The as-dealloyed np-Cu6Sn5 has an ultrafine ligament size of 40 nm and a high BET-specific area of 15.9 m2 g−1. The anode shows an initial discharge capacity as high as 1200 mA h g−1, and it remains a capacity of higher than 600 mA h g−1 for the initial five cycles at 0.1 A g−1. After 100 cycles, the anode maintains a stable capacity higher than 200 mA h g−1 for at least 350 cycles, with outstanding Coulombic efficiency. The ex situ XRD patterns reveal the reverse phase transformation between Cu6Sn5 and Li2CuSn. The Cu6Sn5/Sn composite presents a similar cycling performance with a slightly inferior rate performance compared to np-Cu6Sn5. The study demonstrates that dealloyed nanoporous Cu6Sn5 alloy could be a promising candidate for lithium-ion batteries.The volume expansion during Li ion insertion/extraction remains an obstacle for the application of Sn-based anode in lithium ion-batteries. Herein, the nanoporous (np) Cu Cur3–0.6 V) ,8,9. How3–0.6 V) ,10.−3 for over 200 cycles at 0.5 C. The high cycling stability could be attributed to the 3D nanostructured design that accommodated the volume expansion during charge/discharge [−1 after 100 cycles at a current density of 100 mA g−1 [2/graphene frameworks were designed, showing enhanced lithium storage capability [−3 over 200 cycles at 0.5 C, and a capacity greater than 1200 mA h cm−3 at 10 C [3Sn2 microcage showed stable capacity of 534 mA h g−1 after 1000 cycles at the current density of 1 C [2 nanospheres with uniform small particle size have been synthesized, showing better electrochemical performances than other nano-spherical intermetallic compounds such as Cu6Sn5, CoSn3 and Ni3Sn4, due to its crystal structure [Several approaches have been proposed to overcome the volume expansion issue. One is to reduce the size of the Sn-based materials, i.e., construction of nanostructures. For instance, a 3D nanocomposite consisted of 3D structured Sn scaffold and a hollow Sn sphere was fabricated as the LIBs anode . The anoischarge . Anotherischarge . A Sn/grischarge . Nanocab0 mA g−1 . In addipability ,16. Liu  at 10 C . Combini at 10 C . A Ni3Sny of 1 C . Single-tructure . In additructure .6Sn5 possesses the high theoretical capacity of 605 mA h g−1 [6Sn5 nanoparticles, film, or nanowires [6Sn5 powers [6Sn5 alloy on the Cu foil, and the alloy electrode showed a discharge capacity of 462 mA h g−1 [6Sn5 nanowire was synthesized via an electrodeposition process, and the anode showed excellent rate performance even at high current density of 20 C [6Sn5 nanostructure for fabrication LIB anode [17Sn7Al76 alloy in a 1 M NaOH to obtain a 3D nanoporous Cu-Sn electrode. The electrode presents a capacity of 566 mA h g−1 at a current density of 1670 mA g−1 [+ ions. A nanoporous Cu6Sn5/Cu composite was generated via a similar method by dealloying Al10Cu3Sn alloy [In addition to the above-mentioned materials, the most studied Sn-based materials are Cu-Sn alloys, as copper owns high conductivity and elasticity, and CumA h g−1 ,23,24,25anowires ,27,28,29mA h g−1 . A core- of 20 C . RecentlIB anode ,25,28. LSn alloy ,32. The Sn alloy .6Sn5 alloy and Cu6Sn5/Sn composite as the LIB anode. The as-dealloyed anode was found to have excellent cycling stability and rate performance due to the nanoporous structure. Moreover, the anode material has good thermal stability due to the formation of intermetallic Cu6Sn5, favoring the cycling. The ex situ XRD proved the reverse phase transformation between Cu6Sn5 and Li2CuSn during charge/discharge process.In our previous report, we found that by changing Cu and Sn ratio in the Mg-Cu-Sn alloy, Cu-Sn alloys or composite with different phase compositions can be achieved via dealloying . Herein,67Cu18Sn15 and Mg66Cu10.2Sn23.8 (at.%) precursor ribbons were prepared from rapid solidification. Pure Mg, Cu and Sn metal blocks (purity: 99.9 wt.%) were melted in an alumina crucible using a resistance furnace  under the protection of covering agent at about 800 °C. The melt liquid was cast into an alloy rod in a module. The obtained alloy rod was cut into ingots and remelted at 800 °C using a high-frequency induction furnace  in a quartz tube with a pinhole below. The alloy melt was blasted onto a copper roller spinning at 1000 revolutions per minute from the pinhole by an Ar blow. The precursor ribbons obtained were collected for dealloying. The dealloying of Mg-Cu-Sn precursor ribbons was performed in a 1 wt.% tartaric acid (TA) at room temperature until no bubbles emerged. The as-dealloyed ribbons were rinsed with deionized water and dehydrated alcohol for further characterizations and applications.MgThe microstructures of the as-dealloyed samples were investigated with scanning electron microscope  and transmission electron microscope . The crystalline nature was probed using high-resolution TEM  and selected-area electron diffraction . Nitrogen adsorption/desorption isotherms were measured with a surface area and porosity analyzer  at 77 K. Specific surface area was determined using the Brunauer–Emmett–Teller (BET) method. Pore size distribution was calculated from the adsorption branch by the Barrett–Joyner–Halenda (BJH) method using the corrected form of Kelvin equation. The ex situ XRD patterns of the discharged/charged samples were determined by a XD-3 diffractometer  equipped with Cu-Ka radiation as described before ,34. The 6Sn5 or Cu6Sn5/Sn powders (80 wt.%) were ground with acetylene black  and polyvinylidene fluoride (PVDF) binder (10 wt.%) in N-methyl-2-pyrrolidinone (NMP) solvent to make a slurry. The slurry was coated onto Cu foil and further dried at 80 °C for 12 h under vacuum. The coated Cu foil was punched into disk shape of 12 mm in diameter, with the low mass loading of around 0.5 mg/cm2. The as-prepared foil was used as the working electrode. A Li foil was used as both the reference electrode and counter electrode and a polypropylene (PP) film (Celgard 2325) was utilized as the separator. The electrolyte was made by mixing 1.0 M LiPF6 with ethylene carbonate (EC) and DMC (1:1 by volume). The CR2032-type coin cells were assembled in an argon-filled glove box , with oxygen and moisture levels below 0.1 ppm. The galvanostatic discharging/charging tests were conducted at various current densities at the voltage window between 0.01 and 2.00 V (vs. Li+/Li) using a LANDCT2001A test system. Cyclic voltammetry (CV) was conducted at a scan rate of 0.1 mV s−1 between 0.01 and 2.00 V (vs. Li+/Li) on an electrochemical workstation (CHI 660E).The as-dealloyed samples were ball-milled to fine powders with zirconia beads at a rotation speed of 300 rpm for 3 h. The Cu67Cu18Sn15 (66Cu10.2Sn23.8 (2Sn (PDF # 65-2997) and Mg2Cu (PDF # 65-5460) phases. Only a minor amount of Mg2Cu is present, which indicates that substantial Cu enters the lattice of Mg2Sn to form Mg2 phase. The dealloying of Mg67Cu18Sn15 (66Cu10.2Sn23.8 (6Sn5 (PDF # 65-2303) alloy and Cu6Sn5/Sn (PDF # 65-2303/PDF # 65-0296) composites, respectively, as described in ref [6Sn5 involved the selective dissolution of Mg and co-diffusion of Cu and Sn [6Sn5 derived from Mg67Cu18Sn15 has ultra-fine bi-continuous ligament-channel morphology. EDS spectra confirms the co-existence of Cu and Sn  crystal plane (0.34 nm) of Cu6Sn5  are shown in 6Sn5 anode shows a peak at 0.77 V (vs. Li+/Li), due to the irreversible reactions in the initial cycle [6Sn5 anode was evaluated using galvanostatic discharge–charge cycling. +/Li) and 2.0 V (vs. Li+/Li) of the dealloyed Cu6Sn5 anode at the current density of 0.1 A g−1. For the first cycle, the discharge process shows a plateau at around 0.25 and 0.77 V (vs. Li+/Li), corresponding to the CV curve in +/Li), in correspondence with the slight anodic peak from CV curves. The charge processes have plateaus at around 0.5 and 0.75 V (vs. Li+/Li), which were also found in other Cu6Sn5-based materials [6Sn5 clearly shows the peaks indexed to Cu6Sn5 phase (PDF # 65-2303)  emerges at around 24.5 °C and 40.6 °C. The XRD pattern corresponding to the fully charged state of the second cycle confirms the transition from Li2CuSn to Cu6Sn5 again. Furthermore, the second fully discharged process proves the reversible conversion from Cu6Sn5 to Li2CuSn. The ex situ XRD patterns indicate the reversible conversion of Cu6Sn5 to Li2CuSn during the charge/discharge process. 6Sn5 alloy at the current density of 0.1 A g−1. The initial discharge capacity is as high as 700 mA h g−1, slightly higher than that from electrodeposited Cu6Sn5 alloy film [6Sn5 ligaments [−1 for at least 20 cycles and keep at ~200 mA h g−1 for the following 400 cycles with the Coulombic efficiency of nearly 100%. The residual stress of electrodes is related to the performance and stability of energy generation devices such as LIBs here, and np-Cu6Sn5 with bi-continuous ligament-channel structure own strain relaxation, favoring the cyclability of LIBs [6Sn5 anode is expected to be improved by adjusting the constituent of alloy precursors and dealloying conditions in the future. The np-Cu6Sn5 anode also presents excellent rate performance, as illustrated in −1, the capacity is ~900 mA h g−1 for the first cycle and maintains at higher than 700 mA h g−1 for 10 cycles. With the current density increasing to 2.0 A g−1, the capacity gradually decreases to ~380 mA h g−1. The anode recovers to a capacity of around 600 mA h g−1 when the current density is reversed to 0.1 A g−1. The results demonstrate that the nanoporous structure gives an excellent rate performance to the Cu6Sn5 anode, even after experiencing the high current density of 2.0 A g−1. The anode was also performed under 0.5 A g−1 to investigate its possible application under a higher current density. As shown in −1 for at least 1000 cycles, demonstrating excellent stability.CVs at a scan rate of 0.1 mV sal cycle . The irraterials ,23,24,2665-2303) c. After igaments ,9. The c of LIBs . The dec6Sn5/Sn composite was also tested. The initial charge capacity is higher than 800 mA h g−1, better than that of Cu6Sn5, suggesting the contribution of Sn in the composite (−1 for at least 550 cycles, comparable to that of np-Cu6Sn5. At a higher current density of 0.5 A g−1, the capacity could maintain 150 mA h g−1 (6Sn5/Sn composite also shows an inferior rate performance, as shown in −1, the capacity could only maintain around 60% of its initial capacity at 0.2 A g−1. The inferior cycling performance might be attribute to the Sn particles with large volume expansion during charge/discharge process [The charge/discharge cycling performance of Cuomposite a. The camA h g−1 . The Cu66Sn5 alloy and Cu6Sn5/Sn composite were prepared via dealloying and their performance as lithium-ion battery anodes were tested. The dealloyed np-Cu6Sn5 presented an ultrafine ligament-channel structure with ligament size below 40 nm and BET specific area of 15.9 m2 g−1. The 3D nanoporous structure and high specific area endowed the anode with high lithium-ion battery performance. The anode displayed a capacity of higher than 600 mA h g−1 for the initial five cycles at 0.1 A g−1. It also had excellent cycling performance for at least 350 cycles at 0.1 A g−1, with a reversible capacity of higher than 200 mA h g−1. The ex situ XRD patterns reveal the reverse phase transformation between Cu6Sn5 and Li2CuSn. Similar performance was obtained from Cu6Sn5/Sn composite. This study provided a practice for fabricating highly stable Sn-based anode in lithium-ion batteries.In summary, nanoporous Cu


Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity. Human cytochrome P450 (CYP) 2C9, a member of the CYP2C subfamily of CYP enzymes, is an important drug metabolizing enzyme and represents as much as ~20% of the total hepatic cytochrome P450 content ,2,3. It The amino acid arginine at 150 (R150) in CYP2C9 wild-type (WT) or histidine (R150H) in CYP2C9*8 is located on the surface of the protein at the D-helix region and away from the active site. The R150 in WT is highly conserved across different species (181 protein sequences) . The effThe structure of CYP2C9*8 complexed with losartan clearly illustrates the amino acid change from R to H at position 150 (R150H) located on the surface of the protein . The sidThe structural overlay of the active site of the *8 and the WT complex of losartan is shown in the 4β loop, where the sidechain of F476 in the *3 complex rotates by ~90° into the access channel  value for losartan binding to CYP2C9*3 was 24.5 ± 2.7 µM, which was more than three-fold weaker than the WT enzyme with a previously published DK value of 6.8 ± 0.7 µM for losartan binding [DK for losartan binding to CYP2C9*8 was 8.95 ± 0.97 µM, which was comparable to that yielded previously by the *2 variant (9.0 ± 0.5 µM). Differential Scanning Fluorimetry (DSF) was performed in order to measure the thermal denaturation or “melting” point (mT) of CYP2C9 WT and the *2, *3 and *8 variants using SYPRO Orange in the presence and absence of losartan in similar buffer conditions as with ITC. The results show that there are small differences between the thermal stability of the ligand-free WT and variant proteins ethyl)phosphoryl)ethyloxy)-cholane (FA-7/234-chol) is a facial amphiphile used as described previously [Tryptone enzymatic digest, yeast extract, glycerol, potassium phosphate dibasic, potassium phosphate monobasic, L-histidine monohydrochloride, ampicillin sodium salt, phenylmethylsulfonyl fluoride (PMSF), sodium chloride, ethylenediaminetetraacetic acid (EDTA), chloramphenicol, Terrific Broth, deoxyribonuclease I and ribonuclease A were purchased from Sigma-Aldrich . Difco Luria-Bertani broth lennox from Becton Dickinson . Isopropyl β-F-1-thiogalactopyranoside (IPTG) Dioxane-Free and 5-aminolevulinic acid hydrochloride were obtained from BOC Sciences . CHAPS(2-2-Cholamidopropyldimethylammonio)-1-propanesulfonate and Tris(2-carboxyethyl)phosphine hydrochlorate (TCEP) were obtained from Soltec venture . HisPur Ni-NTA resin was from ThermoFisher Scientific . CM Macroprep was from Bio-Rad . 5-Cyclohexyl-1-hexylpentyl-β-D-maltoside (CYMAL-5) was obtained from Anatrace . The 3eviously . PEGRx sE. coli Rosetta2 competent cells, which supply tRNAs for rare codons on a compatible chloramphenicol-resistant plasmid. Autoclaved Luria-Bertani broth, supplemented with chloramphenicol (25 μg/mL) and ampicillin (100 μg/mL), was inoculated with a plasmid glycerol stock of CYP2C9*8, *2 or *3 and grown overnight in a shaking incubator at 225 rotations per minute (RPM) and 37 °C. The following day, autoclaved Terrific Broth, supplemented with chloramphenicol (25 μg/mL) and ampicillin (100 μg/mL) as well as TB salts, was inoculated with 15 mL of the overnight culture. The culture was subsequently grown at 37 °C with shaking at 230 RPM until reaching an optical density at 600 nm wavelength of 0.7–0.8. The protein expression was induced by adding IPTG and 5-aminolevulinic acid at 1 mM and 0.5 mM, respectively. The cultures were incubated in the shaking incubator at 30 °C and 190 RPM for 65 h. E. coli cells were harvested by centrifugation at 4000× g for 10 min at 4 °C.The CYP2C9*8 (R150K), CYP2C9*2 (R144C) and CYP2C9*3 (I359L) were generated from the CYP2C9 wild-type construct, which was a gift from Dr. Keiko Maekawa, Doshisha Women’s College of Liberal Arts, Kyoto, Japan. As described previously, the amino acid residues from 1 to 23 in the CYP2C9 sequence that represents the N-terminal transmembrane anchor domain were replaced with residues MAKKT, and the C-terminal or last residue at position 490 was mutated from valine to isoleucine (V to I), followed by a His-tag or four histidine residues for purification purposes . The altv/v) glycerol, 1.25 mM TCEP and 1 mM PMSF. Lysozyme (0.3 mg/mL) was added to the resuspended cells and stirred for 30 min at 4 °C, followed by centrifugation at 5640× g for 30 min. The spheroplasts were resuspended in 5% original culture volume in 500 mM potassium phosphate, 20% (v/v) glycerol, 1.25 mM TCEP, 0.5 mM PMSF, ribonuclease A (10 μg/mL) and deoxyribonucelase (10 μg/mL). The homogenized mixture was sonicated at 40 pulses per minute for 1 min for a total of four minutes. The detergent CHAPS was added , and the lysate was stirred for 90 min (4 °C) followed by centrifugation at 33,000× g for 2 h at 4 °C.The pellet was resuspended in 10% original culture volume (400 mL for 4 L culture) with 20 mM potassium phosphate (pH 7.4), 20% (v/v) glycerol . The protein supernatant was added to the equilibrated resin and stirred overnight to promote protein binding. The protein-bound resin was collected by centrifugation for 3 min at 1940× g and added to a Bio-Rad Econo-Column. The Bio-Rad NGC Quest chromatography system was used to wash the resin on the column with ten column volumes of 100 mM potassium phosphate buffer with 20% glycerol, 0.5 mM PMSF, 2 mM TCEP, 100 mM NaCl, 0.5% CHAPS and 5 mM histidine. The protein was eluted with 10 mM potassium phosphate, 20% glycerol, 100 mM NaCl, 2 mM TCEP, 0.5 mM PMSF, 0.5% CHAPS and 50 mM histidine at a rate of 1.5 mL/min. Using a UV spectrophotometer, the absorbance of each of the collected fractions was measured at 415 nm and 280 nm. Those fractions with a 415/280 ratio greater than 1 were pooled, and the concentration was measured. The pooled protein was diluted 5 times (by volume) with a 5 mM potassium phosphate buffer with 20% glycerol, 0.5 mM PMSF, 2 mM TCEP, 1 mM EDTA and 0.5% CHAPS.Equilibration of the nickel NTA resin was performed by using 50 mM potassium phosphate buffer with 20% , was performed using 10 mM potassium phosphate buffer with 20% glycerol (1 mL resin/100 nmol protein). The CM resin was incubated overnight with the diluted protein (4 °C). The resin was collected by centrifugation at 2880× w/v). Finally, losartan was added to a final concentration of 1 mM and the complex was filtered prior to crystallization using the Amicon filtration units. Crystal screening was performed using sitting drop vapor diffusion where drops were equilibrated against 50 μL well-reservoir by mixing screen solution (PEGRx from Hampton Research) and CYP2C9*8-losartan complex in a 1:1 ratio. The plates were incubated at 18 °C, and the crystals of CYP2C9*8 grew over the course of 1–2 weeks in drops containing 0.1 M HEPES pH 7.5 and 30% w/v Polyethylene glycol 1000. Crystals for the CYP2C9*8-losartan complex were collected in a crystallization buffer by using 20% sucrose as cryo-protectant, and then it was flash frozen in liquid nitrogen before shipping them to the Stanford Synchrotron Radiation Light source.The pooled protein was diluted to 18 μM using the sucrose buffer . A stock solution of losartan, dissolved in water, was prepared at 40 mM concentration and added to a final concentration of 180 μM. The protein–ligand solution was incubated on ice overnight. The following day, the protein–ligand complex was concentrated 10 times by volume using Amicon Ultra centrifugal filters (10 kDa molecular weight cut off) and then diluted once again to 18 μM with the addition of losartan to 180 μM. This concentration/dilution process was repeated twice more. The protein–ligand complex was then concentrated to a final concentration of 500 μM. CYMAL-5 (detergent) was added to 4.8 mM final concentration followed by the addition of 234-chol facial amphiphile  incubated with 50 pmol human cytochrome P450 reductase on ice for 5 min ,43. An aDifferential Scanning Fluorimetry was performed in a 96-well clear plastic plate using an Eppendorf Master cycler Realplex2 qPCR machine to monitor thermally induced protein denaturation by measuring changes in fluorescence of a dye SYPRO Orange, which binds to hydrophobic regions of proteins exposed by unfolding. The CYP2C9 WT, *2, *3 and *8 protein samples were in the buffer containing the 50 mM potassium phosphate, pH 7.4, 500 mM NaCl, 1 mM EDTA and 1 mM TCEP. Two control experiments included are (1) the buffer with 5 μM SYPRO orange dye and (2) the buffer with 5 μM SYPRO orange dye with 50 μM losartan. The experimental setup included samples with or without losartan. One set of samples included the WT or *2 or *3 or *8 with 5 μM SYPRO orange dye in the buffer, and another set of samples included the WT or *2 or *3 or *8 with 5 μM SYPRO orange dye and 50 μM losartan in the buffer. Each sample was made to a total volume of 300 μL. Each well had 98 μL sample volume, and the assay was performed in triplicates. The plate was sealed with an optically clear adhesive tape to prevent sample loss during heating. The temperature gradually ramped up from 25 to 95 °C while monitoring the change in fluorescence intensity of the dye. GraphPad Prism software was used to analyze the data and used to calculate the melting point of the protein sample by applying nonlinear regression using the melting Boltzmann equation. The standard error was calculated from triplicates of two individual experiments.DK and N values. The active fraction of CYP2C9*8 or *3 in the sample cell was calculated based on a 2:1 ratio for the enzyme in complex with losartan as observed in the crystal structure of these variants. The raw data were integrated, baseline adjusted, normalized for active fraction concentration, and then analyzed with a one set of sites binding model. The experiments were performed in triplicate, and the overall method was consistent with the experiments performed previously with CYP2B6 [Isothermal titration calorimetry (ITC) data were obtained using a MicroCal ITC200 . Experiments were performed at 25 °C with 900 μM losartan in the syringe titrated into 83 μM CYP2C9*8 or *3 in the sample cell. CYP2C9*8 or *3 was snap-frozen in liquid nitrogen for storage after purification and then thawed out and dialyzed into ITC buffer containing 50 mM potassium phosphate, 500 mM NaCl, 1 mM EDTA and 1 mM TCEP at pH 7.4. Losartan (soluble in water) was diluted to 900 μM for titration using the ITC dialysis buffer. ITC experiments were performed by titrating a total volume of 40 μL of losartan in the syringe into 200 μL of CYP2C9*8 or *3 in the sample cell over the course of 28 injections. The first injection was 0.5 μL followed by a delay of 180 s. Subsequently, 27 injections of 1.4 μL with a delay of 150 sec between injections were titrated into the sample cell. All injections had a duration time of 1 s. The syringe paddle was constantly spinning at 750 RPM in the sample cell for the duration of the experiment. ITC binding isotherms were analyzed using Origin 7.0552 software  in order to perform a Marquandt nonlinear least squares analysis assuming one set of sites model. The initial injection was omitted from the h CYP2B6 .


