Defective regulation of the alternative complement pathway (AP) causes excessive activation and promotes the inflammation and renal injury observed in atypical hemolytic-uremic syndrome (aHUS). The usefulness of heat-inactivated Factor B (HFB) in reducing AP activation was evaluated in: fluid-phase reactions, using purified complement proteins and Factor H (FH)-depleted serum; and in surface-activated reactions using human endothelial cells (ECs). C3a and Ba levels, measured by quantitative Western blots, determined the extent of fluid-phase activation. In reactions using C3, FB, and Factor D proteins, HFB addition (2.5-fold FB levels), reduced C3a levels by 60% and Ba levels by 45%. In reactions using FH-depleted serum , Ba levels were reduced by 40% with HFB added at 3.5-fold FB levels. The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated ECs. Fluorescent microscopy was used to quantify endogenously released C3, FB, and C5 attached to EC-secreted ultra-large VWF strings. HFB addition reduced attachment of C3b by 2.7-fold, FB by 1.5-fold and C5 by fourfold. Our data indicate that HFB may be of therapeutic value in preventing AP-mediated generation of C3a and C5a, and the associated inflammation caused by an overactive AP. Following the cleavage of FB by Factor D (FD), C3-H2O-Bb complexes are formed2. The Bb in these complexes proceeds to cleave additional C3 to C3b (releasing C3a) to form transient C3bFB complexes. FB is rapidly cleaved by FD (releasing Ba) to produce the C3 convertase, C3bBb. The AP regulatory protein, Factor H (FH) prevents any unwanted C3 convertase formation by displacing either FB or Bb that is bound to C3b or to C3-H2O. In the absence of FH, C3 convertases react rapidly with C3 and FB, resulting in unrestricted generation of C3a and Ba4.The alternative complement pathway (AP) is initiated in the fluid phase when Factor B (FB) binds to hydrolyzed C3  produce both the complement components and the associated fluid-phase and surface regulatory proteins necessary to control complement over-activation12.The fluid-phase generated C3b indiscriminately binds to surfaces through an internal thioester that is exposed in C3 after its cleavage by Bb. The extent of C3b surface deposition and further activation, depends on whether the surface is an activator of the AP. C3 convertase formation on host, non-activating surfaces, are either prevented with the displacement of FB by FH, or bound C3b are inactivated by Factor I (FI) cleavage in conjunction with FH. On foreign surfaces of invading microorganisms, further deposition of C3b near previously formed C3bBb complexes, promotes C5 binding and formation of C5 convertases (C3bBbC3b). Convertases on surfaces with higher densities of C3b preferably bind C5 and induce the formation of terminal membrane attack complexes (C5b-9)14. A major cause of aHUS is deficiency of functional FH resulting from gene mutations or acquired function-blocking antibodies16. Normally, FH prevents fluid-phase AP activation and the formation of activation complexes on host cell surfaces3. Impairment of additional regulatory proteins, including FI, the five FH-related proteins, CD46 (membrane cofactor protein), and CD141 (thrombomodulin) have also been associated with development of aHUS20. Additionally, gain-of-function mutations in C3 and CFB, producing protein complexes more resistant to regulation, have been reported23.Defective regulation of the AP causes excessive activation and promotes the inflammation, microvascular thrombosis, and renal endothelial injury observed in atypical hemolytic-uremic syndrome (aHUS)25. ECs also secrete ULVWF strings in response to stimulation by various agents, including histamine, lipopolysaccharide, and inflammatory cytokines26. In situations of excessive EC stimulation and inadequate cleavage by ADAMTS-13 (the VWF-specific protease), ULVWF strings persist on EC surfaces and participate in the accumulation of complement proteins and activation of the AP27. The AP response is exacerbated in conditions of deficiencies in functional complement regulatory proteins. Many studies have also shown connections between reduced levels of ADAMTS-13 activity in conjunction with AP regulatory dysfunction in patients with aHUS33.To promote hemostasis and thrombosis, ECs secrete multimeric ultra-large von Willebrand factor (ULVWF) in hyper-adhesive long string-like structures that instigate platelet adhesionIn this study, we evaluated the effect of heat-inactivated FB (HFB) as a new type of agent to modulate AP activation. We investigated the use of HFB to reduce AP activation in early fluid-phase reactions; and additionally, to restrict the assembly of C3/C5 convertases on EC-secreted ULVWF multimeric strings.Western blot analysis was used to measure C3a and Ba generated in fluid-phase reactions as indicators of AP activation. Prior to experimental analyses, several protein concentrations of C3, C3a, FB, and Ba were tested using Western blots to determine the range of linear signal detection. Western blot membranes with lanes containing 7.5 to 240 ng/lane of reduced C3 or C3a-desArg (C3a) were analyzed, using fluorescent intensity measurement for both total protein and specific C3/C3a antibody detection. Reduced dilutions of C3 verified that the entire range C3 protein levels were within the linear range of detection using either the total protein stain or anti-C3a antibody detection . C3a lane amounts of 60 ng and below were linearly proportional to signal intensities in both types of measurements  and FD (0.07 ng/µl) were constant, using normal serum ratios, whereas the normal concentration of FB (10 ng/µl) was either decreased by twofold (to 5 ng/µl), or increased by twofold (to 20 ng/µl)  incapable of supporting further activation.The extent of AP activation was quantified on Western blots that included a range of C3a and Ba protein levels (to generate standard curves) in order to interpolate values . Correlation plots show that the generation of AP activation products C3a and Ba decreased linearly with increasing concentrations of HFB until a plateau was reached with 26 ng/µl HFB  and concentrations of HFB ranged from 10.4 to 31.2 ng/µl  . Detection of vitronectin allowed the serum amounts to be visualized in lanes that appeared blank using FH detection antibodies. FH signals were below the limits of detection in each FH-D serum dilution tested, whereas vitronectin detection was positive  in MgCl+2 ions (or Ca+2 ions), further verifying that the C3a and Ba generated in FH-D serum resulted from AP activation . HFB concentrations were 2-, 2.5-, 3-, and 3.5-fold above reaction FB levels of 10.4 ng/µl  were stimulated with histamine and ULVWF strings were secreted from Weibel-Palade bodies rapidly (within 2 min) onto cell surfaces. The antibodies added to detect VWF (15 min) also impaired EC-released ADAMTS-13 from cleaving the surface ULVWF strings. Additionally, the 15 min period allowed endogenous complement proteins to be released in sufficient amounts to measure their attachment to the ULVWF strings. Subsequently, the GMVECs were fixed and antibodies were added to detect C3, FB, C5 and C4 proteins.In order to substantiate that GMVEC-released complement proteins attached to the ULVWF strings, detected intensities for C3, FB, C5 and C4, were measured along a single GMVEC-secreted string. Graphs of the measured intensities versus the length of the ULVWF string were generated. Each complement protein Fig. . The attIn parallel experiments, GMVECs were both histamine-stimulated and stained with VWF antibodies, in the presence of HFB. Detected signals for C3, FB and C5 were also synchronized with VWF detection along the string with the addition of HFB  of the ULVWF strings. These intensity data were compiled from over 800 microscope images. Mean intensity values measured for C3 (1770/µm), C5 (1312/µm), and FB (500/µm) attachment to the ULVWF strings were 16-fold, 12-fold, and 4.5-fold higher than mean intensity values for C4 (109/µm) attachment , FB and C5 was measured with HFB (20 µg/ml) present during histamine stimulation and VWF antibody incubation. FB, after heat-inactivation at 56 °C, retains binding capacity to C3b, although HFB cleavage by FD is either reduced or absent. , FB attachment by 1.5-fold (322/µm) and C5 attachment by fourfold (325/µm). In the presence of HFB, for each FB or HFB attached to the ULVWF strings, there were 2 components of C3 and 1 component of C5. These are the proportions of the C5 convertase complex after C5 binding [(C3bBbC3b)-C5]  and rapid heating. In typical immunoassays, measurement samples are exposed for extended periods to mild conditions (in order to promote antibody binding and substrate detection). These latter conditions also allow further activation. An excess of EDTA added to samples prevents further convertase formation; however existing convertases continue to cleave C3 and generate C3a39). HFB effectively competed with native FB for binding to C3b. Because of heat-induced impaired enzymatic functions, HFB-C3b complexes were unable to promote further activation. . On our Western blots, the migration pattern of HFB indicated that it is composed of dimers and tetramers of FB monomers (93 kDa). Clear bands of HFB are shown in the blot detected for FB in Supplemental Fig. The usefulness of HFB in reducing over-activation of the AP was evaluated in this study. Initial reactions, with constant C3 and FD levels and varying FB concentrations, were used to test the possibility that AP activation could be reduced by lowering FB levels. The addition of HFB effectively lowered the FB concentration because the two proteins compete for binding to C3b. Heat-inactivated FB was prepared from FB that had been purified from human plasma and then exposed to controlled heating at 56 °C. HFB was determined to be inactivated if activation products were not generated in reactions consisting of C3, HFB and FD, without the addition of FB  that allows FB binding and C3 convertase formation. In the fluid phase, FH prevents any unwanted C3 convertase formation by displacing either FB or Bb that is bound to C3b or to C3(H2O). In the absence of FH, the AP in human serum is activated spontaneously and precipitately upon the addition of mM levels of magnesium ions4. Because of this unique property, FH-D serum was used as a reagent to test the effectiveness of HFB in inhibiting AP activation in the presence of the full spectrum of proteins in normal serum (including complement regulators), with the exception of FH . This addition of FH  brought the FH-D serum within the range of FH  observed in many patients with aHUS resulting from heterozygous mutations in FH or FH autoantibodies29. FH-D serum was initially shown to be devoid of FH by blot analysis using samples with twofold the serum amounts that were used in the FH-D serum AP reactions . Although basal levels of C3a and Ba in the FH-D serum preparations contributed slightly, the increased activation probably resulted from the capacity of properdin to bind and stabilize C3/C5 convertases. The short (2–3 min) half-life of C3bBb can be increased tenfold with properdin binding42.In the absence of HFB, AP activation was mostly higher in the FH-D serum-containing reactions than in reactions using purified AP proteins. Levels of C3a (10 ng/ml) and Ba (9 ng/ml), generated in reactions of purified AP proteins, were nearly half the levels produced in FH-D serum upon addition of Mg+2 ions (or Ca+2 ions)  and Ca+2 ions (for C1 complex formation) are present. In contrast, AP activation, specifically FB binding to C3b, requires only Mg+2 ions38.Negligible amounts of C3a and Ba were produced in FH-D serum reactions in EGTA buffer without added MgIn the reactions using FH-D serum (supplemented with low FH levels), HFB reduced Ba generation, whereas C3a generation was mostly unchanged. In the fluid phase and in the absence or insufficient levels of regulatory proteins, C3 convertases (C3bBb), rapidly cleave C3, producing C3b and releasing C3a. HFB competes with native FB for binding sites on newly formed C3b. We propose that HFB is resistant to cleavage by FD, and therefore, C3b-HFB complexes do not produce