparative studies of how these lectins recognize microorganisms, specially pathogenic fungus, in order to provide subsidies to reduce the pathogen’s impact on subsequent mammalian infections and promote approaches for future therapeutic interventions and mycological diagnosis, the main challenges in the current scenario.http://www.ebi.ac.uk/pride; PXD031121.The datasets presented in this study can be found in online repositories. The link to the repository and accession number can be found below: All authors contributed to conception and design of the study. MF, SM, DG, CR, LH, LR, and AG performed the experiments. MF, SM, DG, and AG organized the database. MF, SM, DG, and AG performed the statistical analysis. All authors designed experiments and participated in scientific discussions. MF, SM, LN, and AG wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.AG was supported by grants from the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico  and Fundação Carlos Chagas de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ E-26/202.696/2018).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


An oversupply of nutrients with a loss of metabolic flexibility and subsequent cardiac dysfunction are hallmarks of diabetic cardiomyopathy. Even if excess substrate is offered, the heart suffers energy depletion as metabolic fluxes are diminished. To study the effects of a high glucose supply, a stably glucose transporter type 4 (GLUT4)-overexpressing cell line presenting an onset of diabetic cardiomyopathy-like phenotype was established. Long-term hyperglycaemia effects were analysed. Rat cardiomyoblasts overexpressing GLUT4 (H9C2KE2) were cultured under normo- and hyperglycaemic conditions for long-term. Expression profiles of several proteins were compared to non-transfected H9C2 cells (H9C2) using RT-qPCR, proteomics-based analysis, or Western blotting. GLUT4 surface analysis, glucose uptake, and cell morphology changes as well as apoptosis/necrosis measurements were performed using flow cytometry. Additionally, brain natriuretic peptide (BNP) levels, reactive oxygen species (ROS) formation, glucose consumption, and lactate production were quantified. Long-term hyperglycaemia in H9C2KE2 cells induced increased GLUT4 presence on the cell surface and was associated with exaggerated glucose influx and lactate production. On the metabolic level, hyperglycaemia affected the tricarboxylic acid (TCA) cycle with accumulation of fumarate. This was associated with increased BNP-levels, oxidative stress, and lower antioxidant response, resulting in pronounced apoptosis and necrosis. Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycaemia resulted in metabolically stimulated proteome profile changes and metabolic alterations on the TCA level. Diabetic cardiomyopathy (DC) is characterised by marked structural and morphological changes at the myocardial level, resulting in left ventricular dysfunction . It deve2+ signalling and titin phosphorylation are also observed, providing a beneficial scenario for the development of HF , 2 mM GSH , and 2 mM MG  were mixed and conserved at 37 °C for 10 min prior to being incubated with samples. A total of 245 μL of blank or test reagent was added to the protein. The increase in absorbance at 240 nm was measured over time and the activity was calculated in relation to the amount of protein used. The absorbance at 240 nm was measured over a period of 30 min at a measuring interval of 1 min and at a temperature of 37 °C. The absorbance of the blank and the reaction mixture were measured based on the blank, and the reaction mixtures were determined in triplicates. In this experiment, the photometric measurement generated hemithioacetal in situ and it was performed in a microplate reader . The enzyme activity of GLO1 can be calculated with the following formula:Glo1 activity was measured in H9C2KE2 cells according to Arai et al. . BrieflyA240 nm/min (Test)—Measured value minus corresponding blank value;A240 nm/min (Test − 1)—Measured value minus corresponding blank value 1 min before A240 nm/min test;V—Volume in mL per well;D—Dilution factor formed from the volume of the protein sample in relation to the total volume of the assay;−1 ∗ cm−1;∆ε240 nm—Extinction coefficient: 2.86 µmol ∗ mLM—Total amount of protein used (mg) per well;d—Layer thickness when measured in 96-well microtitre plate (0.7 cm).6 cells (>2 µg of protein) [g, 4 °C, 10 min) and the supernatant was transferred to another reaction tube. Protein concentration was determined via Bradford assay. Digestion of proteins into peptides was carried out. In brief, 10 µg of lysate were added to the digestion buffer (50 mM ammoniumbicarbonate). Samples were reduced with 15 mM dithiothreitol for 30 min at 56 °C and alkylated with 5 mM iodacetamide for 30 min in the dark at room temperature. Digestion was carried out over night at 37 °C, using trypsin as protease (ration 1:50). Digestion was stopped by acidification. Prior to mass spectrometry, the peptide concentration was determined by amino acid analysis and 200 ng of peptides were taken for the measurements [m/z and a resolution of 30,000. From each full scan, the Top 20 ions were selected for collision-induced dissociation (CID) fragmentation, with a normalised collision energy of 35%. The dynamic exclusion was set for 30 s. The subsequent data analysis was carried out using MaxQuant (v.1.6.10.43) [Cell lysis was carried out as described using approx. 1–2 × 10protein) . Cell peprotein) . The resurements . Mass spurements . Briefly6.10.43) . The int6.10.43) . iBAQ vav/v) NP-40 , 0.25% (v/v) Na-Deoxycholat , 150 mmol/L NaCl , 1 mmol/L EGTA , 1 mmol/L Na3VO4 , 1 mmol/L NaF , and 50 mmol/L Tris , pH 7.4). The cells were manually detached using a cell scraper. An ultrasound  treatment was applied 3 times, providing the samples with a temperature shock on liquid nitrogen. Then, the samples were centrifuged at 3200× g for 10 min in 4 °C. Protein content was measured by bicinchoninic acid assay  using bovine serum albumin  as a standard curve. Equal amounts of total protein  were loaded on 4–12% Bis-Tris gel  for electrophoresis, and transferred onto nitrocellulose membranes . Precision Plus Western C Standard  or precision Plus Standard Dual colour  was used as a molecular weight marker. Membranes were blocked by 5% non-fat dried milk  or 5% BSA in Tris-buffered saline , according to the antibody needs, for 1 h at room temperature. Then, they were washed and incubated overnight at 4 °C with anti-alpha Tubulin ; anti-Fumarate Hydratase ; anti-GAPDH ; anti-GLO1α ; anti-GLUT4 ; anti-Nrf2 total ; anti-p-Nrf2 ; anti-PDH ; anti-PDH phospho ; anti-PGC1 alpha ; anti-Pyruvat Carboxylase ; anti-Succinat Dehydrogenase B ; and anti-ß Actin  antibodies. Following incubation, membranes were washed and incubated with secondary ECL-anti-rabbit-HRP linked antibody  or HRP-goat anti-mouse Ig . Antibodies were diluted in 0.5% BSA/TBST  or dry milk powder solution, depending on antibody needs. HRP substrate Western Bright chemiluminescent Substrate Quantum  was used and recorded by a Charge Coupled Device (CCD)-camera system .All cells were initially washed with PBS . Then, proteinase inhibitor cocktail  and phosphatase inhibitor  was diluted in RIPA buffer . Oligonucleotide sequences are available in the Cells were washed with ice-cold PBS and detached using an RLT buffer  containing β-mercaptoethanol  for cell lysis; they were then transferred to QIAshredder columns . An RNeasy Fibrous Tissue Kit  was used for purifying total RNA and a NanoDrop 2000  was used for quantifying RNA concentrations. After reverse transcription, purified cDNA was determined via quantitative real time PCR using StepOne Plus  with fluorescent SYBRg), and suspended again in 0.5 mL HBSS buffer. Fluorescence intensity was measured by flow cytometry  [Cells were washed twice with Hank’s buffered saline solution  and 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA)  was added. Cells were incubated at 37 °C for 30 min in the dark. Then, cells were harvested using trypsin-EDTA , suspended in HBSS buffer, centrifuged  .Cells were double-stained with an annexin A5–FITC kit and propidium iodide (PI)  according to the manufacturer’s instructions. The fluorescence was immediately analysed by flow cytometry . The proportion of apoptotic or necrotic cells was calculated as the percentage of the number of annexin V- or propidium iodide (PI)-positive cells .g for 10 min to remove insoluble materials. Then, the supernatant was collected and deproteinised with a 10 kDa MWCO spin filter prior to addition to the reaction. Next, the reaction solution was made by addition of 50 μL of the supernatants and 50 μL of