The packing involves three borderline C—H⋯N contacts and a pairing of the triazole rings across an inversion centre.In the crystal structure of the title compound, the triazole ring exhibits interplanar angles of  17H14N4O2, the triazole ring exhibits interplanar angles of 63.86 (2) and 76.96 (2)° with the phenyl and benzothiazole planes, respectively. The C—C—C angle at the methylene group is rather wide at 114.28 (4)°. The packing involves three borderline C—H⋯N contacts, two of which combine to form layers parallel to ac, and a pairing of the triazole rings across an inversion centre [interplanar distance of 3.1852 (2) Å].In the structure of the title compound, C A selection, mostly involving the heteroatoms, is presented in Table 1imine contact in N-[3-(benzo[d]thiazol-2-yl)-6-bromo-2H-chromen-2-ylidene]-4-methylbenzenamine , 3.1852 (2) and 0.94 Å, respectively. This feature is reinforced by the other C—H⋯N interaction, which involves the same operator.The molecular packing displays few significant features. There are three borderline C—H⋯N interactions Table 2, two of ne Fig. 3. The tri4.et al., 2002et al., 2016The searches employed the routine ConQuest -1,3-benzothiazole  to 445 values in the range 106–122°, with a mean value of 114 (5)°. However, restricting one ring to be a C2-substituted thiazole gave only three hits, with four values of 109.6–112.9° for the angle at the methylene groups. These all involved two planar ring systems of the benzo[d]thiazole type, but with different heteroatoms in some cases  and phenyl isothiocyanate 2 (0.01 mol) was stirred for 30 min in ethanol (25 mL) in the presence of sodium ethoxide (0.01 mol). After cooling, methyl iodide (0.015 mol) was added. The reaction mixture was stirred for 30 min at room temperature, then refluxed for 1 h. The resulting precipitate was filtered off, washed with water, dried, and recrystallized from ethanol. The title compound was isolated as a white solid; yield 75%; m.p. 429 K; IR : ν 3053 (Ar—CH), 2928 , 1594 (C=N); 1H NMR : δ 2.60 , 4.57 , 7.36–7.50 , 7.89 , 8.01 ; Analysis calculated for C17H14N4S2 (338.45): C 60.33, H 4.17, N 16.55, S 18.95. Found C 60.66; H 4.15; N 16.40; S 18.90%.A mixture of 2-benzothiazolyl acetohydrazide 6.aromatic = 0.95 Å, C—Hmethylene = 0.99 Å). The U(H) values were fixed at 1.5 × Ueq of the parent carbon atoms for the methyl group and 1.2 × Ueq for other hydrogens.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023007041/yz2038sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989023007041/yz2038Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023007041/yz2038Isup3.cmlSupporting information file. DOI: 2287438CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure of the title bis-urea derivative, the host molecules are linked by N—H⋯O=C hydrogen bonds and C—H⋯O contacts with  26H26N4O6·2C4H8O, representing a bis-urea with terminal phenylalanine units, crystallized with two tetrahydrofuran (THF) molecules. The main molecule is located on a crystallographic twofold axis, while the solvent molecule is disordered over two positions, with occupancies of 0.571 (15) and 0.429 (15). The host molecules are linked by N—H⋯O=C hydrogen bonds and C—H⋯O contacts with R21(6) and R21(7) ring motifs. The THF molecules enclosed in the crystal are connected to the bis-urea compound via O—H⋯O and C—H⋯O interactions.The title compound, C The angle between the planes of adjacent urea moieties is 83.7 (1)°, thus they are nearly perpendicular to one other. Such a motif is also well known in the literature  and 2.32 (3) Å; Table 13 (corresponding to about 29% of the unit-cell volume). The cavities are bounded by the nonpolar phenylene and arene units of the title compound. In addition, the carboxy groups point into the interior of these cavities and form O—H⋯O hydrogen bonds with the THF O atom [H⋯O = 1.73 (3) Å]. Further stabilization of the molecular network, each involving the THF molecules, is realized by C—H⋯O contacts with the carboxy group of an adjacent molecule (H⋯O = 2.78 Å) and weak C—H⋯π contacts (H⋯Cg = 2.61–3.00 Å) with the central benzene core or peripheral arene substituents.The crystal structure exhibits cavities which are occupied by THF molecules requiring about 961 Ået al., 2012The synthetic and spectroscopic details for the title compound have been reported previously (Stapf Uiso(H) values were fixed at 1.2 times the equivalent Ueq value of the parent C atoms. The THF solvent molecule is disordered over at least two positions [refined occupancies 0.571 (15) and 0.429 (15)]. Therefore, the solvent molecule was refined using ISOR for C16A, C16B, C17A and C17B (approximate isotropic behaviour) and SADI (same distances over pairs of bonded atoms) restraints  I. DOI: 10.1107/S2414314623007435/bh4077Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623007435/bh4077Isup3.cmlSupporting information file. DOI: 2290568CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The first high-quality crystal structure of unmodified 2′-deoxyguanosine is reported. The isolated crystals are the dimethyl sulfoxide disolvate. 10H13N5O4·2C2H6OS, which is of interest with respect to its biological activity, at 183 K has orthorhombic (P212121) crystal symmetry. The structure displays a network of intermolecular N—H⋯N, N—H⋯O and O—H⋯O hydrogen bonds. 2′-Deoxyguanosine molecules are linked to each other and to the two dimethyl sulfoxide solvent molecules by hydrogen bonding.The title compound, C Understanding the properties of DNA is crucial for our knowledge of its reactivity in cellular processes of replication and transcription to yield transfer RNA  crystallized in the orthorhombic Sohnke space group P212121, with four formula units per unit cell and one per asymmetric unit .Nucleobase -one. The cocrystal structures of (actinomycin D)·2(2′-deoxyguanosine)·12H2O (ACTDGU) and (7-bromoactinomycin)·2(2′-deoxyguanosine)·11H2O (BRAXGU) at room temperature were also reported , is the only purine nucleoside solvate in the CSD  were obtained upon slow evaporation of 2′-deoxyguanosine  from DMSO.Single crystals of  values were set at 1.2 times  or 1.5 times (for methyl and OH groups) the Ueq value of the parent atom. The Flack parameter x was −0.00 (6) by classical fit to all intensities and 0.022 (14) by Parsons’ method  I, global. DOI: 10.1107/S2056989023007405/zv2028Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023007405/zv2028sup3.pdfFigures S1 and S2. DOI: 2290127CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The molecular conformation is stabilized by two intramolecular N—H⋯N interactions. In the crystal, molecules are also linked via C—H⋯π interactions forming a three-dimensional network.In the title compound, [24]dithiaamethyrin(1.0.0.1.0.0); C 50H44Cl4N4S2 were obtained from the reaction of 2,5-bis(4-propyl-1H-pyrrol-2-yl)thiophene and 3,5-dichlorobenzaldehyde in the presence of trifluoroacetic acid for 3 h and subsequent addition of p-chloranil. The macrocycle in the title compound can be described as a highly planar structure wthe the average deviation of the 32 macrocyclic atoms from the least-squares plane being 0.0416 Å. Its molecular conformation is stabilized by two intramolecular N—H⋯N bonds and a three-dimensional network is formed by C—H⋯π interactions.Purple crystals of the title compound, C Neighboring molecules form dimers via intermolecular C—H⋯Cl and C—H⋯π interactions owing to the H6⋯Cl1 (2.93 Å), H17A⋯Cl2 (3.34 Å), and C3—C11 (3.36 Å) distances, as shown in Fig. 2The molecular structures and electronic properties of hexapyrrolic expanded porphyrins with different numbers of π-electrons and et al. (20152Cl2 (ca 300 mL) under an Ar atmosphere, to which 3,5-dichlorobenzaldehyde (68.4 µL) and trifluoroacetic acid (160 µL) were added. The reaction mixture was stirred for 3 h, p-chloranil (544 mg) was added to it, and the mixture was stirred overnight at ambient temperature. Then, the mixture was neutralized with an aqueous NaHCO3 solution, and the crude products were passed through an alumina column. Finally, the products were purified by chromatography on a silica gel column using chloroform as elute. The third blue (2%) fraction afforded the title compound. The compound was recrystallized from a mixture of hexane and chloroform. Purple plates of suitable quality for diffraction were obtained by slow-diffusing hexane into chloroform. 