Alzheimer’s disease (AD) is a central nervous system degenerative disease, with no effective treatment to date. Administration of immune checkpoint inhibitors significantly reduces neuronal damage and tau hyperphosphorylation in AD, but the specific mechanism is unclear. Here, we found that programmed cell death-receptor 1 (PD1) and its ligand PDL1 were induced by an intracerebroventricular injection of amyloid-β; they were significantly upregulated in the brains of APP/PS1, 5×FAD mice and in SH-SY5Y-APP cell line compared with control. The PD1 and PDL1 levels positively correlated with the glycogen synthase kinase 3 beta (GSK3β) activity in various AD mouse models, and the PDL1-GSK3β immune complex was found in the brain. The application of PD1-blocking antibody reduced tau hyperphosphorylation and GSK3β activity and prevented memory impairments. Mechanistically, we identified PD1 as a critical regulator of GSK3β activity. These results suggest that the immune regulation of the PD1/PDL1 axis is closely involved in AD. PD1, together with its ligand PDL1, constitutes a critical component in the induction and maintenance of autoimmune tolerance . The PD1gulation . These rgulation . Despitegulation . Functiogulation  and othegulation , suggestgulation , suggestin vitro and in vivo. We also showed that the PDL1/GSK3β immune complex exists in the brain and hypothesized that the PD1-PDL1-GSK3β axis plays a vital role in tau hyperphosphorylation in AD. We found that the application of anti-PD1 blocking antibody reduced tau hyperphosphorylation and improved the memory ability of 5×FAD mice. Overall, our study highlights an important role of PD1 regulation in the treatment of human AD and provides a classic theoretical explanation for the immunotherapy of AD.In the present study, we demonstrated that PD1 and PDL1 are upregulated in AD models Anti-hamster IgG was obtained from SouthernBiotech, and anti-PD1 blocking antibody (G4) and anti-PDL1 antibody (10B5) were produced in-house. Detailed information on the antibodies used in the present study is presented in For this study, we used PD1 knockout (KO) mice, which has been described previously , APP/PS1Synthetic β-amyloid (1–42) peptides (corresponding to the human Aβ sequence) were provided by China Peptides Co., Ltd. The Aβ1-42 powder was dissolved in sterile saline solution to a concentration of 1 μg/μl. The solution was then aged for 96 h at 37°C. Aβ aggregates or the corresponding vehicle (4 μl/5 min/mouse) was injected into the lateral ventricle (i.c.v.) using a microsyringe at the following position: −0.2 mm anteroposterior (AP), +1.0 mm mediolateral (ML), and −2.4 mm dorsoventral (DV) relative to the bregma . The conThe dosing program of 5×FAD transgenic mice was based on a previous study . The mic2O2 and 10% fetal bovine serum (FBS) were successively added on the sections. Subsequently, the sections were incubated with the appropriate concentration of primary antibody overnight at 4°C, and then with HRP-conjugated secondary antibody for 30 min at room temperature. 3,3ʹ-Diaminobenzidine was added on the sections under a microscope and allowed to react for 2–5 min; the reaction was then stopped by adding H2O. The sections were then stained with hematoxylin and dehydrated with a gradient series of alcohol. After permeabilization with xylene, the sections were covered with permanent mounting medium. Positive staining was detected and calculated using Image-Pro Plus software for Windows operating system.For immunohistochemical evaluation of the mouse brain tissues, the mice were sacrificed and their brains were harvested, postfixed in 4% buffered paraformaldehyde overnight, embedded in paraffin, and cut into sections. Xylene was used to deparaffinize the paraffin-embedded tissue sections. The resulting sections were rehydrated with a gradient series of alcohol, and antigen retrieval was performed with citric buffer at 120°C for 10 min. To eliminate the influence of endogenous peroxidase and protein, 3% HMouse hippocampal extracts were prepared by homogenizing the tissue in ice-cold RIPA lysis buffer supplemented with 1% (v/v) phenylmethanesulfonyl fluoride, as previously described . Boiled The brains of WT C57BL/6 mice were harvested and homogenized with a lysis buffer (Beyotime Biotechnology). Nonspecific IgG (Beyotime Biotechnology) or PDL1 antibody was added to the lysates, which were then incubated for 3–4 h. Thereafter, 40 μl of protein A/G beads (Santa Cruz Biotechnology) was added to the lysates and incubated for another 1–2 h at 4°C. The precipitates were washed with washing buffer at least five times and then separated by 10% SDS-PAGE for immunoblotting.SH-SY5Y and SH-SY5Y-APP cell lines were provided by Professor Tae Ho Lee; these cell lines have been described elsewhere . The celThe cells were harvested and incubated with different primary antibodies for 30 min at 4°C, and then washed with PBS (1% FBS). The samples were analyzed using FACSVerse, and the data were analyzed using FlowJo software. APC-conjugated PD1 antibody and APC-conjugated PDL1 antibody were purchased from Biolegend.post-hoc test when appropriate or paired Student’s t-test was used to calculate statistical significance.All data are presented as mean ± SD and analyzed using GraphPad Prism version 8.0 software for statistical analysis. A one-way or two-way analysis of variance followed by Dunnett’s in vitro model to mimic AD pathology. We used flow cytometry and western blotting to compare the expression of PD1 and PDL1 between SH-SY5Y and SH-SY5Y-APP cells. There was an increase in the expression of PD1 and PDL1 in SH-SY5Y-APP cells, as shown in in vitro and in vivo.Under normal physiological conditions, human primary neuroimmune cells express very low levels of PD1/PDL1, but neuronal PD1/PDL1 can be immediately induced by drug abuse or non-toxic doses of alcohol to contribute to neuroinflammation and neurodegeneration . The AD cis-trans isomerase NIMA-interacting 1 . To further verify the relationship between GSK3β and PD1 and PDL1, two other transgenic mice were used in the following experiments. In APP/PS1 mice, decreased p-GSK3β (Ser9) level . Moreover, activated GSK3β level in 5×FAD mice was significantly increased compared to that in WT littermates . Moreover, co-immunoprecipitation and double immunofluorescence assay revealed the PDL1/GSK3β immune complex in the brain and in SH-SY5Y-APP cells, respectively  GSK3acting 1 , GSK3β iacting 1 . In our acting 1  and inve9) level  inverseltermates . Furtherectively , suggestAs PD1 is considered a regulator of GSK3β phosphorylation in different AD models, we aimed to clarify whether the upregulation of GSK3β activity in AD conditions is associated with PD1. PD1 deficiency tends to increase pS473-AKT level in normal Kupffer cells and restores AKT activation after murine polymicrobial sepsis attack, suggesting that PD1 KO protects cells from injury stimuli . UnexpecNext, we examined the effect of PD1-blocking antibody on Aβ-induced GSK3β activation and tau hyperphosphorylation. PD1-blocking antibody increased the expression of phosphorylated GSK3β at Ser9 and reduced the p-tau Thr231 and Ser396 levels after Aβ42 administration compared with those in the control, as shown in p < 0.01). Moreover, the average escape latency of the anti-PD1+5×FAD group was significantly lower than that of the IgG+5×FAD group (p < 0.05). However, there was no significant difference in the mean swimming speed during the training phase among the three groups (p > 0.05). In the probe trial, the number of platform crossing was determined for 1 min on day 6 of the test. As expected, 5×FAD mice treated with control IgG had fewer platform crossings than the normal control subjects, and this was reversed by PD1-blocking antibody treatment (To determine the potential role of PD1 blockade in the learning and memory abilities in 5×FAD mice, hippocampus-dependent cognitive performance was evaluated using the MWM 1 month after the antibody treatment. As shown in reatment . These rWe aimed to develop a suitable model wherein PD1 regulates GSK3β activity, which may be related to tau hyperphosphorylation and cognitive dysfunction in AD . InhibitPD1/PDL1 checkpoint blockade exerts a potent protective effect against cognitive impairment and tau hyperphosphorylation in various mouse models of AD  and stroIn conclusion, our study lays a solid theoretical and experimental basis for the immunotherapy of AD, and immune checkpoint inhibitors are expected to become potent tools for the treatment of AD.


However, the sensitivity of this approach is limited by the natural abundance of 13C, which masks detection of 2H by mass spectrometry. Here we report that high-resolution Orbitrap gas-chromatography mass-spectrometry resolves 2H and 13C fatty acid mass isotopomers, allowing DNL to be quantified using lower 2H2O doses and shorter experimental periods than previously possible. Serial measurements over 24-hrs in mice detects the nocturnal activation of DNL and matches a 3H-water method in mice with genetic activation of DNL. Most importantly, DNL is detected in overnight-fasted humans in less than an hour and is responsive to feeding during a 4-h study. Thus, 2H specific MS provides the ability to study DNL in settings that are currently impractical.De novo lipogenesis (DNL) is disrupted in a wide range of human disease. Thus, quantification of DNL may provide insight into mechanisms and guide interventions if it can be performed rapidly and noninvasively. DNL flux is commonly measured by  Fat synthesis is necessary for normal physiology, but its dysregulation contributes to the pathology of many diseases. Here, the authors report a high-resolution mass spectrometry approach that quantifies fat synthesis flux in humans and mice following a brief and low dose of deuterated water. Lipogenic genes are regulated by sterol regulatory element-binding protein-1c (SREBP-1c)2 and carbohydrate-responsive element-binding protein (ChREBP)3 in response to cell signaling and glycolytic intermediates. Overactivation of these transcription factors during obesity and insulin resistance triggers non-alcoholic fatty liver disease4 (NAFLD) by elevating hepatic DNL7. NAFLD carries additional risk for dyslipidemia, cardiovascular disease, steatohepatitis, cirrhosis, and hepatocellular carcinoma8. DNL is also elevated in some types of cancer cells9, may be disrupted in central nervous tissue during neurological disease10, and plays a role in the transformation of immune cells11. In addition to its role in nutrient metabolism, DNL generates endogenous ligands essential for cell signaling pathways such as PPARα13, and small amounts of DNL in mitochondria are necessary for normal mitochondrial function14. Hence, the ability to monitor the function of DNL in settings ranging from organelles, cells, tissue biopsies, mouse models, and humans will facilitate the discovery of new molecular physiology and potential interventions for many different diseases.De novo lipogenesis (DNL) is the synthesis of fatty acids from non-lipid nutrients, such as carbohydrates, and is essential for long-term calorie storage and the maintenance of cell membranes. This process occurs in the cytosol of most cells but is highest in liver and adipose tissues+ captured from water. Thus, each 2-carbon unit of a new fatty acid originates from acetyl-CoA, and its hydrogens originate predictably from NADPH, H2O, and the original acetyl/malonyl-CoA methyl group  and secreted into circulation as VLDL. Thus, palmitate isolated from liver TG or circulating TG is used as an analyte for hepatic DNL measurements.As with most metabolic pathways, measuring DNL flux by isotope tracers requires that precursors and products of the pathway be well defined. DNL begins with the carboxylation of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase. Fatty acid synthase (FASn) condenses acetyl-CoA and sequential units of malonyl-CoA and, after each elongation, reduces carbonyl groups by NADPH-dependent reductases and Houp Fig. 15. The p13C and 14C) labeled substrates that form acetyl-CoA or hydrogen isotopes (2H and 3H) of body water are candidate tracers of DNL16. Lipogenic 14C-substrates that form acetyl-CoA  detect DNL with high sensitivity, but quantification is complicated since dilution of the lipogenic acetyl-CoA pool is difficult to assess. More sophisticated 13C approaches, such as mass isotopomer distribution analysis (MIDA), provide acetyl-CoA dilution and fractional DNL by a statistical analysis of palmitate mass