the master reaction mix. This mixture was incubated for 30 min at room temperature in the dark. Fluorescence was measured using a microplate reader . The relative level of pyruvate in all groups was calculated and normalised to protein concentration. The control group was assigned a value of 1, and the hyperglycaemic group was then calculated relative to the control group [Pyruvate formation was quantified using a Pyruvate Assay Kit . Cells were homogenised in 4 volumes of pyruvate assay buffer and the samples were centrifuged at 13,000× ol group .g for 10 min to remove insoluble materials. Then, the supernatant was collected. Next, the reaction solution was made by addition of 40 μL of the supernatants and 40 μL of the master reaction mix. This mixture was incubated for 30 min at room temperature in the dark. Absorbance at 450 nm was measured using a microplate reader . The relative level of fumarate in all groups was calculated and normalised to protein concentration. The control group was assigned a value of 1, and the hyperglycaemic group was then calculated relative to the control group [Fumarate formation was quantified using a Fumarate Assay Kit . Cells were homogenised and the samples were centrifuged at 13,000× Cells were centrifugated at 1500 rpm for 10 min at 4 °C and the supernatant was collected and quantified following the manufacturer’s protocol from the BNP-32 rat ELISA kit . A microplate reader  was used for reading the samples with a wavelength of 450 nm. Correction of optical imperfections by subtracting readings at 570 nm was performed. After the measurements, the results were obtained based on the standard curve values and the multiplication of the dilution factor.t-test or Mann–Whitney test, respectively. Namely, for protein expression of GLUT4, glucose cell consumption, fumarase levels, protein expression of GAPDH, protein expression of PDH1α, protein expression of Nrf2, and protein expression of PGC1α, an unpaired t-test for normally distributed data was applied. For GLUT4 surface, NBDG levels, GLO1 activity, pyruvate carboxylase and pyruvate levels, SDHB, fumarate, pNrf2, ROS, apoptosis, and necrosis, a Mann–Whitney test for abnormally distributed data was applied. All of these analyses were performed using GraphPad Prism version 9.0.0 for Windows . p-values < 0.05 were considered as statistically significant. N describes independent biological experiments.Results of the experimental studies are reported as mean ± SD. Group comparisons among glucose concentration and cell type were analysed by one-way ANOVA followed by Tukey’s multiple comparison post-test. A Shapiro-Wilk test was applied for checking the normality distribution. Direct comparisons between KE2 and WT cells in different conditions were analysed by unpaired


ABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4). Binomial analysis for damaging missense or loss-of-function variants identified the ABCA1 and LDLR genes at genome-wide significance. In conclusion, whole-exome sequencing of individuals with low HDL-C showed the burden of damaging rare variants in the ABCA1 and LDLR genes is particularly high and revealed numerous occurrences in HDL candidate genes, including many genes identified in genome-wide association study reports. Many of these genes are involved in cancer biology, which accords with epidemiologic findings of the association of HDL deficiency with increased risk of cancer, thus presenting a new area of interest in HDL genomics.Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4–36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. We found 120 occurrences of probably damaging variants  among 45 of 104 recognized HDL candidate genes. Those with the highest prevalence of damaging variants were  Clinical arteriosclerotic coronary heart disease (CHD) is a multifactorial disorder. Circulating lipid and lipoprotein levels, notably, triglycerides (TGs), low density lipoproteins (LDLs), and high density lipoproteins (HDLs), are independent risk factors, and other components such as TG-rich remnants and prebeta-1 HDL play important roles too. Here we report the results of a research discovery genomic study designed to understand better the genetic causes of inherited hypoalphalipoproteinemia, low levels of plasma HDL-cholesterol (HDL-C). Over forty years ago, epidemiologic studies established that low levels of HDL-C are independently associated with an elevated risk of CHD and carotid disease , 3, 4. THDL also has a signaling role that is important to endothelial and platelet function, lymphocyte trafficking, and angiogenesis that involves HDL-bound sphingosine 1-phosphate , 11, 12.2) of HDL-C concentration in plasma is high, at about 0.67 (APOA1), ABCA1, lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), and hepatic lipase (LIPC) (PCOLCE2) (WWOX) (LPL), for example, that result in a sizable elevation of TGs are associated also with decreased HDL-C. Much of the lowering of HDL-C here is due to the well-known entropically driven transfer of cholesteryl esters, facilitated by CETP, from the core of HDL particles to TG-rich lipoproteins  . AdditioPCOLCE2)  and WW d) (WWOX) . Genome-) (WWOX) , 26, 27.proteins . This trWe performed whole-exome sequencing on 204 individuals with low levels of HDL-C. These were selected from 21,639 individuals in the UCSF Genomic Resource in Arteriosclerosis (GRA) , 30 for The sequencing data were analyzed by two separate approaches. Firstly, statistical gene burden testing was used to identify potentially causal rare variants, particularly from novel genes. Secondly, after preliminary filtering to identify rare coding variants, we filtered against two lists of candidate genes. The first list consisted of 594 lipid metabolism related genes, and this included all 23 lipid-related genes sequenced by Geller and colleagues in their study of subjects with low HDL-C . Our lisOur aim in these studies was to provide a valuable resource for future cardiovascular research by compiling a list of rare gene variants that have a high likelihood of being functionally damaging and in many cases clearly pathogenic. We believe this study is unique in applying exome sequencing specifically to a cohort with HDL deficiency. Others have used targeted sequenced of limited numbers of candidate genes, and we have compared our findings to these previous studies , 31.A total of 204 individuals (70 women and 134 men) who had been recruited into the GRA , 30 wereBlood samples were collected, after an overnight fast, in tubes containing 0.1% EDTA. Genomic DNA was extracted using the Wizard purification kit . Plasma was obtained after centrifugation at 3,000 rpm for 20 min at 4°C. Levels in plasma of total cholesterol (TC), HDL-C, and TG were measured using an automated chemical analyzer  as previously described , 33. HDLGenomic DNA samples were sequenced at the Yale Center for Genome Analysis (n = 192) or at the UCSF Genomics CORE (n = 12) as previously described , 38.Samples sequenced at Yale were exon-captured using IDt xGen target capture kit followed by 99 base-paired-end sequencing on the Illumina platform.Samples sequenced at the UCSF were exon-captured by Roche NimbleGen SeqCap EZ library probe, and the captured libraries were sequenced on the HiSeq2500. Processing of image files was performed using a standard protocol. Raw image files were analyzed and converted to base calls by real-time analysis using the recommended default settings. Real-time analysis output base call files (∗.bcl) were converted to FASTQ files with consensus assessment of sequence and variation using bcl2fastq pipeline.http://broadinstitute.github.io/picard/) (v2.5.0) was used to sort mapped reads into coordinate order and to ensure all mate-pair information was properly updated. Picard was also used to mark duplicate and low-quality reads, defined as those with low mapping quality score, that were unpaired or unmapped, or that failed a platform/vendor quality check. Five callers were then used for variant calling: Genome Analysis Tool Kit (Blue Collar Bioinformatics (Bcbio v1.0.0) was used to run the QC pipeline. We used Burrows-Wheeler Aligner  (BWA v0.Tool Kit  (GATK v2Tool Kit  (v1.0.2.Tool Kit  (v1.3.1)Tool Kit   >40; a read depth (DP) ≥20; a genotype quality (GQ) of ≥60 if called by GATK, GATK haplotype caller, or Freebayes; variants with >1 caller. A random selection of 12 with only 1 caller were all shown to be artifacts by Sanger sequencing. Annovar  version databaseFor binomial analysis, variants were called using GATK Haplotype Caller and annotated using Annovar. High-quality variants which passed GATK variant quality score calibration with a read depth (DP) ≥8, a genotype quality