1H NMR : δ (ppm) 24.0 , 7.20– 6.87 , 5.05 , 4.61 , 0.76 , 0.52 , 0.32 ; MALDI–TOF MS found = 904.254, monoisotopic mass = 904.176 calculated for C50H44Cl4N4S2. UV–vis (CH2Cl2): λ = 387, 500 nm.The title compound was prepared by a modified previously reported method by Ishimaru  al. 2015. 2,5-BisCrystal data, data collection, and structure refinement details are summarized in Table 210.1107/S2414314623007666/xu4052sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314623007666/xu4052Isup2.hklStructure factors: contains datablock(s) I. DOI: 2292415CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, chains of molecules extending along the  28H34ClN3O9S, is disordered over two closely spaced sets of sites; the site occupancy of the major component = 0.542 (3). The conformation of each component is approximately U-shaped with the chlorobenzene ring forming the base and the indolinyl and sulfamoyl groups the sides; an intramolecular C—H⋯Cl hydrogen bond possibly contributes to the stabilization of the conformation. In the crystal, a corrugated layer structure parallel to the ab plane is formed by C—H⋯O and C—H⋯Cl hydrogen bonds together with C—H⋯π(ring) interactions.The majority of the title molecule, C Missioui et al., 2022Indapamide, is a dihydro-indole-based thiazide-like diuretic used to manage heart failure and treat hypertension. Various activities are associated with indole derivatives, including antiviral  = 0.247 (8) Å and φ(2) = 327 (2)° for the major component and Q(2) = 0.399 (9) Å and φ(2) = 329.6 (16)° for the other. The dihedral angle between the mean planes of the C1–C6 and C15–C20 rings is 86.2 (4)° while that between the two disordered components of the C1–C6 ring is 3.8 (5)° and that between the two disordered components of the C15—C20 ring is 5.68 (6)°.The molecule adopts an approximate U-shaped conformation Fig. 1 with thed Table 1 may helpd Table 1 of the tA⋯Cl1 hydrogen bonds  and potassium bicarbonate  were dissolved in dimethylformamide (10 mL), to which was added ethyl 2-bromoacetate . Under reflux, the reaction was stirred for 3 h at 80°C. When the starting reagents had reacted completely, distilled water (100 ml) was added. The product precipitated in solid form, was filtered, dried and recrystallized from ethanol solution to afford colorless blocks.N-(2-methylindolin-1-yl)glycinate portion of the molecule is disordered over two partially resolved sets of sites in a 0.542 (3):0.458 (3) ratio. In addition, the C23—C24 ethyl group is disordered over two sets of sites in a 0.526 (12):0.474 (12) ratio. The two components of each disorder were refined with restraints that their geometries be comparable.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623006995/tk4094sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314623006995/tk4094Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623006995/tk4094Isup3.cmlSupporting information file. DOI: 2280340CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title hydrated salt features a dense array of hydrogen bonds, forming a three-dimensional network. 5H5Cl2N2+·C6H4NO3−·H2O, the pyridine N atom of the cation is protonated and an intramolecular O—H⋯O hydrogen bond is observed in the anion, which generates an S(6) ring. The crystal packing features N—H⋯N, O—H⋯O, N—H⋯O, C—H⋯Cl and C—H⋯O hydrogen bonds, which generate a three-dimensional network.In the title hydrated salt, C In the extended structure, the cations, anions and water molecules are connected by N—H⋯N, O—H⋯O, C—H⋯Cl, N—H⋯O and C—H⋯O hydrogen bonds  was mixed with a hot aqueous solution of 4-amino 3,5-dichloro pyridine (34 mg). The mixture was cooled slowly and kept at room temperature. After a few days, colourless block shaped crystals were obtained.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623008210/hb4452sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314623008210/hb4452Isup2.hklStructure factors: contains datablock(s) I. DOI: 2294939CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


To provide the correct values of UCCR, the UCCR was calculated again by converting cortisol and creatinine in the same measurement unit (nmol/L).Section 3 Analytical Procedures, first paragraph, last sentence, should be as follows. “The UCCR was calculated by dividing the urine cortisol concentration (nmol/L) by the urine creatinine concentration (mmol/L).”Reference 29 is removed from the References list.−6 (1.04‐3.81 × 10−6), 32.09 × 10−6 (7.68‐245.5 × 10−6), and 10.55 × 10−6 (3.47‐54.05 × 10−6) in dogs with HA, dogs with DMHA and healthy dogs, respectively.”Section 4 Results, UCCR subsection, first sentence should be as follows. “The median UCCR was 2.03 × 10−6 (7.68‐245.46 × 10−6).Section 4 Results, UCCR subsection, fifth sentence should be as follows. “The median UCCR in dogs with DMHA and BSC ≤2 μg/dL (≤55 nmol/L) was 27.14 × 10Section 4 Results, UCCR subsection, last sentence should be as follows. “A cut‐off value of UCCR <4.4 revealed 100% sensitivity (95% CI: 69.1‐100) and 97.3% specificity (95% CI: 85.8‐99.9) in diagnosing HA.”Figure 1, to be replaced with this new Figure 1.FIGURE 1 Scatter scale plot comparing urinary corticoid: creatinine ratio (UCCR) of dogs with hypoadrenocorticism , dogs with disease mimicking hypoadrenocorticism  and healthy dogs . The horizontal bars represent the median, the maximum, and the minimum value of each group.


The crystal structures of two benzothiophene derivatives are described along with an analysis of the intermolecular contacts in the crystals performed using Hirshfeld surface analysis and two-dimensional fingerprint plots. 26H19NO2S2, (I), and C25H19NO2S2, (II), the benzothiophene rings are essentially planar with maximum deviations of 0.026 (1) and −0.016 (1) Å for the carbon and sulfur atoms in compounds (I) and (II), respectively. In (I), the thiophene ring system is almost orthogonal to the phenyl ring attached to the sulfonyl group, subtending a dihedral angle of 88.1 (1)°, and the dihydropyridine ring adopts a screw–boat conformation. In both compounds, the molecular structure is consolidated by weak C—H⋯O intramolecular interactions formed by the sulfone oxygen atoms, which generate S(5) ring motifs. In the crystal of II, molecules are linked via C—H⋯O hydrogen bonds, generating C(7) chains running along the [100] direction. No significant intermolecular interactions are observed in I.In both of the title compounds, C Thiophene derivatives possess pharmacological and biological activities including antibacterial  attached to an N--N-prop-2-yn-1-yl benzenesulfonamide group (C9–C15/N1/S2/O1/O2) , 19.0 (1) and 33.9 (1), respectively, in compound I and 0.7 (2), 38.1 (2) and 87.6 (2)°, respectively, in compound II with the C1–C6, C11–C16 and C17–C22 phenyl rings. The benzothiophene ring system is almost orthogonal to the C17–C22 phenyl ring attached to the sulfonyl group in I, subtending a dihedral angle of 88.1 (1)°, while the dihydropyridine ring (C10/C11/C16/C23/C24) adopts a screw–boat conformation, as is evident from the Cremer–Pople puckering analysis of the six-membered heterocyclic ring The molecular structure of compound I Fig. 1 comprise2) Fig. 2. In bothI and 119.9 (2)° in II], with a simultaneous decrease in the N1—S2—C17 angle [108.5 (1)° in I and 107.6 (1)° in II] from the ideal tetrahedral value (109.5°) are attributed to the Thorpe–Ingold effect  Å in II] and N1—C16 . No significant intermolecular interactions or C—H⋯π interactions with centroid distances of less than 4 Å are observed in the structure.In the crystal of II, molecules are linked via C25—H25⋯O1 hydrogen bonding, generating C(7) chains  Å where Cg3 is the centroid of the S1/C1/C6–C8 ring; symmetry code: (ii) −x, 2 − y, 1 − z] and C—H⋯π interactions . are also present. Packing view of the title compound are shown in Figs. 3In the crystal of 4.et al., 2019et al., 2007dnorm, curvedness and shape index and 2D fingerprint plots) were performed with CrystalExplorer17  through white to blue . The dnorm surface was mapped over a fixed