Piroxicam (PRM) and meloxicam (MEL) are two nonsteroidalanti-inflammatorydrugs, belonging to the Biopharmaceutics Classification System ClassII drugs. In this study, six novel pharmaceutical salts of PRM andMEL with three basic organic counterions, that is, 4-aminopyridine(4AP), 4-dimethylaminopyridine (4DMP), and piperazine (PPZ), wereprepared by both slurrying and slow evaporation. These salts werecharacterized by single-crystal and powder X-ray diffraction, thermalanalysis, and Fourier transform infrared spectroscopy. All six salts,especially MEL-4DMP and MEL-4AP, showed a significantly improved apparentsolubility and dissolution rate in sodium phosphate solution comparedwith the pure APIs. Notably, PRM-4AP and PRM-4DMP salts exhibitedenhanced fluorescence, and the PRM-PPZ salt showed weaker fluorescencecompared with that of pure PRM due to different luminescence mechanisms. Six novel pharmaceutical salts of piroxicamand meloxicamwith three basic organic counterions were synthesized. All salts showeda significantly improved apparent solubility and dissolution ratein sodium phosphate solution compared with the pure APIs. Notably,piroxicam salts exhibited different luminescent properties comparedwith pure piroxicam due to different luminescence mechanisms. For the preparation of pharmaceutical salts, ΔpKa is one of the most important factors whenconsidering salt formation, and salts can be distinguished from cocrystalsby the degree of proton transfer between the components. Generally,systems with ΔpKa < 0 lead tococrystals, and ΔpKa > 3 resultsin salts, while 0 < ΔpKa <3 can form either of them [where ΔpKa = pKa (base) – pKa (acid)].14 In addition, complementaryhydrogen bond acceptors/donors should exist in the structures of thedrug molecule and salt formers.15 It isalso important that the salt formers should be pharmaceutically acceptableor on the Generally Recognized as Safe (GRAS) and the European FoodSafety Authority (EFSA) lists to ensure that the resulting pharmaceuticalsalts are safe. Based on the presence of acidic or basic functionalgroups in ionizable APIs, potential counterions can be selected. Forbasic drugs, chloride and sulfate are typically the most popular inorganiccounterions, while acidic drugs usually form salts with simple inorganiccations such as sodium, magnesium, and potassium.16 Recently, pharmaceutically acceptable organic counterionshave received increasing attention because they tend to have preferabledissolution behaviors. There is no common ion effect in gastric mediafor the systems compared to the hydrochloride salts, and salt disproportionationmay be reduced owing to the regulation of the microenvironmental pHby the organic counterions.18Over the past few decades, the wide applicationof high-throughputscreening in drug design and discovery has resulted in more drug candidatesexhibiting low aqueous solubility. Many approaches, including theuse of nanoparticles,20 Moreover, PRM and MEL are zwitterionic compounds with pKa values of pKa1 = 1.86 (hydroxylgroup) and pKa2 = 5.46 (pyridyl group)21 and pKa1 = 1.09(hydroxyl group) and pKa2 = 4.18 ,22 respectively. When forming salts with acid counterions, the nitrogenatom of the pyridine ring or the thiazole ring from PRM or MEL isdeprotonated, whereas when basic counterions are involved in the saltformation, the phenolic hydroxyl group is deprotonated  of MEL have been reported,25 andthe zwitterionic form of MEL in the solid form can be found in formIV26 and its monohydrate.27Both PRM and MEL are polymorphic. PRM has six polymorphs,20 Wilson et al. demonstratedthat the PRM molecule can exist in two possible tautomers when cocrystallizingwith mono-substituted benzoic acids.28 Subsequently,they synthesized different multicomponent molecular crystals  of PRM with N-heterocyclesand haloanilic acids, and significantly enhanced solubility can befound in some crystals.29 Zaworotko andco-workers synthesized 12 cocrystal forms of MEL with carboxylic acidsvia crystal engineering and the supramolecular synthon approach. Solubilitytests and pharmacokinetics studies on these MEL cocrystals revealedthat 9 out of 12 cocrystals exhibited a greater apparent solubilityand higher oral bioavailability compared to that of pure MEL.30 In addition to PRM and MEL, two more oxicam drugs  have had multicomponent forms synthesized andtheir physicochemical properties investigated. Nangia et al. synthesizeda series of cocrystals and salts of LRM and TNM, indicating the solubilityadvantages of those new multicomponent crystalline forms.32 The known multicomponent forms of these four oxicam drugs are shownin Childs and Hardcastle investigated the cocrystalformation of PRMwith pharmaceutically acceptable carboxylic acids using a crystalengineering approach.59 and there has been recent interest in the solid-stateluminescent properties of organic multicomponent crystalline materials,with applications including organic light-emitting diodes, semiconductorlasers, and fluorescent sensors.63It is known that PRM exhibits luminescence in solution,66 In 2001, Tang’sgroup reported that silole derivatives exhibited significantly enhancedfluorescence in the aggregated state and proposed the concept of aggregation-inducedemission to explain this phenomenon.67 Recentyears have witnessed the wide application of solid-state fluorescencein the pharmaceutical, food, chemical, and optoelectronic industries.For example, Tang’s group developed a real-time, on-site, andnondestructive fluorescence imaging technique to monitor the crystalformation and transformation based on the crystallization-inducedemission properties of (Z)-1-phenyl-2--ylidene)ethenone. Based on the in-depth analysis of thecrystal structures of two crystalline polymorphs, Li et al. demonstratedthat the mechanoluminescence performance is related to the molecularpacking rather than the chemical structure.68In many conventional systems,fluorophores that exhibit intensivefluorescence in the solution state can experience partial or completeemission quenching in the aggregate state. This is due to the effectsof excited-state energy transfer in the solid state and is known asaggregation-caused quenching (ACQ).To date, the reports of cocrystals/salts of PRM or MEL withorganicbasic cocrystal/salt formers are rare, and the solid-state luminescentproperties of PRM have not been reported. Therefore, one motivationfor this study is to explore whether the crystal landscape of PRMand MEL can be expanded with more bases and, if so, is there any improvementin solubility. Another motivation is to investigate the solid-stateluminescence behavior of PRM and its salts and expand the understandingof crystal engineering in modifying the luminescent properties oforganic materials.In this work, six novel pharmaceutical saltsof PRM or MEL with4-aminopyridine (4AP), 4-dimethylaminopyridine (4DMP), or piperazine(PPZ) 3 wereprPRM (form I) and MEL (form I) were purchasedfrom Fluorochem and used as received without further purification.All salt formers were obtained from Sigma-Aldrich and used as received.Solvents were purchased from commercial sources and used as received.PRM  and 4AP  in a 1:1 molar ratio were dissolved in 5 mL of methanolby heating. Red plate-like crystals were obtained by slowly evaporatingthe filtrated solution for 3 days. Bulk materials were made by slurryinga stoichiometric amount (1:1) of PRM  and 4AP  in 3 mL of methanol at room temperature for 3 days. Theresulting suspension was allowed to dry in the fume hood. The powderedproduct was isolated and analyzed by powder X-ray diffraction (PXRD).PRM  and 4DMP  in a 1:1 molar ratio were dissolved in 10 mL of acetoneby heating. Yellow plate-like crystals were obtained by slowly evaporatingthe filtrated solution for 3 days. Bulk materials were made by slurryinga stoichiometric amount (1:1) of PRM  and 4DMP  in 3 mL of methanol at room temperature for 3 days. Theresulting suspension was allowed to dry in the fume hood. The powderedproduct was isolated and analyzed by PXRD.PRM  and PPZ  in a 2:1 molar ratio were dissolved in 5 mL of nitromethaneby heating. Yellow needle-like crystals were obtained by slowly evaporatingthe filtrated solution for 5–8 days. Bulk materials were madeby slurrying a stoichiometric amount (2:1) of PRM and PPZ  in 3 mL of methanol at room temperaturefor 3 days. The resulting suspension was allowed to dry in the fumehood. The powdered product was isolated and analyzed by PXRD.MEL  and 4AP  in a 1:1 molar ratio were dissolved in 10 mL of acetoneby heating. Yellow needle-like crystals were obtained by slowly evaporatingthe filtrated solution for 5–8 days. Bulk materials were madeby slurrying a stoichiometric amount (1:1) of MEL and 4AP  in 3 mL of acetone at room temperature for3 days. The resulting suspension was allowed to dry in the fume hood.The powdered product was isolated and analyzed by PXRD.MEL  and 4DMP  in a 1:1 molar ratio were dissolved in 5 mL of methanolby heating. Yellow plate-like crystals were obtained by slowly evaporatingthe filtrated solution for 8–10 days. Bulk materials were madeby slurrying a stoichiometric amount (1:1) of MEL and 4DMP  in 3 mL of methanol at room temperaturefor 3 days. The resulting suspension was allowed to dry in the fumehood. The powdered product was isolated and analyzed by PXRD.MEL  and PPZ  in a 2:1 molar ratio were dissolved in 10 mL of DMF–EtOAc by heating. Yellow needle-like crystals were obtained byslowly evaporating the filtrated solution for 5–8 days. Bulkmaterials were made by slurrying a stoichiometric amount (2:1) ofMEL  and PPZ  in 3 mL of methanolat room temperature for 3 days. The resulting suspension was allowedto dry in the fume hood. The powdered product was isolated and analyzedby PXRD.–1 under a continuously purged dry nitrogen atmosphere. Thermogravimetricanalysis (TGA) data were collected using a TA Instruments Q500 thermogravimetricanalyzer. The sample was placed in an aluminum sample pan and heatedunder nitrogen at a rate of 20 °C min–1 from25 to 500 °C. IR spectra were recorded on a PerkinElmer UATRTwo spectrophotometer using a diamond attenuated total reflectanceaccessory over a range of 400–4000 cm–1.An average of four scans was taken for each spectrum obtained witha resolution of 4 cm–1. PXRD data were collectedusing a STOE STADI MP diffractometer with Cu Kα radiation usinga linear position-sensitive detector (PSD) over the 2θ rangeof 3.5–45.5° with an increment of 0.05° at a rateof 2° min–1. The samples were prepared as transmissionfoils, and the data were viewed via STOE WinXPOW POWDAT software.69 Single-crystal XRD (SCXRD) data were collectedon a Bruker APEX II DUO with monochromated Cu Kα radiation forPRM-4AP and MEL-4DMP (λ = 1.54184 Å) and Mo Kα radiationfor PRM-4DMP, PRM-PPZ, MEL-4AP, and MEL-PPZ (λ = 0.7107 Å),respectively. All calculations and refinements were made using BrukerAPEX software with the SHELXL program.71 Nonhydrogenatoms were refined anisotropically. All hydrogen atoms were placedin geometrically calculated positions using the riding model, withC–H = 0.93–0.97 Å and N–H = 0.86–0.89Å and Uiso (H) (in the range 1.2–1.5times Ueq of the parent atom). For PRM-PPZand MEL-PPZ, there was disorder in the methyl group, which was modeledin two conformations in a 50:50 ratio. DIAMOND was used for creatingfigures,62 and PLATON was used for theanalysis of potential hydrogen bonds and short-ring interactions.72 Crystallographic parameters are listed in Differential scanning calorimetry(DSC) data were collected using a TA Instruments Q1000. Samples (2–6mg) were crimped in nonhermetic aluminum pans and scanned from 25to 300 °C at a heating rate of 10 °C min73 Density functional theorycalculations using the Gaussian 09 program package employing the RB3LYP functional with the 6-31G  basis set were performedon PRM, MEL, and the six obtained crystals without conducting structuraloptimization.74 The molecular orbitals were viewedusing the Multiwfn 3.8 program and plotted using VMD.76Hirshfeld surface analyses andtwo-dimensional (2D) fingerprint plots were obtained using the CrystalExplorer21.5 program.Solubility experiments wereconducted in sodium phosphate buffer solutions at pH 6.5 and 37 °Cto simulate intestinal physiological conditions. For each experiment,an excess crystalline solid was sieved through a 300 μm sieveand introduced into a flask with a screw top containing 100 mL ofthe medium. The solution was stirred at 200 rpm using a magnetic stirbar for 48 h to reach the equilibrium state. Sampling was performedat 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240, 300, 360, 540, 720,1440, 2160, and 2880 min. After each sampling, the volume of the liquidremoved from the suspension was not compensated. The withdrawn suspensionwas filtered through 0.2 μm nylon filters and diluted priorto high-performance liquid chromatography (HPLC) analysis. The solubilityexperiment for each crystal form was repeated in triplicate. Afterthe last sample collection, the remaining solid material in the suspensionwas filtered, dried, and characterized by IR and PXRD.