score (GQ) ≥20, a mapping quality (MQ) ≥40, at least three supporting reads, and not falling in the low-complexity regions were kept . Rare vaFor the candidate gene analysis approach, we evaluated the NGS data after the post-variant calling analysis to determine the degree to which individual variants were causative, firstly in 594 candidate genes including those in lipid metabolism pathways plus GWAS hits . This isBecause this was a research discovery study, and we were more interested to look at the overall deleterious gene burden in this cohort with low HDL-C, and it was not designed to provide individual clinical assessments, we did not strictly follow the American College of Medical Genetics (ACMG) guidelines . Rather,i’ denotes the ‘ith’ gene and ‘N’ denotes the total number of damaging dominant variants. The de novo mutability for each type of variant in each gene was calculated based on trinucleotide contexts as described by Samocha et al.  gene ontology analysis  to funct−3 in 1000 Genome and DECIPHER as well as an in-cohort frequency ≤10% were kept.Copy number variations were called using XHMM (eXome-Hidden Markov Model) software . GATK DeP-values were calculated using an unpaired t-test for parametric variables and using Fisher’s exact test for categorical variables.Lipid, lipoprotein, demographic, and clinical data were analyzed using PASW Statistics for Apple Macintosh . Continuous variables were checked for normality, and those with skewed distributions mathematically transformed prior to testing. Body mass index (BMI), TC, and TG were log-transformed and LDL-C square root transformed. Clinical and other characteristics of the study participants are presented in The participants, as self-assessed, were primarily of white European ancestry (76.5%) but included those of Hispanic, East Asian, South Asian, and African-American descent. The self-assessed ethnicity percentages agree very closely with those determined by principal component analysis (Data not shown). LPL p.G215E) (ABCA1 (n = 20), LDLR (n = 15), LPA (n = 14), CYP1A1 (n = 12), ABCC1 (n = 11), PDIA2 (n = 11), ABCC3 (n = 9), STAB1 (n = 9), ABCC2 (n = 8), ACACB (n = 8), APOBEC1 (n = 8), CPS1 (n = 8), OSBPL1A (n = 8), BRCA2 (n = 7), CD36 (n = 7), PCCB (n = 7), ABCA8 (n = 6), AGPAT2 (n = 6), APOB (n = 6), BCMO1 (n = 6), CREBBP (n = 6), LRP1 (n = 6), and MYL5 (n = 6). A selection of rare variants, 48 in total, were all confirmed to be real by Sanger sequencing.Among the whole cohort of 204 participants, after filtering against 594 lipid metabolism candidate genes , we detep.G215E) . There wABCA1 variants among 38 (18.6%) of the 204 individuals studied, with 20 of the variants considered to be potentially damaging or pathogenic. These 20 mutations (one person carried two ABCA1 variants) are listed in ABCA1 variants, when compared to noncarriers, had a lower level of LDL-C . There were no other significant differences in lipid or lipoprotein measurements, including prebeta HDL .We detected a total of 34 rare ABCC1, ABCC2, or ABCC3 transporter genes (P = 0.689).Participants with deleterious variants among one or more of the er genes , when coLDLR mutations on lowering HDL-C among these two groups.Among the 104 HDL-C candidate genes , there wABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), and ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4) , but the frequency here (gnomAD MAF 0.0176) is above the inclusion cutoff we used for rare heterozygous variants and the damaging prediction score was borderline at the 80th percentile cutoff. However, the frequency was considerably higher in our cohort (MAF 0.0466), with 17 heterozygotes and 1 homozygote. Data relating to this LCAT SNP are included in LPL mutations, p.Asp36Asn (TG 295 mg/dl) and p.Gly215Glu (TG 406 mg/dl). The first of these has an equivocal entry in ClinVar, and the second is listed as pathogenic.The 11 HDL genes with the highest occurrence of damaging variants were  (n = 4) . All the in LCAT . None haLILRA3 gene (leukocyte immunoglobulin–like receptor A3) in a person of Japanese ancestry. These were p.Leu131fs  and c.86-1G>C .Two homozygous damaging mutations (frameshift and acceptor splice mutants) were discovered in the LDLR gene among 14 participants. One variant was found in two individuals, and one other carried two variants. The subjects with these variants are listed in LDLR variants are potentially deleterious mutations, with two having borderline damaging prediction evaluations and equivocal ClinVar entries. There were two frameshift, two nonsense, and one deletion mutation. The 12 individuals with LDLR mutations unequivocally classified as either pathogenic or probably damaging each have an LDL-C above 160 mg/dl and are among those with subphenotypes 2 or 3  and of TC . There were no significant differences in other lipid or lipoprotein measurements, including prebeta HDL . Among the 47 individuals with elevated LDL . There were two mutations in each of ABCG5, APOA1, HPR, and IRF2BP2.We found a total of 14 different rare variants in the s 2 or 3 . Those wated LDL . The LDLLPL and three LRP1 mutations and two each in the LMF1, APOA1, GCKR, PPARA, INSR, PEPD, CYP26A1, ATG4C, and CAPN3 genes. Of note, in this respect, no damaging variants were seen in three key genes: APOA5, APOC2, or GPIHBP1.For the 101 individuals with elevated levels of TG, we filtered against 61 TG candidate genes . We found 32 potentially damaging variants in 21 genes among 31 individuals . There wABCC1, ABCC2, and ABCC3  . In the ClinVar record for this variant (rs80358257), it is described as pathogenic and linked to Niemann-Pick type C disease.As part of our post variant calling analysis, we filtered for all genes with MAF <0.05 for homozygosity of damaging variants. Of note, there were two individuals with a damaging homozygous mutation  in the  SCARB1) , an HDL ABCA1, LDLR, HK3, and CFTR genes with genome-wide statistical significance  heterozygous D-Mis or LoF variants identified the ificance . This wate genes . ABCA1 ite genes .Fig. 3EnHK3 and CFTR) are less convincing than those for ABCA1 and LDLR because of the inflation in the Q–Q plot.Hexokinase 3 (HK3) phosphorylates intracellular glucose to produce glucose-6-phosphate, the first step in most glucose metabolism pathways. Cystic fibrosis transmembrane conductance regulator (CFTR) is also known as an ATP-binding cassette, subfamily C, member 7 (ABCC7). The results for these two genes  software. A total of 49 occurrences of duplications  is a very likely cause of low HDL-C in one subject. This person has the phenotype of abetalipoproteinemia , a disorder caused by defects in this gene .In this research discovery study, we aimed to gain a better understanding of the genetic basis of low levels of plasma HDL-C. Binomial analysis revealed four genes to carry potentially damaging rare variants (MAF ≤ 0.001) with genome-wide statistical significance among our cohort with low levels of plasma HDL-C. These were ABCA1 and LDLR. A total of 38 among the 204 studied carried rare ABCA1 variants in our study (18.6%). This is somewhat lower than the 26.9% found in a recent report , that is, 9.3% of the cohort of 204.For the potentially damaging variants we discovered that were on our list of lipid metabolism–related candidate genes, the most prevalent were among t report , though LDLR variants (12 of 204 participants) than a recent similar study of subjects with low HDL-C (4 of 202) . Among 18 kindred with FH and known LDLR gene deleterious mutations in our GRA collection, we have analyzed the baseline levels of LDL-C and HDL-C for a total of 128 genotyped individuals. Among these were 54 wild type, 69 heterozygotes, and five homozygotes. LDL-C values were, respectively, 127 ± 30 mg/dl (range: 63–203), 295 ± 77 mg/dl (range: 168–509), and 817 ± 102 mg/dl (range: 719–925) (ANOVA P < 0.001). Corresponding values for HDL-C were 54.6 ± 16.7 mg/dl (range: 25–107), 47.6 ± 15.4 mg/dl (range: 20–99), and 34.4 ± 8.0 mg/dl (range: 25–45) (ANOVA P = 0.005). The HDL-C values are presented as box plots in We detected a higher prevalence of damaging, rare  of 202) . Howeverl range: –203, 295ABCA1, there were numerous other probably damaging or pathogenic variants found among other HDL candidate genes. Notable were the nine occurrences in STAB1, eight in each of OSBPL1A and CPS1, and six in each of LRP1 and ABCA8. STAB1 codes for stabilin 1, a multifunctional scavenger receptor . It is a multifunctional endocytic receptor thought, pertinently here, to be involved in the clearance of chylomicron remnants by the liver  is widely expressed, notably in the heart, skeletal muscle, and liver and codes for a lipophilic xenobiotic transporter. It has recently been shown in mice to be responsible for the efflux of cholesterol and taurocholate across the hepatic sinusoidal membrane  in 18 subjects at a frequency (MAF 0.047) here higher than in GnomAD (0.017), ExAC (0.018), or TOPMED (0.017). This SNP has been previously reported to associate with HDL levels in two Danish studies , almost all the apoA1in plasma is found in the form of small prebeta HDL particles , it often lacks sensitivity. More advanced techniques such as Linked-Read whole-genome sequencing allow for much higher success in structural variant detection as we have recently found in other studies , 87. A nAll relevant data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