Bb or generate Ba. Consequently, the C3b-HFB complexes would be incapable of further C3 cleavage; however, C3a would continue to be generated from the previously formed C3 convertases.10 and surface regulatory proteins necessary to control complement over-activation12. EC surface proteins that function to regulate activation include: CD55 (to displace the enzyme subunits of convertases formed on its membrane); CD59 (to prevent polymerization and attachment of C9 into C5b-8 complexes); as well as, CD46 and CD141 (to inactivate cell membrane-bound C3b in association with co-factors FH and FI)43. CD141, also known as thrombomodulin, is present exclusively on EC surfaces and participates substantially in controlling coagulation44.The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated human GMVECs. ECs produce complement components and the associated fluid-phase12. The GMVEC-secreted ULVWF strings remained on EC surfaces because the added detection antibodies against VWF impeded string cleavage by ADAMTS-13 released from ECs12.Fluorescent microscopy was used to analyze and quantify the attachment of complement proteins to the GMVEC-secreted ULVWF strings. The polyclonal antibodies made against human proteins were shown previously to identify each specific complement component and their activated formsThe endogenously released AP components, C3, FB and C5, were bound to the ULVWF strings in numerical ratios consistent with the formation of active C3 convertases (C3bBb) and C5 convertases (C3bBbC3b). There was minimal binding of the classical component C4 to the ULVWF strings, indicating little activation of the classical and lectin pathways. The presence of HFB reduced the binding of C3 (as C3b) and C5 to the ULVWF strings by 2.5-fold and fourfold, respectively. Heat-inactivated FB is resistant to FD cleavage; however, HFB retains binding capacity for C3b. The HFB competes with endogenous FB for binding to C3b and suppresses further cleavage of C3 to activated C3b. This resulted in reduced C3 and C5 convertase assembly on the GMVEC-secreted ULVWF strings.5. Reduced C5 binding, translates into: a decreased number of C5 convertases; fewer C5b-6 complexes ; and reduced amounts of generated C5a. C5a is a potent anaphylatoxin, and among other biological properties, induces the release of histamine and tumor necrosis factor, potentiating inflammation and cell injury47.The addition of HFB reduced C5 binding to the ULVWF strings by fourfold, probably as a result of the reduced amount of C3b-ULVWF attachment (2.5-fold lower) in the presence of HFB. Surfaces with high densities of C3b deposition have an increased affinity for C5 bindingHFB inhibits an early reaction of AP activation and, therefore, may be of therapeutic value in preventing AP-mediated generation of C3a and C5a. This inhibitory function may help to suppress the associated inflammation caused by an overactive AP in aHUS and related disorders.C3 (A113) and C3a-desArg (A119) proteins, ranging from 7.5 to 240 ng/lane, were analyzed by Western blotting under reducing conditions. Membranes were initially stained with Revert 700 Total Protein Stain  and subsequently incubated with rabbit anti-human C3a (A218) plus donkey anti-rabbit IRDye-800CW  for specific detection of C3 and C3a. Parallel experiments, using non-reduced FB (A135) and Ba (A154) proteins ranging from 7.5 to 240 ng/lane, were also analyzed by Western blotting, stained for total protein, followed by specific detection with goat anti-human FB (A235) plus donkey anti-goat IRDye-800CW . Complement proteins and antibodies were purchased from Complement Technology.2). C3 was the last protein added to the 25 µl reaction mixtures. Reaction concentrations of C3 (60 ng/µl) and FD (0.07 ng/µl) remained constant , whereas FB concentrations were 20, 10 and 5 ng/µl. After C3 addition, the tubes were mixed and incubated at 37 °C for 15 min. Reactions were stopped by the addition of 5 mM EDTA and 25 µl of Laemmli sample buffer , followed by heating at 95 °C for 5 min. AP proteins were measured by fluorescent blot analysis using rabbit anti-C3a + donkey anti-rabbit IRDye-800 and goat anti-FB + donkey anti-goat IRDye-680.AP activation experiments were conducted on ice using pre-cooled reagents. C3 aliquots were only used once. AP proteins C3, FB, and FD  were diluted in Tris/EGTA-Mg buffer  was prepared from human plasma-purified FB , inactivated by controlled heating at 56 °C for 30 min using a thermocycler and stored at 4 °C until use.2). C3 was the last protein added to the 25 µl reaction mixtures. Final reaction concentrations were: C3 = 60 ng/µl; FB = 10.4 ng/µl; and FD = 0.07 ng/µl. Final concentrations of HFB were: 10.4, 15.6, 20.8, 26, and 31.2 ng/µl. After C3 addition, the tubes were mixed and incubated at 37 °C for 15 min. Reactions were stopped by the addition of 5 mM EDTA and 25 µl of Laemmli sample buffer , followed by heating at 95 °C for 5 min. AP proteins were measured by fluorescent blot analysis with standard curves for C3a and Ba.AP activation experiments were conducted on ice using pre-cooled reagents. C3 aliquots were only used once. AP proteins C3, FB, FD and HFB were diluted in Tris/EGTA-Mg buffer  under non-reducing conditions. Gels included standard lanes of C3a-desArg (32–1.25 ng/lane) and Ba (20–2.5 ng/lane) proteins for quantification and a pre-stained protein ladder  for approximating size. Gels were transferred using a semi-dry method  onto low-fluorescence PVDF membranes (IPFL 10100). Remaining steps followed protocols recommended by LI-COR using TBS-based Intercept Blocking Buffer  for membrane blocking and antibody dilution. Primary antibodies were diluted in Intercept buffer containing 0.2% Tween-20 and diluent for fluorescent secondary antibodies contained 0.2% Tween-20 plus 0.01% SDS. Ba protein was detected using goat anti-human FB (4.3 µg/ml) plus donkey anti-goat IRDye-680RD . C3a protein was detected using rabbit anti-human C3a (6.9 µg/ml) plus donkey anti-rabbit IRDye-800CW . Dried membranes were scanned on a LI-COR Odyssey DLx Imager (9142-01P), intensities were measured at 700 and 800 nm, and data were analyzed using Empiria Studio software .2 buffer to provide final concentrations of 5 mM MgCl2.Factor H-depleted serum  was prepared from human serum by immuno-depletion of FH. FH-D is supplied in 0.1 mM EDTA and contains normal levels of each complement protein except FH. C3 levels in FH-D serum were estimated at 1200 µg/ml, based on the supplier statement that FH levels of 500 µg/ml is required for 100% AP function, and that in normal serum C3 levels are 2.4-fold higher than FH levels. In the reactions, FH-D serum was further diluted in 0.1 mM EDTA, in order to produce C3 concentrations of 60 ng/µl and 10.4 ng/µl FB. The reactions were supplemented with EGTA/MgClFH levels in FH-D serum were measured by fluorescent Western blot analysis. FH-D serum was diluted 20-. 30-, 40-, 60-, 80-, and 120-fold directly into Laemmli sample buffer containing 5 mM EDTA. FH protein  standards in gel lanes ranged from 15, 30, 45, 60, 75, 90 and 120 ng. Blots were detected for FH using goat anti-FH  plus donkey anti-goat IRDye-680 and for the serum-containing protein, vitronectin, with rabbit anti-vitronectin  plus donkey anti-rabbit IRDye-800CW.Single-use aliquots of FH-D serum were diluted 1:4.8 in 0.1 mM EDTA to produce C3 levels of 250 ng/µl. To initiate AP activation, a constant volume of diluted FH-D serum was added to tubes containing Tris/EGTA-Mg buffer alone, or dilutions of FH protein. Reactions contained 60 ng/µl of C3 and 10.4 ng/µl FB in a total volume of 25 µl. FH protein, diluted in Tris/EGTA-Mg buffer, ranged from 0.78 to 25 ng/µl to produce FH levels equivalent to 3–100% of normal serum levels. Reaction mixtures were incubated for 15 min at 37 °C. The reactions were stopped by the addition of 5 mM EDTA in Laemmli sample buffer and heating at 95 °C for 5 min. Levels of AP activation proteins, C3a and Ba, were measured by fluorescent blot analysis.FH-D serum was supplemented with 3.13 ng/µl FH  to study the effect of HFB on AP activation in FH-D serum. This chosen concentration of FH allowed enough generation of C3a and Ba to assure that C3a and Ba levels would remain within measurable ranges during HFB inhibition of AP activation. Additions of ≥ 12.5 ng/µl FH were found to substantially prevent AP activation , were grown to confluence on gelatin-coated coverslips in MCDB-131 basal medium plus 120 units/ml penicillin; 100 µg/ml streptomycin; 2 mM L-glutamine; 0.25 µg/ml amphotericin and microvascular growth supplement . GMVECs, used at passages 4–7, were removed from tissue culture flasks non-enzymatically, prior to seeding on coverslips, by incubation with 5 mM EDTA in CaOur microscope system consists of a Nikon Diaphot TE300 microscope equipped with CFI Plan Fluor 60 × oil, numerical aperture (NA) 1.4 and CFI Plan Apo Lambda 100 × oil, NA 1.45 objectives, a 10 × projection lens and a Prior motorized stage. Fluorescent images were obtained with a SensiCamQE CCD camera (Cooke) using dual filter wheels (Prior) with single band excitation and emission filters for FITC/TRITC/CY5/DAPI (Chroma). Cell images were captured using IP Lab software version 3.9.4r4  and processed using iVision-Mac Scientific Image Processing version 4.5.5r1 (Biovis.com). Images acquired using the 60 × objective have dimensions of 78 × 58 µm. Calibration bars on images are 10 µm.GMVECs on coverslips (7–15 days after seeding) were washed with PBS and stimulated with 100 µM histamine in 1 ml PBS with or without 20 µg/ml HFB for 2 min. Cells were then stained with rabbit anti-VWF plus chicken anti-rabbit Alexa Fluor (AF)-488  for 15 min, washed with PBS and fixed with 1% p-formaldehyde/PBS for 10 min. Following fixation, the cells were stained with one of the polyclonal goat antibodies against human complement; anti-C3 (A213), anti-C4 (A205), anti-C5 (A220), and anti-FB (A235) (Complement Technology) (diluted 1:100 in PBS containing 1% BSA) plus secondary donkey anti-goat AF-647  antibody at 20 µg/ml for 15 min. These two fluorophore dyes, AF-488 and AF-647, have widely separated, non-overlapping, absorption and emission spectra. Cell nuclei were detected with DAPI  that was included in the mounting medium.The GMVEC-secreted ULVWF strings, detected with rabbit anti-VWF plus chicken anti-rabbit AF-488, were electronically traced as lines in 488 nm (green)-captured images at 60X. The ULVWF string length (major axis) and integrated fluorescent intensity was measured along the line. The x- and y-coordinates of the traced ULVWF line were transferred to the corresponding 647 nm (red)-captured images obtained using goat antibodies against C3, FB, C4 and C4 plus donkey anti-goat AF-647. The fluorescent intensity at 647 nm from each detected complement component was measured and integrated at the transferred line coordinates. Background intensities were measured from identical line coordinates translocated from original positions within the same 647-detected image. The quantity of each complement component attached to the ULVWF strings was expressed as complement component intensity at 647 nm, minus the background intensity at 647 nm, divided by the ULVWF string length in microns. Single channel images have maximum intensity values of 4096. Image dimensions at 60 × are 78 µm × 58 µm, or 688 pixels × 512 pixels, (1 pixel = 0.114 µm).Intensities from 488 nm  and 647 nm  channels were measured along the ULVWF strings from merged images. The x-axis is the length of the VWF string in pixels (100 pixels = 11.4 µm) and the y-axis is the measured intensity. Multiple-channel merged images have maximum values of 256. The background 647 nm data was measured from the same merged image where the x- and y-pixel coordinates for the ULVWF string had been relocated to another position within the image.GraphPad Prism v 9.4.1 software (GraphPad.com) was used for standard curve interpolation, Pearson’s correlation coefficient measurements, and for determining significance of differences using 1-way ANOVA and Dunnett’s multiple comparison tests, and 2-way ANAOVA with Tukey’s multiple comparison with an alpha value of 0.05.Supplementary Information.