colour scale of −0.085 (red) to 1.564 (blue) for compound I and −0.286 (red) to 1.374 (blue) for compound II. The red spots indicate intermolecular contacts involved in hydrogen bonding.The Hirshfeld surfaces of compounds I, they reveal that the principal intermolecular contacts are H⋯H , H⋯C/C⋯H , O⋯H/H⋯O , C⋯C , S⋯H/H⋯S , S⋯C/C⋯S  and N⋯H/H⋯N . For compound II, they reveal a similar trend, with the principal intermolecular contacts being H⋯H/H⋯H , H⋯C/C⋯H , O⋯H/H⋯O , C⋯C , S⋯H/H⋯S , S⋯C/C⋯S , C⋯O/O⋯C  and S⋯O/O⋯S . In both compounds, the H⋯H intermolecular contacts predominate, followed by C⋯H/H⋯C and O⋯H/H⋯O contacts.The fingerprint plots are illustrated in Figs. 65.I: A solution of N-propargylbenzenesulfonamide (0.50 g) in xylenes (20 mL), MnO2 (0.50 g) was added and the reaction mixture was refluxed for 24 h. It was then filtered through a celite pad and washed with hot xylenes (2 × 10 mL). The combined filtrate was concentrated under vacuum and then triturated with MeOH to afford dibenzo[b]thiophene  as a dull white solid. Finally, compound I was crystallized using ethanol.Compound II: To a solution of (E)-N-{2-[2-(benzo[b]thiophen-2-yl)ethenyl]phenyl}benzenesulfonamide  in CH3CN (10 mL), K2CO3  and propargyl bromide  were added and the mixture was stirred at room temperature for 12 h. After completion of the reaction (monitored by TLC), it was poured into crushed ice (50 g) containing conc. HCl (5 mL), extracted with ethyl acetate (2 × 20 mL) then washed with water (2 × 20 mL) and dried (Na2SO4). Removal of the solvent in vacuo followed by crystallization from methanol (4 mL) afforded compound II as a white solid.Compound 6.Uiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023003821/zn2028sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989023003821/zn2028Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023003821/zn2028IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989023003821/zn2028Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023003821/zn2028IIsup5.cmlSupporting information file. DOI: 2259716, 2259715CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG‐generating sphingomyelin synthase (SMS)‐related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N‐terminal cytosolic region of SMSr binds to the N‐terminal half catalytic domain of DGKζ. However, the C‐terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways. The sterile α motif domain (SAMD) of sphingomyelin synthase (SMS)‐related protein (SMSr) binds to the C‐terminal half catalytic domain of diacylglycerol kinase (DGK) ζ. Moreover, the C‐terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. SMSr‐SAMD associates with DGKδ‐SAMD and activates DGKδ. However, SMSr‐SAMD moderately suppresses the activity of DGKζ in contrast to DGKδ. CPESceramide phosphoethanolamine synthaseDGdiacylglycerolDGKdiacylglycerol kinaseFLfull lengthHRPhorseradish peroxidasePAphosphatidic acidPAPphosphatidic acid phosphatasePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPLCphospholipase CPSphosphatidylserineSAMDsterile α motif domainSMSsphingomyelin synthaseSMSrsphingomyelin synthase‐related proteinTEVtobacco etch virusTMDtransmembrane domainTSTwin‐Strepl‐lactate dehydrogenase A, and creatine kinase muscle type [Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to convert it to phosphatidic acid (PA) , 3, 4, 5cle type , 15, 16.Diacylglycerol kinase isozymes function in a wide variety of physiological events, including immunity, cell proliferation, and the central nervous system , 18. ForThere are three isoforms of sphingomyelin synthase (SMS) , 37, SMSWe recently searched for an upstream enzyme (a DG supply enzyme) of DGKδ. Consequently, we found that DGKδ interacts with SMSr , which sIn the present study, we comprehensively searched for interactions between DGK isozymes and SMS isozymes. We found that DGKζ binds to SMS1 and SMSr but not SMS2. Moreover, DGKζ interacts with SMSr and SMS1 in different manners. These results suggest that, beyond our expectations, DGK isozymes and SMS isozymes form a complex network.Mouse monoclonal anti‐V5 antibody  was obtained from Thermo Fisher Scientific . Rabbit polyclonal anti‐GFP antibody (#598) was purchased from Medical and Biological Laboratories . Mouse monoclonal anti‐FLAG–tag antibody (F1804) was obtained from Sigma–Aldrich . Mouse monoclonal anti‐GFP antibody (sc‐9996) was purchased from Santa Cruz Biotechnology . Mouse monoclonal anti‐Strep II (M211‐3) and rabbit polyclonal anti‐GST (PM013) were obtained from Medical and Biological Laboratories . A horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG antibody was obtained from Bethyl Laboratories . Goat anti‐rabbit IgG‐HRP was purchased from Jackson ImmunoResearch .Plasmids for expressing N‐terminal 3×FLAG‐tagged human or rat DGK isozymes  and for We used the following nomenclature for epitope‐tagged proteins: TagX‐(protein) and (protein)‐TagY means that TagX and TagY are located at the N and C termini of the protein, respectively.EcoRI/SalI sites. The amplicons were generated using the following primers: SMS1‐NT (aa 1–134) using 5′‐CTCAAGCTTCGAATTATGAAGGAAGTGGTTTATTG‐3′ (forward) and 5′‐CCGCGGTACCGTCGATCACTTGCCCCACTCCATG‐3′ (reverse); SMS1‐CT (aa 348–413) using 5′‐CTCAAGCTTCGAATTCACACTATGGCCAATCAGC‐3′ (forward) and 5′‐CCGCGGTACCGTCGATTATGTGTCATTCACCAGCC‐3′ (reverse); SMSr‐NT (aa 1–151), 5′‐CTCAAGCTTCGAATTATGGCAGGTCCTAATC‐3′ (forward) and 5′‐CCGCGGTACCGTCGATCACTTCCAGTATTCTGGGTC‐3′ (reverse); SMSr‐CT (aa 364–415), 5′‐CTCAAGCTTCGAATTCATACTCTGGCCAATACC‐3′ (forward) and 5′‐ CCGCGGTACCGTCGATCATCCAATTAGTCTTTTC‐3′ (reverse).The N‐terminal (NT) or C‐terminal (CT) cytosolic regions of SMS1 and SMSr were subcloned into the pAcGFP‐C1 vector  via In‐Fusion cloning (Clontech‐Takara Bio) at the S‐transferase (GST)‐tagged SMS1‐CT and SMSr‐NT were generated by in‐fusion cloning. The pGEX‐6P‐1 vector  was linearized at the EcoRI and SalI sites, and amplified gene with 15 bp extensions homologous to vector ends. We generated SMS1‐CT using 5′‐GGGATCCCCGGAATTCCACACTATGGCCAATCAGC‐3′ (forward) and 5′‐GTCGACCCGGGAATTCTATGTGTCATTCACCAGCC‐3′ (reverse) and SMSr‐NT using 5′‐GAATTCCCGGGTCGAATGGCAGGTCCTAATCAAC‐3′ (forward) and 5′‐GGCCGCTCGAGTCGATCACTTCCAGTATTCTGGGTCC‐3′ (reverse).Glutathione The cDNAs encoding DGK isozymes  that were subcloned into the expression plasmid, p3×FLAG‐CMV (Sigma–Aldrich), for expression in mammalian cells were generated as described .EcoRI/SalI sites of the 3×FLAG CMV 7.1 vector. The following DGKζ mutants were generated using the following primers: DGKζ‐NT (aa 1–283), 5′‐GGTGGTGAATTCAATGGAGCCGCGGGACGG‐3′ (forward) and 5′‐ACGCGTCGACCTAGAAGGGTCTCCAGCGGCC‐3′ (reverse); DGKζ‐CD (aa 284–640), 5′‐CCGGAATTCAATCATCAGGCCCACCCCC‐3′ (forward) and 5′‐ACGCGTCGACCTACACCGGCTGCTGGTCG‐3′ (reverse); DGKζ‐CD‐a (aa 284–641), 5′‐CCGGAATTCAATCATCAGGCCCACCCCC‐3′ (forward) and 5′‐ACGCGTCGACCTACTCAGGCCCTGCCTCGG‐3′ (reverse); DGKζ‐CD‐b (aa 433–640), 5′‐CCGGAATTCAGACCGAGATGAAGGCGCC‐3′ (forward) and 5′‐ACGCGTCGACCTACACCGGCTGCTGGTCG‐3′ (reverse); DGKζ‐CT (aa 641–928) using the primers 5′‐CCGGAATTCACCAGAGCAGTTGCGCATCC‐3′ (forward) and 5′‐ACGCGTCGACCTACACAGCCGTCTCCTGGTC‐3′ (reverse).N‐terminal 3×FLAG‐tagged human DGKζ mutants were generated by PCR and inserted into the 2‐GGSA‐WSHPQFEK) was cloned into the XhoI/BglII site of the pCAGGS vector [BglII site of pCAGGS‐C‐TEV‐Twin‐Strep via In‐Fusion cloning. The following primers were used to amplify FL DGKζ: forward, 5′‐TTTTCAAGGCAGATCTATGGAGCCGCGGGACG‐3′; reverse, 5′‐AGAGGGAAAAAGATCTCTACACAGCCGTCTCCTGG‐3′. N‐terminal Twin‐Strep‐tagged DGKζ was subcloned into pOET3 vector . The following primers were used, forward, 5′‐TTTTCAAGGCAGATCTATGGAGCCGCGGGACG‐3′; reverse, 5′‐TTATTAATTAAGATCTCTACACAGCCGTCTCCTGG‐3′.The plasmid expressing N‐terminal Tobacco Etch Virus (TEV) protease cleavable Twin‐Strep‐tag (ENLYFQGS‐WSHPQFEK‐(GGGS)S vector  to gener−1 penicillin/100 μg·mL−1 streptomycin  at 37 °C in an atmosphere containing 5% CO2. The plasmids were transiently transfected using PolyFect  according to the manufacturer's instructions or using polyethylenimine Max  [−1, pH 8.0) were preincubated for 10 min at a 1 : 3 ratio (20 μg DNA: 60 μL polyethylenimine Max) in 750 μL of Opti‐MEM before