POLE) in endometrial cancer (EC) and to investigate the effects of EDMs on tumor cell behavior and catalytic activities of Polϵ.This study was aimed to profile hotspot exonuclease domain mutations (EDMs) of the DNA polymerase ϵ gene (POLE sequencing was performed in tumor tissue samples from patients with EC to identify hotspot EDMs. Bioinformatics tools were used to select the potential pathogenic EDMs. The association of EDMs with the clinical outcomes of patients was assessed. EC cells were transfected with wildtype POLE or POLE variants to examine the effects of the EDMs on EC cell behavior, including cell cycle, migration, and invasion. Co-immunoprecipitation was employed to obtain FLAG-tagged wildtype and mutant catalytic subunits of Polϵ, followed by the assessment of polymerase and exonuclease activities.POLE hotspot EDMs of EC. Patients in EDM group had significantly better clinical outcomes than the rest of the cohort. Compared with wildtype POLE, overexpression of POLE variants promoted cisplatin resistance, G0/G1 cell cycle arrest, and cell migration and invasion in EC cells. Overexpression of POLE variants significantly increased the abundance of 3’-OH and upregulated the expression of DNA mismatch repair genes in HEK293T cells. Compared with wildtype Polϵ, Pol ϵ mutants exhibited undermined polymerase and exonuclease abilities in the presence of mismatched nucleotides in HEK293 cells.In addition to previously reported P286R and V411L, R375Q and P452L were identified as novel, and deleterious POLE hotspot EDMs in endometrial cancer. These hotspot EDMs are associated with the malignant behavior of endometrial cancer cells in vitro and favorable prognosis in patients, suggesting that POLE affects a wide range of cellular processes beyond DNA replication and proofreading.We characterized the of hotspot exonuclease domain mutations in the DNA polymerase ϵ gene and identified P286R, V411L, R375Q, and P452L as pathogenic  POLE)-encoded Polϵ is one of the three DNA polymerases that are required for eukaryotic genome replication. The 140 kDa N-terminal catalytic subunit of Polϵ harbors 5’–3’ polymerase and 3’–5’ exonuclease activities, playing critical roles in DNA replication and proofreading in the newly synthesized DNA strand (POLE exonuclease domain mutations (EDMs) occur in 7–12% of endometrial cancer cases, with P286R and V411L representing the most common pathogenic POLE EDMs, followed by S297F, A456P, and S459F  samples. Among these mutations, there were 49 POLE EDMs, comprising 1 nonsense/stop-gain mutation and 48 missense mutations, in 32/138 (23.2%) samples. To analyze the association of the EDMs with clinical outcomes, we divided the patients into EDM (n = 32), non-EDM (n = 37), and non-mutation (n = 69) groups and performed Kaplan-Meier analysis. The EDM group had a significantly better PFS than the non-EDM  and non-mutation groups  and a significantly better OS than the non-mutation groups  , V411L (n = 3), S297F (n = 2), G304D (n = 2), R375Q (n = 2), P452L (n = 2), and S459F (n = 2). Among them, P286R, V411L, S297F, and S459F had been reported as the most common To characterize the hotspot EDMs, we stably silenced POLE in HEK-293T cells. HEK293T cells were used to optimize the knockdown of POLE  and decreases in the proportions of S-phase cells , compared with overexpression of the wildtype POLE  of patients carried POLE EDMs. Kaplan-Meier analysis showed that patients with EDMs had significantly better PFS and OS than the rest of the cohort, suggesting a complex relationship between POLE mutations and the pathogenesis of endometrial cancer. To reveal the molecular mechanism underlying the role of POLE mutations in endometrial cancer, we selected four hotspot EDMs, including P286R, V411L, R375Q, and P452L, and assessed their pathogenicity based on the effects on catalytic activities of Polε and tumor cell behavior.Endometrial cancer subtypes HGCC, SC, and CCC exhibit higher proportions of metastases and recurrences as well as poorer 5-year survival rates than low-grade endometrial endometrioid carcinomas . In thisPOLE mutation impairs genomic stability by disabling proofreading. For example, Shinbrot et al. have shown that compared with wildtype POLE, the P286R variant essentially inactivates proofreading, whereas the V411L variant reduces exonuclease activity without completely abolishing it  from January 2006 to December 2015, including 160 high-grade endometrial cancer (HGEC), 73 serous adenocarcinoma (SC), and 46 clear cell adenocarcinoma (CCC). Clinicopathological characteristics and paraffin-embedded cancer specimens of these patients were analyzed retrospectively. After excluding the patients with diagnostic errors, incomplete clinicopathological information, or low-quality/quantity cancer specimens, 88 HGEC, 29 SC, and 29 CCC tumor samples were eligible for inclusion. Progression-free survival (PFS) was the time from operation to recurrence or progression. The overall survival (OS) was the duration from operation to death or the last follow-up. This study was approved by the Institutional Ethics Committee of Fudan University Shanghai Cancer Center. A written consent was obtained from each patient.2 at 37 °C.Human embryonic kidney (HEK)293T cells and endometrial adenocarcinoma cell lines (HEC-1A and AN3CA) were purchased from the Cell Bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences . HEK-293T, HEC-1A, and AN3CA cells were maintained in DMEM , McCoy’s 5A , and RPMI 1640 (Corning) medium, respectively, supplemented with 10% fetal bovine serum (Gibco) and 1% ampicillin/streptomycin  in a humidified atmosphere of 5% COPOLE sequencing were summarized in Genomic DNA was extracted from paraffin-embedded tissue samples using a QIAamp DNA FFPE tissue kit  following the manufacturer’s instruction. The DNA quality was analyzed using an Agilent 2100 bioanalyzer instrument . The primers for POLE was constructed using I-Tasser  was cloned into a pHB-CMV-MCS-EF1-Puro vector as previously described (via PCR. The recombinant FLAG-protein was expressed in 293T cells. Co-immunoprecipitation (Co-IP) was performed to isolate the FLAG-conjugated catalytic subunit using the FLAG-beads  following the manufacturer’s instruction. The FLAG-tagged protein band was visualized using Coomassie blue staining and Western blot analysis with an anti-FLAG antibody . The FLAG-tagged wildtype and mutant catalytic fragments were purified from the immunoprecipitated complexes using the 100 kDa Amicon®Ultra-0.5 filter .The N-terminal half of escribed . The plaBsaJI restriction site sequence (bold and underlined) for 2 min at 92°C in the presence of 150 mM NaAc.All oligonucleotides were purchased from Genewiz (China). The DNA polymerase and exonuclease substrates P50/T80a and P51T/T80 were prepared by annealing P50   at 60°C for 100 min. The reactions were stopped by adding the same volume of nucleic acid electrophoresis buffer. Samples were analyzed by electrophoresis in 8 M urea-12.5% polyacrylamide gels at 130 V for 55–60 min. Images were acquired using a GE Typhoon laser scanner .To determine the polymerase activity, 100 nM substrate was incubated with 6.25 nM purified wildtype or mutant Polϵ, dNTPs, and PCR buffer at 30°C for 30 min. To determine the exonuclease activity, the mixture was digested with POLE  or shRNA against POLE or POLE variants. Cells were treated with different concentrations of cisplatin  for 48–72 h. MTT  was added to each well following the manufacturer’s instruction. After 4 h of incubation at 37 °C, the MTT-containing medium was removed, and 150 μL of dimethyl sulfoxide (Sigma-Aldrich) was added to each well. The absorbance values were measured using a microplate reader  at a wavelength of 490 nm.HEC-1A cells were seeded in a 96-well plate at a density of 4,000 –6,000cells/well and cultured overnight. Cells were transiently transfected with lentiviral vectors expressing wildtype POLE or POLE variants. A total of 2.5×104 cells were loaded into a matrigel-coated upper