This review study reports a useful cyan-emitting phosphor based on CLHAO composition with a garnet structure to have a cyan-to-green emitting color with good energy transfer. It could be employed as cyan filler in warm-white LED manufacturing. Due to its stability, ability to dope with various ions suitable for their desired qualities, and ease of synthesis, this garnet-like compound is a great host material for rare-earth ions. The development of CLHAO cyan-emitting phosphors has exceptionally high luminescence, resulting in high CRI and warm-white LEDs, making them a viable desire for LED manufacturing. The development of CLHAO cyan-emitting phosphors with diverse synthesis techniques, along with their properties and applications in white LEDs, are extensively covered in this review paper.Future generations of solid-state lighting (SSL) will prioritize the development of innovative luminescent materials with superior characteristics. The phosphors converted into white light-emitting diodes (white LEDs) often have a blue-green cavity. Cyan-emitting phosphor fills the spectral gap and produces “full-visible-spectrum lighting.” Full-visible spectrum lighting is beneficial for several purposes, such as light therapy, plant growth, and promoting an active and healthy lifestyle. The design of cyan garnet-type phosphors, like Ca The white LEDs obtained because of this combination generate poor-quality white light due to excellent light generation because of the deficiency of the red color component. The poor color quality limits the use of this combination for general illumination  known phosphors plays an essential role in developing optically driven white LEDs . The commination . AnotherThe phrase refers to evaluating a light source’s ability to faithfully replicate the colors of diverse objects compared to a perfect reference light . The CRI values range from 0 to 100. The color quality of the white light produced will be lower when the CRI is lower. Similarly, CRI values over 80 are typically required for general lighting. There is a substantial demand for high-CRI (Ra>90) light sources in numerous fields, including photography, movies, museums, and art galleries. The emission spectrum of a light source must be broad enough to achieve a high CRI value. “Full-visible spectrum illumination” has been suggested as a fresh idea for solid-state white lighting . It aims2O2N2:Eu2+ phosphor  of 32 nm has recently been reported for a narrow band BaSiphosphor . However4]: Eu2+ . However3B2C3O12, which has different cation feasibility for A, B, and C ions and garnet type structure with different surrounding environments for Ce3+ and Eu2+ doped ion occupation. Because these dopants (Ce3+ and Eu2+) exhibit a 5d - 4f spin-permitted transition with tunable emission throughout the visible range, the Ce3+ or Eu2+ ions are frequently used as activators in various phosphors. Furthermore, this type of composition has high stability against moisture and heat. More interestingly, the Ce3+-activated garnet phosphors may exhibit much lower near-UV and blue light absorption, making them suitable for use with the most commercially available near-UV and blue excitable white LEDs. For example, the BaLu2Al4SiO12:Ce3+ garnet phosphors emit green emission peaking at 513 nm under 450 nm blue light excitation  are shown in 2+/Lu3+, Hf4+, and Al3+ in Wyckoff sites 24c, 16a, and 24d. There was strong octahedral and tetrahedral coordination between Hf4+ and Al3+, in which the octahedrons [HfO6] and the tetrahedrons [AlO4] were joined by O2- ions. As a result, eight O2- anions were coordinated with the Ca2+/Lu3+ cations, yielding a [(Ca/Lu)–O8] dodecahedron. This compound may exhibit good thermal stability because of the structure’s compactness and rigidity. 2LuHf2Al3O12 calculated from the refined crystal structure. The CLHAO compound was found to have a direct bandgap of 4.15 eV at the G point of the Brillouin zone, located between the maximum valence band and the minimum conduction band. The calculations show that CLHAO is an appropriate host material because it offers enough band gaps to occupy Ce3+ to serve as emission centers. As a result of the absence of phonons in the transition, the direct band gap is also more likely to be advantageous to luminescence than the indirect band gap  light irradiation . The surface morphology of the synthesized materials was checked with field-emission scanning electron microscopy (FE-SEM). Under near-UV (400 nm) light irradiation, a broadband emission in the cyan spectral region (477–493 nm) is highly efficient.Zhang and others developed Ceadiation . The Ce33+ phosphors was excellent. To find the potential of the synthesized phosphors in the generation of white LEDs, a prototype of white LEDs was developed with a CRI ranging from 83.2 to 89.4.Interestingly, the thermal stability of the synthesized CLHAO:xCe3+-doped CLHAO (CLHAO:0.02Ce3+) phosphors is illustrated in 2GdZr2Al3O12 (COD ID-4338781). There is an impurity peak of HfO2 at around 30.36°. All the remaining peaks match well with those in the standard PDF card number. This result meant that the phases of samples are independent of the Ce3+ doping. The minute amount of the HfO2 impurity phase should not impact the optical characterization    (The Cend Lu3+)  and Ca2M+, Lu3+)  phosphor+, Lu3+) . For exarum LEDs . Thermalrum LEDs , the strrum LEDs , the incrum LEDs , the coand TiO2) , and thend TiO2) .3+ phosphors. The excitation (PLE) is broadband in the 300–450 nm range, peaking at 339 and 400 nm. The dominant peak was observed at 400 nm, attributed to 4f to 5d absorption. Under 400 nm near-UV light irradiation, the CLHAO:0.02Ce3+ phosphors show a broadband emission peak at 480 nm associated with a spin-allowed 5d to 4f transition. It was observed that the emission band is a dual-band emission under Gaussian fitting, leading to a difference of 1537 cm-1 attributed to the 5d → 2F5/2 and 2F7/2 transitions of Ce3+ dopants. 3+ ion energy level system in CLHAO:0.02Ce3+ phosphors. The electrons of the Ce3+ ion go into the 5d excited state by absorbing the excitation energy at wavelengths below 400 nm and close to UV stimulation. After that, a non-radiative mechanism leads the excited electrons to relax to the lowest energy level of the 5d excited state. When electrons go through the lowest exciting levels of type 5d and then transition back to their ground states of types 4f (2F5/2 and 2F7/2), they emit bright emissions in blue and cyan spectral regions.xCe3+ phosphors  are shown in 3+ concentrations are broadband in the 300–450 nm range, with a maximum peak at 400 nm. The higher intensity was observed at x = 0.02. Similarly, the PL  to 97 nm (at x = 0.08), which is similar to other garnet-structured phosphors like (Gdx1-Lux)Al5O12:Ce3+ (FWHM = 100–116 nm) and YAG:Ce3+ (FWHM = 106 nm) . The luminescence of the CLHAO:xCe3+ samples continuously rises as the Ce3+ ion doping concentration increases from x = 0.01 to x = 0.08  . Becausex = 0.08 . A conce3X2C3O12, where the A site coordinates with eight O ions, the X site coordinates with six O ions, and the C site coordinates with four O ions  phosphor is tuned with various cations at the X site  and log are 1.10781, as shown in 3+ concentration. Additionally, the PL spectra of CLHAO:yCr3+ phosphors  triggered by 460 nm blue light excitation exhibit a shift from 769 nm for CLHAO:0.01Cr3+ to 797 nm for CLHAO:0.11Cr3+ is shown in 3+ ions are extensively used as dopant because of their strong line emission in the green spectral region (at a wavelength of roughly 543 nm) caused by the 5D4→7FJ transition . However, they have drawbacks, such as the ultraviolet (UV) and near-UV absorption spectra of Tb3+ ions being very low because of the spin-forbidden character of their 4f→4f transitions. As a result of their poor luminous strength and absorption efficiency, this significantly restricts their usage in white LEDs. Therefore, green phosphors that have solely been doped with Tb3+ ions are typically inappropriate for use in white LEDs.The trivalent Tb3+ ions by using some of the energy from the spin-allowed absorption of Eu2+ and Ce3+ ions. Despite the potential for improving the absorption spectrum by co-doping Eu2+ and Tb3+ ions, the Eu2+/Tb3+ pair has several clear restrictions that harm the luminescence quality and applications of Tb3+-triggered phosphors. (1) Eu2+ ions normally have an excitation band outside the visible range; (2) Tb3+ doping concentrations in co-doped phosphors with Eu2+/Tb3+ ions are usually low. Consequently, full Tb3+ green emissions are not possible. (3) When Eu2+ and Eu3+ ions are present together in phosphors co-doped with Eu2+/Tb3+ ions, Eu3+ ions can kill Eu2+ ion luminescence, reducing light output. The high absorption efficiency of Ce3+ ions in the near-ultraviolet range makes up for the absence of substantial absorption of Tb3+ ions in the area. This is because Ce3+ ions have a spin-allowed 4f →5d transition. The PL and PLE spectra of the phosphors made from CLHAO: 0.5Tb3+, further explained in the energy transfer (ET) section.Energy transfer (ET) is a method that may be used to boost the brightness of TbSolid-state lighting, erasable optical data storage, and temperature sensors are just a few of the many uses that inorganic materials might be put to. This potential is demonstrated by the ability to achieve broadband, adjustable, and tunable emission via various energy transfer processes assisted by various co-dopant activators.3+ ions