–1 was employed, and the column temperature was set at 25 °C.The binary mobile phase consisted of acetonitrile and sodium phosphatebuffer (pH 6.5) in a volume ratio of 25:75. The samples were dilutedappropriately with the mobile phase, and the absorbance was measuredat 347 nm. The retention times of PRM and MEL were 7.1 min and 10.9min, respectively.The HPLCmethod was developed to determine the concentration of PRM and MELusing an Agilent 1260 series Infinite HPLC system . A C18 HPLC column  with a flow rate of 1 mL minSolid-state UV–visspectra were recorded on a Shimadzu 3200 UV spectrometer. Solid-statefluorescent spectra were collected using a Cary Eclipse fluorescencespectrometer  with 365 nm excitation light.The fluorescence quantum yield values were measured using a HamamatsuPhotonics C9920-02G instrument .Ka values. The ΔpKa values are greaterthan 3 in all cases since 4AP, 4DMP, and PPZ are strong organic bases cation. As shown in R32(8) motif is formed between PRM– and 4APH+ through two discrete hydrogenbonds  and intramolecular hydrogen bonding interactions . There are two moreintramolecular interactions within the PRM– anion forming a S(6) motif and a S(5) motif, respectively.Notice that the bond angle of the methyl C–H···Ointramolecular interaction is smaller than that of other interactions,which is reasonable since five-membered ring intramolecular hydrogenbonds usually have the smallest angles and the longest distances comparedto the six-eight-membered intramolecular hydrogen bonds and are withinthe geometric limits of a hydrogen bond as defined.80 Moreover, the angle (τ) formed between the methylhydrogen and the plane of a sp2 oxygen in the PRM– anion is 45.4°, which falls in the required range (<50°).81 This methyl C–H···O intramolecularinteraction in the PRM– anion can also be foundin PRM-4DMP and PRM-PPZ salts, and their dihedral angles (τ)are listed in Figure S6. The basic unitis extended via two discrete hydrogen bonding interactions, that is,N5–H5N···O4 (2.65 Å) and C19–H19···O1(3.28 Å), resulting in a double-layer 2D network (Table S1).PRM-4AP crystallizes in the C2/c space group and contains, in the asymmetricunit, one PRM– anion and one 4DMPH+ cation.As shown in – anion. The two PRM– anions and two4DMPH+ cations form a R44(22) motif around an inversioncenter via two discrete N5–H24···N1 (3.15 Å)and C22–H29···O1 (3.40 Å) hydrogen bondinginteractions. The discrete N5–H24···O4 (2.69Å) hydrogen bond is also responsible for the construction ofthe tetramer. This tetramer is then extended by four C–H···Ohydrogen bond interactions, displaying the 3D hydrogen bonding network(Table S2). As shown in Table S2, the 3D structure is further stabilized by π–πinteractions between the two pyridyl rings of PRM and 4DMP .PRM-4DMP crystallizes in the monoclinic P21/c with one PRM– anion and half of thePPZH22+ dication in the asymmetric unit. Asshown in 22+ dication is located on an inversioncenter (protons abstracted each from two PRM molecules), and the twoPRM– anions are at the general position in the unitcell. A R22(11) motif is formed between PPZH22+ and PRM– through the discrete N–H···Ohydrogen bond interaction  and C–H···O hydrogen bond interaction. Along the c axis, there are discrete N–H···Ohydrogen bonds  betweenPPZH22+ and another PRM– anion.No significant π–π interactions participate instabilizing the 3D structure of PRM-PPZ salts (Table S3).The PRM-PPZ salt crystallizes in the monoclinicsystem with the space group – anion and one 4APH+ cation arepresent in the asymmetric unit of a monoclinic Cc crystal structure. As shown in R21(6) supramolecular heterosynthonis formed between one 4APH+ and one MEL– via N–H···O and C–H···Odiscrete hydrogen bond interactions . Themethyl C–H···O intramolecular interaction canbe observed in three MEL salts, and their dihedral angles (τ)are listed in Figure S14. Along the a axis, the MEL– anion in this unit isfurther connected with other MEL– and 4APH+ through C12–H12···O3 (3.26 Å) and N1–H1···O1(2.69 Å) hydrogen bonds, respectively, extending into a 2D sheet.These 2D sheets are further linked through N2–H21···N5(3.02 Å) and C17–H17···O2 (3.29 Å)hydrogen bonds to construct a 3D network, as shown in Table S5).MEL-4AP salt crystallizes as a 1:1 salt,consistent with the acid–base donor–acceptor ratio.One MELP1̅ space group. The asymmetric unit contains oneMEL– anion and 4DMPH+ cation  contribute to the extended 3D structure (Table S6).The MEL-4DMP salt crystallizes in the + cation 8a. The tP21/c space group. The asymmetricunit contains one MEL– anion and half of the PPZH22+ dication. As shown in the top of 22+ dication connects to two MEL– anions viaN–H···O and N–H···S  discretehydrogen bond interactions, generating a R12(6) motif  discretehydrogen bond interactions, stabilizing the 3D hydrogen bonding network.The neighboring layers are further held together via π–πinteractions (∼4.2 Å) to generate a 3D structure (Table S7).The MEL-PPZ salt crystallizes in the monoclinic 32 Comparing the crystal structures of the four oxicam-PPZ salts revealsthat they all have a 2:1 stoichiometry. The PPZH22+ dication lies over an inversion center, with a proton abstractedfrom each of the oxicam molecules, and it bridges the two oxicam anionsthrough N+–H···O– hydrogen bonds. Notably, for the MEL-PPZ salt, the PPZH22+ dication is involved in more hydrogen bonding interactionsthan the other three oxicams.These are the first examples of the oxicam saltswith 4AP and 4DMP;the salts of LRM and TNM with PPZ are known.Figures S4 and S12, respectively. For all six salts, the experimentalPXRD patterns match the theoretical patterns obtained from the SCXRDanalysis, revealing that these salts were reproduced in bulk quantitiesby the slurry method.The PXRD patterns of thePRM system and the MEL system are shown in Figures S3 and S11, respectively.The new crystalline solids exhibit different vibrational frequenciescompared with those of the pure API and the salt formers, for example,for PRM, the characteristic absorption peak at 1628 cm–1 assigned to the C=O stretching vibration is red-shifted to1626, 1618, and 1615 cm–1 in PRM-4AP, PRM-4DMP,and PRM-PPZ salts, respectively. For MEL, the C=O stretchingvibration is red-shifted from 1617 to 1614 (MEL-4AP), 1611 (MEL-4DMP),and 1616 cm–1 (MEL-PPZ). Furthermore, the unencumbered−NH stretch in PRM (3337 cm–1) and MEL (3287cm–1) is lost in the PRM or MEL salts, revealingthat the −NH group is engaged in the formation of hydrogenbonds. These changes suggest the reconstruction of hydrogen bond networksin those solids and indicate the formation of new crystalline solids.FT-IR spectra of PRM and MEL systems areshown in Supporting Information . Eachsalt shows a single sharp endothermic peak, suggesting that each productis in a homogeneous phase. Moreover, the TGA traces indicate thatno solvent or water molecule is involved in the crystal lattice ofthese salts.DSC and TGA studies were conducted on the six salts and their individualcomponents to obtain the melting points and decomposition temperatures.The melting trace and decomposition behavior of each salt and thestarting materials are given in the 82 Therefore,solubility tests of PRM, MEL, and their salts were performed in sodiumphosphate buffer solutions (pH = 6.5) to investigate the ability ofsalt formation to improve the solubility of the poorly water-solubleAPIs. As shown in –1) at 60 min. Subsequently, the concentration of purePRM decreases slowly over time due to the crystal transformation fromPRM anhydrate to PRM monohydrate and forms a plateau (0.14 mg mL–1). Similar behavior is observed for theophylline andcaffeine.83 All the PRM salts exhibit the“spring and parachute” phenomenon,84 in that they dissolve faster than pure PRM and reach theirmaximum solubility within 5 min, and then the solubility decreasesslowly over time owing to the transformation of the salts into theless soluble PRM monohydrate in solution .The dissolved PRMs of PRM-4AP, PRM-4DMP, and PRM-PPZ salts are 1.08,1.07, and 0.43 mg mL–1, which are 2.8, 2.8, and1.1 times higher than that of the anhydrous PRM, respectively (PRM is dissolved and absorbed mainlyin the intestine (in pH 6–8), and MEL undergoes significantdegradation at lower pH (<3).ectively 4. Lookin–1) at 90 min. The “springand parachute” phenomenon is also observed for all the MELsalts, with PXRD analysis of the solid residues collected after thesolubility experiments indicating that these undissolved solids hadtransformed to MEL . The timeto maximum dissolved concentration of the salts is extended to 5 min,demonstrating the remarkably improved dissolution rate in comparisonwith that of pure MEL. The dissolved MEL of MEL-4AP, MEL-4DMP, andMEL-PPZ salts is 8.1, 10.2, and 3.3 times higher than that of thepure MEL, respectively. Furthermore, MEL-4DMP and MEL-4AP can maintainthe supersaturation state for more than 200 and 300 min, respectively,indicating that the two salts of MEL could be promising formulationsfor achieving extended release without using polymers.86 These two salts have better solubility behavior when compared tomost reported MEL cocrystals and salts, although the MEL aspirin cocrystalhas significantly improved solubility compared to all other systems in the crystalstate, possibly indicative of fluorescence quenching caused by aggregation(ACQ).64 The MEL-4AP, MEL-4DMP, and MEL-PPZsalts present almost no fluorescence , again indicating quenching due toaggregation.MEL canexhibit fluorescence in the solution state;In summary, six new pharmaceutical saltsof PRM and MEL with threeorganic counterions  were successfully synthesizedand characterized by various solid-state analytical techniques, includingSCXRD, PXRD, DSC, TGA, and IR. In the solubility tests, the apparentsolubility of all six salts was enhanced relative to that of the parentmolecule (PRM/MEL), and the dissolution rate of all of six salts wasalso improved significantly. The salts exhibit similar solid-stateluminescent properties to those of PRM and MEL. The proton-transfer-inducedenhanced luminescence with a large red shift could be used to explainthe luminescence mechanism of PRM-4AP and PRM-4DMP. For PRM-PPZ, themechanism could be the combination of the proton transfer processwith some quenching process. Hirshfeld surface analysis and HOMO–LUMOanalysis were also employed to further investigate the different luminescentbehaviors of PRM solids. Overall, this study revealed that salt formationby using organic counterions is an effective approach to improve thesolubility behavior of poorly water-soluble APIs. Furthermore, theluminescent properties of organic fluorophores can be altered andmodified by forming salts involving proton transfer. In this example,exposure of samples under UV illumination provides a convenient anduseful tool to examine the synthesis of new crystalline materials.