isotopomers17. However, the method requires constant infusion to maintain steady-state precursor enrichment, which is impractical for extended experimental periods. Hydrogen isotope tracers of water overcome this limitation because its enrichment diminishes very slowly following initial administration and can be maintained for months with enriched drinking water6. When present, hydrogen isotopes of body-water incorporate into new palmitate at known positions and stoichiometry  labeled FAs is sensitive and straightforward18, but safety and logistical considerations limit the use of radiolabeled tracers in preclinical models and virtually prohibit their use in humans. In contrast, administration of 2H2O is safe, inexpensive, convenient, and has been used extensively to estimate DNL in vivo1.Carbon isotope  with high sensitivity, and 2H incorporation is commonly measured as mass shifts in the spectra 16. The 2H labeling of a new lipid molecule is a function of DNL flux, body-water enrichment, and the total number of exchangeable hydrogens. Palmitate has 31 hydrogens, but only 22 are exchangeable during synthesis19. Hence, the 2H fractional enrichment of the palmitate pool increases over time towards a maximum enrichment that is proportional to DNL and 22 x body-water enrichment. Typical 2H2O doses in mice enrich body-water to ~5%20, but ~0.5% enrichment is more common in human studies19. To reach this body-water enrichment, human subjects must drink ~200 mL of 70% 2H2O, which is usually divided over several hours to avoid nystagmus and nausea21. This staggered administration prolongs the study period, limits throughput, and convolutes the first several hours of labeling during an acute 2H2O study. Here, we investigate the use of high-resolution Orbitrap gas chromatography mass spectrometry (HR-Orbitrap-GC–MS) to resolve 2H and 13C fatty acid mass isotopomers. We report that this approach allows 2H2O doses that are 50-times lower than the typical mouse dose and two times lower than the typical human dose, and that tracer incorporation into plasma TG-fatty acids can be detected 1-h after administration. The combination of lower doses and rapid detection will allow DNL flux to be investigated in clinical and basic studies that were previously impractical.Gas chromatography-electron ionization-MS (GC-EI-MS) detects palmitate  makes tracer doses of less than 0.5% theoretically possible, but quantitation of low 2H enrichment by GC–MS is limited by natural abundance isotopes of other nuclei in the analyte16. Most notably, the 1.1% natural abundance of 13C produces a background M + 1 that is ~18% of the M + 0 palmitate ion. Natural abundance correction algorithms provide reliable 2H mass enrichments, if excess enrichment in the mass isotopomer distribution can be detected16. Under ideal conditions, this approach is limited to >0.1% molar excess 2H in palmitate19. A binomial model of 2H enrichment16 predicts that a fractional DNL flux of 10% in the presence of 0.3% body water will produce 0.7% molar excess 2H enriched palmitate, which is consistent with previous DNL measurements in humans under these parameters19. However, the challenge of making this measurement is illustrated by simulation data that predicts nearly imperceptible increases in M + 1 (+3%) and M + 2 (+5%) as the fraction of new lipid  ranges from 0 to 10%  mass shifts can be resolved with a minimum resolving power of 165,00022. Baseline separation of 13C1 and 2H1 has been demonstrated for palmitate and peptides using Fourier transform ion cyclotron resonance mass spectrometry23, an instrument that can achieve a resolution >600,000, but requires long scan times (~4 s), a high-field superconducting magnet, and is generally restricted to MS technology development laboratories. More recently, high-resolution Orbitrap MS detection has become commercially available, making it feasible to resolve 13C and 2H mass shifts in metabolism labs. To establish whether this approach provides an advantage over unit resolution MS, we simulated ultra-high-resolution MS (HRMS) mass isotopomers of 2H-palmitate across a 0–10% range of fractional DNL and a 0.3% body water enrichment. In contrast to unit resolution M + 1 . Pentafluorobenzyl bromide was used to derivatize palmitate prior to analysis in electron capture negative ion (ECNI) mode. ECNI detection of the pentafluorobenzyl ester produced an unfragmented palmitate-ion  with higher efficiency and a 1000-fold higher sensitivity than EI detection of the methyl ester  at different AGC targets . The M + 1 of 2H1 and 13C1 of palmitate standards were well-resolved by both methods  and precision (coefficient of variation <10%) 26 and 2H NMR27 can detect 2H in water at, or below, natural abundance, but compared to GC–MS, these specialized instruments are uncommon in metabolism labs. A simplified technique allows 2H2O enrichment to be measured following base-catalyzed hydrogen exchange into acetone, with subsequent detection of deuterated acetone by GC–MS29. Again, the sensitivity of this approach is limited by background 13C  natural abundance28. However, HR-Orbitrap-GC–MS easily meets the minimum resolution  required to resolve 2H and 13C isotopomers of acetone22. The 2H of water could be detected as M + 1 2H1 acetone at natural abundance levels , and 5 µL blood samples were examined. The 2H2O dose equilibrated with body-water nearly completely in 0.5-h  could be used to quantify DNL in mice. Mice were fasted overnight and refed their normal chow diet 2-h before the administration of 2H2O. Plasma was collected at 0.5,1, 2, 2.5, 3, 4, and 24-h. HR-Orbitrap-GC–MS provided excellent resolution of palmitate 13C1 and 2H1 isotopomers of M + 1  , and the number of exchangeable hydrogens on palmitate. This latter value was estimated to be ~22 from the ratio of fractional 2H1 (0.091) to 2H2 (0.027) in palmitate for the 20 µl/g group  between 2H palmitate enrichment in plasma and liver TGs, suggesting that TG-palmitate sampled from blood reflects hepatic DNL in mice.We next tested whether these + 1 Fig. , and 13C+ 2 Fig. . 2H isoture Fig. . Most pa/g) Fig. . This dioup Fig. , which wDNL Fig. , confirmion Fig. . There w2H1 enrichments. Desaturase products, like palmitoleate (16:1) and oleate (18:1) had roughly half the M + 1 2H1 enrichments compared to their saturated counterparts  is 1.5 to 9-fold lower than doses used in other human studies25 and produced a body water enrichment of ~0.2%  at the 2-h time-point. This intervention caused a transient decline in fractional DNL, likely due to absorption and esterification of meal fat, and was followed by resumption of palmitate eal Fig. . The low2H mass enrichment from other isotopes, to study lipogenesis. Direct detection of 2H mass isotopomers in palmitate prevents convolution with 13C natural abundance at low enrichments. Thus, near-natural abundance 2H can be detected, which is not possible using unit resolution MS. DNL could be detected in 1-h and with lower doses of 2H2O than previously feasible. The dose can likely be lowered another 3-fold, if longer experimental durations are used. Thus, lipogenesis can be evaluated during a short clinical examination, providing the opportunity for large-scale translational studies, or using ultra low doses in vulnerable patient populations. The capacity to collect detailed kinetic data will provide insights into pool sizes and turnover that are lost in a single fractional DNL measurement6, particularly if taken outside of steady-state30. It is also significant that high-resolution detection allows simultaneous 13C and 2H tracer experiments without mass interference. We note that new fatty acid incorporation into specific high molecular weight subclasses of lipid esters  can be detected by HR-LC-MS or direct infusion analyses with HR accurate MS33. Recently DNL was determined from the 2H enrichment of fatty acyl fragments in the MS/MS spectra of some of these lipid esters, providing insight into the synthesis of specific bioactive lipids33. However, the general application of HR-LC-MS to DNL is complicated compared to HR-GC–MS analysis due to the many combinations of fatty acid species and lipid backbones that are detected32. While less applicable to large complex lipids, the HR-Orbitrap-GC–MS is simple and generalizable to all FAs, sterols or other metabolites that form fragments below ~300 Da. The ability to measure 2H incorporation at very low enrichment without 13C interference will allow 2H tracer approaches to be used in metabolic studies that were previously impractical.In this study we introduce a HR-Orbitrap-GC–MS approach, which resolves 2H2O dose and timing for palmitate 2H measurements in plasma TG were determined in C57BL/6 J mice purchased from the UT Southwestern Mouse Breeding Core. Mice were maintained on a 12-h/12-h dark/light cycle, in a room with an average temperature of 22 degrees Celsius, 50-70% humidity and unrestricted access to food and water unless otherwise noted. Male mice, 16-18 weeks of age, were fasted 16 h prior to being refed by providing ad libitum access to standard chow . After 2 h of refeeding, mice received IP injections of either 20, 5, 2.5, or 1 μL/g body weight of 2H2O (n = 5 per group). Animals were provided access to 2H2O labeled drinking water  corresponding to the expected body-water enrichment estimated from previous studies20. Blood was collected from the tail vein at 0, 30, 60, 120, 150, 180, and 240 min after tracer administration. At 24-h post injection, mice were anesthetized with isoflurane, the liver was excised, flash-frozen in liquid nitrogen, and a final blood sample was collected.Animal protocols complied with and were approved by the Institutional Animal Care and Use Committee at the UT Southwestern Medical Center (APN: 2018-102548). The optimal 35. Samples were vortexed for 1 min and then centrifuged for 5 min at 1635 × g. The DCM layer was transferred to a new tube and dried under N2. Samples were saponified with 1 mL 0.5 M KOH in methanol at 80 °C for 1 hr. Resulting FAs were extracted with 2 mL DCM/water  for 1 min, and the solvent was evaporated under N2. Dried lipid extract was resuspended in 50 µL of 1% triethylamine/acetone and reacted with 50 µL of 1% PFBBr/acetone for 30 min at room temperature. To this solution, 1 mL of isooctane was added before MS analysis.Plasma samples (5 µL) were treated with 2 mL  methanol/dichloromethane (DCM) to precipitate proteins and extract triglycerides, similar to previously reported methods13C] palmitate was added to the equivalent of 1 mg of liver extract for concentration assays when applicable. Samples were dried under N2 before saponification, fatty acid extraction, and derivatization were performed as described above for plasma samples.Approximately ~20 mg of the liver was weighed and homogenized with 1 mL methanol/DCM  for 1 min in 2.0-mL pre-filled Bead Ruptor Tubes . The tubes were washed twice with 1 mL methanol/DCM and all solutions were combined. In a new tube, a known amount of [U-2H‐enrichment of palmitate  was determined using a Q Exactive GC-Orbitrap MS (Thermo Scientific). A 1 μL sample was injected onto a HP-5ms capillary column  in split mode. The helium gas flow rate was 1 mL/min for 13 min, followed by 0.4 mL/min for 3 min and then 1 mL/min for the remainder of the run. The GC injector temperature was 250 °C and the transfer line was 290 °C. The column temperature program was 150 °C for 1 min, followed by a 30 °C/min ramp to 250 °C for 12 min, and finally 5 min at 310 °C. Samples were analyzed at 70 eV in ECNI mode with methane gas at 1.5 mL/min. Full scan and t-SIM methods were investigated at different AGC target values  at 240,000  resolution to determine the accuracy and repeatability of palmitate enrichment measurements. In full scan, a scan range of m/z of 250-265 was selected. In t-SIM method, the quadrupole was set to pass ions between m/z 254.2 and 260.2. For both methods, the maximum injection time was 54 ms.The 2H-mol fraction excess standards for validation experiments were prepared by mixing weighed samples of unlabeled and 99.9% enriched 2H1-palmitate. Biologically labeled palmitate 2H enrichment was determined from mass isotopomers M + 1 (2H1), M + 2 (2H2) and M + 3 (2H3). A small correction for 2H1 natural abundance was made using the MID of a biological sample (collected before 2H2O administration) and a matrix correction algorithm24. Palmitate 2H enrichment = 2H1 + (2H2 x 2) + (2H3 x 3). Higher mass isotopomers may also contribute to body water enrichments above 5% ; standard curves containing known concentration ratios of unlabeled and internal standards were run with the samples.Palmitate concentrations were calculated from the ratio of peak areas corresponding to palmitate (m/z 255) and to [U-36 for direct comparison to the palmitate pentafluorobenzyl ester derivative  to 250 °C at a rate of 20 °C/min, then to 275 °C at a rate of 10 °C/min. The final temperature was held for 3 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. GC–MS analysis, with 70 eV EI, was carried out in full-scan acquisition mode over a m/z range of 100–300 at 15,000 mass resolution.Methyl palmitate was prepared as previously described2H species dependent on body water enrichment, the number of exchangeable hydrogens and the theoretical natural abundance of 13C and 2H as a function of %DNL. A spreadsheet with the calculations shown in Fig. High resolution data was simulated as a binomial distribution of 29. Briefly, 5 μL of plasma, 2 μL of 10 M sodium hydroxide, and 5 μL of acetone were added to a GC vial with a PTFE/silicone threaded cap. Samples were incubated overnight at room temperature prior to analysis. Calibration standards of known 2H-mol fraction excess were prepared by mixing weighed samples of naturally labeled and of 99.9% 2H2O.Plasma and acetone were incubated under alkaline conditions as previously reported4 with a maximum injection time of 54 ms.Negative chemical ionization mode (NCI) was used with t-SIM acquisition (m/z 55.5-60.5) and 60,000 mass resolution  on the same Q Exactive GC-Orbitrap MS instrument as described above. 