are often utilized as the light-emitting core of green-emitting phosphors due to their high green light emission (at around 543 nm), which is filled by electronic transitions from 5D4→7FJ   . On the ite LEDs . Hence, ite LEDs .3+/Tb3+ co-doped phosphors were developed with high temperature solid-state reactions. The PL and PLE spectra of the CLHAO: 0.02Ce3+ single doped phosphor is shown in 3+ phosphors exhibit a broad excitation band ranging from 300 to 470 nm due to the 4f→5d electronic transition of Ce3+ ions. The peak excitation occurs at 408 nm, with a secondary peak at 348 nm  , 7F6→ 5G6, and 7F6 →5D4 transitions , was discovered to be 90 nm. The PL spectrum is surrounded by two bands that suit a Gaussian distribution. Cectively) . The cal000 cm-1 . The PLEansition . With pensitions .3+ ions when they were doped singly. Still, because their 4f→4f transitions were spin-prohibited and their absorption intensity was incredibly low, their usage in near-UV-excited white LEDs was severely constrained. The CLHAO:0.5Tb3+ garnet phosphors emit green light when illuminated at 264 nm, and the PL spectra show a series of strong emission peaks at 492, 543, 591, 628, 643, 663, and 680 nm, which correspond to the 5D4→ 7F6, 5D4→ 7F5, 5D4 →7F3, 5D4 →7F2, 5D4→ 7F1, and 5D4→F0  and PL (λex = 408 nm) spectra are shown in 3+ ions create a robust and broad excitation band with a peak at 408 nm and a shoulder at 337 nm, as seen in the PLE spectra. The 4f → 5d and 7F6 → 5H7 transitions of Tb3+ ions were also assigned to cause the very low excitation peaks at 265 and 309 nm.The PLE and PL of CLHAO with single-doped Ce3+ ions lead to the highly efficient green emission of CLHAO:0.02Ce3+,0.5Tb3+ co-activated phosphors, confirming the efficient energy transfer from the trivalent Ce3+ to Tb3+ ions in the CLHAO phosphors host lattice. The CLHAO:0.02Ce3+,0.5Tb3+ phosphors generated a dazzling green light with a definite emission peak at 543 nm when stimulated at 408 nm. 3+ ion transitions as a series of strong emission peaks at 492, 543, 591, 628, 643, 663, and 680 nm. There are also 5D4 →7F6, 5D4 →7F5, 5D4 →7F4, 5D4→ 7F3, 5D4 →7F2, 5D4 →7F1, and 5D4 →7F0 in addition to 5D4→ 7F6. Based on these findings, co-doping with Ce3+ ions may allow Tb3+ ions to expand their near-UV absorption band. As a result, when exposed to near-UV light, the CLHAO:0.02Ce3+,0.5Tb3+ phosphors work well as green components to produce white LEDs.The distinctive excitation band Ce3+ to the co-doped Tb3+ ions in CLHAO:Ce3+, Tb3+ co-activated phosphors. The electron shifts to a 5d excited state from 2F5/2 of Ce3+ ions absorbing photons at 408 nm wavelengths. Excited electrons from the most excited 5d state moved via non-radiative transitions to reach the lowest excited 5d state. A blue emission with a peak at 483 nm was produced when the electrons of the lowest 5d excited state (Ce3+ ions energy levels) changed back to their 4f ground state. Using the ET method, the lower 5d excited state electrons of other Ce3+ ions transferred their energy to the Tb3+ ions’ 5D4 excited state in the interim. The excited 5D4 state electrons in the Tb3+ ions were released after being radiated back to the ground state 7FJ (J = 1→6), which led to a series of recognizable vivid green emissions because of the 5D4→7FJ (J = 1→6) transitions. 3+ doping concentrations makes it possible to change the CIE color coordinates of CLHAO:0.02Ce3+, xTb3+ phosphors from cyan  to green  due to the efficient Ce3+, Tb3+ ET process. Aside from that, the CLHAO:0.02Ce3+, xTb3+ digital pictures of  phosphors driven by 365 nm light are given in 3+ phosphors and the CLHAO:0.02Ce3+, xTb3+  phosphors’ intensity values of Ce3+ ions at 457 nm are expressed by IS0 and IS, respectively. C indicates the overall concentration of Ce3+ and Tb3+ ions, and the electric multipolar contact type indicated by n during energy transfer ET, where n = 6, 8, and 10 are, respectively, connected to dipole-dipole, dipole-quadruple, and quadruple-quadruple interactions. Finally, as shown in IS0/IS are reliant on the line fitting results of R2 for 3+→Tb3+ energy transfer ET process in the CLHAO:Ce3+→Tb3+ phosphors were a quadruple-quadruple interaction mechanism as the best linear fitting relationship between IS0/IS and Cn/3 was found at n = 10.3+, xTb3+ phosphors with  based on their PL spectra at 408 nm. 3+ doping rose. This change may be attributed to the effective energy transfer between Ce3+→Tb3+. The CLHAO:Ce3+,Tb3+ garnet phosphors exhibit high Ce3+→Tb3+ energy transfer and generate green near-UV light. A solid-state procedure carried out at a high temperature was used to create the green phosphor CLHAO:Ce3+,Tb3+. Between Ce3+ and Tb3+ ions, quadruple-quadruple interactions have been linked to the ET process. The best green phosphors have internal and exterior quantum efficiencies of 77.1% and 55.8%, respectively, for CLHAO:0.02Ce3+,0.5Tb3+ phosphors compositions.We also determined the CIE color coordinates for the CLHAO:0.02Ce3+, 0.5Tb3+ phosphors were employed as green-emitting color converters. To construct a white LED device with near-UV pumping, CLHAO:0.02Ce3+ phosphors to fulfil the cyan color gap in the fabrication of white LEDs for the applications of solid-state lighting, a white LED device was fabricated with the addition of the prepared cyan emission with the combination of blue, green, and red phosphors. The broad emission band of CLHAO:Ce3+ cyan emitting phosphors effectively filled the cyan gap, which led to a white light generation with high CRI values  that were noticeably higher than those of red, green, and blue phosphors converted LED .To determine the possibility of CLHAO:Ce3+ phosphors effectively filled the cyan gap, and the CRI values of LED2   were noticeably higher than those of LED1 . Compared to LED1, LED2’s CCT value  was very similar .These two white LEDs produced bright, warm white light when each was supplied with 120 mA. 3+ cyan phosphors hold enormous promise for usage in high-color-rendering white LEDs. 2LuHf2Al3O12:0.02Ce3+ cyan phosphors that are near UV excitable and have internal quantum efficiency (IQE) and external quantum efficiency (EQE) values of 84.3% and 60.8%, respectively, are efficient for filling the cyan gap and producing white LEDs with outstanding color rendering.Digital images and CIE chromaticity graph coordinates for the LED1 and LED2 devices are shown in 3+ garnet phosphors can be efficiently tuned in the cyan and green color for the desired spectral region. More specifically, phosphor materials with extraordinary photoluminescence capabilities must be created for the subsequent development of high-quality solid-state white illumination. The broadband cyan-emitting phosphor is crucial to achieve “full-visible-spectrum lighting” and close the spectral gap since the emission spectrum of conventional phosphor-converted (w-LEDs) comprises a blue-green cavity. The synthesis of the thermally stable cyan-emitting Ca2YHf2Al3O12:Ce3+ garnet phosphor was doped with Ce3+. The prepared CYHAO: xCe3+ phosphor excitation band spans a wide range of wavelengths, from 360 to 460 nm, with a maximum peak at 408 nm. As a result, it can work with an LED chip that produces near-ultraviolet (NUV), which has a wavelength shorter than 400 nm and is produced by an LED chip. The best sample of CYHAO: 0.03Ce3+ showed robust broadband cyan emission when irradiated at 408 nm. The wavelength and bandwidth of the emission were 493 nm and 100 nm, respectively. The sample has a high internal quantum efficiency (IQE) of 89.5% despite having a low external quantum efficiency (EQE) of just 69.1%  varied as the concentration of Ce3+ doping increased. The dominant peak of the photoluminescence (PL) was observed at 485 nm at x = 0.005 and moved to 504 nm at x = 0.10, moving the peak point 19 nm toward the longer wavelength. The following is an explanation for the phenomenon of redshift.The normalized photoluminescence emission spectrum of CYHAO:3+ ions (r = 1.019 Å) were replaced by the bigger Ce3+ ions (r = 1.143 Å) in the phosphors CYHAO:xCe3+. The Ce3+-O2- bond may be compressed in the hard garnet structure when the Ce3+ concentration increases due to a possible reduction in the distance between ligands and light centers. An increase in Ce3+ 5d crystal field splitting thus caused the red shift in emission spectra. There was also the possibility that the Ce3+ activators might transfer energy, which would explain the redshift. An increase in low-energy emission and a shift of the emission maxima to a longer wavelength were ultimately caused by a greater energy transfer from higher 5d level Ce3+ ions to lower-level Ce3+ ions as the Ce3+ concentration enhanced. The emission bands of CYHAO:xCe3+ phosphors also grew wider when the Ce3+ doping concentration enhanced from x = 0.005 to x = 0.10. xCe3+ garnet phosphors. By increasing the concentration of Ce3+ ions from x = 0.005 to x = 0.10, it is possible to modify the emission colors from cyan to green with CIE chromaticity coordinates ranging from  to , , and so on. The inset of The smallest Yx1-LuxHAO:Ce3+ garnet phosphor has been created based on the solid-solution design of chemical cation substitution in Ca2Lax1-LuxHf2Al3O12:Ce3+ garnet phosphors  of 118.7 nm. Two Gaussian sub-bands in the CaLaHAO:Ce3+ phosphor’s PL emission band correspond to ion transitions caused by Ce3+ ions 5d→2F5/2 and 5d→2F7/2, respectively,  phosphors upon 408 nm excitation are shown in 3+ concentration increases (517 nm at x = 0–506 nm at x = 0.5). 