Domain 1 of CD147 participates in matrix metalloproteinase (MMP) production and is a candidate for targeted therapy to prevent cancer invasion and metastasis. A functional mouse anti-CD147 monoclonal antibody, M6-1B9, was found to recognize domain 1 of CD147, and its respective mouse single-chain variable fragment (ScFvM61B9) was subsequently generated. The EDLGS epitope candidate for M6-1B9 was identified using the phage display peptide technique in this study. For future clinical applications, humanized ScFv specific to domain 1 of CD147 (HuScFvM61B9) was partially adopted from the hypervariable sequences of parental mouse ScFvM61B9 and grafted onto suitable human immunoglobulin frameworks. Molecular modelling and simulation were performed in silico to generate the conformational structure of HuScFvM61B9. These results elucidated the amino acid residues that contributed to the interactions between CDRs and the epitope motif. The expressed HuScFvM61B9 specifically interacted with CD147 at the same epitope as the original mAb, M6-1B9, and retained immunoreactivity against CD147 in SupT1 cells. The reactivity of HuScFvM61B9 was confirmed using CD147 knockout Jurkat cells. In addition, the inhibitory effect of HuScFvM61B9 on OKT3-induced T-cell proliferation as M6-1B9 mAb was preserved. As domain 1 is responsible for cancer invasion and metastasis, HuScFvM61B9 would be a candidate for cancer targeted therapy in the future. CD147 is overexpressed in cancer and plays a fundamental role in regulating the tumor microenvironment and cancer progression by several mechanisms, including proliferation, invasion, metastasis, angiogenesis, glycolysis, and chemoresistance3. CD147 was shown to stimulate the production of MMP-1, MMP-2, MMP-3 in melanoma4, MMP-9 in breast cancer5, MMP-11 in colorectal cancer7 and MMP-2 and MMP-9 in hepatocellular carcinoma10. CD147 also plays a crucial role in T cell activation and proliferation; however, CD147 seems to display different functional activities at different phases of T cell responses14.CD147, a transmembrane glycoprotein in the immunoglobulin superfamily, is widely expressed in various cell types and tissues and exerts a diverse range of physiological and pathological processes15. The disruption of CD147 dimerization can prevent cancer cell invasion and metastasis8. In comparison to the strengths of other therapeutic antibodies, immunotargeting of the CD147 functional domains is considered a promising candidate for cancer immunotherapy in preventing cancer invasion and metastasis24. A therapeutic mouse anti-CD147 monoclonal antibody (mAb), HAb18, recognizes the residue 39LTCSLNDSATEV50 in domain 1 of CD147 and exerts an anti-metastatic activity in hepatocellular carcinoma through the reduction of MMPs17.Domain 1 (IgC2 domain) of CD147 is important in matrix metalloproteinase (MMP) production, which leads to the degradation of the basement membrane and extracellular matrix and promotes tumor proliferation, invasion, and metastasis26. Subsequently, mouse single-chain fragment variable of M6-1B9 (ScFvM61B9) was generated from the cDNA of hybridoma clone M6-1B9 to be expressed as soluble ScFvM61B9 and ScFvM61B9 intrabody forms27. Soluble ScFvM61B9 inhibited T-cell proliferation after stimulation with an anti-CD3 mAb28. ScFvM61B9 activity has not yet been studied in solid tumors. However, the ScFvM61B9 intrabody was previously found to inhibit the cell surface expression of CD147 and decreased the aggressiveness of various cancers via specific mechanisms32. Although the ScFvM61B9 intrabody did not affect either MMP-2 or MMP-9 activities in the cervical cancer HeLa cell line, it suppressed urokinase-type plasminogen activator (uPA) and inhibited HeLa cell metastasis31. In addition, the ScFvM61B9 intrabody affected the α3β1-integrin and MCT-1 function and suppressed the progressive phenotype in the colorectal cancer Caco-2 cell line29. Moreover, the ScFvM61B9 intrabody promoted intracellular acidosis and induced apoptosis in the Caco-2 cell line30. Correspondingly, immunotargeting of CD147 domain 1 is feasible for cancer immunotherapy.The functional epitope of M6-1B9, a mouse anti-CD147 mAb, was formerly characterized as residing on domain 1This present study aimed to verify the epitope on CD147 extracellular domain recognized by M6-1B9 mAb. In addition, the peptide sequences of heavy and light chain frameworks of its mouse single chain variable  were replaced with human frameworks (HuScFvM61B9) and the biological activity of this humanized scFv was assessed for future clinical applications.After three rounds of bio-panning, 16 phage clones were randomly chosen to identify the bona fide binding partner to M6-1B9 mAb via phage enzyme-linked immunosorbent assay (ELISA)  ≤ 3.5 Å, θ(D-H…A) ≥ 90°. Atom-π, π-π, and hydrophobic van der Waals interactions were analyzed using the centroid–centroid and between heavy atoms criteria with a maximum distance of 6.0 Å.Amino acid residue contributions and interaction types were examined to understand the intermolecular interactions that can improve discovery. We estimated a possible intermolecular residue interaction based on the atomic coordinates from the selected candidate. We identified hydrogen bonds of the D-H…A type (donor atom D and acceptor atom A) using the following distance and angle criteria: The original mouse ScFvM61B9 and the predicted HuScFvM61B9 structures using BioLuminate were compared Fig. a. The anvan der Waals interactions are essential in this protein–protein association. The crucial side-chain residues on CDRs of HuScFvM61B9 and domain 1 (composed of EDLGS epitope) at the binding surface are labeled in Fig.  van der Waals interactions.This atomistic picture of the structural assembly was in accordance with the epitope mapping analysis results. The complex structure suggested that hydrogen bonding and Escherichia coli (E. coli) Origami B (DE3) and purified via affinity chromatography. The results revealed that the purified HuScFvM61B9 protein containing a His6x tag was successfully produced. The recombinant protein at approximately 26 kDa was detected via SDS-PAGE  and human CD147-BCCP. The Western blot analysis of the produced CD147-BCCP was shown  were expressed in E. coli strain Origami B to assess the specificity of HuScFvM61B9 by Western immunoblotting. The HuScFvM61B9 was specifically reacted to the band at ~ 35 kDa of CD147WT-BCCP only while the band at ~ 35 kDa of CD147Δ32DL33-BCCP was absent  and mutant CD147  significantly inhibited the binding of purified HuScFvM61B9 to immobilized CD147 extracellular domain-human IgG Fc fusion protein (CD147Rg) in a concentration-dependent manner Fig. b. These Flow cytometric analysis was performed to evaluate the functional activity of HuScFvM61B9 against CD147 on SupT1 cells. The HuScFvM61B9 was firmly bound to CD147 on the cell surface of SupT1 Fig. c, compar34. At day 11 post-nucleofection, the edited cell pool was collected to determine the CD147 expression using 40 μg/ml HuScFvM61B9 and 40 μg/ml M6-1B9 mAb. The flow cytometric analysis indicated approximately 39.7% and 33.4% of CD147-negative cells in CD147-Jurkat KO when stained with HuScFvM61B9 and M6-1B9, respectively  was established. Jurkat cells were nucleofected with the ribonucleoprotein (RNP) complex using the sgRNA designed to target an exon 1 of the CD147 gene28. Thus, the biological activity of HuScFvM61B9 was also assessed on anti-CD3 (OKT3)-mAb induced T-cell proliferation in this study. Peripheral blood mononuclear cells (PBMCs) from a healthy donor were activated with immobilized OKT3 in the presence or absence of HuScFvM61B9. The M6-1B9 mAb was used control. The results showed that HuScFvM61B9 inhibited the OKT3-induced T-cell proliferation as its parental, M6-1B9, in a dose-dependent manner -mAb induced T-cell proliferation in previous reportsner Fig. . However35, our data suggested that the immunogenicity of HuScFvM61B9 was reduced and could potentially decrease the chance of HuScFvM61B9 immunogenicity in humans. Using the T20 humanness score, complicated and time-consuming in vitro experiments for immunogenicity prediction of HuScFvM61B9 were not required35.The T20 humanness scores for the heavy and light chains of HuScFvM61B9 increased over those of the parental sequence. As a weak correlation between the T20 score of 65 therapeutic antibodies and their available immunogenicity data37. Although a linear epitope can be mapped directly to the target protein, it must be combined with computer modelling to identify binding sites on the natural target. The amino acid sequences of phage clones 10, 12, and 14 were aligned with the human CD147 sequence and shared less than three continuous amino acid residues identical to the human CD147 sequence registered in GenBank. This indicated that these peptides were likely the mimotope of CD147. The EDLGS motif was an antigenic determinant, as it was not present in mouse CD147. These data suggested that M6-1B9 mAb recognized the epitope different from other anti-CD147 mAbs, including HAb18, which is used as a therapeutic antibody in hepatocellular carcinoma17. In addition, the M6-1B9 mAb recognized the EDLGS, which resides in the residues 22AAGTVFTTVEDLGSKILLTCSLNDSATEV50 in domain 1 of CD147; these residues have been suggested to play a critical role in MMP secretion and tumor invasion17.Phage display combinatorial peptide libraries have been successfully and efficiently used to identify a wide variety of epitopes and mimotopes from a wide variety of mAbsFurthermore, the complex structure predicted by the ClusPro docking algorithm provided crucial residues for the antigen–antibody at the binding site. The analyzed RMSD was 0.758; by comparison, the Å values of mouse ScFvM61B9 and HuScFvM61B9 indicated a similar conformation between these two ScFvs. Interestingly, the predicted complex from ClusPro was similar to the structural coordinate generated by the Rosetta SnugDock protocol (data not shown).32DL33-BCCP. While HuScFvM61B9 and M6-1B9 specifically interacted with wild-type CD147, the immunoreactivity was absent with CD147Δ32DL33-BCCP. This phenomenon supports that the specificity of HuScFvM61B9 was retained for the EDLGS motif in domain 1 of CD147. Domain 1 of CD147 is crucial for MMP production, which leads to the degradation of the basement membrane and extracellular matrix and promotes tumor proliferation, invasion, and metastasis15. Taken together, HuScFvM61B9 would possess the necessary functional activities, including the ability to reduce MMPs, needed to ensure a feasible targeted cancer immunotherapy regimen.The amino acids of each HuScFvM61B9 CDR interacting with the epitope on domain 1 of CD147 are listed in Fig. –8) was lower than its parental mAb, M6-1B9 (3.11 ± 1.28 × 10–9). This phenomenon was similar to the previous report of anti-Her2 scFv38. Considering the biological function of HuScFvM61B9, the immunoreactivity of HuScFvM61B9 revealed that HuScFvM61B9 could specifically bind to CD147 at the same epitope