chamber filled with serum-free medium. The lower chamber was filled with 600 μL medium containing 20% FBS. After incubation at 37°C for 24–48 h, cells remaining in the upper chamber were removed with a cotton swab. The invading cells adhering to the lower surface were fixed and stained with crystal violet (0.1%). The stained cells were counted in 3 randomly selected fields under an inverted light microscope  at 200× magnification.Cell invasion was measured using the Transwell chamber assay. HEC-1A and AN3CA cells were transiently transfected with lentiviral vectors expressing wildtype 5 cells per well and cultured overnight. Cells were transfected as above mentioned. A 10 µL micropipette tip was used to generate a 2 mm-wide scratch line in the cell monolayer. Cells were allowed to migrate for 6, 12, 24, 48, or 72 h. Images were captured under an inverted light microscope .Cell migration was measured using the wound healing assay. Cells were seeded in a 6-well plate at a density of 5 × 105 per well. Cells were transiently transfected with lentiviral vectors expressing wildtype POLE or POLE variants. Cells were collected at 48 h after transfection, resuspended in 300 µL phosphate-buffered saline (PBS), and fixed with 100% ethanol overnight at -20 ˚C. After propidium iodide staining, cell cycle was analyzed using a MoFlo XDP flow cytometer .POLE-knockdown HEC-1A cells were seeded in a 6-well plate at a density of 2×10The abundance of mismatched base pairs was measured using T7 endonuclease I mismatch cleavage assay. Cells were fixed with 4% formaldehyde for 30 min, followed by incubation with T7 endonuclease 1 (Vezyme) at 37 °C for 45 min to cleave the mismatched base pairs. The produced 3′-OH DNA ends were stained with TUNEL using a TUNEL kit  following the manufacturer’s instruction. The samples were immediately analyzed on the flow cytometer.Total RNA was isolated using TRIzol  according to the manufacturer’s instructions. cDNA was synthesized using a reverse transcription kit (Sangon Biotech) following the manufacturer’s protocol. Amplification was performed using a SYBR master mixture (Sangon Biotech) and gene-specific primers  and centrifuged at 13,000 rpm for 15 min at 4 ˚C. The supernatant was collected, and the protein concentrations were determined using a Bradford method. The protein samples were separated on a 10% SDS-PAGE gel and then transferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk at room temperature for 90 min, the membrane was incubated with primary antibodies against POLE , MLH1 , MSH2 , E-cadherin , N-cadherin , Slug , Snail , Cyclin A , Cyclin B , Cyclin B1 , Cyclin D1 , CDK4 , CDK6 , P16 , P21 , or β-actin  overnight at 4°C. After incubation with a horseradish peroxidase-conjugated secondary antibodies (mouse or rabbit) (Sigma-Aldrich) for 1.5 h at room temperature, the protein bands were detected using a Tanon gel imaging system . The results were quantitatively analyzed using a GelCap software.t test. A P value < 0.05 was considered statistically significant.Data were expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS 19.0  or Flowjo 7.6.2 . The graphs were generated using Graphpad 5.0 . Differences between groups were compared using one-way analysis of variance, followed by Student’s The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Fudan University Shanghai Cancer Center. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.WT and ZJ designed the concept and experiments. JW and JM collected the data. RB, YR, and BS participated in the project design and manuscript discussion. GY and HW revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Science Foundation of China (No. 82002758 to WT) and the Yangfan Plan of Shanghai Science and Technology Commission (No. 22YF1404700 to ZJ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tgfbr1; Alk5), in macrophages, resulted in increased HO, with a trend toward decreased tendinous HO. To bypass the effect seen by altering the receptor, we administered a systemic treatment with TGF-β1/3 ligand trap TGF-βRII-Fc, which resulted in decreased HO formation and a delay in macrophage infiltration to the injury site. Overall, our data support the role of the TGF-β1/ALK5 signaling pathway in HO.Transforming growth factor–β1 (TGF-β1) plays a central role in normal and aberrant wound healing, but the precise mechanism in the local environment remains elusive. Here, using a mouse model of aberrant wound healing resulting in heterotopic ossification (HO) after traumatic injury, we find autocrine TGF-β1 signaling in macrophages, and not mesenchymal stem/progenitor cells, is critical in HO formation. In-depth single-cell transcriptomic and epigenomic analyses in combination with immunostaining of cells from the injury site demonstrated increased TGF-β1 signaling in early infiltrating macrophages, with open chromatin regions in TGF-β1–stimulated genes at binding sites specific for transcription factors of activated TGF-β1 (SMAD2/3). Genetic deletion of TGF-β1 receptor type 1 ( Transforming growth factor–beta (TGF-β) signaling is essential for normal tissue-specific regeneration and aberrant wound healing. The response to injury following a traumatic event can be divided into hemostasis, inflammation, proliferation, maturation, and remodeling . In eachAlk5 deletion in monocytes has been shown to inhibit proinflammatory and promote antiinflammatory macrophage markers of expression  further corroborated the autocrine effect of TGF-β receptor signaling on macrophages, which was not observed with deletion of Alk5 in MPCs (T2Hoxa11CreER). Due to some additional effects seen in macrophage receptor deletion, we opted to treat upstream of the receptor by targeting the TGF-β ligands. Treating injured mice systemically with a ligand trap, TGF-βRII-Fc, which blocks both TGF-β1 and TGF-β3 ligand, resulted in attenuated HO formation and in delayed macrophage infiltration. Taken together, these findings suggest that macrophage ALK5 signaling potentiates aberrant bone formation and that pharmacological inhibition of TGF-β1 with the ligand trap TGF-βRII-Fc is a potential therapeutic agent to prevent HO.In the current study, we investigated the impact of TGF-β signaling in macrophages and the MPCs in the local wound environment. Utilizing a mouse model of HO and single-cell transcriptomic and epigenomic analyses, we found specific increases in TGF-β–stimulated gene expression as well as open chromatin regions at p-SMAD2/3 binding sites in TGF-β1–stimulated genes in macrophages and MPCs. Overall, the findings are suggestive of an autocrine effect of TGF-β1 in macrophages. Targeted deletion of https://doi.org/10.1172/jci.insight.144925DS1). Both MPCs and macrophages express the receptor for and can respond to TGF-β1; therefore, we analyzed TGF-β1 signaling by p-SMAD3 specifically in these populations. MPCs, marked by PDGFRα, had an increased percentage of cells that were p-SMAD3 positive at 3 weeks (89.7% ± 3.8%) compared with 1 week  (P = 0.8657) (Increased TGF-β activity has been shown to be present in human HO tissue . When TG 0.0819) . Alterna 0.8657) , A–C. InPdgfrα and Prrx1  or Tgfbr3 . Ma. MaPdgfrand Cd14 . The MPCnd Trem1 . Analysid Kif26b . Becaused Kif26b . In the d Kif26b . Conversr Tgfbr3 . In factTgfb1, Tgfbr1, Tgfbr2, corresponding to our scRNA-Seq findings  on cells from the HO site of day 0 (uninjured) and 7 after injury. We evaluated the accessibility of chromatin around the known DNA binding sequence for p-SMAD2/3 in the genes for the TGF-β ligands and receptors. In the Macs, there was openness in promoter regions near SMAD2/3 binding sites for findings . The MPCfindings . Next, wfindings . TogetheBased on our previous data, Mac infiltration into the HO site is at its peak 3 days after injury ; therefoTo get a better understanding of the putative autocrine TGF-β signaling that might be occurring in these early infiltrating Macs, we performed receptor-ligand pairing analysis. To do this, a list of all potential ligands and receptors was adapted from humans to mice  and was Alk5. To do this, we targeted the TGF-β1 receptor type 1, TGF-βRI, encoded by Alk5, using fl/flAlk5 mice. To delete Alk5 in MPCs, we chose to cross our fl/flAlk5 mice with the T2Hoxa11CreER mouse line, creating T2 Alk5fl/flHoxa11CreER mice . We found that +/– Alk5fl/flLysMCre mice developed increased volume of HO compared with –/– Alk5fl/flLysMCre littermate controls . In our tenotomy model of HO, ectopic bone forms at 2 distinct anatomic sites: i) growing off the calcaneus (bone-associated HO) and ii) growing off the proximal cut end of the tendon  in both the endosteum and periosteum  to determine if the bone-associated effect observed could be