transfection. The cells overexpressing recombinant proteins were harvested after 24 h and the pellets were resuspended in 40% (v/v) glycerol diluted in phosphate‐buffered saline. The cell samples were flash‐frozen in liquid nitrogen and stored at −80 °C until use.HEK293 cells  were maintained in Dulbecco's modified Eagle's medium  supplemented with 5% FBS (Thermo Fisher Scientific) and 100 U·mLPA, USA) . The exp2 in the dark. Volume of the medium was kept at 20–30% of flask volume. To generate recombinant baculovirus was generated using pOET3 vector and the flashBAC system (Oxford Expression Technologies) as described previously [Sf9 cells were maintained in Sf‐900 II serum‐free medium  in sterile Erlenmeyer flask at 120 r.p.m. and 28 °C without COeviously .HEK293 cell lysates expressing V5‐tagged SMS1, SMS2, SMSr, or their AcGFP‐tagged mutants and 3×FLAG‐tagged DGKs  or their mutants were subjected to immunoprecipitation with anti‐V5 (MA5‐15253) or anti‐GFP (#598) antibody and Protein A/G PLUS‐agarose beads (Santa Cruz Biotechnology) as described previously .S‐transferase‐fused SMS1‐CT and SMSr‐NT were bacterially expressed and highly purified using glutathione‐Sepharose beads . Twin‐Strep (TS)‐tagged DGKζ (TS‐DGKζ) was expressed by mammalian cells and highly purified using Strep‐Tactin XT beads .Glutathione S‐transferase pull‐down assays were performed as previously [m Tris–HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1% (v/v) Triton X‐100, and 1 mm phenylmethylsulfonyl fluoride. Purified Twin‐strep tagged DGKζ was incubated with the beads for 2 h at 4 °C with constant rocking. Then, the beads were washed five times with buffer. The washed beads washed were then boiled in SDS sample buffer, and the extracts were analyzed by western blotting.Glutathione eviously . Purifiem Tris–HCl, pH 7.4, containing 150 mm NaCl, 10% (v/v) glycerol, 1 mm PMSF, 0.1 mm DTT, 20 μg·mL−1 aprotinin, 20 μg·mL−1 leupeptin, 20 μg·mL−1 pepstatin, and 20 μg·mL−1 soybean trypsin inhibitor) containing detergents (1% (w/v) n‐dodecyl‐β‐d‐maltoside (DDM) and 0.2% (w/v) cholesteryl hemisuccinate (CHS)). The supernatant (1% DDM soluble fraction) was isolated by ultracentrifugation at 100 000 g for 30 min at 4 °C and then purified using Strep‐Tactin XT beads. The beads were washed with the lysis buffer containing 0.05% DDM and 0.01% CHS. Subsequently, the bound proteins were eluted with the lysis buffer containing 0.05% DDM, 0.01% CHS, and 2.5 mm d‐desthiobiotin (Sigma–Aldrich). TS‐tagged DGKζ was purified using Strep‐Tactin XT beads without detergents.C‐terminally TS‐tagged human SMS1 and SMSr (SMS1‐TS and SMSr‐TS), and N‐terminally TS‐tagged DGKζ (TS‐DGKζ) were expressed in HEK293 cells. SMS1‐TS and SMSr‐TS were lysed via lysed via homogenization on ice with ice‐cold lysis buffer (20 mDiacylglycerol kinase activity was measured using liquid chromatography–tandem mass spectrometry (LC–MS/MS) as previously described .Western blotting was carried out as previously described . Equal qt test for the comparison of two groups or one‐way ANOVA followed by Tukey's or Dunnett's post hoc test for multiple comparisons using graphpad prism 8  to determine any significant differences. P < 0.05 was considered significant.Data are represented as the means ± SDs and were analyzed by the Student's We first examined the interaction of all DGK isozymes with SMSr Fig. . We confNext, we determined the interaction between 10 DGK isozymes and SMS1 Fig. . As prevWe next examined the interaction between 10 DGK isozymes and SMS2 Fig. . As prevWe next attempted to determine a DGKζ‐interaction region in SMSr. AcGFP‐tagged N‐terminal (AcGFP‐SMSr‐NT) and C‐terminal (AcGFP‐SMSr‐CT) cytosolic regions of SMSr were generated Fig. , and theA DGKζ‐interaction region in SMS1 was also identified. For this purpose, AcGFP‐tagged N‐terminal (AcGFP‐SMS1‐NT) and C‐terminal (AcGFP‐SMS1‐CT) cytosolic regions of SMS1 were made Fig. . InteresTo narrow the DGKζ‐interaction area in the N‐terminal cytosolic region of SMSr, the DGKζ‐interaction activity of SAMD alone of SMSr was tested. Figure To determine an SMSr‐interaction region in DGKζ, we divided the protein into three parts, DGKζ‐NT, DGKζ‐CD, and DGKζ‐CT Fig. , and detNext, the interaction of DGKζ‐NT, DGKζ‐CD, and DGKζ‐CT with SMS1‐CT was examined. Among them, 3×FLAG‐DGKζ‐CD most strongly interacted with AcGFP‐SMS1‐CT Fig. . AlthougWe further divided the catalytic domain of DGKζ into CD‐a and CD‐b Fig.  and deteWe next examined whether DGKζ directly binds to SMSr and SMS1. GST‐fused SMSr‐NT and SMS1‐CT Fig.  were bacin vitro [in vitro in the presence of 34:1 (16:0/18:1)‐DG. Intriguingly, SMSr, but not SMS1, moderately inhibited DGKζ activity  in vitro . Moreoveity Fig. . Moreoveity Fig.  also attity Fig. . These rIn the present study, we demonstrated for the first time that DGKζ interacts with SMS1 and SMSr but not SMS2 Figs . DGKδ1 aWe previously reported that DGKδ associates with SMSr via the interaction between DGKδ‐SAMD and SMSr‐SAMD . AlthougSMSr, but not SMS1, inhibited DGKζ activity Fig. . TherefoIn the case of the SMSr–DGKζ interaction, SAMD in the N‐terminal cytosolic region of SMSr binds to DGKζ Fig. . In contSMSr‐SAMD interacted with DGKζ‐CD‐a Fig. . Howeverhttps://www.proteinatlas.org/ENSG00000149091‐DGKZ/tissue), SMSr (https://www.proteinatlas.org/ENSG00000156671‐SAMD8/tissue), and SMS1 (https://www.proteinatlas.org/ENSG00000198964‐SGMS1/tissue) are ubiquitously expressed in a variety of tissues [Although SMSr is a DG‐generating enzyme, its CPES activity is very low . We rece tissues . These rIn summary, in the present study, we demonstrated for the first time that DGKζ interacts with SMS1 and SMSr but not SMS2 Fig. . DGKδ alThe authors declare no conflict of interest.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/2211‐5463.13628.The peer review history for this article is available at MF primarily designed and conducted the experiments and analyzed the data. CM, YN, and RS designed and conducted the experiments and analyzed the data. CM, FS, and MF wrote the manuscript. CM and FS conceived the research. All authors revised the manuscript and approved its final version.Fig. S1. Sequence alignment of SMSr‐SAMD and SMS1‐SAMD. (A) Sequence alignment of SMSr‐SAMD (aa 12–78) and SMS1‐SAMD (aa 7–70). Sequence alignment was created using Clustal Omega provided by EMBL's European Bioinformatics Institute (EMBL‐EBI). Compared with SMSr‐SAMD, white letters on a black background indicate fully conserved residues, and black letters on a gray background indicate strongly similar residues. (B) Amino acid identities between the SAMDs of SMSr and SMS1. Amino acid identity and similarity were determined using Pairwise Sequence Alignment provided by the European Molecular Biology Open Software Suite (EMBOSS).Fig. S2. Multiple sequence alignment of the C‐terminal regions of SMS1, SMS2, and SMSr. (A) Multiple sequence alignment of the C‐terminal regions of SMS1‐CT (aa 348–413), SMS2‐CT (aa 292–365), and SMSr‐CT (aa 364–415). Multiple sequence alignment was created using Clustal Omega provided by EMBL's European Bioinformatics Institute (EMBLEBI). Compared with SMS1‐CT, white letters on a black background indicate fully conserved residues, and black letters on a gray background indicate strongly similar residues. (B) Amino acid identities between the C‐terminal regions of SMS1, SMS2, and SMSr. Amino acid identity and similarity were determined using Pairwise Sequence Alignment provided by the European Molecular Biology Open Software Suite (EMBOSS).Fig. S3. Sequence alignment of DGKζ‐CD and DGKι‐CD. (A) Sequence alignment of DGKζ‐CD (aa 293–622) and DGKι‐CD (aa 374–702). Sequence alignment was created using Clustal Omega provided by EMBL's European Bioinformatics Institute (EMBL‐EBI). Compared with DGKζ‐CD, white letters on a black background indicate fully conserved residues, and black letters on a gray background indicate strongly similar residues. (B) Amino acid identities between DGKζ‐CD and DGKι‐CD. Amino acid identity and similarity were determined using Pairwise Sequence Alignment provided by the European Molecular Biology Open Software Suite (EMBOSS).Click here for additional data file.