as its parental mAb, M6-1B9  was successfully constructed and retained its immunoreactivity and functional activity of its parental M6-1B9 mAb. The development of HuScFvM61B9-Fc fusion (HuScFvFcM61B9) or fully humanized M6-1B9 IgG will be attempted in future research to improve the binding activity of monovalent ScFv.41. Briefly, three rounds of biopanning were performed by gradually reducing the amount of the M6-1B9 antibody, ranging from 10 to 5 and 2 µg for each consecutive round of affinity selection. After three rounds of biopanning, 16 individual phage clones were randomly chosen and amplified to assess their binding activity against the M6-1B9 mAb by phage ELISA, as previously reported42. The phagemid DNA from positive phage clones was prepared for nucleotide sequencing through automated DNA sequencing services using the -96gII primer (5′-CCC TCA TAG TTA GCG TAA CG-3′). The amino acid sequences were analyzed using SnapGene software.Bio-panning of a 12-mer phage display random peptide library (SUT12) against the anti-CD147 mAb (clone M6-1B9) was performed as noted in previous research27. Upon submitting the amino acid sequence of ScFvM61B9 heavy and light chain variable domains, a PDB number 6N4Q crystal structure containing a mouse Fab template was selected for conforming to the possible structure. Subsequently, the human frameworks of PDB number 5HYS were automatically retrieved to substitute the frameworks of the modelled ScFvM61B9 structure to retain the canonical shape of antibody CDR loops. The structure comparison and RMSD calculation of the designed HuScFvM61B9 versus parental ScFvM61B9 were performed using UCSF Chimera 1.15 software. The CDR regions of the generated HuScFvM61B9 structure were deduced using the PyIgClassify Web server43. The humanness of HuScFvM61B9 was calculated using the T20 score analyzer tool35.The suitable frameworks in the variable domain of HuScFvM61B9 were assigned from parental ScFvM61B9 using the antibody humanization process of the BioLuminate 4.0 demo software 44. The antibody mode option was selected to automatically mask non-CDR regions in this in silico protein–protein docking process.The generated structure of HuScFvM61B9 from BioLuminate was submitted to the ClusPro 2.0 Web server along with the CD147 domain 1 extracted from PDB number 5X0TE. coli. The HuScFvM61B9 coding sequence containing 5′ NheI and 3′ HindIII restriction sites was synthesized . The synthesized polynucleotide was subsequently digested with NheI and HindIII and cloned into the NheI and HindIII sites of the pET-21a plasmid vector to generate the pET-21a-HuScFvM61B9(HIS6X) plasmid.The amino acid sequence of modified ScFvM61B9 was reverse transcribed and optimized using the GenScript web service for proper expression in HuScFvM61B9(HIS6X) plasmid was transformed into competent E. coli Origami B (DE3) cells to produce a humanized single-chain variable fragment of M6-1B9 containing a His6x tag (HuScFvM61B9). A single colony was picked and grown in a 5 ml super broth medium starter culture overnight at 37 °C. Then, the culture was inoculated into a 500 mL SB medium containing 0.05% glucose and 100 µg/mL ampicillin at 37 °C until an OD600 of 0.8 was reached. Protein expression was induced by adding 50 µM IPTG, and this process continued for 16–18 h at 20 °C. The induced bacteria expressing HuScFvM61B9 were washed twice with PBS and lysed via three sonication times of 5 min each at 0.5 cycles with 80% amplitude on ice. The lysed bacteria were subjected to freeze–thaw cycling, followed by centrifugation at 4000g for 30 min at 4 °C. The cell lysate was collected, and HuScFvM61B9 was purified via affinity chromatography on a HisTrap HP column  using ÄKTA Prime plus. The collected fractions were analyzed via SDS-PAGE on a 15% gel with PageBlue staining to determine the purity of the HuScFvM61B9 proteins. The collected fractions were subjected to Western blot analysis. Proteins were separated by SDS-PAGE, transferred onto a PVDF membrane, and subsequently probed with an HRP-conjugated anti-His-tag antibody  at 1:3000 dilution. The reaction was developed using a chemiluminescent substrate detection system.The pET-21a-E. coli strain Origami B harboring pAK400cb-CD147-BCCP as described previously45. The CD147-BCCP was validated by Western immunoblotting using either M6-1B9 mAb and followed by HRP-conjugated anti-mouse Igs antibody or HRP-conjugated Streptavidin, and stored at − 20 °C for further analysis. Two hundred microliters of 2 mg/ml crude CD147-BCCP were immobilized on Streptavidin biosensor tip (Sartorius FortéBio) and then placed to buffer (0.05% Tween-20 in PBS) to generate the baseline. To determine the association signal, the tips were subsequently placed into either mouse anti-CD147 mAb clone M6-1B9 or HuScFvM61B9 at 20, 10, and 5 μg/ml. After that, the tips were placed into the buffer to generate the dissociation signal. The constants of association (ka) and dissociation (kd) were analyzed, and the KD was calculated via the same formular by BlitZPro 1 program.Biolayer interferometry was used to analyze the binding affinity of HuScFvM61B9 to human CD147-BCCP and compared to its parental mouse mAb, M6-1B9. The CD147-BCCP was in situ biotinylated in 32DL33-BCCP) were generated. Briefly, deletion of 32DL33 in EDLGS of pAK400cb-CD147-BCCP was performed using QuickChange Lightning Multi Site-Directed Mutagenesis kit (Agilent) following the manufacturer’s instruction. Mutated primer DNA sequences are listed as follows.Fw_del32-33 5′-GCA CAG TCT TCA CTA CCG TAG AAG GCT CCA AGA TA-3′.Rv_del32-33 5′-TAT CTT GGA GCC TTC TAC GGT AGT GAA GAC TGT GC-3′.To assess the specificity of HuScFvM61B9 by Western immunoblotting, wild-type CD147-BCCP (CD147WT-BCCP) and mutant CD147-BCCP  agar containing 25 μg/ml of chloramphenicol. The plasmid from transformed E. coli DH5α was prepared by QIAGEN Miniprep kit (QIAGEN) and sequenced. The corrected pAK400cb-CD147-BCCP-del32DL33 was transformed to E. coli strain Origami B to produce CD147Δ32DL33-BCCP, and validated as described previously45. Crude lysate protein at 1:2 dilution of both CD147WT-BCCP and CD147Δ32DL33-BCCP were subsequently separated by SDS-PAGE, transferred onto a PVDF membrane, and subsequently probed with HuScFvM61B9 (1 μg/ml) followed by HRP-conjugated anti-His-tag mAb at 1:1000 dilution, M6-1B9 mAb (0.5 μg/ml) followed by HRP-conjugated anti-mouse Igs  at 1:2000 dilution and HRP-conjugated Streptavidin  at 1:500 dilution. The reaction was developed using a chemiluminescent substrate detection system.The pAK400cb-CD147-BCCP-del32DL33 was subsequently transformed into competent To investigate the binding activity of HuScFvM61B9 to CD147, the indirect ELISA was performed. The microtiter plates were immobilized with 50 µL of 10 µg/mL CD147Rg and incubated overnight at 4 °C in a moist chamber. The other steps were performed at ambient temperature in a moist chamber. The coated wells were washed three times with a washing buffer (0.05% Tween 20 in PBS), followed by blocking with 2% skim milk in PBS for 1 h. After washing, purified HuScFvM61B9 protein was added at various concentrations to the wells and incubated for 1 h. After washing three times, 50 µL of HRP-conjugated anti-His-tag mAb at 1:5000 dilution was added and incubated for 1 h. The reaction was developed using a TMB substrate, and then the reaction was ceased with 1 N HCl. Absorbance at 450 nm was measured using an ELISA reader.Inhibition binding activity of purified HuScFvM61B9 was assessed using the inhibition ELISA. Fifty microliters of 10 µg/mL CD147Rg were immobilized on a microtiter plate and incubated overnight at 4 °C in a moist chamber. The other steps were performed at ambient temperature in a moist chamber. Non-specific protein binding was blocked using 2% skim milk in PBS. Fifty microliters of 1 and 5 µg/mL mouse anti-CD147 mAb (M6-1B9 and M6-1E9) were added and incubated for 1 h. The microtiter plate was washed three times with 0.05% Tween20 in PBS and 50 μL of 10 µg/mL purified HuScFvM61B9 was added to the well. After 1 h of incubation, the microtiter plate was washed three times with 0.05% Tween20 in PBS. HRP-conjugated anti-His-tag mAb at 1:5000 dilution was subsequently added to the well and incubated for 1 h. TMB substrate was then added to develop the reaction. The absorbance at 450 nm was measured using a microplate reader after adding 1 N HCl.25. SupT1 cells were washed three times with a FACS buffer and adjusted to 107 cells/mL with 10% human AB serum for 30 min on ice to block the Fc receptor. Fifty microliters of 10 µg/mL HuScFvM61B9 or M6-1B9 mAb was added into 50 μL of blocked cells and incubated on ice for 30 min. The cells were washed twice with 1% FBS in PBS. After washing, a PE-conjugated anti-His-tag antibody  at 1:20 dilution or conjugated anti-mouse Igs was added and incubated on ice for another 30 min. Finally, the cells were washed three times and fixed with 1% paraformaldehyde in PBS. The fluorescence intensity of the stained cells was analyzed using a BD FACSCelesta instrument and FlowJo software.Flow cytometric analysis was performed to evaluate the functional reactivity of HuScFvM61B9 against CD147 on a human T-cell lymphoblastic lymphoma cell line, SupT1, compared to its parental M6-1B9 mAb34 was generated using the GeneArt Precision gRNA Synthesis kit . The RNP complex was prepared by mixing 150 μg/mL of SpCas9 (Integrated DNA Technologies) and 90 μg/mL of sgRNA at a 1:3 molar ratio, then incubated for 10 min at room temperature. 1 × 106 Jurkat cells were nucleofected with RNP using the Cell Line Nucleofector Solution V  and the program X-005. On day 11, the edited cell pool (CD147 knockout Jurkat cells) was used to analyze the specificity of HuScFvM61B9 by immunofluorescence analysis using the protocol described above except 40 μg/ml concentration was used. Untransfected Jurkat cells were used as a positive control.For the generation of CD147 knockout Jurkat cells, the sgRNA targeting CD147 molecule7 PBMCs/ml with PBS. CFSE (Sigma-Aldrich) at a final concentration of 1 μM was added to PBMCs for 10 min at 37 °C. Excess CFSE was quenched with cold 10% FBS-RPMI. CFSE-labelled PBMCs were washed twice with RPMI. To determine the effect of HuScFvM61B9 on T-cell proliferation in PBMC after anti-CD3 activation, triplicate aliquots of 105 CFSE-labeled PBMCs were cultured with immobilized CD3 mAb OKT3 (12.5 ng/mL) with various concentrations of purified HuScFvM61B9. Mouse M6-1B9 mAb was used as a control. The culture was incubated for 5 days in a 5% CO2 incubator at 37 °C. Cells from each treatment were then washed twice with PBS, fixed with 1% formaldehyde in PBS and analyzed using BD Accuri C6 flow cytometer and FlowJo software.T cell proliferation was assessed using 5-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling. PBMCs were washed with PBS 3 times and adjusted to 10The study protocol was approved by the Ethics Committee, Faculty of Associated Medical Sciences, Chiang Mai University, Thailand (AMSEC-64EX-016).


