Noticeably, BN-doped graphene supporting a single metal atom  activates the N2 molecule with a red shift in the N–N stretching frequency . This red shift is corroborated by an increase in the N–N bond length (1.23 Å from 1.09 Å) and charge transfer to an N2 molecule from the catalyst.Using the periodic density functional theory–based methodology, we propose a potential catalytic system for dinitrogen activation, viz., single metal atoms  supported on graphene-based sheets. Graphene-based sheets show an excellent potential toward the anchoring of single atoms on them  with adsorption energies ranging between 1.048 and 10.893 eV. Factors such as defects and BN doping are noted to enhance the adsorption energies of single metal atoms on the support. The adsorption of a dinitrogen molecule on metal atom–anchored graphene-based supports is seen to be highly favorable, ranging between 0.620 and 2.278 eV. The adsorption is driven through a direct hybridization between the  Though the availability of dinitrogen (N2) is abundant in air, it requires high energy for fixation and activation owing to its existence of inert triple bonds between nitrogen atoms. Currently, the well-known Haber–Bosch process invented more than a century ago is used for converting dinitrogen (N2) in the atmosphere into NH3 in the presence of the iron catalyst at an extreme temperature (500°C) and pressure (200 atm)  . The eneemission . Neverthrganisms . Researc V atoms , yet the2 activation on potential inorganic metal clusters (2) activation using infrared multiphoton dissociation (IR-MPD) on neutral Ru clusters. Similarly, −1 on solid Lin (2 < n < 8) clusters, particularly the Li8 metal cluster showing an exothermic trend in splitting the N–N bond completely. In the midst of metal clusters for evaluating N2 activation reaction, Al clusters play a remarkable role. Previously, Jarrold et al. observed low energy barriers for N2 activation on Al44 and Al100 clusters at high temperatures using concerted experimental and theoretical techniques  methodology   represents the energy of the optimized single transition-metal cluster  and the designed surface supports. E(M) and E(system) represent the energy of a single metal and surface support, respectively.The ground state geometries of single transition-metal clusters  are adsorbed on the above-mentioned supports and the complexes optimized. The adsorption energy of Mo, V, and Fe on these supports is calculated as follows:2 molecule is adsorbed on these active metal clusters  on graphene-based surface supports. A parallel mode of adsorption  is used as this mode has been found to be more effective as compared to the vertical mode. In the vertical mode, only one N atom in the N2 molecule interacts with the metal leading to weak activation  represents the energy of the dissociated N2 molecule on the catalytic systems. E(N2) and E(M--system) represent the energy of the N2 molecule and metal-adsorbed various surface supports, respectively. Nudged elastic band (NEB) calculations were performed toward prediction of energy barrier of N2 activation on metal-adsorbed BN-doped graphene-based substrates.The dissociated adsorption energy of the NGraphene-based 2D materials which act as an electron reservoir are used as the support for adsorbing the single atom cluster  which increases the catalytic activity of the metal center. The five graphene-based supports are designed, viz., 1) pristine graphene (50 carbon atoms), 2) defective graphene (49 carbon atoms with a single vacancy at the center), 3) BN-doped graphene (4% heteroatom doping in which boron and nitrogen are substituted instead of carbon in the pristine graphene), 4) BNC-ring graphene (8% heteroatom doping), and 5) BN-ring graphene (12% heteroatom doping). All these graphene-based supports are designed and optimized to the local minima as shown in Thus, we have tried to establish the relative reactivity of single atom clusters  chemisorbed on the above-mentioned surfaces. The optimized structure of adsorption of Mo (gray), Fe (red), and V (purple) on various surface supports is shown in 2 activation. The total density of states and projected density of states of a single metal atom  on graphene-based supports are shown in The carbon–metal (C–M) interatomic distance of Mo, Fe, and V on pristine graphene, defective graphene, BN-doped graphene, and BNC-ring graphene is 2.200–2.213, 2.069–2.080, and 2.147–2.170 Å;1.932–1.956, 1.766–1.768, and 1.863–1.873 Å; 2.145–2.261, 2.012–2.473, and 2.079–2.192 Å; and 2.072–2.305, 1.947–2.148, and 2.072–2.306 Å, respectively. The boron–metal (B–M) interatomic distance of Mo, Fe, and V on BN-doped graphene, BNC-ring graphene, and BN-ring graphene is 2.258, 2.303, and 2.215 Å; 2.279–2.28, 2.106–2.218, and 2.279–2.28 Å; and 2.216–2.219, 2.063–2.124, and 2.17–2.227 Å, respectively. The nitrogen–metal (N–M) interatomic distance of Mo, Fe, and V on BN-doped graphene, BNC-ring graphene, and BN-ring graphene is 2.211, 1.861, and 2.172 Å; 2.224–2.226, 2.01–2.225, and 2.224–2.226 Å; and 2.267–2.273, 2.073–2.197, and 2.204–2.226 Å, respectively. The interatomic distances and adsorption energies of Mo, Fe, and V on various graphene-based supports are shown in 2 on a single metal atom  on pristine graphene, defective graphene, BN-doped graphene, BNC-ring graphene, and BN-ring graphene are 1.739, 1.334, and 1.996 eV; 0.887, 0.620, and 0.628 eV; 1.844, 2.278, and 1.988 eV; 1.870, 1.544, and 2.116 eV; and 1.868, 1.510, and 2.258 eV, respectively. Comparatively, the adsorption energies of N2 on a single metal atom  on the defective graphene support are less compared to those on the rest of the support. Moreover, there is an eventual increase in adsorption energies of N2 on V on supports  due to more vacant d orbitals , which is vice versa in Fe  on the same supports. The optimized structure of adsorption of N2 on a single metal atom  on various surface supports is shown in The adsorption energies of N2 on a single metal atom  on pristine graphene, defective graphene, BN-doped graphene, and BNC-ring graphene is 2.228–2.313, 2.077–2.162, and 2.190–2.264 Å; 1.946–2.013, 1.777–1.847, and 1.871–1.925 Å; 2.194–2.257, 2.096–2.144, and 2.133–2.234 Å; and 2.111–2.319, 2.004–2.172, and 2.048–2.241 Å, respectively. The boron–metal (B–M) interatomic distance of N2 on a single metal atom  on BN-doped graphene, BNC-ring graphene, and BN-ring graphene is 2.313, 2.18, and 2.319 Å; 2.316–2.407, 2.219–2.222, and 2.303–2.342 Å; and 2.244–2.41, 2.141–2.216, and 2.256–2.371 Å, respectively. The nitrogen–metal (Ndoped–M) interatomic distance of N2 on a single metal atom  on BN-doped graphene, BNC-ring graphene, and BN-ring graphene is 2.32, 2.202, and 2.223 Å; 2.239–2.328, 2.165–2.173, and 2.23–2.242 Å; and 2.274–2.341, 2.083–2.234, and 2.264–2.297 Å, respectively.The carbon–metal (C–M) interatomic distance of Nad–M) interatomic distance of N2 on a single metal atom  on pristine graphene, defective graphene, BN-doped graphene, BNC-ring graphene, and BN-ring graphene is 2.039–2.117, 1.92–1.923, and 1.911–1.994 Å; 2.22–2.221, 1.964–2.078, and 2.161–2.218 Å; 2.027–2.091, 1.902–1.907, and 1.918–2 Å; 2–2.057, 1.889–1.89, and 1.908–1.979 Å; and 2–2.068, 1.9, and 1.869–1.928 Å, respectively. The interatomic distances and adsorption energy of N2 on a single metal atom  on various substrate systems are shown in 2 on a single metal atom  on the graphene-based support are shown in The nitrogen–metal  on the graphene-based support, we investigated the spectral range of 1,300–2,300 cm−1, which covers the typical frequencies of the different N2 species known to exist on transition-metal surfaces. The stretching frequency of the unbound N2 molecule is attributed to 2,330 cm−1, and the N–N bond length is 1.09 Å  on pristine graphene, defective graphene, BN-doped graphene, BNC-ring graphene, and BN-ring graphene are 1.20 Å (1735 cm−1), 1.18 Å (1823 cm−1), and 1.21 Å ; 1.17 Å (1907 cm−1), 1.16 Å (2009 cm−1), and 1.16 Å (1997 cm−1); 1.20 Å (1701 cm−1), 1.19 Å (1802 cm−1), and 1.20 Å (1711 cm−1); 1.21 Å , 1.19 Å (1777 cm−1), and 1.21 Å ; and 1.21 Å , 1.19 Å (1796 cm−1), and 1.23 Å , respectively.To probe the stretching frequency of the adsorbed Ns 1.09 Å . The N–N2 activation are listed in 2 molecule adsorbed on Mo, Fe, and V on various graphene supports are shown in The Bader charge analysis  and the sp2 orbitals of unsaturated carbon atoms of various designed graphene-based supports. The catalytic performance of a single metal atom  on graphene-supported catalysts is explored for the activation of molecular nitrogen. The adsorption energies of the nitrogen molecule on a graphene-supported single atom cluster  range between 0.620 and 2.278 eV, which is attributed to the interacting environment of the active metal centered on the support and the p orbital of adsorbed molecular nitrogen. Bader charge and density of states analyses corroborate an enhanced hybridization between the d states of the single metal atoms  and adsorbed molecular nitrogen for activation. The N–N stretching frequencies are found which are considerably red-shifted ranging from 2009 cm−1 (1.16 Å) to 1,597 cm−1 (1.23 Å) compared to that of the unbound N2 molecule ). Thus, from the results, we understood that even a single metal atom  with functionalized (BN-doped) graphene supports can highlight the excellent potential for nitrogen activation.In this work, we explore the potential of various graphene-based 2D materials, viz., pristine, defective, BN-doped graphene, etc., as a support for a single atom cluster . These graphene-based supports show excellent potential toward the anchoring of a single atom cluster  with adsorption energies ranging between 1.048 and 10.893 eV. Thus, the adsorption energies vary substantially with respect to the graphene-based supports, viz., pristine, defective, BN doped, etc. This is attributed to the size and nature of hybridization between the