The crystal structure of bucetin, an analgesic and antipyric similar to phenacetin, is presented. N-(4-ethoxyphenyl)-3-hydroxybutanamide], C12H17NO3, the molecule is in an extended conformation as illustrated by the C—O—C—C torsion angle [170.14 (15)°] in the ethoxy group and the subsequent C—N—C—C [−177.24 (16)°], N—C—C—C [170.08 (15)°] and C—C—C—C [171.41 (15)°] torsion angles in the butanamide chain. In the crystal, the O—H group donates an intermolecular O—H⋯O hydrogen bond to the amide carbonyl oxygen atom and also accepts an intermolecular N—H⋯O hydrogen bond from an adjacent N—H group. The former forms 12-membered dimeric rings about inversion centers, and the latter form chains in the [001] direction. The overall hydrogen-bonded network is two-dimensional, with no propagation in the [100] direction.In the title compound, racemic bucetin [systematic name:  N-(4-Ethoxyphenyl)-3-hydrobutanamide, popularly known as bucetin, is an analgesic and antipyric that is similar in structure to phenacetin [N-(4-ethoxyphenyl)acetamide]. Once thought to be a better substitute for phenacetin hydroxylamine and 1-ethoxy-4-nitrosobenzene) on PGE2 synthesis and a possible reduction of COX-2 expression acetamide  and 2.8611 (19) Å for N—H⋯O was obtained from Sigma-Aldrich, St. Louis, MO and was used without further purification. Single crystals of racemic bucetin were prepared by slow cooling of a nearly saturated solution of bucetin in boiling deionized water.The title compound, CCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623002316/vm4059sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623002316/vm4059Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623002316/vm4059Isup3.cmlSupporting information file. DOI: 2247342CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure, the Th atom (site symmetry 3∞[ThBr4/2Br4/2]. In comparison with the previous crystal structure refinement , the current rerefinement resulted in much higher preciscion of the lattice parameters and the atomic coordinates.Single crystals of α-ThBr al. 1974. J. Less A comparison of the structural parameters of the original crystal structure refinement and of the current rerefinement is given in Table 1The crystal structure of α-ThBra, site symmetry f, site symmetry 1) in the asymmetric unit. The Th atom is surrounded by eight Br atoms to form a tetragonal-disphenoidal coordination polyhedron. The Th—Br bond lengths of 4 × 2.9100 (4) Å and 4 × 3.0107 (4) Å are in good agreement with previously reported values of 2.909 and 3.020 Å , with values of 2.85 and 3.12 Å  Å in α-ThBr4 is shorter compared to β-ThBr4, with a value of 4.8774 Å  Å and 4 × 6.70680 (19) Å.Fig. 1−3 mbar; 1 bar = 105 Pa) at least three times before use. Aluminium bromide  was sublimed in vacuo before use; β-ThBr4 was prepared according to a literature protocol  using a fine-vacuum line and a glove-box (MBraun). Silica ampoules were flame-dried under dynamic fine vacuum  and CuBr , and sealed under vacuum. The ampoule was heated in a furnace to 753 K at a rate of 1 K min−1 and kept at this temperature for 480 h for the reaction to take place. Afterwards, it was cooled to 330 K at a rate of 50 K d−1. Several colourless crystals of α-ThBr4 were obtained.A silica glass ampoule was loaded with β-ThBrCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623008908/wm4200sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2414314623008908/wm4200Isup2.hklStructure factors: contains datablock(s) I. DOI: 2300477CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The carbamate group has key bond lengths of 1.404 (6) Å (N—C), 1.330 (5) Å (O—C), and 1.201 (6) Å (C=O). The crystal contains intermolecular π–π interactions, as well as both type I and type II intermolecular I⋯I interactions.The molecular structure of  These interactions have Cg⋯Cg distances of 3.484 (3) and 3.589 (3) Å with slippages of 1.028 and 1.376 Å and angles of 0.00 (3)° . The supramolecular pillars are held together via both type I and type II intermolecular I⋯I interactions  with an angle of 147.68 (13)° and an I⋯I distance of 3.6630 (5) Å. The type II interaction is found between atoms C11—I2⋯I1 with an I⋯I distance of 3.8332 (5) Å and an angle of 46.69 (13)°.In the crystal, molecules of the title compound form pillars 4.et al., 2016et al., 2015I with iodine atoms at the C5 and C11 positions, but where the nitrogen atom has been alkylated with either a butyl or benzyl group. Structure ECUNUM bears bromine atoms at the C5 and C11 positions with a phenylcarbamate group on the nitrogen atom  = 1.2Ueq(C) for aromatic hydrogen atoms and Uiso(H) = 1.5Ueq(C) for the hydrogen atoms of the methyl group.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S205698902300230X/wm5676sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902300230X/wm5676Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902300230X/wm5676Isup3.cmlSupporting information file. DOI: 2247288CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-chromen-4-ylamino)benzoate, has been synthesized and characterized.A new coumarin derivative, methyl 2-(2-oxo-2 H-chromen-4-ylamino)benzoate, C17H13NO4 (1), was prepared by condensation between 4-hydroxycoumarin and methyl 2-aminobenzoate. It crystallizes in the orthorhombic space group Pca21 at 300 K. The molecule of compound 1 consists of the 2H-chromen-2-one part connected by an amine moiety (–NH–) to the methyl benzoate ring. The supramolecular array is formed by hydrogen bonds between the aromatic ring and the O atoms of the lactone and ester portions. The structural details match the spectroscopic data acquired from NMR and IR spectroscopy.Methyl 2-(2-oxo-2 These sDipteryx odorata Wild; Fabaceae family) by Vogel in 1820. Since then, more than 1300 coumarins have been identified from natural sources benzoate, 1 benzoate molecule , although the C4—N1—C9 angle at 130.9 (4)° is about 7° larger than in those molecules, presumably due to the intramolecular N1—H1⋯O2 hydrogen bond (Table 1The average C—C bond distance in the aromatic portion of the coumarin is 1.374 (7) Å, while the C9—C13, C9—C10 and C10—C11 bond lengths in the lactone portion are 1.450 (7), 1.353 (7) and 1.412 (7) Å, respectively, because of the partial localization of π-bonding within the ring. The C11—O3 and C12—O3 bond lengths are equivalent at 1.374 (7) and 1.373 (7) Å, respectively, while the C11=O4 distance is 1.204 (7) Å. The sum of the angles about N1 is 359 (3)°, implicating involvement of its lone pair in N—C π-bonding. This is supported by the N1—C9 and N1—C4 distances of 1.351 (6) and 1.391 (6) Å, respectively. Similar geometrical parameters are found in closely related structures °.1H and 13C NMR confirms the product as methyl 2-(2-oxo-2H-chromen-4-ylamino)benzoate. In the 1H NMR spectrum, there is a singlet at δ 3.74 ppm attributable to the methoxy group of the ester, the coumarin vinylic H atom appears at δ 5.31 ppm and a singlet is seen at δ 9.67 which can be assigned to N—H. In addition, there are eight aromatic H atoms between δ 7.44 and 8.14 ppm. In the 13C NMR spectrum, the methoxy group appears at δ 52.47 ppm, the two carbonyl C atoms at δ 166.50 and 161.40, and the vinylic and aromatic C atoms between δ 114 and 154 ppm.The NMR spectra are shown in Figs. 43.B⋯O4i) and the H atom from the aromatic ring (C7—H7⋯O4ii)  Table 1. These bon Fig. 6. The cryon Fig. 6 involves4.et al., 2016H-chromen-2-one -2H-chromen-2-one  was heated in a 50 ml Becher at 453 K for 1 h. A solution comprised of 30 ml of hot methanol and 30 ml of aqueous NaOH (1 mol l−1) was then added to the solid. This mixture was stirred for 30 min at 333 K and then filtered. The solid was washed with water, dried and used without further purification.The reaction was carried out according to the literature  I. DOI: 10.1107/S2056989023007351/mw2198Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023007351/mw2198Isup3.cmlSupporting information file. DOI: 2289922CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structure features C—H⋯O and C—H⋯N hydrogen bonds, which link the molecules into layers parallel to the (100) plane. IC—H⋯π interactions and weak van der Waals interactions occur between the layers. 