Although Jaw1 is known to interact with inositol 1,4,5-triphosphate receptor (ITPRs), Ca2+ channels on the endoplasmic reticulum, the function of Jaw1 in the Ca2+ dynamics with physiological stimulation remains unclear. In this study, using inducible Jaw1-expressing HEK293 cells, we showed that Jaw1 increases Ca2+ influx by GPCR stimulation via changing the Ca2+ influx oscillation pattern. Furthermore, we showed that Jaw1 increases the Ca2+ release activity of all ITPR subtypes in a subtly different manner. It is well known that the Ca2+ influx oscillation pattern varies from cell type to cell type, therefore these findings provide an insight into the relationship between the heterogeneous Ca2+ dynamics and the specific ITPR and Jaw1 expression patterns.Ca Intracellular Ca2+ is stored in the endoplasmic reticulum (ER), and cell surface receptor-mediated extracellular stimuli trigger Ca2+ influx into the cytoplasm to initiate a physiological response4.Intracellular Ca2+ channels on the ER membrane and cell surface has been described in stimuli-dependent Ca2+ dynamics regulation. Inositol 1,4,5-triphosphate receptors (ITPRs), Ca2+ channels on the ER membrane, release Ca2+ from the ER into the cytoplasm in an IP3-dependent manner when the IP3 is produced upon G protein-coupled receptor (GPCR)-related stimulation6. The Ca2+ efflux from the ER subsequently leads to Ca2+ depletion in the ER, triggering the activation of stromal-interaction molecule (STIM), a Ca2+ sensor protein on the ER8. The activated STIM interacts with Orai, a plasma membrane Ca2+ channel, resulting in Ca2+ influx from the extracellular source into the cytoplasm, called store-operated calcium entry (SOCE). The cytoplasmic Ca2+ is retrieved by sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), a Ca2+ pump on the ER, resulting in Ca2+ replenishment in the ER9. This Ca2+ dynamics series is repeated and observed as a cytosolic Ca2+ concentration ([Ca2+]c) oscillation. Interestingly, recent studies revealed that the oscillation, as well as the cytoplasmic Ca2+ influx level, is important for Ca2+-dependent protein activation11.To date, the involvement of several basic Ca15. We previously reported that Jaw1 is localized on the ER and the outer nuclear membrane, and it is associated with nuclear shape maintenance16. Furthermore, Jaw1 was recently reported to interact with all ITPR subtypes via its coiled-coil domain, and the deletion of Jaw1 in mouse embryonic fibroblast (MEF) reduces Ca2+ influx into the cytoplasm upon ionomycin stimulation, a reagent that increases cell membrane ion permeability13. However, whether and how Jaw1 is involved in the calcium dynamics under physiological conditions, such as GPCR stimulation-related aspects, remains unclear. Furthermore, physiological differences have been reported among ITPR1–3, three ITPR isoforms, such as IP3 affinity, Ca2+ release activity, and in vivo expression patterns20, but it remains elusive whether Jaw1 affects the ITPR activities subtype-specifically.Jaw1, also known as lymphoid restricted membrane protein (LRMP), is a type II integral membrane protein with specific expression in immune cells, taste cells, pancreatic acinar cells, and small intestinal tuft cells2+ influx upon GPCR stimulation, leading to changes in the Ca2+ influx pattern. Furthermore, our data indicated that Jaw1 interacts with all ITPR subtypes, increasing their Ca2+ release activity with subtle differences among each subtype. These results contribute to our understanding of the physiological significance of the heterogeneous Ca2+ dynamics among cell types and the involvement of Jaw1, as an ITPR regulator, in the uniqueness of Ca2+ dynamics in tissues or cells.In this study, we showed that Jaw1 increases cytoplasmic Ca2+ dynamics upon physiological stimulation, we created HEK293 Flp-In T-REx Jaw1 inducible-expressed cells with doxycycline (Dox) treatment-inducible Jaw1 expression. As previously reported, endogenous Jaw1 is expressed in HEK293 cells at a low level21. First, we generated HEK293 Flp-In T-REx Jaw1 KO cells (Jaw1 KO cells) by CRISPR/Cas9 , we prepared cells expressing Jaw1 at medium and high levels  among the cell lines  suggest that the Jaw1–ITPR interaction on the ER increases the Caa2+ Fig. A. The tolls Fig. B,C, indiype Fig. D. Ca2+ iity Fig. E,F. Howells Fig. E,G, indi24. To confirm whether Jaw1 interacts with all ITPR subtypes, we first prepared Jaw1 KO, Jaw1 IE, and Δcoil IE cell lysates and performed co-immunoprecipitation. The results showed that all ITPR subtypes co-immunoprecipitated with Jaw1, but none with Δcoil, suggesting that Jaw1 interacts with all subtypes via its coiled-coil domain , expressing exclusively a single subtype of each ITPR , a Jaw1 homolog, is also known to interact with ITPRs but represses their activity unlike Jaw12+ influx activates the Ca2+ adaptor proteins and enzymes in the cytoplasm, which transmit various signals4. Importantly, recent studies revealed that the oscillation, as well as the cytoplasmic Ca2+ influx level, is important for downstream protein activation30, suggesting that the oscillation-mediated duration of high [Ca2+]c above a certain threshold could be a key factor to induce the physiological responses. Indeed, several cellular events, such as secretion and gene expression, reportedly take a few minutes to initiate a response after the stimulation31. Of interest, as shown in Figs. 2+ imaging in Jaw1 IE cells demonstrated that each kinetic curve is more oscillated than that of Jaw1 KO cells. Particularly, the curve for steady reduction type showed a trend of being highly oscillated, although each ratio was depending on the concentration of ligand as well as the expression of Jaw1. Thus, more frequent Ca2+ peaks brought by the expression of Jaw1 increase the residential time of Ca2+ in the cytosol at single cell level. These data provide us a new hypothesis that the augmentative effect of Jaw1 on the sustained Ca2+ influx contributes to downstream response activation in Jaw1 expression-specific cells. To verify this concept, the Ca2+ dynamics in taste or tuft cells, expressing Jaw1 abundantly under physiological conditions, should be investigated in future studies.The cytoplasmic Ca2+ influx analysis focusing on each ITPR subtype, Jaw1 expression changed the cytoplasmic Ca2+ influx oscillation pattern in R1 and R2 SE cells, but not in R3 SE cells. The ability of ITPR3 to induce continuous cytoplasmic Ca2+ influx is reportedly the lowest among the three subtypes32, which might result in the smaller effect of Jaw1 on the residential time in the R3 SE cells. On the other hand, the Jaw1 expression had more augmentative effects on the maximum amplitude in R3 SE cells. Interestingly, the amino acid sequence homology among the subtypes is approximately 65–70%33, and their interactors are different depending on the subtypes34. These factors might also contribute to the above-described phenotypic differences among the subtypes, although further analyses would be required to clarify these aspects. Intriguingly, the ITPR subtype expression patterns were tissue- and cell-dependently heterogenous. Jaw1 and ITPR2 are expressed in small intestinal tuft cells, chemosensory cells that detect parasitic infection, Jaw1 and ITPR3 in tongue epithelial taste cells that perceive taste, and Jaw1 and ITPR2/3 in pancreatic acinar cells that secrete digestive enzymes. The non-uniform Jaw1 effect on the Ca2+ dynamics in each cell type and Jaw1 depletion in MEF reportedly reduced the Ca2+ influx while it increased that in pancreatic acinar cells15. This heterogeneous Jaw1 effect on each ITPR subtype might explain the non-uniform effect of the Jaw1 depletion on Ca2+ dynamics. It would be also important to investigate the biological significance of how the ITPR subtype-specific Ca2+ influx, as well as ITPR and Jaw1 expression patterns, are involved in the physiological responses in each tissue or cell in vivo.In the Cal-glutamine , 100 U/mL penicillin, and 100 µg/mL streptomycin . Cells were grown in 5% CO2 at 37 °C.Each HEK293 Flp-In T-REx cell line  was cultured in DMEM supplemented with 10% fetal bovine serum , 5.84 mg/mL Fect™A plus . After incubation for 48 h, the cells were subjected to selection by treatment with 2 µg/mL puromycin . The cells were then single-colonized by the limiting dilution cloning method in a 96-well plate and expanded. For the verification of the mutated sequence in each cell line by Sanger sequencing, genomic DNA was purified from each cell line using QIAamp DNA Micro Kit , and the amplified PCR products containing the target region were submitted to the DNA sequence contract service (Genewiz). Two clones for each cell line  from the cells with mutated sequences were randomly selected and used for following assay to remove the bias behind genetic background between clones. The numbers with cell name are original experimental clone number. Of note, there was no differences in the results between two clones. For the HEK293 Flp-In T-REx Jaw1 IE