circumvented. Before use in vivo, we first tested the equilibrium binding constant of the TGF-βRII-Fc using surface plasmon resonance. Our results showed TGF-β1 and -β3 had a r TGF-β2 . Subsequr TGF-β2 . OverallAlk gene family members, in our model of HO  by subcutaneous injection for 3 weeks. We chose to treat these mice for the 3 weeks based on our previous paper using inhibitors of BMP signaling receptors, similar el of HO . Furtherel of HO . HO siteel of HO . Safraniel of HO . Next, w+ cells that are nuclear p-SMAD3+) in the TGF-βRII-Fc–treated group compared to vehicle-treated controls  (P = 0.1628) as well as a decrease in cell count of PDGFRα+ cells  and a decrease in the percentage of total cells that were PDGFRα+  that were Ki-67+ was not different with TGF-βRII-Fc treatment compared with control  . Further+ cells  cell counts and overall percentage of cells across time points, independent of treatment group (P = 0.03), with a trend toward decreased monocyte numbers earlier with treatment  with the added benefit of being a more feasible treatment option.Selective genetic deletion of ALK5 in  peptide . Unlike lization . The Macignaling . Alternaignaling , 61. Anofl/flLysMCre Alk5 mice. Previous research has shown inhibition of ALK5 alone can impair monocyte migration toward TGF-β1  treatment, we noted decreased proximal/tendinous HO. Our IF and flow cytometry data looking at the HO anlagen site 1 week after injury demonstrated there were decreased monocyte/Macs at the site with ligand treatment, suggesting a delay in monocyte/Mac migration to the injury site. Though not statistically significant, a similar decreasing trend of HO was seen in the d TGF-β1 . Our finIt is well documented in the literature that TGF-β1 is an important factor in chondrogenesis , cartilaGiven how the literature is so demonstrative toward TGF-β’s prochondrogenic effects, it raises the question of why in our study the loss of TGF-βRI signaling specifically in MPCs resulted in no significant change in HO formation after traumatic injury. Data presented by Wang et al. where TGF-βRI functions in cartilage to block BMP signaling in resting growth plate chondrocytes do support our findings . TherefoCurrently, effective management of HO is limited, with prophylactic NSAIDs or radiation therapy offering only modest benefit . SurgicaWe demonstrate that Macs are the critical target of TGF-βRI signaling for aberrant wound healing after traumatic injury. We also show that systemic treatment with TGF-βRII-Fc modulates TGF-β signaling upstream of the TGF-βRI, impairing monocyte/Mac migration, such that HO formation is attenuated, particularly in the proximal region. These data signify that targeting TGF-β ligands and Mac autocrine signaling after traumatic injury is an effective future therapeutic target to improve wound healing and prevent aberrancies such as muscle fibrosis  or HO.Mice were housed in standard conditions. All animals used were C57BL/6 background mice. C57BL/6 mice were purchased from The Jackson Laboratory (000664). All mice received preoperative and 48 hours postoperative subcutaneous buprenorphine (0.06 mg/kg) for analgesia. Animals were anesthetized with inhaled isoflurane. Mice received 30% total body surface area partial-thickness dorsal burn. The dorsal burn was induced using a metal block heated to 60°C in a water bath and applied to the dorsum for 18 seconds continuously. Tenotomy was performed by transection of the left Achilles tendon. Animals were assigned to the vehicle control or ligand trap group. Vehicle control was 1× PBS and ligand trap was muTGFbRII-mFc, shortened to TGF-βRII-Fc, supplied by Acceleron Pharma, Inc. Treatment began the day of surgery (day 0). Mice were administered vehicle or ligand trap (10 mg/kg) subcutaneously twice weekly for 3 weeks. Mice were euthanized for experiments at 1, 3, or 9 weeks after injury for experimental analysis.T2Hoxa11CreER and LysMCre were bred in-house with fl/flAlk5. The T2Hoxa11CreER line was obtained from Deneen Wellik at the University of Wisconsin, Madison, Madison, Wisconsin, USA. These were then crossed with the fl/flAlk5 line that was obtained from Katherine Gallagher at the University of Michigan to produce T2 Alk5fl/flHoxa11CreER mice. To induce the deletion of Alk5, T2 Alk5fl/flHoxa11CreER mice were placed on tamoxifen chow for 3 weeks when they were 5 weeks of age. LysMCre mice were crossed with fl/flAlk5 mice, both from The Jackson Laboratory, to generate fl/flLysMCre Alk5. Littermates of both crosses were used as controls. Mice underwent the burn/tenotomy injury described above. Legs were harvested at 9 weeks and MicroCT analysis was performed. CD169Cre mice were obtained from Riken Group (RBRC06239)  to obtain double-heterozygous mice. They underwent our injury model and tissue harvested for IF histology.RC06239) , 73. CD1Single-cell data replicates from day 0 (uninjured), 3, 7, and 21 postinjury were taken from GEO GSE126060 . We consTwo analyses were set up in Seurat 3.1.4 , consideTo perform receptor ligand pairing analysis on our Mac cluster, a list of all potential ligands was adapted from a human ligand-receptor database  for micehttps://github.com/timoast/signac; commit ID fa23843) as previously described by our group using GEO GSE150995 (https://timoast.github.io/sinto/) and deeptools  before the end of the association phase (which is close to a plateau) is plotted against the ligand concentration, which ranges from 0.12 nM to 90 nM for TGF-β2 binding.All analyses were performed with a Biacore T100 instrument . An anti-mouse Fc-specific antibody was immobilized on a Series S CM5 sensor chip through amine coupling by following the manufacturer’s instructions . HBS‑EP buffer  supplemented with 350 mM NaCl and 0.5 mg/mL bovine serum albumin (BSA) was used as running buffer. TGF-βRII-Fc was captured on the experimental flow cell at a density of approximately 50 response units. TGF-β1 and TGF-β3 (0.013–10 nM) and TGF-β2 (0.04–90 nM) were injected in 3-fold serial dilution over the captured protein for 300 seconds, followed by 600 seconds dissociation time at a flow rate of 70 μL/min with buffer blanks injected periodically for double referencing. The chip regeneration was performed with 10 mM glycine pH 1.7. All sensorgrams were processed by double referencing (subtraction of the responses from the reference surface and from an average of blank buffer injections). To obtain kinetic rate constants, TGF-β1 and TGF-β3 data were fit to 1:1 interaction model that includes a term for mass transport using BIAevaluation software . A concentration range of 0.013–1.1 nM for both TGF-β1 and TGF-β3 was used to fit TGF-βRII-Fc binding sensorgrams. The equilibrium binding constant 4 cells per well in F-12K medium (ATCC) supplemented with 10% FBS and incubated overnight. All incubations were at 37°C, 5% CO2, unless otherwise noted. The cells were transiently cotransfected with both the luciferase vector pGL3 (CAGA)12 in which the firefly luciferase gene expression is under the control of the TGF‑β ligand-responsive (CAGA)12 element, and the constitutively active renilla luciferase plasmid pRL-CMV  to normalize for transfection efficiency. This was done by combining 10 μg pGL3(CAGA)12, 0.1 μg pRL-CMV, and 30 μL XtremeGENE9 (Roche) with 970 μL Opti-MEM (Thermo Fisher Scientific) and incubating the mixture for 30 minutes at room temperature prior to adding 24 mL of assay buffer (F-12K (ATCC) supplemented with 0.1% BSA) and applying to the plated cells (500 μL/well) for an overnight incubation. The next day, 3-fold serial dilutions of TGF-βRII-Fc were made in assay buffer in a separate 48-well plate . The final concentration of TGF-β1, TGF-β2, or TGF-β3 added to the corresponding wells in this plate was 640 pg/mL, 480 pg/mL, and 270 pg/mL, respectively. The receptor-ligand mixture was incubated for 30 minutes prior to applying 500 μL/well to the transfected A549 cells. The cells were harvested after an 18- to 20-hour incubation and assayed using the Dual Luciferase Reporter Assay system  to determine normalized luciferase activity expressed as relative luciferase units.The cell-based assay utilizes A549 cells . A549 cells were seeded in 48-well plates at 6.5 × 10n = 2).Legs were decalcified, embedded, and sectioned as previously described by our lab . SafraniIF was done as previously described . BrieflyImage area quantifications were performed in FIJI . The p-SLeft legs were scanned using Bruker SkyScan 1176 MicroCT. Bone volumes were determined utilizing MicroCT imaging . Scans were analyzed using calibrated imaging protocol as previously described by MicroView micro CT viewer  . Bone reFollowing a burn tenotomy injury, at time points of 5, 