3+ concentration-dependent integrated PL intensity of CaLax1-LuxHAO:Ce3+ phosphors. x1-LuxHAO:Ce3+ (0 ≤ x ≤ 0.5) phosphors. Each of them consists of two bands of excitation. With a peak at 408 nm (caused by the Ce3+ ion’s 4f–5d1 transition), these samples exhibit a broad and strong excitation band in the 350–480 nm spectral range. The intensity of excitation increases as the Lu3+ content (x) increases. This indicates that near-UV LED chips can function effectively in the excitation of CaLax1-LuxHAO:Ce3+ phosphors.The PL spectra of CaLax1-LuxHAO:Ce3+ phosphors samples exhibit a relatively weak excitation band at 300–350 nm wavelengths (due to the 4f–5d2 transition of Ce3+ ions), and the intensity of this band increases with increasing Lu3+ content (x), but the excitation peak position red-shifts from 326 nm for x = 0–332 nm for x = 0.5. The CaLa0.5Lu0.5HAO:Ce3+ solid solution sample has the maximum emission intensity among the CaLax1-LuxHAO: Ce3+ (0 ≤ x ≤ 0.5) garnet phosphors. 0.5Lu0.5HAO:Ce3+ phosphors. As the PLE spectrum shows, Ce3+ ions exhibit spin-and-parity-allowed electronic transitions of 4f–5d1 and 4f–5d2 in the 300–480 nm regions. A prominent cyan-green emission band was observed upon stimulation at 408 nm in the CaLa0.5Lu0.5HAO:Ce3+ phosphors sample. In addition, the emission band can be split into two Gaussian-fitting bands at 493 nm and 542 nm, corresponding to the electronic transitions of Ce3+ ions at 5d→2F5/2 and 5d→2F7/2. According to the experimental results, the energy difference between 2F5/2 and 2F7/2 levels is close to the theoretically calculated value of 1583 cm-1 in CaLa0.5Lu0.5HAO:Ce3+ phosphors.Moreover, all these CaLax1-LuxHAO:Ce3+ phosphors and their CIE chromaticity diagram. CIE chromaticity coordinates show a blue shift as Lu3+ concentration increases, going from  for x = 0 to  for x = 0.5. As x increases, the emission color of CaLax1-LuxHAO:Ce3+ becomes cyan, green, with the cyan component deepening. The PLE and PL characteristics of Ce3+-activated phosphor materials could be fine-tuned by adjusting their coordination environment.9 = 95.9, and R12 = 94.3), novel cyan-green phosphors with a superior quantum efficiency (76.4%) and significantly higher thermal stability have been developed. Due to the induced highly symmetric crystal structure, solid-solution phosphors synthesized from Ca2Lax1-LuxHf2Al3O12:Ce3+ exhibit enhanced cyan-green emission with enhanced thermal stability for full-spectrum white LEDs, as shown in For near-UV-pumped full-visible spectrum white LEDs with ultra-high color rendering indices  and un-doped (424 nm) samples. It is clear from the comparison that the un-doped sample displayed a broadband excitation at the monitored wavelength of 424 nm, ranging from 200 nm to 400 nm, with the dominant peak around 315 nm on the high energy side.In our most recent study, Ca3+-doped Ca2YTaO6 phosphors show impressive broadness. In particular, the PLE spectra of the Bi3+-doped material expanded to longer wavelengths (at least 50 nm red-shifted). They gained an additional peak at 361 nm on the lower energy side because of the absorption of 1S0 to 3P1 in the activated Bi3+ ions. We investigate whether the presence of several luminescence centers in the host lattice of Ca2YTaO6 phosphors may explain the occurrence of multiple excitation peaks, in addition to varied excitation, samples with and without Bi3+ doping yield phosphors with distinct emissions  of optimized Ca2YTaO6:0.02Bi3+ phosphors with various excitations (250–430 nm with a 20 nm spacing). The optimized Ca2YTaO6:0.02Bi3+ phosphors sample’s normalized PL spectra can be shown in 2YTaO6:0.02Bi3+ double perovskite oxide phosphors’ digital images acquired at 254 and 365 nm showed that the color tuning was effective at the two excitation wavelengths (The increase of Bielengths .2LuHf2Al3O12 (CLHAO) are a significant component of the inorganic material family, where numerous cation substitutions may be performed to produce optimal compositions for application in various sectors of illumination. The concepts and techniques of (a) fulfilling of cyan gap in the full spectral region of white-LEDs, (b) cations substitution to accomplish efficient tuning of the emission color and (c) the growing and tuning abilities of sensitizer emission because of the efficient energy-transfer phenomena via doping using different rare-earth (RE) and transition metal (TM) ions, including Ce3+, Cr3+, Tb3+, and Bi3+, were thoroughly examined in this review. The choice of host materials is essential in producing w-LED phosphors and the doped activators. The interaction between the host materials and the doped activators alters the luminous features of this garnet phosphor.In conclusion, phosphors with the general chemical formula CaA major challenge in garnet ceramics for solid-state lighting (SSL) is distinguishing between concentration quenching, thermal quenching, and optical excitation quenching . InvestiThis review article highlights the development of other cyan-emitting phosphors to fulfil the cyan gap in the emission spectrum and fabricate a white LED with high thermal and moisture stability to generate a highly efficient white light source. Developing narrow-band cyan emission will also be promising for the high color gamut displays.


L-lactide) (PLLA) and poly(ε-caprolactone) (PCL) composites reinforced by commercial β-tricalcium phosphate (βTCP) or synthesized carbonated hydroxyapatite with hexagonal and plate-like crystallite shapes  were fabricated using injection molding. pCAp-based composites showed advanced mechanical and thermal characteristics, and the best set of mechanical characteristics was observed for the PLLA-based composite containing 25 wt% of pCAp. To achieve compatibility of polyesters and pCAp, reactive block copolymers of PLLA or PCL with poly(tert-butyl ethylene phosphate)  were introduced to the composite. The formation of a polyester-b-poly(ethylene phosphoric acid) (PEPA) compatibilizer during composite preparation, followed by chemical binding of PEPA with pCAp, have been proved experimentally. The presence of 5 wt% of the compatibilizer provided deeper homogenization of the composite, resulting in a marked increase in strength and moduli as well as a more pronounced nucleation effect during isothermal crystallization. The use of C1 increased the thermal stability of the PLLA-based composite, containing 25 wt% of pCAp. In view of positive impacts of polyester-b-PEPA on composite homogeneity, mechanical characteristics, and thermal stability, polyester-b-PEPA will find application in the further development of composite materials for bone surgery and orthopedics.Composites of synthetic bone mineral substitutes (BMS) and biodegradable polyesters are of particular interest for bone surgery and orthopedics. Manufacturing of composite scaffolds commonly uses mixing of the BMS with polymer melts. Melt processing requires a high homogeneity of the mixing, and is complicated by BMS-promoted thermal degradation of polymers. In our work, poly( Human cortical bone is a complex entity comprising the bone mineral (BM), organic components, and water ,2. The vA major focus of bone surgery is to restore the functionality of the bone in terms of its optimal strength parameters and anatomical shape. Bone healing is a complex morphogenetic process , and its2+, PO43−, and other “biophilic” ions, are seen as prospective bone mineral substitutes (BMS) /[pCAp] ratio; similar patterns were observed previously for HAp/PLLA (BHT-Mg) , 2-chlor(BHT-Mg) , and 2-t(tBuOEP)  were synMn of 107 kDa and ÐM of 2.02; supplied by FDplast, Moscow, Russia) and PCL  were chosen as polyester components. Commercial βTCP  was selected for comparison with CAp.For composite molding, commercial PLLA  was supplied by Cambridge Isotope Laboratories, Inc. . The 1H and 13C NMR spectra were recorded on a Bruker AVANCE III HD 400 spectrometer  at 20 °C. The chemical shifts are reported in ppm relative to the solvent residual peaks.CDClScanning electron microscopy (SEM) images and energy dispersive X-ray (EDX) analysis data were obtained using a Phenom XL microscope  at an accelerating voltage of 15.0 kV.−1). The measurements were recorded with universal calibration based on a polystyrene standard at 40 °C.Size exclusion chromatography (SEC) of the polymers was performed using an Agilent PL-GPC 220 chromatograph equipped with a PLgel column , and THF was used as the eluent  X-ray tube and a 1D D/teX position-sensitive detector. Data were collected at room temperature in the 2θ range 4.5–90° with a 0.02° step size using a Bragg–Brentano setup. The cell parameters were determined with full-profile fitting, as implemented in Bruker TOPAS 5 2  in CH2Cl2 (1.0 mL) was added. After 2 h of stirring at 20 °C, a solution of L-lactide  in CH2Cl2 (4.5 mL) was added, and the mixture was stirred within 12 h. AcOH  was added. The polymer was precipitated using a 10-fold volume excess of dry Et2O, and dissolution in CH2Cl2 and precipitation was repeated three times. The yield was 2.46 g (67%).1H NMR : δ 7.37 ; 5.16 ; 5.04 ; 4.18 ; 1.57 ; 1.50 . 31P{1H} NMR : δ −5.48 (1P); −5.65 (23P).1H NMR spectrum (31P NMR spectrum (tBuOEP)23-b-(L-LA)142.Comparison of the integral intensities of the signals of BnO end-groups and comonomer fragments in the spectrum , and thespectrum , allowed2Cl2 (4.0 mL) were placed under an argon atmosphere into a flame-dried flask. A solution of [(BHT)Mg(μ-OBn)(THF)]2  in CH2Cl2 (0.5 mL) was added. After 90 min of stirring at 20 °C, tBuOEP  was added, and the mixture was stirred within 12 h. AcOH  was added. The polymer was precipitated using a 10-fold volume excess of dry Et2O, and dissolution in CH2Cl2 and precipitation was repeated three times. The yield was 1.15 g (74%).