25H17N3O5S2, intramolecular π–π interactions [centroid-to-centroid distance = 3.5640 (9) Å] are observed between the furan and benzene rings of the 4-cyanophenyl group. In the crystal, molecules are connected via C—H⋯O and C—H⋯N hydrogen bonds, forming layers parallel to the (100) plane. These layers are interconnected by C—H⋯π interactions and weak van der Waals interactions. Hirshfeld surface analysis indicates that H⋯H (30.2%), N⋯H/H⋯N (22.3%), C⋯H/H⋯C (17.9%) and O⋯H/H⋯O (15.4%) interactions make the most significant contributions to the crystal packing.In the title compound, C Reactions with ammonia, and primary and secondary amines are the most widespread. A primary amine will form a soluble sulfonamide salt in the presence of aqueous alkali (either KOH or NaOH). A secondary amine in the same reaction forms an insoluble sulfonamide. The most widely used sulfonylamide is sulfanilamide, an antibacterial drug that was first obtained in 1908 by the Austrian chemist Paul Josef Jakob Gelmo while he was trying to synthesize a dye for textile materials .In the title compound Fig. 2, the angCg1⋯Cg4 = 3.5640 (9) Å; Cg1 and Cg4 are the centroids of the furan (O1/C7–C10) and benzene rings (C19–C24), respectively, of one of the two 4-cyanophenyl)sulfonyl groups, respectively; slippage = 0.793 Å], ensures the stability of the molecular configuration.Intramolecular π–π stacking interactions  between the furan and a phenyl ring of one of the two (3-nitrophenyl)sulfonyl groups result in chains parallel to the a axis. In JOBTIF (space group P21/n), molecules are linked by pairs of C—H⋯O hydrogen bonds, forming inversion dimers. In CEGMIM (space group Pbca), molecules are connected by C—H⋯O interactions into sheets in the ab plane. In the crystal of CEGSUE , molecules associate via N—H⋯N and N—H⋯O hydrogen bonds, forming extended hydrogen-bonded sheets that lie parallel to the bc plane. The N—H⋯N hydrogen bonds propagate along the b-axis direction, while the N—H⋯O hydrogen bonds propagate along the c-axis direction. The crystal structure of OCABUR (space group P21/c) features C—H⋯O hydrogen bonds. In the crystal structure of AYUPUG (space group P21/c), weak C—H⋯O interactions connect the molecules in a zigzag manner along the a-axis direction. In the crystal of PONZIC (space group Pa axis by intermolecular C—H⋯O hydrogen bonds and π–π stacking interactions. In ROGJON (space group Pbca), the crystal structure features weak intermolecular N—H⋯O, C—H⋯O and C—H⋯N hydrogen bonds and π–π interactions.In PIMGUR  was added gradually to a solution of 2-(5-methyl-2-furyl)aniline  in pyridine (7 mL) under stirring and cooling in an ice–water bath. The mixture was stirred for 7 h at r.t. and after completion of the reaction , the mixture was poured into hydrochloric acid . The separated oil was washed with water until its crystallization. The obtained crystals were filtered off, dried, and recrystallized from an ethanol/dimethylformamide (DMF) mixture to give the target disulfonamide as a colourless solid. Single crystals were obtained by slow crystallization from an EtOH/DMF mixture . IR (KBr), ν (cm−1): 1156 (νs SO2), 1329 (νas SO2), 2237 (CN). 1H NMR  : δ 8.08 , 7.90 , 7.72 , 7.56 , 7.36 , 7.04 , 6.61 , 5.93 , 1.96 ; 13C{1H} NMR : δ 153.2, 147.9 (2C), 142.9, 134.0 (4C), 133.7, 132.2, 132.0, 129.6 (4C), 128.8, 128.7, 128.6, 117.9 (2C), 117.4 (2C), 112.0, 108.6, 13.4; MS (ESI) m/z: [M + H]+ 504. Elemental analysis calculated (%) for C25H17N3O5S2 %: C 59.63, H 3.40, N 8.34, S 12.74; found: C 60.00, H 3.27, N 8.56, S 13.03.6.Ueq(C) (1.5 for methyl groups). The hydrogen atoms of the methyl group containing the C11 atom were disordered over two positions with equal occupancies.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023006254/tx2071sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023006254/tx2071Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023006254/tx2071Isup3.cmlSupporting information file. DOI: 2282070CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, N—H⋯O and C—H⋯O hydrogen bonds form chains extending along the b-axis direction. Due to the disorder of the p-tolyl rings, short C⋯C distances are observed between adjacent chains.The quinoxaline moiety in the title molecule, C Lgaz et al., 2015e.g. Abad et al., 2021Among the various classes of nitrogen heterocyclic compounds, quinoxaline derivatives display a broad spectrum of biological and pharmacological activities ° apart in a 0.503 (2):0.497 (2) ratio. In the crystal, N3—H3A⋯O2 and C10—H10B⋯O2 hydrogen bonds (Table 1b-axis direction. Pairs of inversion-related chains show C16⋯C17i and C17⋯C16i  distances of 2.695 (4) Å, which is 0.71 Å less than the sum of the van der Waals radii and is likely due to the disorder involving this ring. The chains stack along the c-axis direction ° between the mean planes through the constituent rings. The s Table 1 form chan Figs. 2 and 3 ▸.H)-one was dissolved in 25 ml of dimethylformamide and 1.15 g (6.24 mmol) of 2-chloro-N-(p-tolyl)acetamide were added, followed by 1.0 g (7.5 mmol) of potassium bicarbonate, and a spatula tip of BTBA (benzyltributylammonium chloride) was used as a phase-transfer catalyst. The reaction was stirred for 2 h under reflux at 353 K. When the starting reagents had completely reacted, 500 ml of distilled water were added and a few minutes later the product precipitated. This was filtered off, dried and recrystallized from hot ethanol solution to yield light-yellow plate-like crystals of the title compound.1.00 g (6.24 mmol) of 3-methylquinoxalin-2(1I/σ(I) > 20 and chosen from the full data set with CELL_NOW :0.497 (2) ratio] of the disordered C12–C17 ring were refined as rigid hexagons.Crystal, data collection and refinement details are presented in Table 210.1107/S2414314623003577/vm4060sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314623003577/vm4060Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623003577/vm4060Isup3.cmlSupporting information file. DOI: 2254194CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Correction: BMC Ophthalmol 23, 279 (2023)https://doi.org/10.1186/s12886–023-03012-1After publication of the original article , the autThe original article has been updated and the correct values are also given below in bold.The second paragraph in page 4 should read:28.6 mg/dL, and apolipoprotein A1 131.6 mg/dL. A total of 782 patients in the alirocumab group had ≥ 2 consecutive LDL-C values < 15 mg/dL (0.39 mmol/L) and were matched to 2346 patients from the placebo group with similar baseline characteristics (Tables 4 and 5). Baseline characteristics of these patients included mean age 59 years, male sex (81%), diabetes (33%); and mean body mass index 27.3 kg/m2, LDL-C 2.0 mmol/L, lipoprotein (a) 18.8 mg/dL, and apolipoprotein A1 129.9 mg/dL.A total of 4305 patients in the alirocumab group had ≥ 2 consecutive LDL-C values < 25 mg/dL (0.65 mmol/L) and were matched to 4305 patients from the placebo group with similar baseline characteristics (Tables 2 and 3). Baseline characteristics of these patients included mean age 59 years, male sex (81%), diabetes (33%); and mean body mass index 28.3 kg/m2, LDL-C 2.1 mmol/L, lipoprotein (a)


The layers are connected by additional C—H⋯O hydrogen bonds and π-stacking interactions.In the title compound, the thiazine ring exhibits a screw-boat conformation. In the crystal, corrugated layers of molecules parallel to the  8H7NO3S, the nitrogen atom has a planar environment, and the thiazine ring exhibits a screw-boat conformation. In the crystal, corrugated layers of molecules parallel to the ab plane are formed by N—H⋯O and C—H⋯O hydrogen bonds together with C—H⋯π(ring) and S=O⋯π(ring) interactions. The layers are connected by additional C—H⋯O hydrogen bonds and π-stacking interactions. Hirshfeld surface analysis indicates that the most important contributions for the crystal packing are from H⋯O/O⋯H (49.4%), H⋯H (23.0%) and H⋯C/C⋯H (14.1%) interactions. The volume of the crystal voids and the percentage of free space were calculated as 75.4 Å3 and 9.3%. Density functional theory (DFT) computations revealed N—H⋯O and C—H⋯O hydrogen-bonding energies of 43.3, 34.7 and 34.4 kJ mol−1, respectively. Evaluation of the electrostatic, dispersion and total energy frameworks indicate that the stabilization is dominated via the electrostatic energy contribution. Moreover, the DFT-optimized structure at the B3LYP/ 6–311 G level is compared with the experimentally determined molecular structure in the solid state. The HOMO–LUMO behaviour was elucidated to determine the energy gap.In the title molecule, C The distorted screw-boat conformation places O2 in an axial position and O3 in a pseudo-equatorial position  Å, S1⋯Cg2 = 4.1655 (5) Å, S1=O2⋯Cg2i = 101.77 (3)°; symmetry code: (i) −x + y + z + Cg2⋯Cg2 = 3.7353 (5) Å, slippage = 1.55 Å] into a tri-periodic network structure  with 4.9% contribution to the HS is viewed at de + di = 3.30 Å. The C⋯C contacts  appearing as a bullet-shaped distribution of points make a contribution of 3.7% to the HS with the tip at de = di = 1.70 Å. The spikes of H⋯N/N⋯H contacts  with 3.2% contribution to the HS are viewed at de + di = 2.75 Å. Finally, the O⋯O  and C⋯N/N⋯C  contacts contribute 1.3% and 0.4%, respectively, to the HS. The Hirshfeld surface representations with the function dnorm plotted onto the surface are shown for the H⋯O/O⋯H, H⋯H and H⋯C/C⋯H interactions in Fig. 8a–c, respectively. The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of H⋯O/O⋯H, H⋯H and H⋯C/C⋯H interactions suggest that van der Waals interactions play the major role in the crystal packing u. Fig. 4, the whits Fig. 7e with 4ts Fig. 7f appearts Fig. 7g with 3⋯O Fig. 7h and C⋯⋯C Fig. 7i contacet al., 2011a,b) and the percentage of free space in the unit cell are calculated as 75.4 Å3 and 9.3%, respectively. Thus, the crystal packing appears compact and the mechanical stability should be substantial.The strength of the crystal packing is important for determining the response to an applied mechanical force. If the crystal packing results in significant voids, then the molecules are not tightly packed and a small amount of applied external mechanical force may easily break the crystal. To check the mechanical stability of the crystal, a void analysis was performed by adding up the electron densities of the spherically symmetric atoms contained in the asymmetric unit  is the sum of electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) energies  were calculated to be  for N1—H1⋯O3,  for C8—H8B⋯O2 and  for C5—H5⋯O1.The intermolecular interaction energies were calculated using the CEB3LYP/631G energy model available in et al., 2015Eele Energy frameworks combine the calculation of intermolecular interaction energies with a graphical representation of their magnitude EHOMO and ELUMO, electronegativity (χ), hardness (η), dipole moment (μ), electrophilicity (ω) and softness (σ) are compiled in Table 3η and σ are essential for assessing reactivity and stability. The electron transition from HOMO to LUMO energy levels is depicted in Fig. 11b]thiazin-3(4H)-one 1,1-dioxide ring. The energy band gap [ΔE = ELUMO − EHOMO] for the molecule is 11.7261 eV, and the energies of the frontier molecular orbitals, EHOMO and ELUMO, are −9.6740 eV and 2.0522 eV, respectively.The highest-occupied molecular orbital (HOMO), functioning as an electron donor, and the lowest-unoccupied molecular orbital (LUMO), acting as an electron acceptor, serve as vital parameters in quantum chemistry. A small energy gap signifies high molecular polarizability and enhanced chemical reactivity. The DFT calculations provided crucial insights into the reactivity and site selectivity of the molecular framework. Parameters such as 7.et al., 2016II was dissolved in 3 ml of acetic acid and added dropwise into a solution of potassium permanganate (1.81 mmol) in 6 ml of water. After stirring for one h at room temperature, a solution of sodium thiosulfate pentahydrate (20%wt) was added to react with excessive potassium permanganate. The precipitate obtained was filtered and recrystallized from ethanol to yield single-crystals suitable for X-ray structure analysis..3,4-Dihydro-29.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S205698902300868X/wm5698sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698902300868X/wm5698Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902300868X/wm5698Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S205698902300868X/wm5698Isup4.cmlSupporting information file. DOI: 2298958CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The various intermolecular interactions, such as N—H⋯O, C—H⋯N and C—H⋯O, were investigated using Hirshfeld surface analysis and the three-dimensional interaction energies were calculated. 29H27F2N3O6, which crystallizes in the monoclinic space group P21/c, the cyclohexenone ring is puckered and adopts an envelope conformation. The crystal structure features various intermolecular interactions, such as N—H⋯O, C—H⋯N and C—H⋯O. These interactions were investigated using Hirshfeld surface analysis and the three-dimensional interaction energies were calculated using the B3LYP/6–31 G energy density model.In the title compound, C The orientation of other two carboxylate groups at the C4 and C5 positions are described by the torsion angles C1—C6—C27—O6 = −15.4 (5)° (–syn-periplanar), C1—C6—C27—O5 = 167.2 (4)° (+anti-periplanar), C5—C6—C27—O5 = −17.2 (6)° (–anti-periplanar), C5—C6—C27—O6 = 160.1 (3)° (+anti-periplanar) and C3—C4—C23—O3 = 44.9 (5)° , C3—C4—C23—O4 = −136.4 (3)° , C5—C4—C23—O3 = −75.8 (4)° , C5—C4—C23—O4 = 102.9 (3)° . The orientation is due to the intermolecular N—H⋯O and C—H⋯O interactions.In the title compound Fig. 1, the cyc3.N⋯O1, C14—H14B⋯N3, and C24—H24B⋯O3 propane-1,2,3-tricarboxylate  was computed using two-dimensional fingerprint calculations  mapped over ns Fig. 5. The res6.CrystalExplorer 17.5 software calculates interaction energies between crystal molecular pairs. Energy calculations were carried out using the B3LYP/6-31G basis set within a default radius of 3.8 Å  is associated with a pair of yellow molecules with the short centroid distance R = 9.29 Å with rotational symmetry −x, y + z + Etot = −17.6 kJ mol−1) was observed for a pair of green molecules interacting at the longer centroid distance R = 12.86 Å; this is in accordance with the classical laws of electrostatics. In each of the energy terms, the dispersion component is dominant over the others.7.2SO4, then concentrated under reduced pressure to get the crude product. This was purified by silica gel column chromatography using n-heptane/ethyl acetate as eluent. The mixture was quenched in cold water and the organic layer was extracted with ethyl acetate, washed with 5% sodium bicarbonate solution, and dried over anhydrous sodium sulfate. Slow evaporation of the solvent lead to crystals of the title compound, which were recrystallized from ethanol solution.Piperidine (6 mmol) was added to ethyl cyanoacetate (30 mmol) and the mixture was stirred for 10 min. Then 4-fluorobenzaldehyde (20 mmol) was added dropwise and during the addition, the temperature of the reaction mass rose to 333 K (it should not be cooled), and the mass was stirred for 30 min. The temperature slowly came down to 293–298 K over 30 min. The progress of the reaction was monitored by TLC and found to be complete. Methylene chloride (30 ml) and water (20 ml) were added and the mixture was stirred for 10 min. The organic layer was separated and washed with sat. aq. NaCl solution and dried over anhydrous Na8.Uiso(H) = 1.2Ueq(C) (1.5 for methyl H atoms).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023003134/ex2065sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989023003134/ex2065Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023003134/ex2065Isup3.cmlSupporting information file. DOI: 2202315CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


However, it remains largely unclear how those cells develop. Here we show that transforming growth factor beta (TGF-β) signaling controls the development of TCRγδ+CD8αα+ IELs. Deletion of TGF-β receptors or Smad3 and Smad2 in bone marrow stem cells caused a deficiency of TCRγδ+CD8αα+ IELs in mixed bone marrow chimeric mice. Mechanistically, TGF-β is required for the development of TCRγδ+CD8αα+ IELs thymic precursors (CD44–CD25– γδ thymocytes). In addition, TGF-β signaling induced CD8α in thymic γδT cells and maintained CD8α expression and survival in TCRγδ+CD8αα+ IELs. Moreover, TGF-β also indirectly controls TCRγδ+CD8αα+ IELs by modulating the function of intestinal epithelial cells (IECs). Importantly, TGF-β signaling in TCRγδ+CD8αα+ IELs safeguarded the integrity of the intestinal barrier in dextran sulfate sodium (DSS)-induced colitis.