T cell engineering strategies offer cures to patients and have entered clinical practice with chimeric antibody-based receptors; αβT cell receptor (αβTCR)-based strategies are, however, lagging behind. To allow a more rapid and successful translation to successful concepts also using αβTCRs for engineering, incorporating a method for the purification of genetically modified T cells, as well as engineered T cell deletion after transfer into patients, could be beneficial. This would allow increased efficacy, reduced potential side effects, and improved safety of newly to-be-tested lead structures. By characterizing the antigen-binding interface of a good manufacturing process (GMP)-grade anti-αβTCR antibody, usually used for depletion of αβT cells from stem cell transplantation products, we developed a strategy that allows for the purification of untouched αβTCR-engineered immune cells by changing 2 amino acids only in the TCRβ chain constant domain of introduced TCR chains. Alternatively, we engineered an antibody that targets an extended mutated interface of 9 amino acids in the TCRβ chain constant domain and provides the opportunity to further develop depletion strategies of engineered immune cells.  The authors provide a GMP-ready solution for the enrichment of human αβTCR-engineered T cells by murinization of 2 aa. An extended murinized epitope with a total of 9 aa could become a novel interface to specifically target TCR-engineered T cells with an anti-mouse αβTCR antibody. Current methods for purification of engineered T cells often depend on the expression of artificial molecules such as truncated CD34,in vivo depletion through myc-specific antibodies.in vivo elimination achieved with a single marker, which has previously been described for CD20,in vivo depletion, and thereby would combine all three properties in one gene: tumor specificity, a selection opportunity of cells expressing the transgene at high levels, as well as an in vivo depletion option, which allows for the elimination of the engineered immune cells in case of toxicities caused by the introduced receptor. Within this context we have explored a strategy based on the recent development of purified T cells engineered to express a defined γδ T cell receptor (TEGs).,The US Food and Drug Administration (FDA) approval of the first engineered T cells expressing chimeric antigen receptors (CARs) has paved the way for new cellular interventions in the clinic.157–165in vivo.The good manufacturing practice (GMP)-grade anti-human αβTCR monoclonal antibody clone BW242/412 (from now on referred to as anti-human αβTCR) recognizes a common determinant of the human TCRα/β-CD3 complex, which has not yet been characterized. In order to allow for further epitope mapping of the interface between the anti-human αβTCR clone BW242/412 and a human αβTCR, we first tested the antibody’s ability to bind to murine αβTCRs. Therefore, Jurma T cells, a TCR-deficient T cell line, were transduced with human αβTCRs directed against the cancer/testis antigen NY-ESO-1Analysis of the sequence of domain 3 of the TCRβ chain constant domain revealed 11 residues that are non-homologous between murine and human species . To dete,,157–165 HLA∗02:01 pentamer+ cells before and after depletion, showing a significant increase in pentamer+ cells after depletion γ release was associated with positivity for the different introduced TCRs  by exchanging the hamster immunoglobulin (Ig)G2 constant domain for the human IgG1 constant domain (referred to as chimeric anti-MuTCRβ). We tested binding of this newly constructed antibody in engineered Jurkat-76 cells, which resulted in specific antibody binding to the 9/11 murinized TCRβ chain expressed on Jurkat-76 cells , a cell cycle inhibitor, using the protease cleavable linker VC-PABin vitro, most likely due to the weaker binding of the engineered antibody to the 11/11 αβTCR , and the T2 cell line (ATCC CRL-1992) were cultured in RPMI 1640 + GlutaMAX  containing 1% Pen/Strep and 10% FCS. Cell lines were authenticated by short tandem repeat profiling/karyotyping/isoenzyme analysis. All cells were passaged for a maximum of 2 months, after which new seed stocks were thawed for experimental use. In addition, all cell lines were routinely verified by growth rate, morphology, and/or flow cytometry and tested negative for mycoplasma using a MycoAlert mycoplasma kit . PBMCs were obtained from Sanquin Blood Bank  and isolated by Ficoll-Paque  from buffy coats. PBMCs were cultured using the previously described rapid expansion protocol (REP)Phoenix-Ampho cells (CRL-3213) were obtained from ATCC and cultured in DMEM  containing 1% penicillin/streptomycin (Pen/Strep)  and 10% fetal calf serum (FCS) . The TCRβ157–165/HLA∗02-specific TCR, respectively named M2.2.3 and M1.KA,4.1, were generated as previously described.http://www.imgt.org). Sequences were codon optimized and ordered in an industrial resistance, gene-harboring vector or as DNA strings . DNA strings were processed using the TA TOPO cloning kit  and cloned into the pCR2.1-TOPO vector, according to the manufacturer’s protocol. All TCR chains were cloned separately into the retroviral vector pMP71 between the EcoRI and NotI restriction sites using the indicated restriction enzymes and T4 DNA ligase . Transformation of ligated constructs was performed in JM109 competent E. coli , and subsequent plasmid DNA isolation was conducted using NucleoBond PC500, according to the manufacturer’s protocol .The minimally murinized Vα16.1 and Vβ4.1 chains from an NY-ESO1157–165/HLA-A∗02-specific TCR , and Jurma or Jurkat-76 cells were transduced twice within 48 h with viral supernatant in six-well plates (4 × 106 cells/well) in the presence of 50 IU/ml IL-2 (PBMCs only), 1.7 × 103 IU/ml IL-7, 1.5 × 102 IU/ml IL-15, and CD3/CD28 Dynabeads at a 1:5 bead-to-T cell ratio (CD4/CD8-selected T cells only), and 6 μg/mL Polybrene  . After transduction, primary T cells were expanded by the addition of 50 μL/well anti-CD3/CD28 Dynabeads  and 50 IU/ml IL-2 or 1.7 × 103 IU/mL IL-7 or 1.5 × 102 IU/mL IL-15.Phoenix-Ampho packaging cells were transfected using FuGENE-HD  with env (pCOLT-GALV), gagpol (pHIT60), and separate pMP71 constructs containing α or β chains from a NY-ESO1+ T cells were depleted by MACS cell separation according to the manufacturer’s protocol .Transduced primary T cells were incubated with biotin-labeled anti-human αβTCR antibody , followed by incubation with an anti-biotin antibody coupled to magnetic beads .4ZDH). The structure of the murinized constant domains when binding H57-597 was modeled on the template of the β chain of the murine N15 T cell receptor (PDB entry code: 1NFD).https://pymol.org/2/).The structure of different murinized constant domains was predicted using SWISS-MODEL,U47294; Promega, Leiden, the Netherlands) using 293fectin transfection reagent  following the manufacturer’s instructions. Antibody-containing supernatant was harvested 4 days after transfection and purified by affinity chromatography using HiTrap protein G HP antibody purification columns .Hamster-human (IgG1) chimeric H57-597 antibody was generated using Lonza expression vectors .DNA sequences of cloning intermediates and final constructs in pMP71 were verified by barcode sequencing . 75 μg of plasmid DNA and 25 pmol of primer specific for the pCR2.1-TOPO vector or pMP71 vector were premixed in a total of 20 μL and sent to BaseClear for Sanger sequencing.157–165 HLA∗02:01 (SLLMWITQV) pentamer . Samples were fixed using 1% paraformaldehyde (PFA) in PBS, measured on a FACSCanto II flow cytometer , and analyzed using FACSDiva  or FlowJo  software.Cells were stained with Vβ4-fluorescein isothiocyanate (FITC) , αβTCR-phycoerythrin (PE) , CD3-Pacific Blue (PB) , CD4-PeCy7 , CD8-allophycocyanin (APC) , CD8-PB , or RPE-conjugated NY-ESO-1Effector and target cells  were incubated for 16 h, after which supernatant was harvested. IFNγ ELISA was performed using an ELISA-ready-go! kit  following the manufacturer’s instructions.Chimeric H57-MC-VC-PAB-MMAE was constructed using a kit from CellMosaic  following the manufacturer’s instructions.Statistical analyses were performed using GraphPad Prism 8.3.0 for Windows . Differences between groups was calculated using a one- or two-tailed paired t test  and 5 or


Reduced 3,4′-bipyrazole-2-carbothioamides are formed in cycloaddition reactions between chalcones and thiosemicarbazide, and these can undergo further cycloaddition reactions to form oxothaazole of thiazole substituents. Structure analysis establishes the regiochemistry of the cycloaddition reactions and shows the very simple patterns of supramolecular assembly in these compounds. H-pyrazol-4-yl)-1-(thiophen-2-yl)prop-2-en-1-ones and thiosemicarbazide leads to the formation of reduced 3,4′-bipyrazole-2-carbothioamides. Further cycloaddition of these intermediates with either diethyl acetylenedicarboxylate or 4-bromophenacyl bromide leads to reduced 3,4′-bipyrazoles carrying oxothiazole or thiazole substituents, respectively. The structures of two representative intermediates and two representative products established unambiguously the regiochemistry of the cycloaddition reactions. The molecules of 3′-methyl-5′-(2-methylphenoxy)-1′-phenyl-5-(thiophen-2-yl)-3,4-dihydro-1′H,2H-3,4′-bipyrazole-2-carbothioamide, C25H23N5OS2 (Ia), are linked by N—H⋯N hydrogen bonds to form simple C(8) chains. The analogous compound 5′--3′-methyl-1′-phenyl-5-(thiophen-2-yl)-3,4-dihydro-1′H,2H-3,4′-bipyrazole-2-carbothioamide hemihydrate crystallizes as a hemihydrate, C24H19Cl2N5OS2·0.5H2O (Ib), and the independent components are linked into a chain of spiro-fused R44(20) rings by a combination of O—H⋯N and N—H⋯O hydrogen bonds. In the structure of ethyl (Z)-2-{2--4-oxo-4,5-dihydrothiazol-5-ylidene}acetate, C31H27N5O4S2 (II), inversion-related pairs of molecules are linked by paired C—H⋯π(arene) hydrogen bonds to form cyclic centrosymmetric dimers, but there are no