ε-Caprolactone  and CH1H NMR : δ 7.32 ; 5.08 ; 4.16 ; 4.03 ; 2.28 ; 1.62 ; 1.48 ; 1.35 . 31P{1H} NMR : δ −5.42 (1P); −5.69 (24P).119-b-(tBuOEP)24 with the analysis of 1H and 31P NMR spectra (The composition of C2 was determined as BnO(εCL) spectra .3  and Na2[EDTA]·2H2O  were dissolved in H2O (1 L). Then, Na2HPO4·12H2O  and Na2CO3  were added. After 1 h at 100 °C, the mixture was filtered to a 1.8 L autoclave placed in a heating bath. After the addition of water , the autoclave was heated at 150 °C within 5 h. After cooling to 20 °C, the precipitate was separated with decantation, washed with H2O (2 × 300 mL), iPrOH (2 × 200 mL), and pentane (2 × 100 mL), and dried in vacuo. The yield was 16.74 g (18.6%).CaCO3  and Na2[EDTA]·2H2O  were dissolved in H2O (1 L). The solution was filtered to a 1.8 L autoclave and pre-loaded with NaHCO3  and KH2PO4 . After the addition of water , the autoclave was heated at 140 °C within 24 h. After cooling to 20 °C, the precipitate was separated with decantation, washed with H2O (2 × 300 mL), iPrOH (2 × 200 mL), and pentane (2 × 100 mL), and dried in vacuo. The yield was 18.70 g (19.6%).CaCOPLLA (7 g) was dissolved in THF (50 mL). BMS  was added. After 1 h of stirring at 20 °C, the mixture was poured into a PTFE cuvette and dried at 100 °C within 12 h. In the presence of C1, the stirring of the reaction components (BMS and C1) was conducted in a closed glass vial at 90 °C within 1 h. Cooled suspension was added to the solution of PLLA in THF (20 mL). After 1 h of stirring at 20 °C, the mixture was poured into the PTFE cuvette and dried at 100 °C within 12 h.PCL (7 g) was dissolved in THF (50 mL). BMS  was added. After 1 h of stirring at 20 °C, the mixture was poured into a PTFE cuvette and dried at 50 °C within 2 days. In the presence of C2, the stirring of the reaction components (BMS and C2) was conducted in a closed glass vial at 90 °C within 1 h. Cooled suspension was added to the solution of PCL in THF (20 mL). After 1 h of stirring at 20 °C, the mixture was poured into the PTFE cuvette and dried at 50 °C within 2 days.Melt homogenization of the composites was carried out using a Polydrive mixer HAAKE R600 . Before mixing, the composite films were shredded manually and dried at 50 °C and 0.01 Torr within 12 h. Molding of the samples was made on HAAKE MINIJet Pro equipment  with the pressure of 600 Bar. For PLLA-based composites, the temperatures of molding were 180–200 °C (cylinder) and 65 °C (mold), for PCL-based composites, 70–90 °C (cylinder) and 30 °C (mold). A Mould Tensile Bar ASTM 0638-V injection mold was used for preparation of the samples for a tensile test , and a MThe results of the mechanical tests are presented as the mean ± standard deviation (SD) and median with the interquartile range. The data were analyzed with SigmaStat 3.5 software .tBuOEP)23-b-(L-LA)142 (C1) and BnO(εCL)119-b-(tBuOEP)24 (C2) were synthesized and used as compatibilizer precursors.In the present work, we assumed that the properties of polyester/BMS composites can be substantially improved when using polymeric compatibilizers comprising long polyester and a phosphorus-containing polyacid block. Having chosen PLLA and PCL as polyester components, we prepared PLLA/BMS composites with 10–60 wt% loading of the inorganic filler and PCL-based composites with 50 wt% of BMS. Micro-sized commercial βTCP and synthesized carbonated apatites hCAp and pCAp were used as BMS. The synthesis of hCAp and pCAp was based on our previous study  but was b-PEPA compatibilizers. Tensile and bending tests of the molded PLLA-based samples showed the better mechanical characteristics for pCAp-based formulations, viz. a lower decrease in σt and σf values, as well as a higher increase in Et and Ef values. An additional improvement of the pCAp-based composite properties was achieved with the addition of 5 wt% of C1. The best set of mechanical characteristics was demonstrated by the sample PL12, prepared with the use of C1: even with the 25 wt% filler content, the material exceeded neat PLLA in strength and moduli values. The positive impact of the use of C2 on the mechanical characteristics of the PCL/pCAp composite was even more pronounced; the composite with 50 wt% of the filler had nearly doubled strength values relative to σt and σf of PCL, with an almost four-time increase in moduli.At the first stage of the composite preparation, all components were dispersed in THF media, and additional heating at 90 °C resulted in formation of polyester-b-PEPA copolymers followed by chemical bonding between the PEPA block and pCAp surface. This bonding was confirmed with FT-IR studies of the mineral residues after dissolution and elimination of the PLLA matrix from the samples PL09 and PL13, prepared without and with the addition of C1; in the latter case, the presence of PLLA, chemically bonded with the pCAp surface, was confirmed by the presence of characteristic signals at ~1800 (>C=O) and 1220 (C–O) cm−1. SEM data for PL13 showed that during composite molding, PEPA-b-PLLA served as an efficient dispersant for pCAp splices, whose structure remained unchanged during preparation of PL09.The effect of C1 and C2 on composite properties was attributed to the formation of polyester-Mw of PLLA in this composite was 12% lower relative to Mw of neat PLLA, whereas in the absence of the compatibilizer, the loss of MW was 35%.The use of C1 provided a more pronounced nucleation effect of the filler during PLLA crystallization (SEC data). Additional experiments on isothermal crystallization at 110 °C showed the immediate onset of crystallization only for samples PL12 and PL13 (25 and 50 wt% of the filler) prepared with the addition of C1. Rheological studies at 175 °C showed a significant reduction in viscosity in the case of pCAp-containing composites that can be attributed to a decrease in the molecular weight of PLLA components. The decline in MW can be caused by the presence of BMS and their impact on thermal degradation of PLLA, and the presence of a compatibilizer could prevent or weaken the effect of the filler. We observed a similar weakening in the example of PL12 with 25 wt% content of pCAp; during melt processing, PLLA-based composite samples, obtained during this study, do not reach the mechanical characteristics of the cortical bone ,11 but o


Introduction: Bulevirtide is a first-in-class antiviral drug to treat chronic hepatitis B/D. We investigated the drug-drug interaction potential and pharmacokinetics of high-dose subcutaneous bulevirtide (5 mg twice daily) with organic anion transporting polypeptide 1B1 (OATP1B1) and cytochrome P450 (CYP) 3A4.Methods: This was a single-center, open-label, fixed-sequence drug-drug interaction trial in 19 healthy volunteers. Before and at bulevirtide steady state, participants ingested a single 40 mg dose of pravastatin. A midazolam microdose was applied to quantify CYP3A4 activity.Results: At bulevirtide steady state, pravastatin area under the concentration-time curve (AUC0–∞) increased 1.32-fold (90% CI 1.08-1.61). The 5 mg bulevirtide twice-daily treatment resulted in a mean AUC0-12 of 1210 h*ng/ml (95% CI 1040-1408) and remained essentially unchanged under the influence of pravastatin. CYP3A4 activity did not change to a clinically relevant extent. As expected, total bile acids increased substantially (35-fold) compared to baseline during bulevirtide treatment. All study medication was well tolerated.Discussion: The study demonstrated that high-dose bulevirtide inhibited OATP1B-mediated hepatic uptake of the marker substrate pravastatin but the extent is considered clinically not relevant. Changes in CYP3A4 activity were also not clinically relevant. In conclusion, this study suggests that OATP1B substrate drugs as well as CYP3A4 substrates may safely be used without dose adjustment in patients treated with bulevirtide. However, in patients using high statin doses and where concomitant factors potentially further increase statin exposure, caution may be required when using bulevirtide. Howeverlication . Until 2lication . The app50 at 50 nM (50 to inhibit the HBV/HDV entry is in the sub-nanomolar range (Bulevirtide consists of a myristoylated peptide containing 47 amino-acids derived from the preS1-domain of the hepatitis B virus (HBV) L-surface protein. Bulevirtide acts as a virus entry inhibitor by binding to and inactivating the hepatic sodium taurocholate co-transporting polypeptide , which is the natural bile acid transporter and also promotes entry of HBV and HDV into hepatocytes . Bulevirat 50 nM , total bat 50 nM . Bulevirar range .Several clinical trials with bulevirtide showed an excellent safety profile and a significant reduction of biomarkers such as HDV RNA in serum and liver and normalization of alanine aminotransferase (ALT) in patients with CHD . In addiThe clinical effect of inhibiting HDV entry is clearer than the inhibition of the HBV entry, where trials failed to show effects on markers of HBV infection. .In vitro, bulevirtide not only inhibits NTCP but also the organic anion transporting polypeptide 1B1 (OATP1B1) at nanomolar concentrations (IC50 = 530 (±90) nM) and to a lesser extent OATP1B3 (IC50 = 8650 (±3740) nM) (740) nM) . Treatme740) nM) . Althoug740) nM) .As a liver-specific uptake transporter, OATP1B contributes to the targeting of many substrate drugs from different classes and most importantly lipid-lowering statins, whose adverse events are closely linked to drug-drug interactions . We asseSLCO1B1*5 and SLCO1B1*15 haplotype  and the competent national authority . All participants provided written informed consent prior to any trial-related procedure.The trial was carried out between 19/07/2018 and 23/11/2018 at the Clinical Research Unit of the Department of Clinical Pharmacology and Pharmacoepidemiology . We conducted a single-centre, open-label, fixed-sequence drug-drug interaction trial in healthy volunteers. The primary objective was to evaluate the effect of a 5 mg dose of bulevirtide, given twice a day (bid) and dosed to steady state on the pharmacokinetics of a single dose of 40 mg pravastatin (sampling over 24 h). The dose of bulevirtide was 5 times higher than the currently conditionally approved daily dose of 2 mg, however it was chosen to serve as a worst-case scenario in a situation where the optimal therapeutic dose remains unclear. Secondary objectives were the evaluation of steady-state pharmacokinetics of bulevirtide with a 5 mg bid regimen , the effect of pravastatin on bulevirtide pharmacokinetics , the pharmacodynamic effects on plasma bile acid concentrations at bulevirtide steady state , and the effect of both drugs on CYP3A4 activity as assessed by administration of an oral 30 µg midazolam microdose suing a limited sampling strategy  . Additio®, Hexal, Holzkirchen, Germany) was assessed at baseline prior to starting bulevirtide and at bulevirtide steady state.The detailed trial flowchart is presented in ® V 5 mg/5 ml solution for injection, Cheplapharm Arzneimittel GmbH, Greifswald, Germany) diluted in 100 ml water, following an established procedure  and c.388A>G  . The comBlood samples for the quantification of bulevirtide, pravastatin, midazolam, and bile acid profiles were taken before and at multiple timepoints up to 48 h after drug intake. Samples were centrifuged at 3600 g and 4°C for 10 min, and plasma was separated and stored at ≤ −20°C until analysis. Backup samples were stored at −80°C. Bulevirtide plasma concentrations were determined by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) as described previously . Pravastmax) and time to reach Cmax (Tmax) were directly obtained from pharmacokinetic data. Area under the concentration time curve from 0 to 12 h  was determined using the predose time point until the last observed timepoint at 12 h. For AUC0–∞ (pravastatin), AUC was determined using the log-linear trapezoidal rule and by adding the extrapolated part until infinity. Half-life (t1/2) was calculated as met) (derived from midazolam AUC2-4) as described earlier . Maximum plasma concentration  variables were evaluated by t-test using the 95% confidence interval and the significance level of ≤0.5. Midazolam AUC2-4, eClmet, and AUC0-12 of bile acids on four study days were evaluated by analysis of variance with adjustments for multiple testing, using a significance level of ≤0.05.Based on the guideline for bioequivalence trials  changes post hoc analysis bulevirtide exposure (AUC0-24) with 5 mg sc bid dosing in this trial was compared to exposure with 10 mg sc bulevirtide once daily as observed in a previous trial ). The extent of the drug interaction in volunteers with SLCO1B1 polymorphisms was evaluated descriptively. In a us trial , by predTwenty healthy volunteers  were enrolled in this trial. One volunteer terminated the trial prematurely due to an unrelated urinary tract infection after having received the first microdose of midazolam; The remaining 19 volunteers completed the trial as planned. Their median age was 23 y (range 18–45), their body weight [mean ± standard deviation (SD)] was 69.6 kg (±15.8), body mass index was 23.4 kg/m2 (±3.4), and their estimated creatinine clearance (Cockcroft-Gault formula) was 133 ml/min (±30.2). Participants had the following SLCO1B1 genotypes: *1A/*1A , *1A/*1B , *1B/*1B (n = 2), *5/*15 (n = 1), *1B/*15 (n = 1), *1A/*15 or *1B/*5 (n = 1), and *15/*15 (n = 1).0-∞ to 1.32-fold  and GMR for Cmax to 1.32  in the overall trial population  for AUC N = 19, , Table 10–∞ of 1.4  and a GMR for Cmax of 1.47 . Homozygous carrier of SLCO1B1*1A/*1A (N = 8) had comparable AUC values to the overall population at baseline and showed an increase in GMR for AUC0–∞ of 1.76 (90% CI 1.17-2.64) and a GMR for Cmax of 1.88 (90% CI 1.11-3.2). The SLCO1B1*5/*15 and SLCO1B1*15/*15 carriers, i.e., with an assumed decreased transport function, had a relatively high pravastatin exposure at baseline (without bulevirtide) with an AUC0–∞ of 331/532 h*ng/ml respectively, and a Cmax 143/316 ng/ml, respectively. The AUC ratio for these two individuals decreased to 0.89/0.72 respectively  showed a GMR for AUC0–12 was 1.01 (90% CI: 0.94-1.09) and of Cmax was 0.93 (0.84-1.02). In a post hoc analysis, the 24-h exposure (AUC0-24) in the present trial with 5 mg bulevirtide bid was 29% larger than after the once-daily 10 mg s.c. administration in a previous trial in healthy volunteers   .0–12 of total bile acids 35.2-fold from 49.8 µmol/l*h  at baseline to 1752 µmol/l*h  unconjugated bile acids 3.62-fold (p = 0.001), taurine-conjugated bile acids 61.5-fold, and glycine-conjugated bile acids 38.0-fold  at baseline, 1107 ml/min (957-1280) with single-dose pravastatin, 1111 ml/min (946-1306) at bulevirtide steady state, and 1176 ml/min (993-1392) at bulevirtide steady state with pravastatin. The corresponding GMR compared to baseline was 1.10 , 1.11 , and 1.17 . Explorative evaluation considering multiple testing revealed a small, but statistically significant difference (p = 0.009) for the GMR at bulevirtide steady state with a single dose of pravastatin  and headache (8 events) being the most common .in vitro , suggesin vitro . FinallyIn the present trial, a new high-dose regimen of 5 mg bulevirtide bid was used. Compared to the steady-state exposure of a once-daily administration of 10 mg, the bulevirtide steady-state exposure with 5 mg bid  was 29% larger . This isIn this study, the average exposure of the sensitive OATP1B marker substrate pravastatin increased by only 32% with bulevirtide at steady state. Pravastatin clearance is mainly dependent on OATP1B uptake. Although the FDA indicates that pravastatin may also be a substrate of NTCP on their list of marker substrates, to our best knowledge there is no data convincingly showing a major role of NTCP in the elimination of pravastatin. . In thisIn vitro, bulevirtide inhibited OATP1B1 and to a lesser extent OATP1B3  than in homozygous or heterozygous carriers of wild-type alleles.We were able to include a small number of carriers of polymorphic SLCO1B1 alleles/haplotypes. Two of these had a genotype with expected lower transport function. Similar to various pharmacogenetic studies , pravastWhile this genetically reduced OATP1B1 activity is associated with statin-induced myotoxicity in patients with *5 or *15 haplotypes , our SCLmax compared to the 10 mg once-daily administration (285 ng/ml versus 423 ng/ml)  . Interes3 ng/ml) . In the 3 ng/ml) . The ovefunction . The bidmet of midazolam decreased to 0.71. However, in this previous trial, bulevirtide was co-administered with tenofovir and it was impossible to differentiate between the influence of bulevirtide and the influence of tenofovir. Therefore, in the present trial we aimed to evaluate the bulevirtide effect on eClmet of midazolam as a marker for CYP3A4 activity and found that CYP3A4 activity remained essentially unchanged with a very small, clinically irrelevant 1.17 increase of eClmet at one time point at bulevirtide steady state, suggesting that CYP3A4 substrates are not affected by bulevirtide to any meaningful extent. Concurrently, this trial confirmed that pravastatin does not modify CYP3A activity.In a previous trial administering once-daily 10 mg bulevirtide for 10 d  the eClmThe tolerability of the still novel drug bulevirtide in this trial was completely in line with the excellent safety profile known so far. The injection site reactions attributed to bulevirtide have been described previously and were all transient .The main limitation of the present trial is that healthy participants and not patients were studied. Therefore, the results may not be completely transferable to patients suffering from comorbidities, such as impaired liver and/or kidney function. Also due to the low sample number conclusions for carriers of known dysfunctional SLCO1B1 cannot be drawn.In conclusion, this trial demonstrated, that the interaction of the novel HBV/HDV entry inhibitor bulevirtide with the drug transporter OATP1B is mild and no clinically relevant impairment of CYP3A4 activity has to be expected. The study suggests that OATP1B substrate drugs as well as CYP3A4 substrates may safely be used without dose adjustment in patients treated with bulevirtide. Only if patients require high statin doses and concomitant factors may potentially further increase statin exposure, close observation may be required when using bulevirtide. The general precautionary warning in the current EMA Summary of Product Characteristics against concomitant use of OATP1B1 substrates should be reconsidered and modified in order not to unnecessarily withdraw HDV patients from other important drugs, that are used, for example, for cardiovascular prevention.