and Δcoil cell generation, HEK293 Flp-In T-REx Jaw1 KO #17 cells were transfected with pcDNA5 FRT/TO Hu SJaw1 or pcDNA5 FRT/TO Hu SJaw1 Δcoil using ScreenFect™A plus. After incubation for 48 h, the cells were selected by treatment with 100 µg/mL Hygromycin B  and expanded.For the generation of HEK293 Flp-In T-REx Jaw1 KO cells and ITPRs SE cells using the CRISPR/Cas9 system, HEK293 Flp-In T-REx cells were transfected with pSpCas9-2A-Puro Jaw1 or ITPRs sgRNA using the ScreenRabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech). The sequence of human Jaw1 was referred from NCBI (NP_00191055). The serum was subjected to affinity chromatography using N-Hydroxysuccunumidyl-Sepharose 4 Fast Flow  coupled with the N-terminal region of Jaw1 encoding amino acids1–144, resulting in purified anti-Jaw1 N-ter antibody.35) was digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector digested with the same enzymes using a commercial DNA ligation kit . For the production of pcDNA5 FRT/TO Hu SJaw1 Δcoil, pcDNA3.1 (+) Hu LJaw1 (previously described35) was first performed by inverse PCR with primer set 1 resulting in pcDNA3.1 (+) Hu LJaw1 Δcoil. pcDNA3.1 (+) Hu LJaw1 Δcoil was then digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector (digested with the same enzymes) using the aforementioned DNA ligation kit, resulting in pcDNA5 FRT/TO Hu LJaw1 Δcoil. Furthermore, pcDNA5 FRT/TO Hu LJaw1 Δcoil was digested with HpaI and XhoI, and the fragment was subcloned into pcDNA5 FRT/TO Hu SJaw1 digested with the same enzymes to swap the N-terminal region of Hu LJaw1 to that of Hu SJaw1.For the production of pcDNA5 FRT/TO Hu SJaw1, pcDNA3.1 (+) Hu SJaw1 -2A-Puro (PX459) vector  digested with g for 30 min. The supernatants were added to SDS-PAGE sample buffer and heated at 95 °C for 5 min. The samples were loaded onto a poly acrylamide gel, electrophoresed, and transferred onto polyvinylidene difluoride membranes . The membranes were blocked with tris-buffered saline containing 0.1% Tween20 (TBS-T) and 3% skim milk  for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Jaw1 N-ter , rabbit anti-ITPR1 , mouse anti-ITPR2 , mouse anti-ITPR3 , mouse anti-α-Tubulin , or mouse anti-GAPDH . The membranes were washed with TBS-T three times and incubated for 3 h with the following secondary antibodies: anti-Mouse IgG, HRP-Linked Whole Ab Sheep  or anti-Rabbit IgG, HRP-Linked Whole Ab Donkey . The membranes were washed with TBS-T three times and reacted with Immuno-star Zeta  or SuperSignal™ West Atto Ultimate Sensitivity Substrate . The bands were detected using an LAS4000 imaging system. The membranes were cut prior to hybridization with antibodies as shown in Supplementary Fig. Each cell line was seeded onto six-well plates  and incubated overnight. The cells were then treated for an additional 24 h with each Dox concentration to induce Jaw1 expression. After that, the cells were harvested by gentle pipetting, washed once with PBS, and lysed with RIPA buffer  containing a protease inhibitor cocktail . The samples were sonicated for 10 min on ice and centrifuged at 12,000 × 16). After the incubation for 1 h, the cells were washed with PBS three times and incubated for 1 h with Hoechst 33342 along with the following secondary antibodies: goat anti-rat IgG conjugated with Alexa Fluor 488  and goat anti-rabbit IgG conjugated with Alexa Fluor 568 . The cells were then washed with PBS three times and observed using fluorescence microscopy .Each cell line was seeded onto 96-well plates  and incubated overnight. The cells were treated with each Dox concentration to induce Jaw1 expression. After incubation for 24 h, the cells were fixed with 4% PFA in PBS buffer for 15 min. The cells were then washed with PBS once and permeabilized with 0.2% Triton-X  in PBS for 10 min. Next, the cells were washed with PBS three times and blocked with 2% bovine serum albumin in PBS for 1 h. The cells were then incubated with the following primary antibodies: rabbit anti-Jaw1 N-ter  and rat anti-Jaw1 coil  containing 0.5% NP-40 and 1 µL of a protease inhibitor cocktail . The lysates were sonicated for 10 min on ice and centrifuged at 12,000 × g for 30 min at 4 °C. The lysates were diluted with lysis buffer into 0.1% NP-40, then incubated with anti-Jaw1 antibody-conjugated beads for 1 h at 4 °C. The conjugated beads were prepared by mixing the anti-Jaw1 N-ter antibody and Protein A Mag Sepharose  for 1 h at 4 °C. The beads reacted with the cell lysate were then collected by a magnetic stand and washed with lysis buffer three times. For elution, the beads were mixed with SDS-PAGE buffer and heated at 95 °C for 5 min. On the magnetic stand, the supernatants were collected and subjected to western blotting.Each cell line was cultured on a six-well plate and incubated overnight. The cells were treated with 200 ng/mL Dox to induce Jaw1 expression. After incubation for 24 h, the cells were collected by gentle pipetting and centrifuged at 500 × jaw1 and gapdh gene detection, primer sets 6 and 7 were used, respectively.Each cell line was cultured on a six-well plate and incubated overnight. The cells were treated with 200 ng/mL Dox to induce Jaw1 expression. After incubation for 24 h, total RNA was isolated using RNAiso Plus . Total RNA was then reverse transcribed using PrimeScript RT Master Mix , and subsequent PCR was performed using TB Green Premix EX Taq II  on Thermal Cycler Dice Real Time System II MRX . For 2+ condition, the cells were incubated for 24 h, washed with PBS once, and incubated with recording buffer  containing 2 µM Fluo-4 AM  or 2 µM Rhod-2 AM  for 30 min. The cells were then washed with PBS once and incubated with HBSS containing 1.25 mM probenecid for 30 min. For the well scanning calcium assay using a plate reader, Varioskan LUX (Thermo Fisher Scientific) was used. The fluorescence values were recorded every second, and ATP  or carbachol  solution was added at 10 s. The data was measured from three independent wells. For the calcium imaging, we used a fluorescent microscope  equipped with a 20X fluor objective. The fluorescent images were captured every 2 s, and ATP solution was added at 10 s. The data was measured from four independent wells. For the calcium assay under Ca2+ free conditions, the cells were incubated with Ca2+ free recording buffer containing Fluo-4 AM for 30 min, washed with PBS once and incubated with Ca2+ free recording buffer. For the well scanning calcium assay for SOCE activity quantification, a plate reader was used and 2 µM thapsigargin solution was added at 10 s and recording buffer with 2 mM CaCl2 at 480 s. For the calcium imaging under Ca2+ free conditions, fluorescence microscopy was used and the 100 µM ATP solution without Ca2+ was added at 10 s.Each cell line was seeded onto 96-well black wall plates  and incubated overnight. The cells were treated with each Dox concentration to induce Jaw1 expression. For the calcium assay with extracellular Ca0 and the relative fluorescent intensity at each time point was calculated as F/F0. The calculated data was then graphed in GraphPad Prism7.The fluorescence intensity at 0 s was defined as FFor the calcium imaging, fluorescent images of the Fluo-4-loaded cells upon ATP stimulation were analyzed and quantified by Fiji. The calcium imaging was performed in the four independent wells and data from 50 cells in each image was used for the quantification. We randomly chose 50 cells from each image, and the center of each cell (8 × 8 pixel) was selected as a representative area. The fluorescent intensity in the chosen area was measured over time for 5 min.2+ influx types as follows: (1) The first group (single type) included the cells with amplitudes only once for 5 min. (2) The second group (oscillation type) included the cells with more than two oscillations. The oscillation was defined as the relative Fluo-4 intensity decreasing below 125% with a peak that rose again during 5 min. (3) The third group (steady reduction type) included the cells that maintained the relative Fluo-4 intensity higher than 125% of the initial values for 5 min. (4) The fourth group (no response type) included the cells maintaining the amplitude less than 300% of the initial value for 5 min. To calculate the ratio, we used 50 cells for each of the four measurements .Relative Fluo-4 intensity of each cell was plotted for 5 min using GraphPad Prism 7. The relative Fluo-4 intensity curves were then transferred to layouts and manually classified into four Cat-test was used to compare two sample groups. To compare more than two groups, one-way ANOVA followed by Tukey–Kramer’s t-test was used. *, **, ***, and **** indicate statistically significant P values of p < 0.05, < 0.01, < 0.001, and < 0.0001, respectively. n.s.: non-significant.All statistical tests were calculated using GraphPad Prism 7, and the data are represented as the mean ± S.D. The sample numbers are included in each figure legend. Unpaired, nonparametric Student’s Supplementary Information 1.Supplementary Information 2.