7, and 14 days, the soft tissue from the posterior compartment between the muscular origin and the calcaneal insertion of the Achilles tendon was dissected out and collected for processing. The tissue was digested for 20–30 minutes in 0.3% type 1 collagenase and 0.4% dispase II (Gibco) in Roswell Park Memorial Institute (RPMI) medium at 37 °C under constant agitation at 180 rpm. The digestions were quenched with 10% FBS in RPMI and then filtered through 40 μm sterile strainers. Specimens were blocked with anti-mouse CD16/32 and subsequently stained using the antibodies Ly6G, CD11b, Ly6C, and/or CD45 listed in t test was used for parametric data, and data are represented by mean ± SEM. Mann-Whitney U test was performed on nonparametric data and represented as median ± interquartile range. For MicroCT, statistical analyses were performed in IBM SPSS Statistics 24 and GraphPad Prism 8. Graphs were created in GraphPad Prism 7 or Microsoft Excel. Shapiro-Wilk test was used to determine the appropriate test. Two-tailed independent Student’s t tests were performed on parametric data at α = 0.05, with P value significance indicated by *. Mann-Whitney U test was performed on nonparametric data at α = 0.05, with P value significance indicated by #. One-way ANOVA with Brown-Forsythe test for correction as well as Dunnett’s multiple comparison was performed on data at α = 0.05.For microscopy quantifications, statistical analyses were performed in GraphPad Prism 8, where associated graphs were also generated. Shapiro-Wilk was applied to assess for normality. Student’s 2-tailed Guide for the Use and Care of Laboratory Animals: Eighth Edition .All animal experiments described were approved by the Committee on the Use and Care of Animals at the University of Michigan, Ann Arbor (PRO0007930), and University of Texas Southwestern, Dallas (2020-102949). All animal procedures were carried out in accordance with the guidelines provided in the Prior publication: NKP, MS, CDH, SL, RK, and BL are authors on an abstract submitted to Plastic Surgery Research Council 63rd Annual Meeting, May 19, 2018; Birmingham, Alabama, USA; for which a small portion of the preliminary data was part of the abstract.NKP was responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing. JHN and MS were responsible for conception and design, collection and/or assembly of data, and data analysis and interpretation. SM was responsible for single-cell data analysis and interpretation and manuscript reading and editing. CAP and ALS were responsible for data analysis and interpretation and manuscript writing. CDH was responsible for data analysis and interpretation. SL was responsible for conception and design. KRP was responsible for snATAC-Seq analysis and pathway analysis. RK was responsible for conception and final approval of the manuscript. ACB, JAG, RN, HAR, and NL were responsible for data analysis and interpretation. KV was responsible for data analysis and study design. AKH was responsible for conception and design, data analysis and interpretation, collection and/or assembly of data, manuscript editing, and final approval of the manuscript. BL was responsible for conception and design, data analysis, and final approval of the manuscript.


Constructing the composite electrode materials with vanadium substitution is a scientific endeavor to boost LFP’s power capacity. Herein, a series of xLiFePO4·yLi3V2(PO4)3 (xLFP·yLVP) composites were fabricated using a simple spray-drying approach. We propose that 5LFP·LVP is the optimal choice for Li-ion battery promotion, owning to its excellent Li-ion storage capacity , strong machining capability (compacted density of 1.82 g·cm−3) and lower raw material cost consumption. Furthermore, the 5LFP·LVP||LTO Li-ion pouch cell also presents prominent energy storage capability. After 300 cycles of a constant current test at 400 mA, 75% of the initial capacity (379.1 mA·h) is achieved, with around 100% of Coulombic efficiency. A capacity retention of 60.3% is displayed for the 300th cycle when discharging at 1200 mA, with the capacity fading by 0.15% per cycle. This prototype provides a valid and scientific attempt to accelerate the development of xLFP·yLVP composites in application-oriented Li-ion batteries.The development of LiFePO The commitment to carbon neutrality and the implementation of a low-carbon economy in the world have become the key to the sustainable development of human society, given the massive population explosion and incremental demand for new or renovated infrastructure ,2. The m4 (LFP) has received substantial attention from academia and industry, because of its relatively high theoretical energy density (580 W·h·kg−1), structural integrity during the Li-ion extraction/insertion process, and low raw cost consumption [+ fast migration in order to address this issue. One of them is to modify LFP by combining it with Li3V2(PO4)3 (LVP), which has three-dimensional pathways for Li+ mobility [Cathode material as a key component determines many aspects of LIBs, including the Li-ion storage capability, lifespan, cost, and safety ,10. In psumption ,12. At psumption ,14. Manymobility ,16. Howemobility ,18,19,204Ti5O12 (LTO) is an appealing anode candidate for high-power LIBs, because of its fast Li+ transport capability, zero volume change during charging cycles, and alleviated lithium deposition [In addition to selecting a cathode material with a high Li-ion diffusion coefficient, pursuing high-power LIBs is still dependent on several factors. Firstly, the anode material should be carefully considered. Liposition ,22. Secoposition ,24, the position .To develop an application-oriented energy storage device with a high power density, material engineering optimization was conducted to manufacture a series of xLFP·yLVP composites including LFP·LVP, 3LFP·LVP, 5LFP·LVP, and 8LFP·LVP utilizing a simple spray-drying approach. 5LFP·LVP (5LFVP) stands out as a strong candidate cathode material for the high-power LIBs, due to its comprehensive superiorities of Li-ion storage capability, machining capability, and raw material cost consumption. Subsequently, a 5LFVP||LTO Li-ion pouch cell was exploited to demonstrate its significant energy storage capability. According to the characterization of Li-ion extraction/insertion capability, this prototype Li-ion full cell presents a reasonable and scientific attempt to promote the development of xLFP·yLVP composites in application-oriented Li-ion batteries.The information on the crystallographic structure of synthetic products was obtained using a D8 Advance X-ray diffractometer  with a non-monochromatic Cu K α X-ray source. The features of amorphous carbon in xLFP·yLVP composites were determined using the Renishaw INVIA micro-Raman spectroscopy system . The Vario EL cube CHNSO elemental analyzer  was employed to examine the carbon content. The information of the valence state was evaluated by XPS measurement via an Ultra DLD with a monochromic Al X-ray source. The Microtrac S3500 laser  particle size analyzer was used for the particle distribution. To determine the morphology of xLFP·yLVP, the JEOL-7100F  with energy-dispersive spectroscopy  was employed. The detailed structural information was obtained using a JEM-2100F microscope .−2. The LAND CT2001A battery test systems were used to characterize the electrochemical performance. To evaluate the electrochemical behavior, cyclic voltammetry (CV) curves were collected using a CHI 760e electrochemical workstation. To achieve a reasonable pouch-cell capacity design, the half cells of LTO||Li were also assembled using the same method described above to assess the specific capacity and cyclability of anode material.Li-ion half cells were assembled for estimating the difference in Li-ion storage capabilities between various xLFP·yLVP composites. In this study, the Li-ion half cells were divided into five components: Li foil, power-type electrolyte, separator films from Celgard company, 2025 coin-type battery shell, and electrode piece with 14 mm diameter. Each electrode piece had a weight ratio of 80:10:10, corresponding to xLFP·yLVP active materials, conductive carbon nanotube paste  and PVDF binder, respectively. The current collector was made of carbon-coated aluminum foil. The mass loading of all xLFP·yLVP electrodes was controlled between 2.0 and 3.0 mg·cmThe pouch-cell fabrication allows a rational way to demonstrate the utilization of xLFP·yLVP composites in application-oriented Li-ion batteries. To meet the requirements of practical application, the ratio of electrode materials, for all types of xLFP·yLVP cathodes and LTO anode, was set to 90.5 wt.