direction-specific intermolecular interactions in 4-(4-bromophenyl)-2--4-thiazole, C32H22BrCl2N5OS2 (III). Comparisons are made with the structures of some related compounds.Cycloaddition reactions between 3-(5-aryloxy-3-methyl-1-phenyl-1 Within er Fig. 7.D—H⋯A angle, and so is unlikely to be structurally significant H-pyrazole-4-carbaldehydes have been reported  and 0.157 (6) and involving some unexpected geometrical features, although all other atoms are reported as being fully ordered. Hence this structure is unlikely to be entirely correct.Structures have been reported recently for a number of compounds related to those reported here, including precursors and intermediates in the synthetic pathways to compounds (I)–(III). The structures of five examples of 5-aryloxy-3-methyl-1-phenyl-1et al., 2019N,N-dimethylformamide .Samples of compounds (Ia)i.e. for (Ia)3), 0.97 Å (CH2) or 0.98 Å , and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms. For the H atoms bonded to N or O atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O), giving the N—H and O—H distances shown in Table 1Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021002310/dx2035sup1.cifCrystal structure: contains datablock(s) global, Ia, Ib, II, III. DOI: 10.1107/S2056989021002310/dx2035Iasup2.hklStructure factors: contains datablock(s) Ia. DOI: Click here for additional data file.10.1107/S2056989021002310/dx2035Iasup6.cmlSupporting information file. DOI: 10.1107/S2056989021002310/dx2035Ibsup3.hklStructure factors: contains datablock(s) Ib. DOI: Click here for additional data file.10.1107/S2056989021002310/dx2035Ibsup7.cmlSupporting information file. DOI: 10.1107/S2056989021002310/dx2035IIsup4.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989021002310/dx2035IIsup8.cmlSupporting information file. DOI: 10.1107/S2056989021002310/dx2035IIIsup5.hklStructure factors: contains datablock(s) III. DOI: 2065478, 2065477, 2065476, 2065475CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A two-step synthesis of 3,6-dihydroxypicolinic acid, its crystal structure and that of a labile intermediate, are described. 6H5NO4 (II), an intermediate in the metabolism of picolinic acid, is described. The crystal structure of II, along with that of a labile intermediate, dipotassium 3-hydroxy-6-(sulfonatooxy)pyridine-2-carboxylate monohydrate, 2K+·C6H3NO7S2−·H2O (I), is also described. Compound I comprises a pyridine ring with carboxylate, hydroxyl (connected by an intramolecular O—H⋯O hydrogen bond), and sulfate groups at the 2-, 3-, and 6-positions, respectively, along with two potassium cations for charge balance and one water molecule of crystallization. These components are connected into a three-dimensional network by O—H⋯O hydrogen bonds arising from the water molecule, C—H⋯O interactions and π–π stacking of pyridine rings. In II, the ring nitrogen atom is protonated, with charge balance provided by the carboxylate group . The intramolecular O—H⋯O hydrogen bond observed in I is preserved in II. Crystals of II have unusual space-group symmetry of type Abm2 in which extended planar networks of O—H⋯O and N—H⋯O hydrogen-bonded molecules form sheets lying parallel to the ac plane, constrained to b = 0.25 (and 0.75). The structure was refined as a 50:50 inversion twin. A minor disorder component was modeled by reflection of the major component across a mirror plane perpendicular to c.A simplified two-step synthesis of 3,6-dihydroxypicolinic acid , C We considered two routes, as shown in Fig. 1We report here a two-step synthesis also starting with 3-hydroxypicolinic acid by an Elbs oxidation . Jerfy & Roy  gives complete hydrolysis in a few minutes. The isomeric 6-oxo-picolinic acid-3-sulfate is much more stable although subject to anchimeric assistance  and 1.2758 (16) Å for C7—O3 and C7—O4, respectively. The longer of these is part of an S(6) intramolecular hydrogen-bonded ring with the hydroxyl group. In the sulfate group, the oxygen atom bound at the 6-position [C6—O2 = 1.3968 (14) Å] is longer [S1—O2 = 1.6343 (9) Å] than the other three S=O bonds [range 1.4361 (9) to 1.4486 (9) Å], as would be expected for this bonding arrangement.The asymmetric unit in I Fig. 3 containsII  hydrogen-bonded ring from I is preserved in II. The 6-position of the ring is occupied by a second hydroxyl group. As discussed in more detail in section 6 (Refinement), there is a small minor disorder component [refined occupancy 4.7 (3)%] and probable inversion twinning.In II Fig. 4, the nitI, strong Ow—H⋯O (w = water) hydrogen bonds exist in which the water molecule acts as a linker between c-glide-related anions. The water oxygen is also coordinated to the K+ cations, both of which are seven coordinate. Around K1, coordination distances range from 2.7376 (9)–2.9102 (10) Å (for K⋯O) and 2.7869 (11) Å (K1⋯N). For K2, coordination distances range from 2.6769 (9) to 2.9525 (10) Å . The coordination geometry about each K+ cation is very roughly pentagonal bipyramidal, with K1 much more distorted than K2. As each K+ cation is coordinated to the water molecule, the KO6N and KO7 polyhedra augment the extended chains that propagate parallel to c ° with a centroid–centroid distance of 3.698 (1) Å. These columns of hydrogen-bonded and π-stacked molecules are interlinked by the aforementioned chains of K+ cations and water molecules into a three-dimensional network  as a consequence of the unusual space-group symmetry of type Abm2. Strong O2—H2O⋯O3i and N1—H1N⋯O4i hydrogen bonds link pairs of 21 screw-related molecules into c. Weaker C4—H4⋯O2iii and C5—H5⋯O1ii  at the 3-position.A search of the Cambridge Structure Database 3-Hydroxypyridine-2-carboxylic acid-6-sulfate dipotassium salt monohydrate  was dissolved in 10 ml of water and cooled. 3-Hydroxypicolinic acid  followed by potassium peroxydisulfate  were added. The reaction mixture was stirred at RT for 24 h. The precipitate was filtered and dried by washing with acetone, yielding 0.46–0.49 g (∼44%) of the dipotassium salt of 3-hydroxypyridine-2-carboxylic acid-6-sulfate monohydrate (I). Compound I migrates on paper electrophoresis at pH 7.5 with pR = 2 as a fluorescent spot. pR is the migration distance relative to picric acid at pR = 1 . Crystals of I grow from aqueous solution when treated as follows: 0.16 g were suspended in 2.0 ml of water, dissolved by heating carefully to about 313 K, and then cooled slowly to RT. Further cooling to 278 K overnight gave 0.12 g of needles. Analysis (%) calculated for C6H5NO8K2S: C, 2l.88; H, 1.53; N, 4.25. Found: C, 21.97; H, 1.44; N, 4.25. IR(Nujol): 3485, 3310, 3096, 1701, 1684, 1624, 1574, 1250, 1059, 957, 860, 833, 768, 716, 638 cm−1. NMR δ 7.27 , 7.45 . UV (in water): λmax, (nm), ɛ (l mol−1 cm−1): 307, 1120; 230, 1200; 205, 4180. Heating behavior: I begins to discolor at about 448 K and then gets darker without melting up to 523 K.II)3,6-Dihydroxypicolinic acid , was suspended in 2 ml of water and then heated to about 313 K to dissolve it. HCl was then added to about pH 2. A heavy precipitate formed immediately. After cooling, the colorless solid was filtered and washed with cold water to yield 50–60 mg of the product (yield 67–80%), pR = 1. The proton NMR spectrum of II agreed with that reported by Qiu et al.  and 7.45 . These are shifted because of the solvent difference from Qiu et al. : calculated for C6H5NO4: C, 46.46; H, 3.25; N, 9.03. Found , C, 46.22; H, 3.20; N, 9.07. (precipitate), C, 45.65; H, 3.16; N, 9.10. IR: Nujol, 3120, 1614, 1540, 1360, 1269, 1100, 845, 810, 760, 621 cm−1. Later, larger crystals were obtained using 88% formic acid as solvent. Upon heating, II carbonizes above 473 K without melting. UV (in water): λmax, (nm), ɛ (l mol−1 cm−1) at pH 7: 346, 5970; 243, 4350; 221.5, 9450. Shukla & Kaul ; Flack & Bernardinelli, 1999et al., 2008et al., 2013y = 0.63 (7); z = 0.62 (10), respectively], however, each gave a much stronger suggestion of twinning by inversion. Since the space group itself has a twofold parallel to its c axis, this results in a mirror operation (i.e. a twofold rotation combined with inversion). Thus, the twinning and disorder in II may effectively be treated by the same operation, not unlike that in uric acid dihydrate  were used to equalize displacement parameters of superimposed groups. Full occupancy (and major component for II) hydrogen atoms were found in difference-Fourier maps. Carbon-bound hydrogen atoms were included using riding models with constrained distances set to 0.95 Å (Csp2H). In I, the water H atoms were refined but subject to distance and angle–distance restraints, while the hydroxyl H atom was refined freely. In II, for O—H and N—H groups, riding models that allowed the bond distance to refine were used. Uiso(H) parameters were set to values of either 1.2Ueq or 1.5Ueq (OH only) of the attached atom. The structures were validated using PLATON and checkCIF  I, II, global. DOI: 10.1107/S2056989021004904/hb7975Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989021004904/hb7975IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989021004904/hb7975Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021004904/hb7975IIsup5.cmlSupporting information file. DOI: 2082685, 2082684CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Myocardial ischemia/reperfusion (I/R) injury is a serious issue during the therapy of myocardial infarction. Herein, we explored the beneficial influence of Epigallocatechin-3-gallate (EGCG) on hypoxia/reoxygenation (H/R)-stimulated cardiomyocyte H9c2 cells damage, along with possible internal molecular mechanism related autophagy related 4C (ATG4C). H9c2 cells were subjected to H/R stimulation and/or EGCG treatment. ATG4C mRNA expression was measured via q-PCR assay. ATG4C overexpression plasmid (OE-ATG4C) was transfected to arise ATG4C level. Cell viability, apoptosis, reactive oxygen species (ROS) production, ATP level were tested via CCK-8 assay, Annexin V-FITC/PI staining, DCFH-DA staining and ATP