Fucosyltransferase 2(FUT2) and its induced α-1,2 fucosylation is associated with cancer metastasis. However, the role of FUT2 in colorectal cancer (CRC) metastasis remains unclear.△IEC mice) were used to investigate the effect of FUT2 on metastasis in colorectal cancer. Quantitative proteomics study of glycosylated protein, UEA enrichment, Co-immunoprecipitation identified the mediator of the invasive-inhibiting effects of FUT2.The expression levels and clinical analyses of FUT2 were assessed in CRC samples. Migration and invasion assays, EMT detection, nude mice peritoneal dissemination models and intestinal specific FUT2 knockout mice (FUT2△IEC mice) develop AMO and DSS-induced tumors and promote EMT in colorectal cancers. FUT2-induced α-1,2 fucosylation impacts the ability of low-density lipoprotein receptor-related protein 1(LRP1) to suppress colorectal cancer invasion.FUT2 is downregulated in CRC tissues and is positively correlated with the survival of CRC patients. FUT2 is an inhibitor of colorectal cancer metastasis which, when overexpressed, suppresses invasion and tumor dissemination in vitro and in vivo. FUT2 knock-out mice (FUT2Our study demonstrated that FUT2 induces α-1,2 fucosylation and inhibits EMT and metastasis of colorectal cancer through LRP1 fucosylation, suggesting that FUT2 may serve as a therapeutic target for colorectal cancer.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01060-0. Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related death worldwide . Despiteα1,2-linkage [Fucosyltransferase 2 (FUT2) is a key enzyme that catalyzes the transfer of fucose to the terminal galactose of type 1 or type 2 disaccharide via -linkage . Aberran-linkage . Recent -linkage . Neverth-linkage . The rol2.The human colorectal cancer cell lines HCT116 and SW480 were purchased from American Type Culture Collection (ATCC). All CRC cell lines were authenticated by short tandem repeat analysis and were negative for mycoplasma. The cells were cultured with DMEM  supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL penicillin and streptomycin  at 37 °C in a humidified incubator in 5% COflox/flox C57BL/6 mice (purchased from GemPharmatech Co. Ltd) were crossed and generated mice with FUT2 gene specifically deleted in intestinal epithelial cell . 29 human samples were obtained from the Endoscopy Center of Union Hospital in 2020. All the samples were obtained with the patients’ informed consent, and the samples were processed histologically.BALB/c nude mice and Wild-type C57BL/6 male mice were purchased from Beijing Huafukang Biological Co., Ltd. and housed under standard pathogen-free conditions in the Experimental Animal Center of Tongji Medical College, Huazhong University of Science & Technology. Pvillin-Cre recombinase transgenic C57BL/6 mice (Pvillin-Cre TG mice) and FUT2△IEC mice were intraperitoneally (IP) injected with 10 mg/kg AOM working solution . On Day 7, mice were provided a continuous supply of 2.5% (2.5 g/100 ml) DSS solution in their drinking water for seven days. On Day 14, mice were again given standard drinking water for two weeks. The abovementioned DSS and water steps were repeated on Days 28 and 49 to provide a second and third cycle of DSS administration. Mice were sacrificed on Day 70, and samples were prepared for assessment.On Day 0, WT and FUT2Lentivirus for FUT2 overexpression and shRNA sequences targeting LRP1 were designed and constructed by Obio Technology Co. Ltd., Shanghai, China. OeRNA and shRNA lentivirus was added to the culture medium of GC cells with HitransG A , and puromycin was used to screen stable clones. The sequence targeting LRP1 was as follows: CATGCTGGACCTCTCCAATAA.Premix Ex Taq (Tli RNaseH Plus) . The 2-(ΔΔCt) method was used to calculate the relative abundance of RNA after normalization to GAPDH. For HCT116 and SW480 cells, cells were suspended and added to the 6-well plates (1.2 × 106 cells/plate). After 24 h, cells grew to 80% fusion and the total RNA was extracted. The primers that were used for PCR are listed in Additional file Total RNA was extracted with RNAiso Plus reagent  and reverse transcribed into cDNA using a verse Transcription system . Quantitative real-time polymerase chain reaction (qRT‒PCR) was performed using TB Green Tissues were embedded and sectioned. After deparaffinization and rehydration, the sections were boiled for 30 min at 100 °C for antigen retrieval, treated with 3% hydrogen peroxide for 4 min and blocked by incubation with 10% goat serum for 30 min. Subsequent staining with primary antibodies was performed at room temperature for 60 min. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, the sections were washed in distilled water, treated with DAB, counterstained with hematoxylin, and dehydrated.4 cells/insert) and added to the upper chamber of the 24-well insert. Medium supplemented with 10% serum was added to the lower chamber. After incubation for 18 h, the cells that migrated were fixed in 4% paraformaldehyde and stained with 1% crystal violet for 10 min. For the invasion assay, chamber membranes were coated with Matrigel .Cell migration and invasion were measured using 24-well inserts . For the migration assay, SW480 and HCT116 cells were suspended in serum-free medium (5 × 10△IEC mice administered with AMO and DSS) were lysed into peptides. Peptides were labeled with the TMT reagent label and mixed into one sample. Then the mixed sample was separated by liquid chromatography, and the distillate was divided into 12 samples in series. An aliquot of each sample was loaded onto an analytical HPLC column using the auto sampler of an EASY-nLC 1000 HPLC. The results were searched against a decoy database and proteins.Colon tissues of mice within both groups(WT and FUT2Immunoprecipitation was performed via a protein A immunoprecipitation kit (Roche) according to the manufacturer’s protocol. SW480 and HCT116 cells were suspended and added to the 10 cm dish. After 24 h, cells grew to 80% fusion and were lysed , 0.5% Triton X-100) and incubated for 1 h on ice. The cell lysates containing the protein were incubated overnight with primary antibody-conjugated beads at 4 °C overnight. The beads were eluted with lysis buffer, and the proteins were resuspended in SDS sample buffer and resolved by Western blotting.SW480 and HCT116 cells were transfected with a FUT2 overexpressing or control vector. Cells were suspended and added to the 10 cm dish. After 24 h, cells grew to 80% fusion and were were lysed with lysis buffer  containing 1% Nonidet P-40. The lysates (1000 μg) were mixed with 100 μl of biotin-conjugated UEA-I (0.1 µg/ml) and incubated with rotation at 4 °C overnight. Then, agarose coupled with streptavidin was added and incubated continuously for 4 h. The samples were extracted and separated by SDS‒PAGE and transferred to PVDF membranes. The membranes were incubated with LRP1, LAMB1 and Integrin β1 antibodies.6 cells/plate). After 24 h, cells grew to 80% fusion. Proteins were extracted from tissues and CRC cells in ice-cold RIPA lysis buffer containing phosphatase and protease inhibitor mixes  at 4 °C for 20 min. The proteins were separated by SDS‒PAGE and transferred to nitrocellulose membranes. The membranes were blocked using 10% milk. The membranes were then incubated overnight with primary antibodies at 4 °C, followed by washing three times in TBST before the addition of secondary antibodies and incubation for 1 h at room temperature. The membranes were further exposed to ECL with Millipore immobilon western chemilum HRP substrate.HCT116 and SW480 cells were suspended and added to the 6-well plates  before injection. Two hundred microliters of cell/Matrigel (1:1) suspensions were then subcutaneously or peritoneally injected into four-week-old male BALB/c nude mice. Mice were sacrificed after 30 days, and peritoneal metastatic nodules were counted.HCT116 cells transduced with LV-FUT2 or LV-CON lentiviruses were harvested and resuspended at a concentration of 5 × 10p < 0.05, **p < 0.01, ***p < 0.001).All the experiments were repeated at least three times, and the results are expressed as the means ± SDs. All the statistical analyses were performed using GraphPad Prism software. Student’s t test or one-way analysis of variance (ANOVA) was used to analyze the data, and the chi-square test was used to analyze differences in other variables, as appropriate. A P value of < 0.05 was considered statistically significant for all datasets.  and the resulting alteration in protein fucosylation were identified as features of metastatic behavior . KeiichiFurther analyses of the FUT2-overexpressing cells indicated a change in EMT. There was a clear increase in the surface expression of β-catenin and a decrease in the expression of the mesenchymal marker vimentin in FUT2-overexpressing cells  were generated by hybridizing FUT2-knockout mice with Villin-CRE transgenic mice. qRT‒PCR analysis of colon lysates from WT and fut2 − / − mice confirmed the reduction or absence of FUT2 expression  were subdivided into two groups and treated with or without AMO and DSS. FUT2△IEC mice developed significantly increased established colon tumors in comparison to WT mice 90 days after AMO and DSS treatment . A total of 324 candidate downregulated α-1,2 fucosylated proteins were identified in the proteomics study of N-glycosylation in FUT2△IEC mice. Enrichment  analysis of genes was performed and revealed enrichment in biological processes relevant to metastasis, such as cell migration and angiogenesis. The proteins that bound to UEA-I and were related to migration include laminin β1 (LAMB1), laminin β2 and laminin γ1, integrin-αV, integrin β1, signal-regulatory protein alpha (Sirpa) and Pro-low-density lipoprotein receptor-related protein 1 (LRP1) (Additional file △IEC mice, while there was no difference in the expression of these proteins in the input samples (Fig. FUT2 can generate α-1,2 fucosylated structures both at the termini of N-acetyllactosamine and on galactose linked with N-acetylgalactosamine, which are epitopes recognized by UEA-I and TJA-II/SNA-II lectins , 25. To LRP1 is a highly glycosylated cysteine-rich protein. Numerous studies have suggested a role for LRP1 in the regulation of cell invasion and migration in several cancers , 27, andModifications of glycosylation at the surface of tumor cells and tissue induce unique features that are characteristic of cancer cells and the tumor microenvironment . SeveralFUT2, an enzyme governing epithelial α-1,2 fucosylation, is associated with various human disorders . FUT2 meNext, we focused functional and mechanistic studies of FUT2. FUT2 was significantly suppressed during the EGF- or bFGF-triggered EMT of colorectal cancer cells . HoweverOne of the most important aspects of our study is related to the involvement of LRP1 in the regulation of EMT and metastasis by FUT2. We identified LRP1 as a mediator of the invasive-inhibiting effects of FUT2 using a proteomics study and immunoprecipitation. LRP1 is a cell surface receptor involved in invasion and neovascularization, processes that drive tumor progression and metastasis . PreviouTaken together, our results highlight the inhibitory potential of FUT2 in colorectal cancer. In addition, we provide a number of solid arguments for a regulatory role of FUT2 and the α-1,2 fucosylation it causes in the function of LRP1. These data may not only enrich our knowledge of fucosylation but also suggest a strategy for therapeutic intervention in colorectal cancer.Our study demonstrated that FUT2 induces α-1,2 fucosylation and inhibits the EMT and metastasis of colorectal cancer through LRP1 fucosylation, suggesting that FUT2 may serve as a therapeutic target for colorectal cancer (Additional file Additional file 1. Table S1. Primers for qRT-PCR.Additional file 2. Figure S1. Expression of FUT2 gene in CRC cell lines.Additional file 3. Table S2. Mass spectrometric analysis of N-glycosylated TMT proteins in migration category of GO analysis from colon tissues of mice in WT and FUT2△IEC mice.Additional file 4. Uncropped images of western blot analysis.