%. The contents of carbon nanotube conductive paste, super P, and PVDF binder in the pouch cells were adjusted subsequently to 1.5 wt.%, 2 wt.%, and 6 wt.%, respectively. Rolls of separator films were used to prevent direct contact between the cathode and anode pieces. Here, we proceeded with the control on a capacity ratio of the anode to cathode, which could fluctuate within the range 1–1.05. Moreover, the roller machine with 15 tons of pressure was performed to achieve the appropriate compacted densities of all types of xLFP·yLVP cathodes and LTO anode pieces. A battery test system of TITANS THCX-IO/5-96-M-A was used to estimate the electrochemical property of pouch cells.In this work, material engineering optimization was conducted to manufacture the xLFP·yLVP composites using a simple spray-drying approach. Here, the molar ratio of x/y was varied from 1:1, 3:1, 5:1, to 8:1 with corresponding products denoted as LFVP, 3LFVP, 5LFVP, and 8LFVP, respectively. 2O5, 1.009 kg of FePO4·2H2O, 0.465 kg of LiOH·H2O, 0.621 kg of NH4H2PO4, 0.5 kg of C6H12O6, and the milling solvent (10 L of methanol and 5 L of water) was mixed to form a uniform precursor liquid. The milling time was set at 2 h. The resulting slurry was then transformed into the precursor powder via the continuous heating exchange from the spray-drying equipment. Finally, the abovementioned precursors were sintered in N2 atmosphere at 700 °C for 12 h, allowing consistent xLFP·yLVP composites. Other xLFP·yLVP composites could be obtained by adjusting the ratio of V2O5 to FePO4·2H2O in the same synthesized procedure. Moreover, according to the theoretical content calculation of LFP and LVP in xLFP·yLVP composites  and monoclinic Li3V2(PO4)3 (JCPDS No. 01-072-7074) [−1 and 1590 cm−1 could be indexed to D and G bands, respectively, indicating graphite defects and stretching vibration from sp2 carbon atom bonds [D/IG ratio for LFVP was only 1.02, lower than that of 8LFVP (1.15), suggesting that the amorphous carbon embedded on the LVP surface was inclined to the higher electron conductivity, or that vanadium may have had a positive impact on the formation of graphitized carbon. Furthermore, XPS measurement was carried out to obtain the valence state information of key elements in xLFP·yLVP composites. All peaks labeled in s, P 2p, P 2s, C 1s, V 2p, O 1s, and Fe 2p. p with two peaks appearing at around 709 eV and 723 eV for Fe 2p3/2 and Fe 2p1/2, respectively [p peaks increased gradually with the increase in the raw material proportion of FePO4·2H2O.72-7074) ,27,28,29om bonds . Howeverectively ,32. In pThe SEM picture of as-prepared xLFP·yLVP samples in 3+/V4+ from the LVP electrode can induce three pairs of peak signals, which are shown in the figures except for a/a′. The positions of redox peaks are shifted gradually, associated with enlarged electrochemical polarization, when the scan rates are increased. Furthermore, it is well documented that the electrochemical kinetics is determined by the Li-ion diffusion coefficients, which can be estimated from CV results. Here, the a/a′ and b/b′ redox peaks were chosen to calculate the Li-ion diffusion coefficients (D+Li) of LFP and LVP, respectively. p vs. v1/2, rooting from the Randles–Sevchik equation [+Li values of xLFP·yLVP composites were obtained and plotted as a contrast diagram (+Li of LFP in 3LFVP was 2.94 × 10−10/3.1 × 10−10 cm2·S−1, faster than that of other xLFP·yLVP composites. Meanwhile, the D+Li of LVP presented similar trend, with 3LFVP having the highest D+Li value of 8.27 × 10−9/7.2 × 10−9 cm2·S−1. Therefore, it is supposed that 3LFVP could provide better reversibility and the fastest Li-ion migration. However, we should note that the Li-ion diffusion rate in LVP exceeded that in LFP by an order of magnitude, but LFP would deliver higher intrinsic specific capacity. The optimal xLFP·yLVP composition would depend on the specific application requirements.To determine the differences in intrinsic kinetics, different scan rates of CV measurements were carried out on the series of xLFP·yLVP composites, and the curves are shown in equation ,40. By c diagram i,j. The −1 for LFVP, 3LFVP, 5LFVP, and 8LFVP electrodes, respectively. Higher specific capacity could be achieved for the 8LFVP electrode at currents less than 2 C. However, when increasing the current rate to 5 C or more, LFVP electrode took the lead due to its high proportion of LVP with a higher ion diffusion coefficient. −1 could be obtained for LFVP, 3LFVP, 5LFVP, and 8LFVP electrodes, respectively. Furthermore, 95.7%, 100%, 97.2%, and 96% capacity retention can be maintained after 1000 cycles, demonstrating excellent structural stability. Although the capacity difference between xLFP·yLVP composites was very small at 1 C, the material energy density varied significantly, which was closely related to discharge medium voltage. As shown in −1. To acquire more information on the electrochemical capacity of as-prepared xLFP·yLVP electrodes, the ultrahigh rate of 20 C was performed to make a critical assessment of the cyclability (−1 and an energy density of 291.3 W·h·kg−1 after 2000 cycles. Significantly, series of xLFP·yLVP electrodes obtained good energy retentions of 77.6%, 86.3%, 88.4%, and 92.1%. Because of the high power capability of xLFP·yLVP electrodes, our effective material design strategy in this work provides an opportunity for improving the rate performance of LFP in application-oriented Li-ion batteries.lability e. As exp50 diameter of 18.66 µm demonstrated an optimal size distribution, which may remedy the interstitial space between particles and achieve an enhanced supporting effect [−3, but a compacted density of only 1.64 g·cm−3. The equivalent two values of 5LFVP were 1.03 and 1.82 g·cm−3, respectively. According to these results, the tap density of an electrode material does not have a distinct impact on the compacted density of electrode pieces, which can be deeply influenced by size distribution.Compacted density is a critical parameter to evaluate whether an electrode material can be used in application-oriented Li-ion batteries. Previous reports claimed that high tap density could lead to higher gravimetric and volumetric energy densities. In other words, electrode materials with high tap density would possess high compacted density, which is not entirely precise. In fact, the compacted density can be determined by a variety of factors, involving the components of electrode pieces, the particle size distribution, and the intrinsic true density of electrode material. Hence, here, we applied a laser particle size analyzer to evaluate the size distribution of xLFP·yLVP. As shown in g effect . As showA concise evaluation of four types of xLFP·yLVP cathode materials was performed on the basis of six aspects: industrial feasibility, cost of raw material, energy density, power density, cycling performance, and machining capability. As shown in 4Ti5O12 with a Spinel structure exhibits superior Li-ion storage capability with long cycling stability at a high rate [Commercialized Liigh rate ,43, ascr−1. Four couples of redox peaks can be clearly observed from the CV curve in the potential range of 1.2–2.8 V (+ ions between the 5LFVP cathode and LTO anode electrodes. It is well documented that assembling Li-ion pouch cells is a more rational way to demonstrate the potential of electrode materials in practical LIBs. As shown in Here, the Li-ion full battery was assembled with 5LFVP as the cathode and LTO as the anode. The electrochemical behavior of 5LFVP||LTO was analyzed by the CV test, in which the scan rate was set at 0.1 mV·s.2–2.8 V a, owing −1, great machining capability with a compacted density of 1.82 g·cm−3, and lower raw material cost consumption. Furthermore, the application-oriented energy storage device of the 5LFVP||LTO pouch cell was exploited to demonstrate the Li-ion storage capability. At both low discharge currents of 400 mA and higher currents of 1200 mA, it exhibited superior electrochemical properties. Significantly, the Coulombic efficiency could be maintained around 100% throughout the charge/discharge progress, implying a great electrochemical reversibility. This prototype provides a reasonable and scientific attempt to promote the development of xLFP·yLVP composites in application-oriented Li-ion batteries. In this paper, material engineering optimization was performed to prepare a series of xLFP·yLVP composites using an uncomplicated spray-drying approach, including LFVP, 3LFVP, 5LFVP, and 8LFVP. Taking all the scientific and practical factors into account, 5LFP·LVP stands out as a strong candidate cathode material in Li-ion batteries, attributed to its excellent power capability with a material energy density of 413.6 W·h·kg