Assay Kit, respectively. Western blotting was performed to test Cleaved-caspase 3, Cleaved-caspase 9, cytochrome C, and LC3B protein levels. H/R stimulation resulted in H9c2 cell viability loss, promoted cell apoptosis, and ROS overproduction, as well as lowered ATP level in cells. EGCG treatment alleviated H/R-resulted H9c2 cell viability loss, cell apoptosis, ROS overproduction, and reduction of ATP level. Moreover, H/R stimulation reduced the ATG4C expression in H9c2 cells, while EGCG raised the ATG4C expression. Overexpression of ATG4C strengthened the beneficial influence of EGCG on H/R-stimulated H9c2 cell viability, apoptosis and ROS production. Besides, ATG4C overexpression weakened the H/R-stimulated H9c2 cell autophagy via reducing LC3B II/I expression. EGCG exerted beneficial influence on H/R-stimulated cardiomyocytes, which protected cardiomyocytes from H/R-stimulated viability loss, apoptosis, and ROS overproduction via enhancing ATG4C expression. As is known to all of us, adequate blood supply is essential for the heart to perform normal physiological functions . When thet al. [et al. [in vitro and in vivo. More studies are still demanded to further exploring the myocardial beneficial activity of EGCG subjecting to I/R stimulation, which will provide experimental basis for EGCG as a new drug for coronary artery disorder therapy.Green tea is a popular beverage. In recent years, the negative correlation between green tea drinking and the incidence of cardiovascular diseases has been widely reported ,8. Epigaet al.  discover [et al.  revealedin vitro can simulate the damage of cardiomyocytes caused by I/R in vivo [H9c2 cell line is a subclone of the cloned cell line of BD1X rat embryonic heart tissue, which is often used to investigate cardiomyocyte damage after I/R or other stimulation ,20. Prev in vivo . We propH9c2 cells were provided by Stem Cell Bank, Chinese Academy of Science , and growth in DMEM  replenishing with 10% (v/v) fetal bovine serum  and 1% (v/v) Penicillin-Streptomycin solution .2 and 95% N2 for 6 h and then grown in a normal chamber with 95% air and 5% CO2 for 12 h.To simulate cardiomyocytes’ damage after I/R stimulation, H9c2 cells were grown in an anoxic chamber with 5% COEGCG  was dissolved in distilled water to 100 mg/L and saved at −4°C. Before experiments, phosphate buffer saline (PBS) was used to dilute EGCG solution to 2, 4, 8, 16, 32, or 64 mg/L. For H9c2 cells both stimulated by H/R and treated by EGCG, EGCG was pre-added into the culture medium for 24 h, after that, the culture medium was changed and cells were received H/R stimulation.TM 2000 Reagent (Invitrogen) [A full-length sequence of ATG4C was inserted into pcDNA3.0 plasmid  to construct OE-ATG4C, which was transfected in H9c2 cells via Lipofectamineitrogen) . After 4TM qPCR RT Kit . Real-time PCR was carried out using BestarTM qPCR MasterMix (DBI Bioscience) with reaction condition: 2 min at 95°C, 40 cycle of 20 s at 94°C, 20 s at 58°C, and 20 s at 72°C. GAPDH expression was served as an internal control. The primer sequences were as follows: 5ʹ-ACCCCAACAATTTCTCTGAAGG-3ʹ (ATG4C-F), 5ʹ-GTCCATACCAATCTCCTGCTTTT-3ʹ (ATG4C-R), 5ʹ-TGTTCGTCATGGGTGTGAAC-3ʹ (GAPDH-F), and 5ʹ-ATGGCATGGACTGTGGTCAT-3ʹ (GAPDH-R). Data were analyzed by the 2−ΔΔCt method.qRT-PCR assay was conducted similarly as stated in the earlier literature . Total R4 cells/well for 24 or 48 h. Then, 10 μL CCK-8 solution was replenished in each well for 2 h. Subsequently, the absorbance value at 450 nm (OD450 nm) of each well was measured through a Micro-plate reader .After relevant stimulation and/or treatment, the viability of H9c2 cells was tested via cell counting Kit-8 (CCK-8) assay  . H9c2 ce4 H9c2 cells were cultivated in a 24-well plate for 24 h. Then, cells in each group were gathered, rinsed using PBS, and dyed with 5 μL Annexin V-FITC and 10 μL PI for 15 min protected from light. The apoptosis rate was measured via a flow cytometer .After relevant stimulation and/or treatment, cell apoptosis was tested via Annexin V-FITC/PI Apoptosis Detection Kit  . A total4 cells H9c2 cells were cultivated in a 24-well plate for 6 h. Then, cells in each group were collected, rinsed using PBS and stained with 10 μM DCFH-DA (Sigma-Aldrich) for 20 min protected from light. The ROS generation was detected via a flow cytometer.2ʹ,7ʹ-dichlorofluorescein-diacetate (DCFH-DA) is a fluorescent probe that can transform non-fluorescent DCFH in cells . ROS can4 H9c2 cells were cultivated in a 24-well plate for 24 h. Then, cells were collected, mixed with cell lysis buffer for 10 min and centrifuged at 12,000 g at 4°C for 5 min. The ATP concentration in supernatant was tested through incubating with 100 μL kit solution and recorded using Luminometer .After relevant stimulation and/or treatment, the ATP concentration in H9c2 cells was tested via ATP Assay Kit (Beyotime Biotechnology) . A totalTotal proteins were detached using RIPA Lysis Buffer (Beyotime Biotechnology). Western blotting was performed similarly as earlier literature reported . PrimaryP values with a significance level of P < 0.05.GraphPad Prism 9.0 software was conducted for statistical analysis. Data were displayed as mean ± standard deviation (SD) from three repeated experiments. One-way ANOVA was performed for calculating P < 0.01), while 4, 8, 16, 32, or 64 mg/L EGCG treatment notably raised the H9c2 cell viability (P < 0.05 or P < 0.01). P < 0.01). Relative to H/R group, the Cleaved-caspase 3 expressions were remarkably reduced in H/R + 2, 4, 8, 16, 32, or 64 mg/L EGCG group (P < 0.01). 8 mg/L EGCG was selected for follow-up experiments. Moreover, P < 0.01), while 8 mg/L EGCG treatment alleviated the reduction of H9c2 cell viability in both 24 and 48 h (P < 0.01). P < 0.01), but 8 mg/L EGCG treatment weakened the H/R-resulted H9c2 cell apoptosis (P < 0.01). These outcomes represented suggested that EGCG suppressed H/R-resulted H9c2 cell viability reduction and apoptosis.Firstly, whether EGCG could exert protective effect on H/R-stimulated H9c2 cells was explored. Following H/R stimulation and 2, 4, 8, 16, 32, or 64 mg/L EGCG treatment, H9c2 cell viability was tested. P < 0.01). Compared to H/R group, the ROS generation was decreased in H/R + 8 mg/L EGCG group (P < 0.01). Moreover, P < 0.01), while 8 mg/L EGCG treatment notably attenuated the H/R-resulted reduction of ATP concentration (P < 0.01). Besides, H/R stimulation elevated the cleaved-Caspase 3, cleaved-Caspase 9 and cytochrome C expressions in H9c2 cells ( P< 0.01). Relative to H/R group, the cleaved-Caspase 3, cleaved-Caspase 9, and cytochrome C expressions were lowered in H/R + 8 mg/L EGCG group (P < 0.01). These above outcomes represented that EGCG lessened H/R-resulted ROS overproduction, ATP loss, and mitochondrial-dependent apoptosis.Then, the influences of H/R stimulation and EGCG treatment on ROS production and ATP concentrations in H9c2 cells were detected.  P< 0.01). However, 8 mg/L EGCG treatment notably attenuated the H/R-resulted decrease of ATG4C mRNA expression (P < 0.01). To overexpress ATG4C in H9c2 cells, OE-ATG4C was transfected in H9c2 cells. P < 0.01). These outcomes implied that EGCG reversed the H/R-resulted H9c2 cell damage possible via raising ATG4C expression.Previous literature reported that ATG4C expression was down-regulated in myocardial tissue of mice with myocardial I/R damage , which iP < 0.01). Relative to H/R+ OE-ATG4C group, the cell viability was hoisted in H/R+ OE-ATG4C+EGCG group (P < 0.01). Moreover, P < 0.01). 8 mg/L EGCG treatment further reduced the H/R-resulted H9c2 cell apoptosis and ROS production, as evidenced by the reductions of apoptotic rate (%) and ROS generation (%) in H/R+ OE-ATG4C+EGCG group. P < 0.01). Relative to H/R+ OE-ATG4C group, the expressions of these three proteins were lowered in H/R+ OE-ATG4C+EGCG group (P < 0.01). Besides, the LC3B II/I protein expression in H9c2 cells was notably increased after H/R stimulation (P < 0.01). OE-ATG4C transfection reduced the H/R-resulted increase of LC3B II/I protein expression in H9c2 cells (P < 0.01). Relative to H/R+ OE-ATG4C group, the LC3B II/I protein expression was further lowered in H/R+ OE-ATG4C+EGCG group (P < 0.01). These outcomes represented that ATG4C participated in the modulation of H9c2 cell autophagy, which overexpression strengthened the beneficial activity of EGCG on H/R-treated H9c2 cells.Finally, whether ATG4C took part in the influence of EGCG on H/R-stimulated H9c2 cell viability, ROS production and mitochondrial-dependent apoptosis were explored. I/R damage is a phenomenon in which myocardial tissue damage is intensified after the blood reperfusion to the ischemia myocardial tissue . This haThe equilibrium among ROS production and consumption is important to maintain normal cell physiological activities . There iet al. [Any activity of the cells requires the support of energy. As the universal ‘currency’ for energy transfer in cells, the stability of ATP levels is also crucial for cardiomyocytes . Earlieret al. , discoveet al., demonstrated that ATG4C expression was reduced in mice with myocardial I/R injury [et al. [et al. [ATG4C, a member of autophagy-related gene family, is a cysteine peptidase that take part in the modulation of LC3 lipidation and de-lipidation, as well as C-terminal peptide cleavage of ATG8, in the cell autophagy process ,45. Sun R injury . What isR injury . For exa [et al. , reveale [et al. , reporte [et al. . Some li [et al. ,51. Some [et al. ,53. In tTaken together, this research confirmed the beneficial activity of EGCG on H/R-stimulated cardiomyocytes. EGCG declined H/R-resulted cardiomyocytes viability loss, apoptosis, and ROS over-production via enhancing ATG4C expression. Considering that ATG4C is a key protein involving in cell autophagy, further investigations concerning the modulatory influence of EGCG on cardiomyocytes autophagy during H/R stimulation are still needed in the future.