ETη) from Si to Er3+ is crucial. In order to achieve high ETη, we used nonthermal plasma to synthesize Si quantum dots (QDs) hyperdoped with Er at the concentration of ~1% . The QD surface was subsequently modified by hydrosilylation using 1-dodecene. The Er-hyperdoped Si QDs emitted near-infrared (NIR) light at wavelengths of ~830 and ~1540 nm. An ultrahigh ETη (~93%) was obtained owing to the effective energy transfer from Si QDs to Er3+, which led to the weakening of the NIR emission at ~830 nm and the enhancement of the NIR emission at ~1540 nm. The coupling constant (γ) between Si QDs and Er3+ was comparable to or greater than 1.8 × 10−12 cm3·s−1. The temperature-dependent photoluminescence and excitation rate of Er-hyperdoped Si QDs indicate that strong coupling between Si QDs and Er3+ allows Er3+ to be efficiently excited.Erbium-doped silicon (Er-doped Si) materials hold great potential for advancing Si photonic devices. For Er-doped Si, the efficiency of energy transfer ( Hence, the ETη plays a significant role in the practical applications of Er-doped Si QDs. The ETη can be described by [τEr−Si QDs and τSi QDs are the PL lifetime of Er-doped and undoped Si QDs, respectively. H. Rinnert et al. reported Er-doped Si QDs embedded in SiO/SiO2 multilayers, whose ETη was estimated as ~50% [ETη (~50%) for Er-doped Si QDs embedded in SiO/SiO2 multilayers [3+ was only found in close proximity to Si QDs inside a dielectric matrix, resulting in a system with a low degree of coupling [3+ energy transfer in Er-doped Si QDs because of the variable distance between Si QDs and Er3+. Notably, the exciton-to-Er3+ energy transfer should be most effective if Er3+ is present in the Si QD in order to facilitate the overlap of electron wavefunctions [3+ is incorporated as a dopant.Silicon (Si) photonics has been intensively investigated due to its excellent compatibility with Si-based complementary metal oxide semiconductor (CMOS) technologies ,2. Whileribed by (1)ηET=1 as ~50% . Timoshetilayers . Despitecoupling . The weaunctions . This mo20 cm−3), surpassing its solubility. Er-hyperdoped Si QDs were found to emit NIR light at 830 and 1540 nm, with the majority of the Er3+ located in the subsurface area. As a result of the effective transfer of energy from Si QDs to Er3+, the Er-hyperdoped Si QDs exhibited an ultrahigh ETη of ~93%, emitting at 1540 nm. Meanwhile, the efficient energy transfer from Si QDs to Er3+ greatly reduced the 830 nm NIR emission. Additionally, a high effective excitation cross section of Er3+ (Erσ) was obtained, with a value of 1.5 × 10−17 cm2. Furthermore, the coupling constant (γ) between Si QD and Er3+ was greater than or equal to 1.8 × 10−12 cm3·s−1. The temperature-dependent photoluminescence and excitation rate of Er-hyperdoped Si QDs indicate that strong coupling between Si QDs and Er3+ allows Er3+ to be efficiently excited.Here, nonthermal plasma was used to fabricate Er-hyperdoped Si QDs at an atomic concentration of 1% ) (99.999%) was purchased from Nanjing Aimouyuan Scientific equipment Co., Ltd. . SiH4/Ar (20%/80% in volume) was obtained from Linde Electronic & Specialty Gases Co., Ltd. . Mesitylene (97%) and 1-dodecene (97%) were purchased from Aladdin . Methanol (≥98.5%), hydrofluoric (HF) acid (≥40%), and toluene (≥99.5%) were obtained from J&K Scientific . Reference solutions of Er (100 µg·mL−1 in nitric acid) and Si (1000 µg·mL−1 in nitric acid) were purchased from Qingdao Qingyao Biological Engineering Co., Ltd.  and Sigma-Aldrich Trading Co., Ltd. , respectively.Er(tmhd)−2 mBar, and a heating strip was used to increase the temperature of the pipeline and the erbium precursor Er(tmhd)3 to 200 °C. The plasma chamber was filled with 4.8 sccm of a 20% SiH4/Ar mixture and 500 sccm of Ar-loaded Er(tmhd)3 3 a. The prr(tmhd)3 b. After An FEI Tecnai G2 F20 S-TWIN  was used to capture the TEM images, operating at an acceleration voltage of 200 kV. High-angle annular darkfield scanning transmission electron microscopy (HAADF-STEM) was performed and element map images were obtained using the FEI Titan G2 80-200  operated at an acceleration voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) was used to examine the oxidation states in each sample . Er concentration was calculated using ICP-MS . The Shimadzu-7000  was used for the X-ray diffraction (XRD) tests. Raman spectra were obtained to analyze the strain . Using FLS1000 PL equipment , we obtained the PL and PLE spectra. A 405 nm laser was used to excite the transient PL, and an NIR photomultiplier  was used to detect it. The effect of temperature on PL was investigated using a cryostat . Electron paramagnetic resonance (EPR) spectroscopy on a Bruker ESRA-300  was used to analyze the Si dangling bond. Details of spectral analysis can be found in .20 cm−3) [18 cm−3 [20 cm−3) . The QD 20 cm−3) . To put [18 cm−3 , and theThe transmission electron microscopy (TEM) images of undoped and Er-hyperdoped Si QD are presented in −1 (−1) [−1 after Er hyperdoping. This blueshift was brought on by the compressive strain caused by the presence of sites of tetrahedral interstitial Er [ε) using [ν denotes the shift of the Raman peak. We observed a 9 cm−1 shift in the Raman peak, which suggests ε = ~1.3%. On the other hand, Raman peak broadening of the Si-Si bonds suggests that these bonds were deformed when Er3+ was doped [x thickness of Si QDs was reduced from 0.8 nm to 0.6 nm after Er hyperdoping, allowing for a corresponding SiOx stoichiometric shift from SiO1.0 to SiO0.7  . The Si-itial Er . It is rε) using (2)ε=(Δνas doped . This pr[(2)ε=Δν61.2×100%as doped , suggesto SiO0.7 . XPS meace of +3 , we may 4I13/2 → 4I15/2 transition of Er3+ [3+ ions, previously seen in the literature, may account for this decrease [2H9/2 → 4I15/2) and 460 nm (4F5/2 → 4I15/2). The synchronicity of the PLE spectrum for the 830 and 1540 nm emissions suggests that Er3+ was excited through an efficient energy transfer from Si QDs to Er3+, which is the critical attribute responsible for the emission of Er3+. The PL lifetime was shown to drop from 275.1 s to 19.5 s due to Er hyperdoping (12 cm−2 (corresponding to 0.6 dangling bonds per QD) whereas that at the surface of Er-hyperdoped Si QDs was 6.9 × 1011 cm−2 (corresponding to 0.4 dangling bonds per QD) . Hence,3+, assuming that no other factors play a role. Using Equation (1), we obtain an ultrahigh ETη of ~93%, which is substantially larger than the previously reported ~50% for Si QDs with neighboring Er3+ [It was shown that the radiative recombination lifetime of Si QDs was not strongly influenced by compression because the compression hardly changed the HOMO–LUMO transition . This meing Er3+ ,11,33.830 nm) increased linearly with the increase of excitation power until the excitation power was so high that the PL tended to be saturated. For Si QDs, Auger recombination is responsible for this saturation [830 nm, the PL intensity at 1540 nm (I1540 nm) was proportional to the power of the 405 nm laser. Since this indicates that the I1540 nm is constrained by the total amount of excitons transferred from Si QDs, this observation provides more evidence for the existence of excitonic energy transfer to Er3+ in Si QDs. We can calculate the effective excitation cross section of Er3+ (Erσ) using [φ represents the photon flux and 3+ in level 4I13/2. By fitting the data in Erσ was obtained as 1.5 × 10−17 cm2, an increase by four orders of magnitude compared with the value obtained for the direct excitation of Er3+ (8 × 10−21 cm2) [onτ) obtained for the 1540 nm light emission [As seen in turation . Like I8r) using (3)I1540−21 cm2) . The folemission (4)1τon=Erσ by fitting Equation (4). We monitored the excitation-power-dependent rise for the 1540 nm light emission over time  between a Si QD and Er3+ was calculated using the formula in the literature [Si QDσ = 10−16 cm2, φ = 1020 cm−2·S−1, −5 s, −5 s and 0A < 1017 cm−3, where Si QDσ is the absorption cross section of Si QD, 0 is the initial state of the Si QD. Finally, we obtained that the γ was comparable to or greater than the value of 1.8 × 10−12 cm3·s−1, an increase by three orders of magnitude compared with the γ in Er-doped silicon-rich silica (3 × 10−15 cm3·s−1) [3+ allows Er3+ to be efficiently excited, resulting in the ultrahigh ETη and Erσ.Hence, we can calculate ver time h. Figureterature  with thecm3·s−1) . Therefo830 nm decreased with the increase of temperature from 77 to 237 K while it increased with the increase of temperature from 237 K to room temperature. However, there was only a monotonic decrease in I830 nm with the increase of temperature in undoped Si QDs . Thus, Erσ is proportional to 1540 nm. Therefore, the temperature-dependent Erσ results in the trend of I1540 nm with the increase of temperature. Moreover, the reverse temperature-dependent PL tendency between I830 nm and I1540 nm demonstrates the effective energy transfer of excitons between Si QDs and Er3+, further supporting the strong coupling mechanism.The dependence of the integrated PL intensity on temperature was studied to further verify the strong coupling. d Si QDs . Thermalnd 237 K . In controm Er3+ . Please onτ) and decay time (dτ) of Si QDs (3+ (R = (1on/τ − 1d/τ), was calculated, showing that Si QDR = 7962 s−1 for Si QDs and ErR = 7768 s−1 for Er3+. The observed ErR is close to Si QDR, which is also in agreement with the strong coupling mechanism [Both the rise time (f Si QDs c and Er3 QDs (3+ d were moechanism .3+. For Er-hyperdoped Si QDs, an ultrahigh ETη and a high Erσ were obtained. Moreover, the quantitative study of the coupling constant demonstrates the strong coupling between Si QDs and Er3+. The strong coupling was also manifested by the temperature-dependent PL and excitation rate of Er-hyperdoped Si QDs. All these findings suggest that Er-hyperdoped Si QDs have great potential for the fabrication of high-performance NIR light-emitting devices.The synthesis of Er-hyperdoped Si QDs was accomplished by employing nonthermal plasma. Both 830 and 1540 nm NIR light were emitted from Er-hyperdoped Si QDs under 405 nm excitation. PLE and PL decay measurements showed that energy transfer occurred between Si QDs and Er


Microbial growth leads to a decrease in apical glucose levels. T1Rs detect this change and liberate bitter taste receptor signaling, initiating an innate immune response to both kill and expel pathogens through releasing antimicrobial peptides and increasing nitric oxide production and ciliary beat frequency. However, chronic inflammation due to disease, smoking, or viral infections causes a remodeling of the epithelial airway. The resulting squamous metaplasia causes a loss of multi-ciliated cells and solitary chemosensory cells, replaced by basal epithelial cells. To understand how T1R function is altered during disease, we used basal epithelial cells as a model to study the function of T1R3 on Ca2+ signaling dynamics. We found that both T1R1 and T1R3 detect amino acids and signal via cAMP, increasing the responsiveness of the cells to Ca2+ signaling stimuli. Either knocking down T1R1/3 or treating wild-type cells with MEM amino acids caused a reduction in ER Ca2+ content through a non-cAMP signaled pathway. Treatment with amino acids led to a reduction in downstream denatonium-induced Ca2+-signaled caspase activity. Thus, amino acids may be used to reduce unwanted apoptosis signaling in treatments containing bitter compounds.T1Rs are expressed in solitary chemosensory cells of the upper airway where they detect apical glucose levels and repress bitter taste receptor Ca N-Acyl homoserine lactones [2+) signaling cascade leading to an increase in nitric oxide (NO) production while also increasing ciliary beat frequency to hasten mucociliary clearance, thus providing a means to both kill and remove ensnared pathogens [Upper airway epithelial cells initiate an innate immune response through taste receptor signaling pathways in retaliation against inhaled pathogens. Bitter taste receptors T2Rs) localized to the motile cilia of multi-ciliated airway epithelial cells  detect “Rs localilactones . While mathogens ,5.3, and Gγ13, which signal for intracellular Ca2+ elevations [Pathogens feed on apical glucose in the airway surface liquid. Solitary chemosensory cells (SCCs) are