1 μL of headspace acetone gas from the GC vial was injected in split mode. The column temperature program was 2 min at 60 °C, increased by 30 °C/min to 200 °C for 2 min. The transfer line temperature was 250 °C leading into the ion source (250 °C), which was operated with a methane reaction gas at 1.5 mL/min. The Orbitrap AGC target was set to 5em/z values representing each isotopomer ion  was performed using TraceFinder 4.1 (Thermo Scientific) with 4 ppm mass tolerance.Thermo Scientific Xcalibur (version 4.2.47) Data System was used for data collection. Extraction of individual high-resolution 25. Briefly, 2H2O quickly equilibrates with the total body water pool, DNL is measured based on the incorporation of 2H into palmitate following the administration of 2H2O. The contribution of palmitate synthesis was determined using Eq. 2H2O, palmitate enrichment is expressed as described above, BW (body water) is the fraction 2H2O in plasma, and “n” represents the number of exchangeable hydrogens. The value for n was measured to be 22 using 2H1 and 2H2 enrichments and Eq. 19.The contribution of DNL to the pool of total circulating palmitate was determined as previously described2H2O. Absolute palmitate synthesis rates in the liver were determined by multiplying DNL by tissue TG-palmitate concentration where noted.DNL/hr is determined by dividing by the time since the administration of 2H2O and 3H2O was performed using mice that express a transgenic, constitutively active form of SREBP-1a (n = 3) and littermate controls (n = 3), generated as previously described18. 11- to 12-week-old mice were maintained ad libitum on Teklad 2018 chow diet. Food was removed 3-h before mice were injected intraperitoneally with 500 µL of either 50 mCi of 3H2O or 99.9% of 2H2O. After 1-h, mice were anesthetized for plasma and tissue collection. Hepatic DNL measurements by 2H2O were performed as described above. To compare these results to DNL measured using the 3H2O method18, fractional DNL was multiplied by the total amount of liver TG content.Validation of DNL measurements by n = 4, Supplementary Table 2H2O administration. Subjects consumed a single oral dose of 70% 2H2O at 2 g/kg body-water (where body-water was estimated to be 0.6 x body weight in men and 0.5 x body-weight in women), or roughly ~1 g/Kg body weight. Blood samples were collected at 0.5, 1, 1.5, and 2-h. Following the 2-h collection, subjects consumed a liquid meal consisting of 8 ounces of Ensure®, which contains 220 Kcal, 6 g fat, 32 g carbohydrates, 15 g sugar and 9 g protein . Blood collections continued at 2.5, 3, 3.5, and 4-h.Human subjects (2H2O in mice. Significant differences (p < 0.05) were determined using a two-tailed t-test. Significant correlations were determined using a two-tailed Pearson correlation. All statistics and graph preparation were performed in Prism 8 (Graph Pad Software Inc.).Results are expressed as means ± standard errors of the mean (SEM). Sample sizes were chosen based on previous experience but without power analysis. All MS data were collected in duplicate or triplicate. Analysis was performed without blinding. Replication of analytical performance is expressed in Supplemental Table Further information on research design is available in the Supplementary InformationReporting Summary


Osteoporosis significantly impacts the normal life of the elderly and is reported to be closely related to dysfunction of osteoblastic differentiation. Runt-related transcription factor-2 (Runx2) is a critical transcriptional factor involved in the regulation of osteoblast differentiation. Omarigliptin is a novel dipeptidyl peptidase-4 (DDP-4) inhibitor and this study proposes to probe into its possible therapeutic function against Osteoporosis by investigating its impacts on osteoblastic differentiation. Osteogenic medium was used to induce osteoblastic differentiation in MC3T3‑E1 cells, and was verified by the increased alkaline phosphatase (ALP) activity, enhanced mineralization, and promoted expression level of osteoblastic differentiation-related factors, including bone morphogenetic protein-2 (BMP-2), ALP, osteocalcin (Ocn), collagen type I alpha 1 (Col1a1), Collagen Type I alpha 2 (Col1a2), Runx2, osterix (Sp7), fibroblast growth factor receptor 2 (Fgfr2), and fibroblast growth factor receptor 3 (Fgfr3), accompanied by the activation of the p38 and Akt pathways. After treatment with Omarigliptin, the ALP activity and mineralization were further promoted, accompanied by the further upregulation of osteoblastic differentiation-related factors, and activation of the p38 and Akt pathways. Lastly, Omarigliptin-induced osteoblastic differentiation, promoted ALP activity, and increased expression levels of Sp7, Fgfr2, Fgfr3, BMP-2, Ocn, ALP, Col1a1, and Col1a2, in the osteogenic medium- cultured MC3T3‑E1 cells were dramatically abolished by the knockdown of Runx2. Taken together, our data reveal that Omarigliptin promoted osteoblastic differentiation by regulating Runx2. Spp1, Ibsp, and Bglap2 [Osteoporosis is an orthopedic disease mainly observed in the elderly and it affects females more than males . Osteopod Bglap2 . Apart fd Bglap2 . In addid Bglap2 . It is iOmarigliptin is an effective and highly selective inhibitor of DPP-4, with much higher inhibitory activity against DPP-4 than Sitagliptin . With a 2 and 37°C. Osteogenic medium  was utilized to culture MC3T3‑E1 cells for 14 days for osteogenic differentiation, with the medium being replaced every 2 days. Omarigliptin was purchased from Invivochem (#V2771), USA.The rodent pre-osteoblastic cell line MC3T3‑E1 cells were obtained from the American Type Culture Collection (ATCC)  and cultured in α-Minimum Essential Medium (α-MEM)  containing 10% FBS at 5% COTo knockdown the expression level of Runx2 in MC3T3‑E1 cells, the lentiviral containing shRNA targeting Runx2  or control shRNA was designed and synthesized with Genscript . Cells were planted in a 6-well plate, followed by adding the lentiviral-Runx2 shRNA or control shRNA and being incubated for 48 h. The transfection was mediated by the reagent lipofectamine 2000 . The knockdown efficacy was determined using the Western blotting assay.After different treatment strategies, cells were treated with 0.25 mg/ml MTT  at 37°C for 3 h, followed by removing the medium and adding the dimethyl sulfoxide to produce blue formazan. Then, the microplate reader  was used to measure the absorbance at 630 nm .fter necessary treatment, 50 μL of the cell culture medium was collected from each well and mixed with an equal amount of 50 µL reaction buffer. OD value was recorded at 450 nm to index the amount of LDH.2, 8 mM p-nitrophenyl phosphate disodium) and incubated at 37°C for 4 minutes. The reaction was stopped with 0.1 N NaOH. Absorbance at 405 nm was used to index ALP activity.Cells were seeded in a 6-well plate and cultured in the osteogenic medium. The activity of ALP was measured using the ALP activity kit . Briefly, after necessary treatment, cells were lysed and the protein concentration was assessed using a BCA kit. Samples were mixed with reaction buffer ; Col1α1 ; Ocn ; BMP-2 ; RUNX-2 ; Col1a1 ; Col1a2 ; Sp7 ; Fgfr2 ; Fgfr3 ; GAPDH .The Trizol reagents were utilized to extract the cellular total RNA from cells and the concentration of RNA was measured by detecting the optical density at 260 nm, followed by being transcribed into cDNA utilizing the PrimeScript RT Master Mix Kit . The Sybr Premix Ex Taq Kit  was used to perform the RT-PCR in the present study. The expression of genes was determined with the 2Cells were planted in a 6-well plate, followed by being treated with different strategies for 28 days. Then, 4% paraformaldehyde was added to fix cells, followed by adding 2% Alizarin Red S  to examine the extracellular matrix calcification for 15 min. Lastly, 400 μl 10% (w/v) cetylpyridinium chloride  and 10 mM sodium phosphate solution were added into the wells. Lastly, the microplate reader  was utilized to detect the absorbance at 562 nm .After isolating total proteins from cells with the lysis buffer, a BCA kit was used to quantify the isolated proteins, and approximately 30 μg samples were added, followed by separation with 10% SDS-PAGE. Then the protein was transferred to the PVDF membrane  and then incubated with 5% skim milk. The membrane was then immersed in the primary antibodies against BMP-2 , Ocn , ALP , Runx2 , Sp7 , Fgfr2 , Fgfr3 , p-p38 , p38 , p-Akt , Akt , and β-actin . Subsequently, the membrane was incubated with the secondary antibody  at room temperature for 1.5 h. Lastly, the bands were visualized using ECL solution, followed by quantification with the Image J software.Data were analyzed using the GraphPad Prism version 8.3.0 and were presented as mean ± standard deviation (S.D.). T-test was used to compared two independent data and the data among groups were compared using the two-way analysis of variance (ANOVA) method, while p < 0.05 was taken as a significant difference.Firstly, the cytotoxicity of Omarigliptin in MC3T3‑E1 cells was measured to screen the optimal concentration. Secondly, the effects of Omarigliptin on ALP activity, mineralization, and the expressions of osteoblastic differentiation biomarkers such as BMP-2, Ocn, and ALP genes in MC3T3-E1 cells were assessed. Lastly, we proved that the beneficial effects of Omarigliptin were mediated by the activation of p38, Akt, and Runx2.To determine the optimized concentration of Omarigliptin to be used for the incubation with MC3T3‑E1 cells, the cells were treated with Omarigliptin at various concentrations  for 14 days, followed by evaluating the cell viability using the MTT assay. We found that when the concentration was lower than 20 μM , the celALP activity and mineralization are critical signs for osteoblastic differentiation . Cells wWe further measured the expression levels of important factors involved in the regulation of osteoblastic differentiation, such as BMP-2 , Ocn 2121, and ACol1a1 and Col1a2 [Col1a1 and Col1a2 in osteogenic medium-cultured MC3T3‑E1 cells were further promoted by 20 μM Omarigliptin  could be dramatically upregulated in osteoblasts and mesenchymal stem cells by the overexpression of Runx2, indicating Runx2 significantly facilitated osteoblast differentiation and function [The directional differentiation of mesenchymal stem cells or pre-osteoblastic cells into osteoblasts can be facilitated by Runx2 . In vitrfunction . Recentlfunction . Our datfunction . In futuOverall, our study identifies a previously unknown pharmacological function of Omarigliptin in promoting osteoblastic differentiation by increasing ALP activity, stimulating mineralization, and enhancing the expressions of the relevant biomarker genes. Additionally, our work on the p38MAPK, Akt, and Runx2 pathways as important modulators for this role of Omarigliptin provides novel insights into the molecular mechanism. These findings suggest that Omarigliptin may be a novel therapeutic agent for Osteoporosis.