IIbβ3 is foundational and paramount to our understanding of integrin structure and activation. αIIbβ3 is essential for the aggregation and adhesion function of platelets in hemostasis and thrombosis. Structural and mutagenesis studies have previously revealed the critical role of αIIbβ3 transmembrane (TM) association in maintaining the inactive state. Gain-of-function TM mutations were identified and shown to destabilize the TM association leading to integrin activation. Studies using isolated TM peptides have suggested an altered membrane embedding of the β3 TM α-helix coupled with αIIbβ3 activation. However, controversies remain as to whether and how the TM α-helices change their topologies in the context of full-length integrin in native cell membrane. In this study, we utilized proline scanning mutagenesis and cysteine scanning accessibility assays to analyze the structure and function correlation of the αIIbβ3 TM domain. Our identification of loss-of-function proline mutations in the TM domain suggests the requirement of a continuous TM α-helical structure in transmitting activation signals bidirectionally across the cell membrane, characterized by the inside-out activation for ligand binding and the outside-in signaling for cell spreading. Similar results were found for αLβ2 and α5β1 TM domains, suggesting a generalizable mechanism. We also detected a topology change of β3 TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of αIIb association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.Studying the tight activity regulation of platelet-specific integrin α Integrins are a large family of cell surface receptors composed of α and β subunits. The combination of 18 α and eight β subunits in human forms 24 integrin heterodimers that interact with multiple extracellular or cell surface ligands and play diverse functions in different cell types . A uniquIIbβ3 . A conserved Gly residue (G708 in β3) in β integrins and two conserved small amino acids  that form the GXXXG motif in α integrins (G972XXXG975 in αIIb) were identified to be critical in maintaining the resting state of αIIbβ3 integrin (A) (A). Structure studies of αIIbβ3 TM-CT heterodimer show a cross-angled straight α-helical structure for both αIIb and β3 (3 subunit (B). A continued backbone hydrogen bonding network maintains the integrity of α-helical structure (B), which may be critical for the transmission of conformational signals across the cell membrane. To test this hypothesis, we performed proline scanning mutagenesis for αIIbβ3 TM domain (C and D), given that when present in an α-helical structure, proline tends to disturb the α-helical conformation by introducing a break or kink due to the lack of backbone hydrogen bonding (E). Among the 14 proline mutations tested for β3 TM domain, most of them showed similar level of ligand-binding activity when measured with ligand-mimetic mAb PAC-1 under the resting condition . Several proline mutations, including β3-V696P and β3-L713P, and the previously identified β3-G708P and β3-K716P . Among the ten proline mutations tested for αIIb TM domain, seven of them showed a similar level of ligand binding as wild type under the resting condition  and three of them significantly rendered αIIbβ3 constitutively active . All the active proline mutations, such as the β3-L706P and -G708P and the αIIb-G972P and -G976P, are located at or close to the αIIbβ3 TM heterodimer interface, which may disturb the αIIbβ3 TM interface leading to integrin activation.Sequence alignment of the TM domains of 18 α and eight β human integrins shows typical TM features with hydrophobic residues that are rich in Leu, Ile, and Val A. A consintegrin A 21). O. OA). A grin (A) , 15, a cgrin (A) , and a cgrin (A) , 24  A. Structb and β3 , 11, 13. subunit B. A conttructure B, which M domain , C and D bonding  Fig. 1EEA. A conIIbβ3 under resting condition, we next tested the effect of these proline mutations on the inside-out activation of αIIbβ3. Several activating mutations such as αIIb-R995D, αIIb-F993A, αIIb-F992A-F993A, and β3-K716A (or K716C), which are known to disturb the cytoplasmic domain association, have been widely used to mimic the inside-out activation of αIIbβ3. When coexpressed with αIIb-R995D, four of the β3 TM proline mutations, including L698P, L705P, L707P, and A711P, significantly reversed the activating effect of αIIb-R995D (A), while the rest of proline mutations either had no effect or further enhanced the activation by αIIb-R995D (A). Similarly, when coexpressed with αIIb-F993A that is more activating than αIIb-R995D, the β3 L698P, L705P, I707P, and A711P significantly reversed the activating effect of αIIb-F993A (B). We next focused on β3 L705P and A711P for further analysis since their deactivating effect is more dramatic than others (A and B). To test if the proline mutation also reverses the activating effect of β3 mutation, we generated L705P and A711P mutations in the context of β3-K716C. Both proline mutations significantly reduced the activation of αIIbβ3 induced by β3-K716C (C). To test if the β3 L705P and A711P mutations have a synergistic effect, we coexpressed the single or double proline mutations with the highly active αIIb-F992A-F993A mutant. Both β3-L705P and β3-A711P reduced the activation by αIIb-F992A-F993A, while the β3-L705P-A711P double mutation further decreased the activation (C), demonstrating a synergistic effect. All the above activating mutations are expected to destabilize the αIIbβ3 cytoplasmic interaction, which also disturbs TM association. We then tested a TM interface disturbing mutation αIIb-G976L. The β3-L705P-A711P also significantly reduced αIIb-G976L-mediated αIIbβ3 activation (C). In contrast to β3 proline mutations, when coexpressed with the activating β3-K716A mutant, almost all the αIIb proline mutations further enhanced αIIbβ3 activation, except αIIb-I982P that reduced the activation (D), indicating that the αIIb TM domain tolerates proline mutation less than β3 TM domain.Having identified some TM proline mutations that reduced the basal level ligand binding of αIb-R995D A, while Ib-R995D A. SimilaIb-F993A B. We nexn others , A and Bβ3-K716C C. To testivation C, demonstivation C. In contivation D, indicaIIbβ3 activation in HEK293FT cells (IIbβ3 activation reported by PAC-1 binding (A). In contrast, the β3-L705P, β3-A711P, and β3-L705P-A711P mutations all completely abolished EGFP-TH-induced αIIbβ3 activation (A). The αIIb-I982P mutation also significantly reduced EGFP-TH-induced αIIbβ3 activation, while αIIb-L974P by contrast further enhanced αIIbβ3 inside-out activation (A). The expression of EGFP-TH was comparable among all the transfections as determined by flow cytometry. These data are consistent with the above results obtained using the activating mutations that mimic αIIbβ3 inside-out activation. Talin1 head induces αIIbβ3 activation through binding to β3 cytoplasmic tail (CT) and disturbing α-β CT as well as TM associations (3 TM extends to the cytoplasmic region (B), we asked if disturbing the integration of cytoplasmic α-helix also affects αIIbβ3 activation. Instead of using proline mutagenesis, we inserted a flexible loop composed of GS or GGGS into the MP region of β3 CT (B). The insertion site is right before the conserved D723, given that the K716-H722 sequence is the minimal requirement for maintaining the resting state . The longer GGGS insertion had more attenuated effect than the shorter GS insertion on αIIbβ3 activation (C). The αIIb-R995A mutation was used to synergistically enhance the activation by EGFP-TH (3 CT as shown by the EGFP-TH pull-down assay (D). As a control, truncation of β3 CT after H722 (β3-H722Tr) completely abolished EGFP-TH binding (D). Altogether, these data suggest that the integrity of the α-helical structure at both the TM and CT domains of β3 is required for the inside-out activation of αIIbβ3.Overexpression of the talin1 head domain is a well-established method to induce integrin inside-out activation . We usedFT cells . Compare binding A. In contivation A. The αItivation A. The exc region B, we askof β3 CT B. The inng state , and theng state . Comparetivation C. The lotivation C. The αI EGFP-TH . The GS wn assay D. As a c binding D. Altoge2 integrin to ask if certain TM proline mutations also affect the inside-out activation of αLβ2 integrin. We screened 13 proline mutations for the C-terminal half of the β2 TM domain, which includes the residues β2-V691 and -L697 that are equivalent to β3-L705 and -A711, respectively (A and 4A). Using the αL-GFFKR to GAAKR mutation to induce inside-out αLβ2 activation reported by soluble ICAM-1 binding, we found that six β2 TM proline mutations reduced αLβ2 activation (A). Most of the inhibitory proline mutations of β2 are equivalent to those found in β3 integrin, such as β2-I690P, -V691P, and -I693P that are equivalent to β3-I704P, -L705P, and -I707P, respectively (A). Similar to β3-L705P, the equivalent β2-V691P has much profound negative effect. However, the β3-A711P equivalent mutation, β2-L697P, only slightly reduced αLβ2 activation (A). To correlate the effect of proline mutations on ligand binding with large-scale conformational changes of ectodomain, we used two mAbs KIM127 and m24 to report β2 extension and headpiece open, respectively (2-V691P and -I693P mutations significantly reduced the αL-GAAKR-induced binding of KIM127 and m24 (B and C), suggesting that the TM proline mutations restrained the conformational activation signal transmitted across the cell membrane. In contrast, Mn2+, an integrin activator for the extracellular domain, still stimulates KIM127 and m24 binding in the presence of the inhibitory proline mutations (B and C). We also tested the β3-L705P and -A711P equivalent mutations on β1 integrin, namely β1-V721P and -L727P. The fibronectin-binding assay showed that the β1-L727P mutation significantly reduced while the β1-V721P significantly enhanced EGFP-TH-induced α5β1 activation (D). These data suggest that the integrity of TM α-helical structure is also important for the inside-out activation of β1 and β2 integrins.We expanded our studies to βectively A and 4A.tivation A. Most oectively A. Similativation A. To corectively , 29. Con and m24 , B and Cutations , B and Ctivation D. These 3-L705P-A711P mutation greatly reduced the inside-out activation of αIIbβ3 integrin, we further asked whether such mutation also affects αIIbβ3 outside-in signaling, in which extracellular ligand binding induces large-scale conformational changes that are transmitted to the CT through the TM α-helix . By contrast, the HEK293-αIIb/β3-L705P-A711P cells had dramatic defects of cell spreading, showing much smaller spreading area than the WT cells (B and F), although there were no obvious differences in the number of attached cells. Interestingly, the HEK293-αIIb-I982P/β3 cells spread as well as the WT (C and F). Consistently, the HEK293-αIIb/β3-L705P-A711P cells also showed defective cell spreading on the physiological ligand fibrinogen (D–F). These data suggest that the integrity of the α-helical structure of β3 TM domain is required for the outside-in signaling.Integrin