a rare epithelial cell type that contain both T2Rs and sweet taste receptors (T1Rs) ,6,7,8. Ievations ,15,16. Tevations . This pa2+) signaling through detection of apical glucose [2+-dependent release of anti-microbial peptides from neighboring multi-ciliated cells into the apical lumen of the airway to combat infections. Thus, in a typical, heathy epithelial layer, T1Rs and T2Rs function as sentinels of the upper airway.In airway SCC’s, T1R2 and 3 are expressed  and hypo glucose . Once pa glucose . T2Rs thHowever, chronic inflammation, caused by diseases such as chronic rhinosinusitis (CRS) , COPD 2, smoking2+ stores. Thus, umami receptor T1R1/3 may play a pivotal role in reducing apoptosis activated by bitter drugs in infected airway cells.While bitter compounds increase NO production, we have also shown that T2R activation induces apoptosis in squamous and/or basal airway cells . MoreoveFor a detailed list of reagents used in this work, please refer to g lysates were estimated by Bradford DC Assay  then 60 µg protein/lane was loaded into a 4-12% Bis-Tris gel. After electrophoresis, the resulting gel was transferred to nitrocellulose and the membrane was blocked in 5% milk in Tris-Tween  for at least 1 h. The primary antibody was diluted 1:1000 in Tris-Tween containing 5% bovine serum albumin and incubated for at least 2 h. The secondary antibody goat anti-rabbit IgG-horseradish peroxidase was diluted 1:1000 in Tris-Tween containing 5% milk. After extensive washing, blots were incubated in Clarity ECL  as per manufacturers’ protocol, and resulting immunoblots were visualized using a ChemiDoc MP Imaging System . Final images were processed using Image Lab Software .For endogenous T1R1 and T1R3 immunoblot visualization, cell pellets were resuspended in RIPA Buffer  and lysed via sonicating water bath. Protein concentrations of post 800× 2+ measurements, cells were loaded with either 5 µM of Fluo-8 AM or Fura-2 AM for 1 h in HEPES-buffered Hank’s Balanced Salt Solution in the dark at room temperature. For intracellular or nuclear cAMP, biosensors were transfected 48 h prior to measurements. Fura-2 AM images were taken using Fura-2 filters  while all other images were taken utilizing FITC or TRITC filter sets (49002-ET or 49004-ET Chroma) on an Olympus IX-83 microscope (20× 0.75 NA objective) with fluorescence xenon lamp and X-Cite 120 LED boost lamp , excitation and emission filter wheels , Orca Flash 4.0 sCMOS camera  and MetaFluor software .For intracellular CaPrimary human sinonasal cells were isolated from residual surgical material acquired in accordance with the University of Pennsylvania guidelines for use of residual clinical material along with the U.S. Department of Health and Human Services code of federal regulation (Title 45 CFR 46.116) and the Declaration of Helsinki. Informed consent from each patient was obtained in addition to institutional review board approval (#800614). Patients were >18 years old and either receiving surgery for sinonasal disease or other procedures.2 for 2 h in PneumaCult-Ex Plus medium  with 100 U/mL penicillin and 100 µg/mL streptomycin to remove non-epithelial cells. After 2 h, unattached cells were then plated into a tissue culture dish and propagated in PneumaCult-Ex Plus medium. Basal cell identity was confirmed by positive staining for basal cell markers CD49f and p63  and transfected with either 10 nM of non-targeting, TAS1R1.6, or TAS1R3.2 RNAi duplex (Integrated DNA Technologies) for 48 h using lipofectamine 3000  as per manufacturer’s protocol. Knockdown was validated using qPCR as described below.Cultured cells were resuspended in TRIzol  and either stored at −70 °C or immediately used. RNA was purified via Direct-zol RNA kit  subjected to RT-PCR using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Resulting cDNA was then utilized for qPCR using Taqman Q-PCR probes in QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific). Data was analyzed using Microsoft Excel and graphed in GraphPad PRISM v8.t-tests was used. ANOVA was utilized for more than two comparisons where Tukey–Kramer’s post-test was used for comparisons of all samples, Dunnett’s post-test was used to compare multiple samples to one control, and Bonferonni’s post-test was used for selective pair comparisons. In all figures, (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001, “n.s.” . All data represent the mean ± SEM of a minimum of 3 experiments.For comparisons of only two data sets TAS1R1, TAS1R2, and TAS1R3, the genes encoding T1R1, T1R2, and T1R3, respectively, were expressed in both the Beas-2B and primary cells in both basal and differentiated cultures  and 1× non-essential amino acids (NEAAs)  , we did ignaling a. Additie Fura-2 b.2+ elevation that was completely inhibited by a 1-hour pretreatment with 20 mM of umami antagonist lactisole (2+ elevations were from 1× MEM AA’s (~92%) while 1× NEAAs had very little contribution (~6%). While this observation is interesting, it remains to be determined if this is physiologically relevant. We first wanted to study the function of T1R1/3 under normal physiological pH, thus we continued experiments with buffers with pH corrected to 7.4 for the rest of this study.Fortunately, we found that the addition of both 1× MEM AA and 1× NEAA’s to HBSS containing 20 mM HEPES decreased the pH to 6.5, and at this pH, we found that both 1× MEM AAs and 1× NEAAs induced an intracellular Caactisole . The add2+ signaling was unaffected by 1× MEM amino acids at pH 7.4, we also evaluated cAMP signaling. Utilizing genetic encoded fluorescent biosensor Flamindo2 to measure intracellular cAMP levels [Though CaP levels , we obseP levels , we alsoTAS1R3 expression in Beas-2Bs  and ryanodine (RYR) receptors [2+ content.Elevations in cAMP alters many downstream signaling pathways, canonically through protein kinase A (PKA) or exchange protein directly activated by cAMP (EPAC). Using Beas-2B cells expressing PKA biosensor AKAR4 ,35 whicheceptors ,38,39,402+ content, we utilized the SERCA pump inhibitor thapsigargin. Without the constant uptake of Ca2+ into the ER, cells treated with thapsigargin will release their ER Ca2+ content into the cytosol where it can be measured using the intracellular Ca2+ binding dye Fluo-8. The Ca2+ will then dissipate into the extracellular space where it will be chelated by EGTA in the surrounding 0-Ca2+ HBSS buffer. This allows us to observe the release of total ER Ca2+ content without re-uptake of Ca2+ across the plasma membrane or ER membranes. The addition of 1× MEM AAs caused a 35% decrease in ER Ca2+ content  then treated with 15 mM denatonium. To our surprise, 1× MEM AA treatment caused a 30% increase in denatonium-induced Ca2+ release based on the population average  also reduced denatonium-stimulated Ca2+ release by ~50% per responsive cell, demonstrating that MEM AA treatment modifies intracellular Ca2+ release rather than Ca2+ influx  ER Ca2+ content decreased, (2) cultures released less calcium per cell in response to denatonium, (3) and the number of cells responding to denatonium increased. Together, from the perspective of ER Ca2+ content, these data suggest that MEM AAs might be disrupting the T1R1/3 activity. If T1R1/3 is providing a baseline regulation of ER Ca2+ content, one explanation is that something in the MEM amino acids may be acting as an inverse agonist. Additional research would be necessary to determine that this decrease in ER Ca2+ content is solely due to amino acid-T1R1/3 interaction and not the action of another receptor. Furthermore, additional, more detailed pharmacological studies, beyond the scope of this study here, would be needed to test inverse agonism of the MEM-amino acid mixture.Here, we show that and T1R1 and T1R3 are expressed in basal airway epithelial cells. There, they likely function to detect components of MEM AAs and signal via cAMP elevation. While knocking down T1R1 or T1R3 leads to a reduction in ER Ca2+ content, forskolin treatment did not. Given these observations, it seems unlikely that cAMP alone is responsible for the reduction of ER Ca2+. However, either knocking down T1R1/3, treating with 1× MEM AA, isoproterenol, or forskolin all led to an increased sensitization of cells to agonists that signal via Ca2+ elevations. Thus, we hypothesize that while cAMP elevations may increase the number of cells that respond to agonists that signal via intracellular Ca2+ pathways, the reduction of ER Ca2+ content may occur through a separate, distinct pathway. Moreover, as there are many transcription factors that are sensitive to cAMP levels, the data showing changes in nuclear cAMP may also suggest a possible role for T1R1/3 in amino acid-induced transcriptional changes, which could be investigated in future studies.While isoproterenol treatment also led to a decrease in ER Ca2+ signaling [T1R3 has been previously characterized to detect some L-amino acids via heterologous expression studies ,42. One ignaling . A separThe physiological agonist (s) activating T1R1/3 in basal airway cells also needs to be determined in order to identify whether this is an immune sensor  or a sensor of host metabolic or nutritional state. An important caveat to our study is that we do not yet know the identity of the endogenous agonists. Future work is needed. However, MEM AAs have been used as a T1R1/3 agonist in other studies’ cells ,32, and 2+ release in response to 1× MEM AA treatment at physiological pH. However, when added to a standard HBSS formulation containing 20 mM HEPES without any pH corrections, 1× MEM AA and 1× NEAA reduced the pH from 7.5 to 6.5. We observed Ca2+ elevations in response to amino acids when the HBSS was at pH 6.5. At this lowered pH, the majority of the Ca2+ response was due to the 1× MEM AAs. This Ca2+ elevation was also inhibited by pretreatment with 20 mM T1R3 antagonist lactisole, which did not alter the final pH of the solution. While this suggests a function of T1R1/3 during conditions where pH is lowered, the physiological relevance is not yet determined. Additionally, while we have not observed Ca2+ elevations in response to amino acids at a physiological pH; this could also be due to variations in G protein subunits bound to T1R1/3 in our cell models used here. The promiscuity of GPCRs for G protein subunits has been well documented [We did not observe an increase in intracellular Cacumented ,44,45,462+ signaling pathway in pancreatic b cells [2+ signaling pathways reported here is likely independent of mTOR [2+ signaling like MEM-AAs. While ERK1/2 and mTORC1 may still be activated in our models, this is a distinct, non-canonical pathway of umami receptor signaling. Parallel effects of ERK1/2 and/or mTORC1 activation will be investigated in future studies.Through detecting amino acids, T1R1/3 was shown to activate ERK1/2 and mTORC1 through a Ca b cells ,32 and i b cells . We show of mTOR ,32 as ra2+ content led to a reduction in intracellular Ca2+ elevations signaled by denatonium. Thus, amino acids may play a protective role in reducing apoptosis via the umami T1R1/3 receptor. When we previously measured NO production from bitter compounds such as denatonium, flufenamic acid [2+-signaled NO response even in the presence of 1× MEM AAs, our current work suggests that the addition of 1× MEM AAs to a topical T2R agonist therapeutic may help to diminish apoptotic signaling pathways without preventing stimulation of NO production. Thus, while we do not know the identity of the physiological agonist (s) activating T1R1/3 in airway basal cells, our data do reveal the potential therapeutic utility of 1× MEM AAs activation of T1R1/3 to protect airway epithelial cells against pro-apoptotic insults such as bitter agonists.Ultimately, this reduction in ER Camic acid , or diphmic acid , we util2+ content. Furthermore, the addition of 1× MEM AAs reduced ER Ca2+ content and greatly impaired apoptosis signaling via denatonium treatment. Therefore, amino acids or other umami receptor agonists may be useful to employ in therapeutics containing bitter compounds to repress unwanted induction of apoptosis.Our findings demonstrate a previously undescribed function for T1R1/3 in airway epithelial cells as a receptor for amino acids that signal through cAMP and as a regulator of ER Ca