The conventional high temperature solid state method was used to prepare a series of CaMoyW1−yO4:Eu3+x/Tb3+1−x materials. In order to obtain phosphors with the most promising luminescent properties, the experiment was planned by taking into account the different composition of the matrix and the concentration of the particular dopant ions . As a result, luminescent materials were obtained with a pure tetragonal crystal structure, the space group of I41/a, confirmed by X-ray diffraction (XRD). The size and shape of the particles obtained from the materials were analyzed based on scanning electron microscopy images. Luminescence spectroscopy  was utilized to characterize the luminescence properties of the as-prepared phosphors. The color change of the emission from green-yellow to orange-red was confirmed using the 1931 Comission Internationale de l’Eclairage (CIE) chromaticity coordinates and color correlated temperature (CCT).Due to the ongoing need to create phosphors with the appropriate emission color for the production of light emitting diodes, we decided to synthesize a series of multicolour microphosphors with tunable visible emissions, depending on the composition of dopant ions. In this work, we investigated the structure, morphology, and luminescent properties of new molybdate–tungstate phosphors co-doped with Tb Many different synthesis methods including sol-gel [3+ ions are well known as phosphors that are applied, for example, in solar cells [3+ ions and tungstate or molybdate matrices, we decided to present new tunable phosphors based on mixed inorganic matrices. Recently, many investigations have focused on the synthesis and characterization of molybdate ,2,3,4,5  sol-gel ,15, sono sol-gel ,17, hydr sol-gel ,19, micr sol-gel ,21 as we sol-gel ,23,24, h sol-gel . Economi sol-gel , and pos sol-gel . Inorganar cells ,29, scinar cells , lasers ar cells , bioimagar cells , and fie3+, and europium, Eu3+, dopant ions, as well as with a different composition of the matrix (different W/Mo ratio) synthesized by the high temperature solid state method. Several techniques were used to characterize as-synthesized phosphors, including using X-ray powder diffraction, scanning electron microscopy, and photoluminescence spectroscopy, respectively. Under the selected excitation wavelength, Tb3+ and Eu3+ ions emit light with a color ranging from green through orange to red. Therefore, the synthesized phosphors have potential in an LED application. In this study, the correlated color temperature was calculated and the color emission was visualized on chromaticity diagrams and schemes.In this work, studies of the structure, morphology, and luminescence properties were carried out using different compositions of terbium, Tb3+ [3+ ions [yW1−yO4:Eu3+x/Tb3+1−x materials. The literature also provides information on tunable fluoride materials doped with Eu3+ and Tb3+ [3+ and Tb3+ [3+/Tb3+ ion dopant system in the creation of novel lighting setups. The most recent articles describing materials for LEDs show that there remains a need for phosphors for the production of efficient and bright lighting LEDs  that are3+ ,37,38 or[3+ ions ,40; howeand Tb3+  or lithiand Tb3+ , which i3+/Tb3+—doped phosphors based on a mixed matrix, the luminescent properties of which are controlled by the matrix composition, as well as the number of dopant ions. As a result, the effects on changes in the correlated color temperature (CCT) and chromaticity parameters are analyzed in detail. According to our knowledge, such detailed data are not known in the literature.The presented manuscript shows EuyW1−yO4:Eu3+x/Tb3+1−x phosphors were synthesized by the traditional solid state reaction method. The complete formula used for the required calculations is Ca0.99MoyW1−yO4:Eu3+x/Tb3+1−x; however, for simplicity, the shorter version will be used in the text. In order to obtain materials exhibiting tunable intense emission, an experiment was planned regarding the matrix composition (CaMoyW1−yO4:Eu3+x/Tb3+1−x) and the concentration of dopant ions as follows: y = 0.1 − 0.9, x = 0.1 − 0.9. The starting materials were purchased from Stanford Materials  , terbium oxide ), Sigma Aldrich , tungsten oxide ) as well as from POCh Gliwice ). In the first step, CaCO3, MoO3, and WO3 were mixed at a 1:y:1−y molar ratio (y = 0.1 − 0.9), while in the second step, appropriate amounts of Eu2O3 and Tb4O7 were added and the total molar concentration of activator (Eu3+x/Tb3+1−x) ions was 1 mol.% (x = 0.001. 0.003. 0.005. 0.007. 0.009). The starting materials were ground thoroughly in an agate mortar for 1 h. Finally, the obtained homogeneous powders were placed in corundum crucibles and sintered in a muffle furnace at 900 °C for 4 h. After the calcination process, the samples were cooled down to room temperature. The structure of the final products was determined by X-ray powder diffraction (XRD)  using Cu-Kα radiation (λ = 0.1541874 nm), registered from 10° to 60° (2θ) with the step of 0.05°. Scanning electron microscopy (SEM) images were obtained with the use of a HITACHI SU3500 equipped with a BSE-3D detector , using an acceleration voltage of 10.0 kV. Excitation and emission spectra were measured on a Hitachi F-7000 fluorescence spectrofluorimeter  and corrected for the light source used (xenon lamp). The CIE 1931 chromaticity coordinates, x and y, were used to determine CCT using the McCamy equation [CaMoequation .yW1−yO4 codoped with Eu3+ and Tb3+ ions was obtained. The representative composition of the matrix CaMo0.5W0.5O4 was selected and it was used in further studies as the most suitable for the introduction of dopant ions and for studying the dependency of Eu3+ and Tb3+ concentration on luminescence parameters. The influence of dopant ions on the structural properties of tungstate–molybdate matrices was analyzed based on XRD patterns for representative samples  atoms coordinate with four O2− atoms and divalent Ca2+ atoms coordinate with eight O2− atoms, respectively [6+ (0.42Å) and Mo6+ (0.41Å) confirmed that the positions of the diffraction peaks hardly changed [3+ (r = 1.07 Å for CN = 8) and Tb3+ (r = 1.04 Å for CN = 8) with the ionic radius of Ca2+ (r = 1.12 Å for CN = 8), and the purity of the samples, indicate that the lanthanide dopant ions are expected to substitute Ca2+ ions [As a result of the solid state reaction, a series of tunable CaMo samples . The str lattice ,44. In tectively ,45. The  changed , while t changed . In addiyW1−yO4:Eu3+x/Tb3+1−x luminescent materials synthesized by the high temperature solid state technique at 900 °C for 4 h. It is revealed that the samples have a topography typical for this method. Despite the tendency towards agglomeration [The scanning electron microscopy (SEM) images shown in meration ,46, it w0.5W0.5O4:Eu3+x/Tb3+1−x measured at wavelengths corresponding to the most intense emission bands of Tb3+  and Eu3+ . Both spectra present broad bands with maxima in the range of 200–350 nm, which is the result of the charge transfer (CT) phenomenon. These bands consist of the individual components such as the transitions O2− → W6+, O2− → Mo6+, O2− → Tb3+, and O2− → Eu3+. The intensity and broadness of the CT bands indicate the effectively activated emission of Eu3+ and Tb3+ ions through energy transfer between WO43− and MoO43− groups and activator ions, after CT between W6+ or Mo6+ and O2− [3+ and Tb3+ , 375 nm (7F0 → 5G2), 380 nm (7F0 → 5G3), 393 nm (7F0 → 5L6), 415 nm (7F0 → 5D3), and 464 nm (7F0 → 5D2) peaks, typical for Eu3+. This means that we found the characteristic excitation from Eu3+ when monitoring the luminescence of Tb3+ , 272 nm (b), and 394 nm (c). The emission spectra , 589 nm , 593 nm , 657 nm  and 705 nm . Apart from Eu3+, under the excitation of 394 nm, there are also emission bands assigned to the Tb3+ ion. This may indicate effective energy transfer from Eu3+ to Tb3+  and shorter lifetime time of Eu3+ (λem = 617 nm) may indicate energy migration from Eu3+ to Tb3+.The decay curves of the Tb= 394 nm g. The slyW1−yO4:Eu3+x/Tb3+1−x materials with different doping concentrations of Tb3+ and Eu3+ were calculated based on the corresponding emission spectra. The CIE chromaticity diagram, chromaticity coordinates, and CCT values for CaMoyW1−yO4:Eu3+x/Tb3+1−x excited at 256 nm are shown in 3+ doping concentration. The CCT values are in the range of 2062–6842 K under λex = 256 nm. yW1−yO4:Eu3+x/Tb3+1−x under UV light in Eppendorf tubes and glass tubes.Commission International de L’Eclairage (CIE) chromaticity coordinates for CaMo3+ and Eu3+ were synthesized successfully using the high temperature solid state method. XRD analysis shows that the synthesized phosphors are single-phase with a tetragonal I41/a structure. The excitation spectra show a charge transfer band between 200 and 350 nm which effectively activates the emissions of Eu3+ and Tb3+ ions through energy transfer between WO43− and MoO43− groups and activator ions, after CT between W6+ or Mo6+ and O2−. Scanning electron microscopy (SEM) images presented a strong tendency for phosphors to agglomerate, which is specific to the high temperature solid state synthesis method we used. At 265 nm excitation, the CaMoyW1−yO4:Eu3+x/Tb3+1−x microphosphors show color shifts from yellow-green to orange-red with an increasing doping concentration of Eu3+. The intense emission upon UV light excitation indicates that the new multicolored tungstate–molybdate microphosphors can be used as an alternative to LED components. Detailed studies of Eu3+/Tb3+-doped phosphors based on the mixed molybdate–tungstate matrix, whose luminescent properties were controlled by the composition of the matrix and the number of doping ions, showed that the obtained values of correlated color temperature (CCT) and chromaticity parameters were similar to those recommended by CIE. In addition, the use of a relatively small number of doping ions, compared to others described in the literature, allows for intense emission of the tested phosphors, which is important from an economic point of view.In summary, multicolor molybdate–tungstate microphosphors co-doped with Tb


Herein, our study demonstrates that DLP 3D printing represents a potential manufacturing platform for fabricating personalised drug-loaded punctal plugs with extended release characteristics for ocular administration.Dry eye disease is a common ocular disorder that is characterised by tear deficiency or excessive tear evaporation. Current treatment involves the use of eye drops; however, therapeutic efficacy is limited because of poor ocular bioavailability of topically applied formulations. In this study, digital light processing (DLP) 3D printing was employed to develop dexamethasone-loaded punctal plugs. Punctal plugs with different drug loadings were fabricated using polyethylene glycol diacrylate (PEGDA) and polyethylene glycol 400 (PEG 400) to create a semi-interpenetrating network (semi-IPN). Drug-loaded punctal plugs were characterised in terms of physical characteristics (XRD and DSC), potential drug-photopolymer interactions (FTIR), drug release profile, and cytocompatibility. In vitro release kinetics of the punctal plugs were evaluated using an in-house flow rig model that mimics the subconjunctival space. The results showed sustained release of dexamethasone for up to 7 days from punctal plugs made with 20%  Vision is considered one of the most important senses . Dry eyeNovel formulation approaches to improve ocular bioavailability include extending the drug residence time or promoting corneal penetration with the use of liposomes, nanoparticles, penetration enhancers, mucoadhesive polymers and/or in situ gelling components, although maintaining stability could be challenging ,7,8. PhyUse of punctal plugs is a common, non-invasive treatment strategy to mitigate dry eye syndromes ,17. TheyThree-dimensional (3D) printing is a versatile additive manufacturing technology that enables rapid prototyping of bespoke objects created using computer-aided design (CAD) software or imaging techniques  across