conformational signal is transmitted bidirectionally across the TM domain. Having found that the β α-helix . We genepreading A. By conWT cells , B and Fs the WT , C and Fbrinogen , D–F. ThE). To measure the potential conformational change of TM α-helix in the cell membrane, we performed the cysteine scanning accessibility assay (A), which will show the burial/exposure status of TM residues. By comparing the resting and active states, the changes of labeling accessibility of the substituted cysteines indicate the changes of membrane embedding of TM α-helix. We first tested whether ligand-induced large-scale conformational change at the ECD induces structural rearrangement of TM domains. The RGD-mimetic high-affinity drug eptifibatide was used to induce the headpiece opening and extension of αIIbβ3  was used to inhibit the potential palmitoylation of C-terminal cysteines (B–E). BM labeling of αIIbβ3 TM N-terminal cysteine mutations showed that the β3-I693C and αIIb-W697C are the last residues giving detectable BM signal (B and C), suggesting they are the N-terminal boundary residues of αIIbβ3 TM domains. BM labeling of β3 TM C-terminal cysteine mutations suggested that the C-terminal TM boundary of β3 is at the residues I721H722 (D). The BM labeling of αIIb TM C-terminus showed an interesting pattern, suggesting that the residues G991C, F992C, F993C, and R995C are all embedded in the cell membrane, while K994C and the residues after R995 are exposed (E). This data is consistent with the loop conformation of αIIb GFFKR motif as determined by structural studies (B and E). Compared with the results without eptifibatide, eptifibatide did not induce obvious changes in the BM labeling patterns for both αIIb and β3 subunits, suggesting no changes in membrane embedding (B–I).Having found that the rigid α-helical structure of TM domain is critical for the bidirectional integrin activation, we next asked if the α-helix performs conformational change within the cell membrane. Such conformational changes may be disrupted due to the helix-breaking effect of a proline mutation E. To meaty assay . The memty assay A, which f αIIbβ3 , 32. Botysteines . The sigysteines , B–E. BMM signal , B and CI721H722 D. The BM exposed E. This d studies , 13 . Ligand-mimetic PAC-1 binding showed that αIIb-L959C/β3-P688C mutations reversed the activating effect of the GFFKR cysteine mutations (B). However, there are no obvious changes in the BM labeling for the GFFKR motif in the presence of αIIb-L959C-β3-P688C disulfide bond (C and D), suggesting that the activating cysteine mutations do not change the membrane embedding of the αIIb GFFKR motif. Thus, the membrane embedding of GFFPR revealed by BM labeling should represent the resting state.Previous studies showed that the cysteine mutations of αy active , suggesty active . To reveparation . Westernparation A. Ligandutations B. Howeveide bond , C and D3 TM domain for the cells spreading on immobilized fibrinogen. Compared with the BM labeling of suspension cells, we consistently observed a decrease of BM labeling for the residues of β3-K689C, β3-P691C, β3-D692C, and β3-I693C that are at the β3 TM outer boundary (A and B), suggesting a change of membrane embedding of β3 TM α-helix in the spreading cells. Talin1 head has been reported to induce conformational change of β3 TM domain . However, compared with the cells with EGFP expression, no obvious changes of BM labeling were detected for the N-terminal MP residues of β3 TM α-helix (C and D). These data suggest that mechanical force but not talin1 head may induce a change of β3 membrane embedding.During integrin-mediated cell adhesion and spreading, in addition to the ligand-induced long-range conformational changes that are transmitted to the CT through TM domain, the tensile force generated by ectodomain interacting with immobilized extracellular ligand and CT interacting with intracellular cytoskeleton is also applied to TM domain , which mboundary , A and BM domain . We also EGFP-TH . EGFP-TH α-helix , C and D3 TM α-helix has been studied as an isolated single-chain peptide  tag, namely β3-tail-TMCT (A). The β3-tail-TMCT construct could be expressed on the cell surface without αIIb subunit as determined by flow cytometry (data not shown), suggesting that it may mimic a free state of β3 TM domain that is fully separated from αIIb subunit. Both the N- and C-terminal MP regions of β3 TM domain were comparably studied by BM labeling for β3-tail-TMCT and β3 full-length constructs (B–E). BM labeling of β3 full-length cysteine mutants coexpressed with αIIb subunit shows that the N-terminal boundary of β3 TM domain is at residue β3-I693 (B). In contrast, the elevated BM labeling of β3-tail-TMCT suggested the changes of TM boundary and membrane embedding (C and F). Consistently, compared with the β3 full-length, there was an obvious increase of BM labeling for the C-terminal residues, such as β3-R724C and β3-K725C in β3-tail-TMCT , suggesting a change of β3 membrane embedding. Our BM labeling data for the full-length β3 complexed with αIIb indicate that the β3 TM α-helix needs to be tilted in the lipid bilayer to accommodate membrane embedding (E). Upon disassociation from the αIIb TM domain, the β3 TM α-helix may adopt a different tilt within the cell membrane as shown by our BM labeling analysis.The current model suggests a separation of α and β TM domains upon integrin activation . The β3  peptide , 20, whiail-TMCT A. The β3nstructs , B–E. BM β3-I693 B. In conmbedding , C and Fmbedding E. Upon dIIbβ3 TM domains , middle (β3-L705P and -I707P), and C-terminus (β3-A711P) of β3 TM domain (A), which displayed different levels of inhibitory effect on αIIbβ3 inside-out activation (B). Similarly, the β3 equivalent TM proline mutations also inhibited the inside-out activation of αLβ2 and α5β1 integrins .The inhibitory βagenesis . StructuM domain , 36. The2A-F993A C.3 TM domain, as determined using the isolated β3 TM-CT fragment in model membranes . This is in general consistent with the model suggested based on structural studies of αIIbβ3 TM heterodimer by NMR in lipid bicelles . However, due to the activating effect of GFFKR cysteine substitutions, it was suggested by the previous study that the BM labeling pattern of the GFFKR motif reflects an active conformation .Our BM labeling results suggest the membrane-embedded portion for α studies , 13, whi studies E. Howeveormation . To comp bilayer E.IIbβ3 inside-out activation . The β3-K716 side chain hydrogen bonding with the backbone oxygens of αIIb GFFKR motif (IIb GXXXG motif favors the tilt topology of β3 TM domain (E). The activation inhibitory proline mutations may further stabilize the β3 TM tilt and αIIbβ3 TM association. A perpendicular and rigid α-helical structure of αIIb TM domain is critical in maintaining the resting state of αIIbβ3 since our mutagenesis study shows that αIIb is much less tolerant to TM proline mutations than β3.The membrane tilt of β3 TM domain has been suggested to play a role in αtivation , 35. Howtivation . NMR stuion pair . HoweverMR study , questioIb GFFKR E. The β3KR motif , along wM domain E. The acIIbβ3 activation involves a disruption of α-β TM association, which may alter the TM topology of either αIIb, or β3, or both  was cloned into a modified pIRES2-EGFP vector with an N-terminal signal peptide derived from murine IgG kappa V followed by a protein C epitope tag. Mutations were made using site-directed mutagenesis with the QuikChange kit (Agilent Technologies). All the introduced mutations were confirmed by DNA sequencing.The plasmids for αd before , 41, 42.d before . The β3-IIbβ3 integrin . H-160 is a rabbit anti-αIIb polyclonal IgG (Santa Cruz Biotechnology). PE-labeled MAR4 (BD Bioscience) is a nonfunctional anti-β1 mAb. PE-labeled TS2/4 (BioLegend) is a nonfunctional anti-αL mAb (2 conformation-dependent mAbs (PAC-1 (BD Bioscience) is a ligand-mimetic mAb (IgM) for the activated αintegrin . AP3 is n . The KIM127 and m24 binding were performed in HBSGB plus Ca/Mg or 0.2 mM CaCl2 and 2 mM MnCl2 (Ca/Mn). The ligand or conformation-dependent mAb binding was presented as normalized mean florescence intensity (MFI), i.e., the ligand or mAb MFI as a percentage of integrin MFI measured by AP3 for αIIbβ3, TS2/4 for αLβ2, and MAR4 for α5β1. When EGFP-TH was coexpressed with integrin constructs, the EGFP and integrin double-positive cells were gated for MFI calculation. The expression of EGFP-TH was measured based on the MFI of EGFP.The detailed protocol of ligand and antibody-binding assay for integrin was as we published before . HEK293Fintegrin . The ligIIbβ3. The single cell clones that have comparable surface expression of αIIbβ3 were selected for cell adhesion and spreading assay as described before . We also compared the maleimide-PEG11-biotin and biotin-maleimide for labeling the cysteine mutations at the extracellular portion of the TM domain in intact cells, or the cysteine mutations at the intracellular portion of the TM domain after physically breaking the cell membrane. No obvious differences were found among the different conditions. We used maleimide-PEG11-biotin for labeling the extracellular TM cysteine mutations and biotin-maleimide for the intracellular TM-CT cysteine mutations. The labeling was performed following the published protocol with some modifications . HEK293FIIbβ3 plus EGFP-TH or EGFP. For the labeling of spreading cells, the cells suspended in DMEM without serum were allowed to spread on the plate coated with 50 μg/ml fibrinogen at 37 °C for 1 h and then washed with PBSCM and labeled on-site at 4 °C for 30 min.For the labeling in the presence of the RGD-mimetic ligand eptifibatide, the cells were incubated with 20 μM eptifibatide at 25 °C for 15 min and then labeled on ice for 30 min. For the labeling in the presence of EGFP-TH, the cells were cotransfected with αIIb), H-160 (for αIIb), H-96 (for β3), AP3 (for β3 under nonreducing condition), or anti-PC (for β3-tail-TMCT) diluted in Intercept (PBS) blocking buffer plus 0.1% SDS and 0.1% Tween 20. The membranes were washed once with 10 ml TBS-T buffer for 15 min and then washed three times with TBS-T buffer for 5 min each and then incubated at 25 °C for 1 h with IRDye 800 streptavidin plus IRDye 680 goat anti-rabbit (or mouse) IgG diluted in Intercept (PBS) blocking buffer plus 0.1% SDS and 0.1% Tween 20. The membranes were washed and scanned with the Odyssey Infrared Imaging System (LI-COR Biosciences).The immunoprecipitated proteins were resolved on 7.5% or 12% SDS–polyacrylamide gels under reducing or nonreducing conditions and transferred to PVDF membranes. The membranes were blocked with Intercept (PBS) blocking buffer (LI-COR Biosciences) at 25 °C for 1 h and then incubated for 1 h at 25 °C with 1 μg/ml each of 314.5 (for αt test was performed between control and treated experimental groups. p-values ≤0.05 were considered significant.Statistical analysis was carried out on at least three individual datasets and analyzed with GraphPad Prism software. Unpaired two-tailed All data are available in the main text.Authors declare that they have no competing interests.


Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These “life” or “death” cell fate decisions often rely on the activity of the tumor suppressor gene  The ability of a cell to sense and to respond to DNA damage is essential to maintain its genetic material and tissue homeostasis. The DNA damage response (DDR) pathway has evolved in eukaryotes to preserve genomic integrity through a set of cellular responses that include cell cycle control, DNA repair, and apoptosis . Cell cyDrosophila) and the downstream kinases Chk1 and Chk2, which are responsible for the cell cycle checkpoint and apoptotic induction , 66sal>Gely Fig. .Fig. 7p5fzr in all wing imaginal cells for 48 h before dissection. In these conditions, very few pH3-positive cells were observed compared to the control  assay to evaluate the direct physical interaction between p53 and Cdk1 in wing imaginal cells , 69. ThiΔC) maintains its ability to interact with VC-Cdk1 as visualized by BiFC signal, this complex is mainly observed in the cytoplasm and it is unable to trigger apoptosis  strongly abolished the p53-A/Cdk1 BiFC signal and the apoptotic induction . The sex of experimental larvae was only considered relevant when selecting for specific mutations that were X-linked. The strains used in this study are summarized in Table The different DBS (DBS-GFP), UAS-Dronc-GFP, tagged hid-GFP, hid20-10-lacZ, hid 5´F-GFP, IRER{ubi-DsRed}, and the Fly-FUCCI reporters ubi-GFP-E2F11-230 and ubi-mRFP1-NLS-CycB1-266 (ubi-flyFUCCI) and UAS-GFP-E2F11-230 and UAS-mRFP1-NLS-CycB1-266. The Gal4 drivers: ap-Gal4, tub-Gal80ts , salEPv-Gal4 , dppblink-Gal4 (dpp>), hh-Gal4, UAS-GFP; tub-Gal80ts , en-Gal4, UAS-GFP; tub-Gal80ts , nub-Gal4 and tub-Gal80ts; tub-Gal4 . The UAS lines: UAS-dap, UAS-stg-i, UAS-Cdk1-i, UAS-fzr, UAS-CycE-i, UAS-E2f1-i, UAS-Rbf280, UAS-CycA-i,UAS-mre-11-i, UAS-okra-i, UAS-tefu-i, UAS-Dcr-2, UAS-rpr, UAS-hid, UAS-GFP, UAS-mcherry, UAS-p53-A-myc, UAS-p53; UAS-Su(var)3-9-i, UAS-miRNA-RHG, UAS-stg, UAS-CycE, UAS-Cdk1AF, UAS-p53-VN, UAS-Cdk1-HA, UAS-dac-VN and UAS-abdm-VC. The following mutant lines were used: lig4169, mei-41D5, mnkp6, and p535A14. Details for the different lines and reagents used in this study can be found in Table Reporters: -Dcr2 was used in combination with different RNAi lines to enhance message knockdown.UAS-Cdk1 lines together to increase the knockdown efficiency. Details for the different lines used in this study can be found in Table Cdk1 downregulation shows a relative mild phenotype visualized by the stalling of cells at the mitosis phase Fig. ; therefots UAS system [The temporal expression of the different UAS lines was restricted when needed using the Gal4/Gal80S system . BrieflyThird instar larvae were dissected in PBS and fixed with 4% paraformaldehyde, 0.1% deoxycholate, and 0.1% Triton X-100 in PBS for 25 min at room temperature. They were blocked in PBS, 1% BSA, and 0.3% Triton for 1 h, incubated with the primary antibody over night at 4 °C, washed four times in washing buffer (PBS 0.3% Triton) and incubated with the appropriate fluorescent secondary antibodies for 1.5 h at room temperature in the dark. They were then washed and mounted in Vectashield (Cat# H-1000 RRID:AB_2336790) for confocal analysis.TUNEL analysis was performed using In Situ “Cell Death Detection Kit” (TMR Red) (#12156792 910) and “Tunel Dilution Buffer” (#11966006001) kits, both from Roche.https://fji.sc) and Adobe Photoshop software.All confocal images were obtained using a Leica LSM510 and LSM710 vertical confocal microscope. Multiple focal planes were obtained for each imaginal disc. Image treatment and analysis was performed using Fiji  and the area defined by the domains of expression the p-Gal4; UAS-GFP-E2F11-230, UAS-mRFP1-NLS-CycB1-266, genotype were subjected to IR and dissected at 1, 3, and 6 h after treatment. Nonirradiated larvae were used as control. Red, green, or yellow cells were manually quantified. For each experiment, at least five wing imaginal discs were used to count an average of 500 cells per disc. The same region of the imaginal disc for each disc was selected for the quantification.For the Fly-FUCCI cell quantification, third instar larvae of the aThe number of discs analyzed in each experiment is given in the figure legends.https://www.graphpad.com). The specific statistical test and the n used in each analysis are noted in the corresponding figure.Statistical analysis was performed using GraphPad Prism software  containing 1 µl of Hoechst  in agitation. Trypsin digestion was stopped by the addition of 200 µl of 1% fetal bovine serum  in PBS. After centrifugation at 1500 × g at 4 °C for 5 min, cells were suspended in 300 µl of 1% FBS and cells were sorted by GFP expression using FACSVantage SE (BD Biosciences). The cell cycle profiles of GFP-positive and GFP-negative cells were determined by Hoechst fluorescence using a FACSCalibur flow cytometer (Becton Dickinson). The cell cycle profile was analyzed using FlowJo 7.5 software and Dean–Jett–Fox model.A total of 50 wing discs were dissected from third instar larvae expressing the corresponding transgene under the Third instar larvae of the indicated genotypes were irradiated in X-ray machine Phillips MG102 at the standard dose of 4000R and dissected at the indicated times depending on the experiment and stated in each figure.TM Express Enzyme  and stored at −80 °C in freezing buffer (85.5 g/l sucrose and 50 mL/l DMSO prepared in 11.8 g/l citrate buffer at pH 7.6) until use.DNA strand breaks and alkali-labile sites were assessed via the alkaline version of the Comet assay. A total of 60 wing imaginal discs cells were enzymatically individualized by incubation 20 min in TrypLEAn estimated 104 cells were embedded in 0.75% low melting point agarose and deposited on precoated slides with 1% agarose. Immediately after agarose solidification (10 min on ice), samples were incubated for 1 h at 4 °C in a cold lysis buffer . The slides were then rinsed in 0.4 M Tris, pH 7.4. Subsequently, DNA was allowed to unwind for 40 min in the electrophoresis buffer  and electrophoresis was carried out for 30 min at 25 V and 300 mA (0.73 V/cm). Slides were neutralized in 0.4 M Tris pH 7.4 and stained with 50 μl of GelRed (Thermo Fisher Scientific). Samples were examined with a Leica DMI 3000B microscope (Germany), equipped with an EL6000 compact light source and a 480–550 nm wide band excitation filter and a 590-nm cut-off filter. Scoring was carried out using the OpenComet plugin for the image-processing platform ImageJ. A total of 250 randomly selected cells were analyzed per condition. The tail moment (tail intensity × length summed over the whole extent of the tail) was used to measure DNA damage.We used a pUASTattB that have the N-terminal (VN: 1-173) and C-terminal (VC: 155–238) moieties of Venus cloned in Xho1 and Xba1 restriction sites. The coding region of p53-A and Cdk1 were PCR amplified from the GH11591 clone (BDGP) and LD38718 clone (BDGP), respectively. Inserts were cloned in Xho1-Xba sites into the pUASTattB VN or VC version including a five amino acids linker region. N- and C-terminal deletions of p53-A were cloned in a similar manner. To ensure similar expression levels, all UAS constructs were inserted into the same attP site (86Fb), except UAS-VC-Cdk1 that was also inserted in 51D.The sequence of all primers used in this study are as follows:UAS-VN-p53-A:Forward:CTCGAGGGCGGCTCAGGCGGCATGTATATATCACAGCCAATGTCGTGGC-3´.5´-CAGTTCTAGATCATGGCAGCTCGTAGGCACG-3´.Reverse: 5´-CAGTΔN (1-83)UAS-VN-p53-ACTCGAGGGCGGCTCAGGCGGCATGGAGAATCACAACATCGGTGG-3´.Forward: 5´-CAGTReverse:TCTAGATCATGGCAGCTCGTAGGCACG-3´.5´-CAGTΔC (283–385)UAS-VN-p53-ACTCGAGGGCGGCTCAGGCGGCATGTATATATCACAGCCAATGTCGTGGC-3´.Forward: 5´-CAGTReverse:TCTAGATCAGGACTTGCGCTTCTTGCTATTGAGCTGGCG-3´.5´-CAGTUAS-VC-Cdk1:CTCGAGGGCGGCTCAGGCGGCATGGAGGATTTTGAGAAAATTG-3´.Forward: 5´-CAGTTCTAGATTAATTTCGAACTAAGCCCGATTGAAAAC-3´.Reverse: 5´-CAGTFor the initial BiFC analysis, we used the UAS-p53-VN flies available at FlyORF (F004757).Visualization and quantification of the BiFC signal was done using identical parameters for image acquisition between the different genotypes and analyzed using Fiji.sal>GFP (negative control); sal>GFP, p53-A (Myc), miRHG (positive control); sal>GFP, p53-A (Myc), Cdk1-i ; sal>GFP, p53-A (Myc), dap  and sal>GFP, p53-A (Myc), fzr . The wing imaginal discs of 100 larvae were dissected for each condition, performing three replicates per ChIP. Larvae were fixed in FA fix solution  for 25 min at RT. Then, the tissue was incubated with Quench Buffer  for 6 min at RT. Larvae were washed with buffer A  and buffer B  consecutively, 20 min each at 4 °C. Both A and B buffers were supplemented with 1 mM PMSF and 1X protease inhibitors cocktail (Roche #11873580001).The following genotypes were used: Wing imaginal discs were dissected in Buffer B on ice. Later, discs were centrifuged at max speed for 3 min at 4 °C. Collected disc pellet that was resuspended in buffer C  supplemented with 1 mM PMSF and 1X protease inhibitors cocktail (Roche #11873580001). The discs were homogenized in this medium before proceeding to sonication. The tissue was sonicated 20 cycles (30″ ON/30″ OFF), at high power and at 4 °C using diagenode bioruptor sonicator. We removed 10% from the samples for input. Samples were precleared with protein G affinity gel (Sigma-Aldrich #E3403) for 1 h on rotator at 4 °C, and then the chromatin was transferred to a fresh tube. The anti-myc affinity gel (Sigma-Aldrich #E6654) and the protein G affinity gel (Sigma-Aldrich #E3403) as negative control of each chip were blocked in 1X RIPA  supplemented with 100 μg/ml salmon sperm DNA and 100 μg/ml BSA overnight at 4 °C.Next day, we pelleted beads at 6000 rpm for 2 min and mixed the chromatin with the blocked beads. The samples were incubated for 4 h at 4 °C. Finally, the beads were washed four times in RIPA 1X for 5 min at 4 °C, pellet at 6000 rpm for 2 min at 4 °C. Beads were washed again in TE for 5 min at 4 °C and pellet at 6000 rpm for 2 min at 4 °C3 at 50 °C.Chromatin was eluted from beads in TE with 1% SDS and 0.1 M NaHCORE for the rpr and hid genes. Results were quantified using the delta Ct method and presented as percentage of input.To reverse