mThe aim of this work was to employ DLP 3D printing to fabricate drug-loaded punctal plugs for controlled drug delivery. DLP 3D printing can be particularly advantageous, since it is possible to prepare various sizes, shapes, and doses of the punctal plugs in a single step. Here, dexamethasone, a corticosteroid, was incorporated because of its anti-inflammatory properties and wide applications in corneal disease treatment, including dry eye symptoms ,62. DiffDexamethasone , phenylbis  phosphine oxide , β-carotene , polyethylene glycol P400 (PEG 400), and polyethylene glycol diacrylate , Dulbecco’s phosphate buffered saline (PBS), trifluoroacetic acid , and acetonitrile  were obtained from Sigma-Aldrich . All materials were used as received.The punctal plug was designed to be inserted in the punctum of the eye with a configuration similar to those of commercially available punctal plugs, which range from 1.0–2.0 mm in length and from 0.2–1.0 mm in diameter. The dimension of the punctal plug was 1.9 mm in length and 1.0 mm in diameter with a cylindrical core diameter of 0.5 mm .w/w) photoinitiator (Irgacure 819) and 1.0% (w/w) photoabsorber (β-carotene) to a total mass of 2.0 g. Dexamethasone, PEGDA, and PEG 400 were added to each photopolymer solution according to the compositions shown in w/w ratio). The photopolymer solutions were kept in amber containers and continuously stirred overnight at room temperature (~25 °C). The solution was then added into the resin tank of the DLP 3D printing.Photopolymer solutions were prepared with 2.0%  equipped with an HD DLP projector, which has a visible light source (400–700 nm) . The priw/v) and 0.0025% (w/v) in ethanol, respectively. The UV-visible spectra were analysed on a Cary 100 UV–vis spectrophotometer  between 200–800 nm at a scan rate of 600 nm/min.Photoinitiator (Irgacure 819) and photoabsorber (β-carotene) solutions were prepared at concentrations of 0.025% (Discs (23.0 mm diameter × 1.0 mm height) were specifically prepared for XRPD analysis for different formulations  using the same printing conditions as the drug-loaded punctal plugs. Powdered samples of pure dexamethasone were analysed by XRPD. XRPD patterns were obtained with a Rigaku MiniFlex 600  equipped with a Cu Kα X199 ray source (λ = 1.5418 Å). The intensity and voltage applied were 15 mA and 40 kV. Samples were scanned between 2θ = 3–60° with a stepwise size of 0.02° at a speed of 5°/min.zero) were used with an average sample size of 3.0–5.0 mg.DSC measurements were performed with a Q2000 DSC  at a heating rate of 10 °C/min from 0 to 300 °C to characterise dexamethasone powder and different punctal plugs . Nitrogen was used as a purge gas with a flow rate of 50.0 mL/min for all the experiments. Data were collected with TA Advantage software for Q series  and analysed using TA Instruments Universal Analysis 2000. TA aluminium pans and pin-holed hermetic lids (Tn = 3).Images of the DLP 3D printed punctal plugs  were captured with a Leica Galen III optical microscope  and photographs were taken with an iPhone  through the eyepiece. The length of the punctal plugs were measured using ImageJ   were attached to a self-adhesive carbon disc mounted on a 25.0 mm aluminium stub, which was sputter coated with 25.0 nm of gold. The images were captured with an FEI Quanta 200 FEG SEM  at 5.0 kV accelerating voltage using secondary electron detection to obtain the images.−1 at a resolution of 1 cm−1 for 6 scans. The spectra of pure dexamethasone drug powder and PEGDA were collected as the references.The infrared spectra of formulations  before and after DLP 3D printing were collected using a Spectrum 100 FTIR spectrometer . All samples were scanned over a range of 4000–650 cmDrug release rates from the punctal plugs were measured using an in-house in vitro dynamic flow cell model  63]. A . A 63]. v/v TFA water. The gradient was: 20% ACN increased to 70% in 10 min, then decreased to 20% in 1 min and held for 4 min prior to the next injection. The sample injection volume was 100 μL operating at 30 °C at a detection wavelength of 240 nm.The concentration of dexamethasone in the DLP 3D printed punctal plugs was determined by cutting the plugs into small pieces. They were stirred in 5.0 mL ethanol at room temperature overnight. The solutions were filtered through a 0.45 μm filter , and drug concentration was determined with HPLC . An Eclipse plus C18 column, 100 × 4.6 mm  was employed with a flow-rate of 1.0 mL/min and a mobile phase gradient consisting of ACN and 0.1% 2 flasks using complete culture medium  and 1% v/v penicillin/streptomycin/fungizone (PSF)) until 80% confluence was reached. Cells were then trypsinized using TripLE solution  and seeded in wells of 24 well plates at a concentration of 3 × 104 cells per well. After 24 h, the discs (n = 5) were placed individually in wells, and plates were returned for incubation at 37 °C for 24 or 48 h. At each timepoint, medium was discarded and cells were washed with PBS. Freshly prepared CCK8 working solution (400 μL) consisting of culture medium (360 μL) and CCK-8 reagent (40 μL)  was added to each well and incubated for 2 h at 37 °C. Absorbance was recorded at 450 nm using a microplate reader .BalB/3T3 cells  were used to evaluate the cytotoxicity of different 3D printed discs (6 mm diameter × 1 mm height) prepared with the drug-loaded formulations  and the control formulations (C-PEGDA and C-PEGDA:PEG). Cells were cultured at 37 °C in 75 cmDifferent dexamethasone-loaded punctal plugs were successfully fabricated via DLP 3D printing with good resolution and reproducibility. The punctal plug was designed to possess a tapered shaft, which exerts horizontal force to keep it in place for easy insertion and removal . The totSEM images showed the surface morphologies of the different punctal plugs . A minorThe DLP 3D printer used in this study was equipped with a visible-light source 400–700 nm), which offers numerous benefits over UV light, such as being more energy efficient with a reduced risk of eye damage and improved biocompatibility and functional group tolerance ,65,66. H0 nm, whiw/w), indicating a greater proportion of drug was crystalline.Dexamethasone and DLP 3D printed drug-loaded discs (23.0 mm diameter × 1.0 mm height) for different formulations  were analysed by XRPD to evaluate the physical state of the drug and the degree of its incorporation in the polymer matrices. The diffraction patterns  show chaDSC thermograms were also obtained for the dexamethasone powder and different punctal plugs , showing the melting peak of dexamethasone at 266 °C , in agre−1 (O–H stretching), 1660 cm−1 (C=O stretching), and 891 cm−1  [−1 (C=O stretching), 1633 cm−1 (C=C stretching), 1408 cm−1 and 810 cm−1 (CH2=CH) [−1, 1408 cm−1 and 810 cm−1 disappeared because of the conversion of C=C bonds to C–C bonds via photocrosslinking [FTIR was performed to investigate potential interactions between dexamethasone and the photopolymer before and after DLP 3D printing. Previously, an unexpected drug-photopolymer reaction has been reported in oral dosage forms prepared using SLA 3D printing . TherefoF group) . These a(CH2=CH) ,76. Afteslinking ,77,78.max value of 12.4 ± 5.3 µg/mL  was observed by day 1 from the D10PEG punctal plugs  as compared to the C-PEGDA control, i.e., 74.3 ± 5.7% for D10 and 52.2 ± 8.4% for D20 in 24 h, and 54.1 ± 9.3% for D10 and 40.8 ± 9.8% for D20 in 48 h. On the other hand, the viability of the fibroblasts significantly decreased to 67.6 ± 8.9% and 61.6 ± 11.6% after incubation in 20% PEG 400 in the C-PEGDA:PEG controls in 24 and 48 h (p < 0.05). A decrease of approximately 50% in cell viability was shown in D10PEG and D20PEG plugs after 24 h, indicating the composition-dependent cytotoxicity of the formulations. From the drug release results  using different compositions of PEGDA and PEG 400. The FTIR spectra confirmed the absence of drug-photopolymer interactions. In vitro drug release results showed that drug release from D10PEG and D20PEG plugs can be sustained for 1 week, while D10 and D20 plugs can prolong the release with 50% of dexamethasone released in 5 and 11 days, respectively. Drug-loaded punctal plugs were less cytocompatible than blank controls, possibly due to the release of dexamethasone and the inclusion of PEG 400. In summary, the possibility presented in this study in terms of fabricating personalised drug-loaded punctal plugs via DLP 3D printing allows us to extend opportunities by adapting other ocular therapeutics for drug delivery to the front eye.Here, we report on a DLP 3D printing platform that enables the fabrication of drug-loaded punctal plugs with prolonged drug delivery to the front of the eye for patients with dry eye disease. All punctal plugs were successfully prepared with two loadings of dexamethasone (10%


Promoter of p16INK4a gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from −466 to −451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts.p16INK4a gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16INK4a. Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16INK4a gene expression. In addition, the enhanced binding of Sp1 to p16INK4a promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level.Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16INK4a expression during cell aging.All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of  The mechanism of increased p16INK4a expression in senescent cells is not well understood. We and others previously reported that some regulatory factors, such as Bmi-1, Ets, Id1, E47, Jun B, p21CIP1/WAF1and ITSE binding factor, were involved in p16INK4a transcriptionINK4ap16 gene expression during senescence. The increase of Sp1 binding activity rather than the protein level in senescent fibroblasts contributes to the high levels of p16INK4a expression during the progress of aging. What's more, the basal level of Sp1 is essential for this effect.It is well known that p16Human embryonic lung fibroblast cell line (2BS cells) obtained from the National Institute of Biological Products , was cultured in DMEM medium, containing 10% fetal bovine serum, 100 U/ml penicillin and 1 µg/ml streptomycinp16INK4a promoter was digested with Rsa I and Hind III from pSIR-870-EGFP, and inserted into pGL3-Basic Luciferase Report Vector (Promega). Site-directed mutagenesis was conducted using Quickchange™ Site-directed mutagenesis Kit (Stratagene) according to the manufacturer's protocol. Five oligo nucleotides carrying mismatched bases: 5′-GGAAGGAAACGGatCcGGGGCGGATTTC-3′, 5′-GGCGGGGGCaGATcTCTTTTTAACAGAG-3′, 5′-GGGAGGCCGGAtccCGGTGTGGGGGG-3′, 5′-CAGAGGGTGGGGgGatCCGAGTGCGCTC-3′ and 5′-GCAGGCAGCGGGatcCGGGGAGCAGC-3′ were used to mutagenize the segments containing GC boxes spanning from nt (nucleotide) −474 to −447, −462 to −435, −380 to −355, −76 to −49 and −26 to −1 upstream of translational start site respectively . For each transfection experiment, 2BS cells were seeded at 12-well plates and grown for about 24 h until they were above 90% confluent, then transfected with an equal amount of reporter plasmid (1.6 µg) and 0.32 µg of pRL-CMV (Promega) as transfection efficiency control, using lipofectamine 2000 (Invitrogen) and following the manufacturer's indications. Five hours later, serum-free DNA-containing medium was replaced by fresh growth medium and the cells were harvested 48h after transfection. Luciferase assays were performed as described . All assays were carried out in triplicate and performed twice for confirmation.INK4a gene-coding region was used as probe labeled with [α-32P] dCTP  by random priming using the Prime-a-Gene Labeling System (Promega). Hybridization was carried out in ExpressHyb™ Hybridization Solution (Clontech) at 68°C for 1 h. The blot was stringently washed with 0.1×SSC, 0.1% SDS at 68°C for 15 min twice. Autoradiography was performed at −80°C. The same blot was stripped after probing and then reprobed with the 32P-labeled GAPDH probe to control for equivalent RNA loading in each lane.Total RNA was prepared from exponentially growing cells using RNeasy Mini Kit . For Northern blot analysis, RNA was electrophoresed in 1.5% formaldehyde-denaturing agarose gel, with the 0.24–9.5 kb RNA Ladder included in one lane as a size marker . RNA was then transferred onto Biodyne B membrane  according to manufacturer's recommendations and fixed. The human p163VO4) were added to a cell pellet. The following protease inhibitors were added: 0.1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin). After incubation on ice for 30 min, samples were centrifuged at 14,000 r.p.m. for 5 min at 4°C to recover the supernatant. After proteins were electrophoresed in a 15% (for p16) or 8% (for Sp1/Sp3) denaturing polyacylamide gel and transferred to a PVDF membrane, the membrane was blocked in 5% nonfat milk-TBS-0.25% Tween 20 for 1 h and incubated with the primary antibody in TBS-0.25% Tween 20 for 1 h at room temperature. Complexes were detected with horseradish peroxidase-linked secondary antibody and enhanced chemiluminescence . The primary and secondary antibodies used in this study were all from Santa Cruz Biotechnology (Santa Cruz).Cell extracts were prepared following standard procedures. Briefly, three to five volumes of lysis buffer  and collected by centrifugation at 3500 rpm at 4°C for 5 min. The cells were resuspended in 400 µl of solution I  and lysed by passing them through a 25 gauge syringe. Nuclei were pelleted at 2,000 r.p.m for 10 min, washed once with solution I and resuspended in 200 µl of solution II . The suspension was rotated at 4°C for 30 min and then centrifuged at 14,000 r.p.m, 4°C, for 30 min. The resulting clear supernatant was stored at −80°C until use.Nuclear extracts from 2BS cells were prepared as follows: About 1×1032P] dATP  and T4 polynucleotide kinase . The probes were purified using QIAquick Nucleotide Removal Kit (Qiagen) and 15,000 cpm were incubated for 25 min on ice with 10 µg of nuclear extract from 2BS cell line, in the presence of 20 mM Tris-HCl pH 7.5, 75 mM KCl, 3.5 mM DTT, 20 nM ZnCl2, 1 µg/µl BSA, 5% glycerol and 1 µg poly (dI-dC)  in a total volume of 20 µl. For supershift experiments, 2 µg of antibody against Sp1 or Sp3  was incubated for 20 min on ice with the nuclear extract before adding the labeled probe. DNA/protein complexes were separated from free DNA on a 5% polyacrylamide gel in TBE  at 4°C for 120 min at 340 V. After electrophoresis, gels were dried and autoradiographed.A 38 bp long oligonucleotide 5′-GGAAGGAAACGGGGCGGGGGCGGATTTCTTTTTAACAG-3′ (oligo I) extending from nt −474 to −437 or a 28 bp long oligonucleotide 5′-CAGAGGGTGGGGCGGACCGAGT GCGCTC-3′ (oligo II) extending from nt −76 to −49 on the 5′ UTR of p16 gene, was used in EMSA and supershift assay. Double stranded DNA fragments were end-labeled with [γ-6 cells were crosslinked by adding formaldehyde directly to cell culture media and incubated for 10 min at 37°C. Wash cells twice with cold PBS and then cells were scraped and resuspended in 200 µl of SDS Lysis Buffer. Chromatin was then sonicated to an average length of 0.5 Kb for three 30 sec. pulses at maximum power. Chromatin extracts were diluted 10 folds in Dilution Buffer and preincubated for 30 min at 4°C with 80 µl of Salmon Sperm DNA/ protein A Agarose. Twenty microlitres of diluted supernatant was kept for isolation of input DNA and to quantitate the DNA in different samples. After pelleting agarose by brief centrifugation, 2 µg of anti-Sp1 antiserum (test group) or 2 µg of β-actin antibody (irrelevant antibody control) was added to the supernatant fraction and incubated overnight at 4°C with rotation. In addition, perform a no antibody immunoprecipitation by incubating the supernatant fraction with Salmon Sperm DNA/ protein A Agarose for 1 h at 4°C. Add 60 µl of Salmon Sperm DNA/ protein A Agarose for 1 h at 4°C to collect the antibody/antigen-DNA complex. The chromatin bound to the protein A Agarose beads was eluted in 500 µl of freshly prepared elution buffer . After reversing crosslinks, the samples were deproteinized and phenol-chloroform extracted, then DNA was ethanol precipitated using yeast tRNA as a carrier. Pellets were resuspended in 50 µl of TE buffer for PCR analysis.ChIPs were performed using the Chromatin Immunoprecipitation Assay Kit  according to manufacturer's instruction. In brief, 1×10INK4ap16 promoter, the sequences of the primers used were as follows: ChIP1, 5′-CCCCGATTCAATTTGGCAGTTAGG-3′ (−505–−491); ChIP2, 5′-CAGCGTTGGCAAGGAAGGAG GAC-3′ (−342–−318). All primers were synthesized at AuGCT Biotechnology Co., Ltd .Each PCR reaction mixture contained 5 µl of immunoprecipitated chromatin in a final reaction volume of 20 µl. PCR mixtures were amplified for 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s. To amplify GC-box containing regions of The target sequence against mRNA of Sp1 is 5′-AUCACUCCAUGGAUGAAAUGATT-3′, which has been reported to be effective in some cell lines Cells were transfected using Lipofectamin 2000 as specified by the manufacturer. The transfection mixture was left on the cells for 4 h, after which DMEM/20% serum without antibiotics was added. For efficient knockdown two more transfections were performed at 24 h and 48 h after the first transfection.2) with freshly prepared SA-β-Gal staining solution.Cells were washed twice in PBS, fixed in 3% formaldehyde, and washed again in PBS. The cells were incubated overnight at 37°C  were performed. Oligo I including GC-I and GC-II and oligo II containing GC-IV were 5′end labeled with [γ-INK4ap16 promoter in living 2BS cells. For this purpose, we performed ChIP assay to monitor Sp1- and Sp3-occupancies in the INK4ap16 promoter using gene specific primers. Consistent with the results of EMSA, ChIP assays showed that Sp1/Sp3 indeed bound to INK4ap16 promoter in vivo and the binding activity in senescent cells was higher than in that of young cells by nearly 5 folds (Sp1) and 3.5 folds (Sp3) respectively , which specifically blocks Sp1 and Sp3 binding to GC boxes, was added to 2BS cells after transfection with pGL3-620. As expected, the INK4ap16 promoter activity was inhibited by MTR in a dose-dependent manner in both young  accumulating. The biological effect of over-expression (by Sp1 expression plasmid transfection) or knockdown (by RNAi approach) of Sp1 in 2BS cells were further evaluated by this method. The results showed that Sp1-overexpressed cells were strongly stained blue versus the control. However, there were only a few dispersed cells that were SA-β-Gal-stained in the Sp1 knocked-down cells . All theINK4ap16, a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and cyclin-dependent kinase 6, has also been implicated in the mechanisms underlying replicative senescence. Many transcription factors are involved in transcriptional regulation via the corresponding elements distributed in the promoter region of INK4ap16. What should be emphasized is that the character of INK4ap16 promoter is GC-rich rather than the common element such as TATA box. In addition, GC-rich boxes represent putative target sites for binding of Sp1 and Sp3 transcription factors INK4ap16 gene expression from the different aspects and different strategies during aging in human embryonic lung fibroblasts, 2BS cells. Among the five GC boxes within the region of 620 bp upstream of the translation start site, the contribution of these five elements to INK4ap16 transcription is not equal, GC-I, II and IV is more important rather than GC-III and V by mutagenesis analyses, especially GC-II is the most crucial element to the INK4ap16 promoter activity, since its mutation abolished INK4ap16 gene transcription in senescent cells. What's more, the effect on the promoter activity in senescent cells is more significant than in young cells. All results mentioned above suggested that GC boxes are more instrumental to INK4ap16 gene expression coupled with aging process and it is the first time to investigate the roles of GC-boxes distributed in INK4ap16 promoter and the relationship between these elements and transcription activity of the target gene meticulously and systemically during aging.INK4ap16 promoter, we co-transfected Sp1 and/or Sp3 expression vectors with pGL3-620 into young and senescent 2BS cells and performed the luciferase assays. The results showed that Sp1 induced the INK4ap16 promoter activity, while Sp3 had little effect. The induction of promoter activity was not as potent as we would expect, which might be due to the high levels of endogenous Sp1 expression and the lower transfection efficiencies of 2BS cells . To further prove the effect of Sp1 on INK4ap16 expression, two strategies in different way were used: MTR, an inhibitor of Sp1/Sp3 binding, and siRNA, the inhibitor of Sp1 expression. The results which reduced the INK4ap16 promoter activity and its expression both in mRNA level and protein level suggest again that Sp1 is both necessary and sufficient for the induction of INK4ap16 gene expression in 2BS cells.Protein binding studies identified Sp1 and Sp3 as the major components of the complexes formed between the nuclear extracts and the oligos containing GC box. Although we cannot preclude involvement of other factors, the Sp1 and Sp3 antibodies blocked each of the specific complexes in vivo and in vitro. Sp1 is a widely studied transcription factor that can bind to and act through the GC boxes. Although it is generally believed that Sp1 is ubiquitously expressed, Sp1 gene expression can show up to 100-fold differences in different cell types and at different stages of development in mouse INK4ap16 increased in senescent human fibroblast cells owing to the regulation of various of transcription factors in previous and our former work , such as E47, Id1, Jun B and RREB etc.INK4ap16 promoter. Taken together the results that mutation of GC boxes had more detrimental effect on the INK4ap16 promoter in senescent cells and knock-down of Sp1 by siRNA down-regulate the expression of INK4ap16, we postulate that Sp1 transcriptional activity, including the binding activity to the GC boxes of INK4ap16 promoter, is enhanced in senescent cells, which in turn plays a role in the elevated INK4ap16 gene expression during senescence. It is applied a reasonable interpretation for the observation of much more potent promoter activities for INK4ap16 in senescent cells.It has been documented that the expression of INK4ap16 expression. Once it is lower than certain level, the expression of INK4ap16 is also weakened.In addition, though Sp1 is considered to be a constitutive transcription factor, Sp1 protein levels can vary significantly in different tissues. To test whether increased Sp1 binding is a result of induced Sp1 expression in senescent cells, we performed Western analyses. The results showed that the protein levels of Sp1 and Sp3 did not change significantly between young and senescent 2BS cells. So the increased binding observed in the senescent cells is likely the result of the augmentation of Sp1 and/or Sp3 binding affinity. On the other hand, the result of RNAi transfection demonstrated that although it need not increase Sp1 expression, however, a basal level is necessary for The alteration of Sp1 activity during senescence may result from different post-translational modification or transcriptional co-factor. The two major types of modification that are thought to be involved in transcription regulation by Sp1 are glycosylation and phosphorylation. O-glycosylation of Sp1 with N-acetylglucosamine has been linked to multiple changes in Sp1 function, including altered self-association, altered interaction with basal transcription factors and modulation of its degradation INK4ap16, whereas, in another aspect, GC box distributed in the promoter of INK4ap16, especially GC- II is also the key element for both the expression of INK4ap16 and binding with Sp1. Only both of them interact properly, do the gene expression and cell physiology act in the normal program.All in all, among the multitudinous transcription factors, Sp1 is also an important and essential member for the transcription and expression of