the crosslinks, we incubated the eluted material at 65 °C overnight, processing the INPUT of each sample in parallel. The next day, we added 50 μg/ml of RNAase and incubated the samples 30 min at 37 °C. Then, 20 μg of proteinase K was added and incubated at 55 °C for 3 h. We added 4 μl NaCl 5 M and 4 μl Tris 1 M to each sample before purifying them by phenol/chloroform method. Finally, we added 1.25 μl of glycogen (Roche #10901393001), 25 μl of NaAC 3 M pH 5.2, and 550 μl of ethanol 100% at −20 °C ON. Samples were spin for 20 min at maximum speed and the pellet was twice with ethanol 70% at −20 °C and centrifuged again for 10 min and let the pellet dry at RT. The samples were resuspended in 30 μl of TE buffer and amplified by qPCR using GoTaq qPCR Master Mix (Promega #A6001), using as amplicons the p53The sequence of the primers used in this study were previously described in ref. :rpr-p53-RE:Forward: 5´-CTACGTTTCCCAGACCCAAGAC-3´.Reverse: 5´-GTCTCCATCCAATTCCCATCTC-3´.hid-p53-RE:Forward: 5´-ACTTTTGTTCTTTTCGCTTTGGAC-3´.Reverse: 5´-GATGACGAAATTCAAGCACACTCT-3´.tubGal80>GFP, tubGal80>Cdk1-i, and tubGal80>fzr. Larvae were kept at 17 °C for 7 days and then transfer at 31 °C for 2 days before dissection. Two biological replicates for each condition were performed.To carry out the ATAC-seq experiment, at least 30 wing discs were dissected from the following genotypes: g for 5 min at 4 °C to isolate the nuclei. Typically, we used 75,000 cells per condition. Next, samples were resuspended in 25 μl of transposition reaction mix as described in ref. [Samples were lysed in lysis buffer . Lysates were centrifuged at 500 ×  in ref.  and incuFollowing the transposition, samples were purified using the Qiagen MinElute Kit following the manufacturer’s instructions.ATAC-seq libraries were generated according to the standard protocol . BrieflyATAC-seq data analysis was performed using the nf-core/atacseq pipeline (v1.2.1), which runs Nextflow (v20.10.0), for quality controls, read alignment against Dm6 assembly using BWA-MEM (v0.7.17), filtering for blacklisted regions, data visualization, peak calling using MACS2 (v2.2.7.1), read count using featureCounts (v2.0.1), and differential accessibility analysis using the DESeq2 R library.t test when comparing the mean of two conditions or one-way ANOVA Dunnett’s test for multiple comparisons more. P values shown on the graphs are indicated with the following asterisk code: 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****). Sample size was described in each figure legend and in the corresponding “Materials and methods” section.Results were analyzed and presented using Prism 8 GraphPad software. We used unpaired two-tail Student’s Supplementary Figure LegendsS1 figS2 FigS3 FigS4 figS5 FigS6 FigS7 FigS1 tableS2 Table


We used middle-to-old age individuals given immune blockade is commonly used in this population. Whilst most Vδ1+subset cells exhibited a terminal differentiation phenotype, Vδ1− subset cells showed an early memory phenotype. Dimensionality reduction revealed eight γδ T cell clusters chiefly diverging through CD161 expression with CD4 and CD8 expression limited to specific subpopulations. Comparison of matched healthy elderly individuals to bronchiectasis patients revealed elevated Vδ1+ terminally differentiated effector memory cells in patients potentially linking this population with chronic proinflammatory disease.γδ T cells are a highly versatile immune lineage involved in host defense and homeostasis, but questions remain around their heterogeneity, precise function and role during health and disease. We used multiThe online version contains supplementary material available at 10.1186/s12979-022-00269-w. Vδ1 cells predominantly reside in mucosal sites and epithelia, while Vδ2 cells comprise 50–90% of γδ T cells in the circulation and are both effectors and antigen-presenting cells (APCs). Vδ3 cells are rare in circulation but enriched in the liver, notably in individuals with an infection or cancer  and infe+, tissue-resident memory cells (TRMs) [+ T cells and CD8+ T cells [CD161 is a C-type lectin-like membrane receptor expressed at high levels on NK, NKT, Th17, Mucosa Associated Invariant T cells (MAITs), non-MAIT CD8s (TRMs) –14, αβ C T cells . CD161 e T cells .Bronchiectasis is a chronic lung condition, defined as the abnormal, irreversible dilatation of the bronchi, where the elastic and muscular tissues are destroyed by acute or chronic inflammation and infection. This damage impairs the natural drainage of bronchial secretions, which can become chronically infected. Unless appropriately managed, the combination of repeated respiratory infection and chronic inflammation results in progressive lung damage . While nWe performed a high-dimensional flow analysis with dimensionality reduction and unsupervised clustering of human γδ T cells from middle aged to elderly healthy individuals revealing a novel cell subset network, distorted in bronchiectasis.t-test, p = 0.005), while the gender ratio was similar (Fishers exact test p = 0.99). Approval was obtained from the ethics review boards of Greenslopes Private Hospital (GSH), Prince Charles Hospitals (TPCH) and the QIMR Berghofer (QIMRB) Medical Research Institute (QIMRB P2058 and GSH 12/12 and 14/14). Informed written consent was obtained from all participants. Whole blood was collected with EDTA anticoagulant tubes . Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll−Paque PLUS  by density gradient centrifugation and resuspended in RPMI-1640 containing 10% foetal calf serum with 10% DMSO for cryopreservation in liquid nitrogen.Blood samples were obtained from 22 healthy adults  and 11 bronchiectasis patients . Patients were significantly older than the controls , anti-CD3 APC-eFluor780 , anti-CD4-BUV395, anti-CD8-BV786, anti-CD27-BUV737, anti-CD45RA-APC-H7 and anti-HLADR-BV650 , anti-CD161-PE, and anti-ϒδ TCR-PE-CY7  and anti-Vδ1-FITC   was used  to gate live/CD3+/γδ TCR+cells. On manually gated γδ TCR+cells, Uniform Manifold Approximation and Projection (UMAP) analysis was performed using Vδ1, CD45RA, CD27, HLA-DR, CD161, CD4, and CD8 as clustering channels [Gating was performed using standard singlet and viability gates. Positives were gated based on fluorescence minus one (FMO) controls. Manual gating was performed using FlowJov10.8. γδ T cells were divided into two populations based on Vδ1 expression. Each population was subgated based on CD45RA and CD27 expression to differentiate naïve and memory cells and then gated based on CD161, CD4 and CD8expression. Cytobank 5.0 , and Prism 9.1.1 . p-values are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Students + cells), Vδ1+ and Vδ1− (as a percentage of γδ T cells) of 8.6% (± SD 6%), 30.6% (± SD 27%) and 69.2% (± SD 27%) respectively which are similar to values published for a cohort that included elderly individuals [−CD161+cells (53% ± 30%), Vδ1−CD161−cells (12% ± 9%), Vδ1+CD161−(18% ± 23%), andVδ1+CD161+cells (16% ± 20%) determined. Four subsets were observed when combining Vδ1 and HLA-DR expression , Vδ1−HLA-DR+cells (11% ± 13%), Vδ1+HLA-DR−cells (27% ± 26%), and Vδ1+HLA-DR+cells (8% ± 8) determined. Such high HLA-DR expression on γδ T subsets implies the operation of complex APC machinery for adaptive system function. CD45RA and CD27 were used to gate CD45RA+CD27+ naïve (TN), CD45RA−CD27+ central memory (TCM), CD45RA−CD27− effector memory (TEM) and CD45RA+CD27− terminally differentiated effector memory (TEMRA) in the Vδ1+ and Vδ1−populations  and one in TEM population (40% ± 22%) with some bleeding into TCM population (18 ± 21%). In contrast, in the Vδ1− population, there was a clearly defined population in TEM cells (53 ± 20%) and a TCM population (27 ± 18%) with some bleeding into the TEMRA population (16 ± 21%). Comparison of CD161 expression between naïve and memory Vδ1+ and Vδ1−populations revealed a significant difference in both TEM cells and TCM cells (p < 0.0001), with Vδ1− cells having the highest surface expression of CD161  or CD161− (meta-cluster 5) , HLA-DR+CD161− (meta-cluster 4) and HLA-DR+CD161+ (meta-cluster 7) population means were 3.7% (SD- 2.4%), 3.3% (SD- 2%) and 6.4% (SD- 4.2%) respectively. The HLA-DR+CD8+CD27+ Vδ1− cell population (meta-cluster 3) was relatively small  which corresponds the very small cell island identified as phenotypically distinct by UMAP analysis. This population, though small, expressing very high levels of HLA-DR, and CD27 may be a highly activated, functionally distinct subset. Overall, the pattern of marker expression on the eight clusters, as detailed in Figs. We next performed the UMAP dimensionality reduction on manually gated γδ T cells, which clustered the population into eight islands Fig. A and B. t-test p = 0.005), to match the bronchiectasis patients age, five samples (age ranging from 51 to 59 years) from the healthy controls were removed for this analysis. Relative to controls, bronchiectasis patients showed higher numbers of Vδ1+ TEMRA cells (p = 0.03). The two cohorts were similar in peripheral blood γδ T cell phenotype in all other aspects tested.We then compared the γδ T cell subtype network in the circulation of healthy individuals with bronchiectasis patients. Since patients were found to be significantly older than the healthy controls . This age cohort was selected due to relevance, with a peak incidence in cancer, lung infections and bronchiectasis. Validation of these results is required in other age groups to determine the temporal dynamics of γδ T cells. Future studies should also address the knowledge gap inCD161 and LLT1 signaling. In summary, we have defined eight novel γδ T cell subsets that show some distortion in bronchiectasis.Additional file 1: Sup Fig. 1. Gating strategy followed for biaxial gating. Sup Fig. 2 CD161 expressing cell percentages in (A) total γδ T cell, (B) Vδ1+γδ T cell and (C) Vδ1−γδ T cell populations. CD161 expression shows no correlation with age in any of the analyzed subsets . Sup Fig. 3 Fluorescence minus one (FMO) staining controls for CD161, HLA-DR, CD45RA and CD27 are shown together with fully stained sample showing staining pattern on γδ T cells.Additional file 2: Supp. Table 1. Flow cytometry staining panel.