γδ intestinal intraepithelial lymphocytes (IELs) constitute the majority of IELs with unique CD8αα T cell receptor (TCR)+ IELs, which express either TCRγδ+ or TCRαβ+ are usually classified into conventional and unconventional IELs according to their distinct phenotype and developmental pathways. γδ IELs are thought to be homed to the intestine immediately after their generation from γδT precursors in the thymus2. They comprise the vast majority of IELs in the small intestine and are essential to maintaining immune homeostasis in the intestinal territory, such as keeping the integrity of the gut barrier, limiting translocation of microbiomes, responding to antigens invasion, and healing tissue damage. Accumulated studies have shown that the function of γδ IELs is tightly related to their crosstalk with IECs; the dynamic movement of γδ IELs surrounding IECs results in more efficient immune surveillance and higher expression of antimicrobial or antiviral genes5.Intestinal intraepithelial lymphocytes (IELs) localize within the intestinal epithelium and predominate in the mucosal immune system; they are typically surrounded by intestinal epithelial cells (IECs) with a ratio of ~1:10 (IELs:IECs) in the small intestine+ homodimers that are considered the main cell resource for the production of cytokines like IFN-γ, IL-10, and IL-13 from γδ IELs1. However, it remains uncertain which factors determine the development of TCRγδ+CD8αα+ IELs. The development of γδT cells in the thymus undergoes several stages from DN1 to DN4 that can be classified by CD44 and CD25 expression, namely CD44+CD25– (DN1), CD44+CD25+ (DN2), CD44–CD25+ (DN3), and CD44–CD25– (DN4)8. However, it has been recently suggested that the Vγ7+ subset of IELs were generated in an extrathymic way, which relies on Butyrophilin-like (Btnl) molecules on IECs9, but this remains to be verified.Unlike systemic γδT cells settled in other sites, roughly 90% of γδ IELs have a specific phenotype of CD8αα12. TGF-β is enriched in the intestinal environment and represents one of the most important regulators in gut immune system as both IECs and immune cells, including IELs, contribute to TGF-β production14. Additionally, we and others have previously found that TGF-β signaling is crucial for the development of TCRαβ+CD8αα+ IELs and the generation of TCRαβ+CD8α+CD4+ IELs16. However, it remains unknown whether TGF-β plays a role in the development of TCRγδ+ CD8αα+ IELs.TGF-β signaling is involved in the development of various immune cells in the thymus and periphery, such as αβT cells, T regulatory cells (Tregs), and Th17 cells+CD8αα+ IELs in a Smad2 and Smad3 (Smad2/3)-dependent manner. Mice lacking TGF-β receptors or Smad2/3 have fewer TCRγδ+CD8αα+ IELs and thymic γδ precursors capable of migrating to the intestine. We discovered that TGF-β induces CD8α but not CD8β expression in DN γδ thymic precursors of TCRγδ+CD8αα+ IELs through the upregulation of RUNX family transcription factor 3 (Runx3) and downregulation of Th-inducing POZ-Kruppel factor (Th-Pok), two transcriptional factors important for CD8+T cell commitment in the thymus. Moreover, TGF-β directly regulates the maintenance of CD8α expression, proliferation, and apoptosis of TCRγδ+CD8αα+ IELs or indirectly influences these γδ IELs by modulating the function of IECs. Finally, we found that mice with TGF-β signaling deficiency in γδT cells were more vulnerable to bacterial attacks and had a worse response to DSS-induced IBD.We here show that TGF-β controls the development of TCRγδ18. The number of γδ IELs gradually increases from the day of mouse birth and remains stable in 1 month19. We generated mixed BM chimeric mice to investigate the role of TGF-β in the development of γδ IELs. BM cells from CD45.1 wild-type (WT) mice (CD45.1) were mixed with BM cells from CD45.2 Tgfbr1f/fEsr1-cre mice that had been pretreated with tamoxifen (R1 KO) or oil (R1 WT) for 5 days in a ratio 1:6 (CD45.2:CD45.1). The mixed BM cells were then transferred into irradiated recombination-activation gene 1-deficient (Rag1−/−) mice. The Rag1−/− mice were sacrificed to examine IELs populations 4–5 weeks later  cells and reside in the intestine after development in the thymusced Fig. , while tsed Fig. . The fresed Fig.  and totased Fig.  were alssed Fig.  and lympsed Fig.  showed nTgfbr2f/fEsr1-cre mice (pretreated with tamoxifen (R2 KO) or oil (R2 WT) for 5 days) and obtained similar results. Specifically, the frequency and total cell number of TCRγδ+CD8αα+ IELs from R2 KO BM-transferred mice were decreased compared to other control mice  or WT control  BM cells and found that TCRγδ+CD8αα+ IELs were significantly reduced in Smad2/3dko mice 20. We next examined subsets of TCRγδ+CD8αα+ IELs from Smad2/3dko BM chimeric mice and found that their CD8αα+ Vγ1  γδT cell population were all diminished in R1 KO BM-derived thymocytes  had no difference between R1 WT and R1 KO mice  and αEβ7 integrin , and γδT cells in the thymus express a high level of CCR9 that is downregulated when they arrive in the intestine accompanied by CD103 upregulation in mice22. We examined CCR9 on thymic DN4 γδT cells from CD45.1 + R1 WT and CD45.1 + R1 KO mixed BM chimeric mice. Both the frequency of CCR9+ cells and MFI of CCR9 expression in DN4 thymic γδT cell precursors  analysis of TCRγδ+CD8αα+ IELs isolated from the mixed BM chimeric mice 4–5 weeks post-BM transplantation as shown in Fig. https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA739380). Consistent with our flow cytometry analysis, the gene expression of CD103 (Itgae) was substantially downregulated in TCRγδ+CD8αα+ IELs from R1 KO BM chimeric mice, while the gene of CCR9 (Ccr9) was comparable  is another factor that potentially impacts the migration of γδ IELs, and RNA-seq revealed no difference of Itga4 in TCRγδ+CD8αα+ IELs  was downregulated in thymic γδT cells by TGF-β1 , Aatk (apoptosis-associated tyrosine kinase), and Bcl2l14  were all upregulated in R1 KO TCRγδ+CD8αα+ IELs according to our RNA-seq data  or tamoxifen (R1 KO)-treated Tgfbr1f/fEsr1-cre mice for 5 days and examined the mRNA expression of Butyrophilin-like (Btnl1), IL-15 and Myd88 in IECs. These genes are expressed on IECs and suggested to be important for TCRγδ+CD8αα+ IELs generation28. In R1 KO IECs, the expression of Btnl1  or Smad3−/− mice with IECs from WT or Smad3−/− mice with a ratio 1:10  and RegIIIβ , such as Il17a, Il23a, and Ifng31 were all upregulated in TCRγδ+CD8αα+ IELs in TGF-β receptor I-deficient mice  approach. The intestinal structure of Smad3−/− mice exhibited more damage with heavier bacteria located within or under the epithelial layer than WT mice  that contained fewer TCRγδ+CD8αα+ IELs  was diminished in the absence of TGF-β signaling, but CD44−CD25+TCRγδ+ thymocytes (DN3-γδT) were not much altered. This suggests that TGF-β might start working in the stage when γδT cells are developing from DN3 to DN4. Intriguingly, we found more CD44+CD25−TCRγδ+ thymocytes, which exhibit DN1-like phenotype, in TGF-β receptor I-deficient mice, suggesting that TGF-β signaling controls this subset of CD44+CD25−TCRγδ+ thymocytes37. However, the mechanism underlying this abnormal increase in this unique type of CD44+CD25−TCRγδ+ thymocytes remains unknown. As TCRγδ+ thymocytes are presented as either DN3 or DN4 by surface staining38, these CD44+CD25−TCRγδ+ cells are likely recirculated peripheral γδT cells, which are activated in the absence of TGF-β or are converted from DN4 CD44−CD25− γδT cells by regaining expression of CD44 due to the Tgfbr1 deficiency. Nevertheless, the decrease in CD44−CD25−TCRγδ+ DN4 thymocyte should contribute to the deficiency of TCRγδ+CD8αα+ IELs in the absence of TGF-β signaling.Consistent with the crucial role of TGF-β in the thymic environment+CD8αα+ IELs was not affected by TGF-β because their expression of the essential chemokine CCR9 and integrin α4β7 for homing to the gut intraepithelial layer was comparable between WT and TGF-β-deficient mice. However, the ability of γδ IELs to reside in the gut could be degraded due to CD103 low expression on TCRγδ+CD8αα+ IEL in TGF-β signaling-deficient mice. As γδ IELs normally express CD103 rather than CCR9 in the gut, CD103 is more important for γδT cells to settle down in the gut. This notion is further supported by the evidence that TGF-β induces more expression of CD103 in normal thymic γδT cells and gut T cells23. Collectively, TGF-β promotes the development of the thymic precursor CD25−CD44−TCRγδ+ cells of TCRγδ+CD8αα+ IELs and increases the expression of their gut-resident integrin CD103, which together provide a crucial step to fulfill a sufficient number of γδ IELs.The migration ability of thymic γδ precursors of TCRγδ+CD8αα+ IELs in the gut. Therefore, we have revealed that TGF-β induces expression of CD8α in thymic γδT cells and maintains CD8α expression in mature TCRγδ+CD8αα+ IELs. For thymic γδT cells, TGF-β induces CD8α by regulating the balance of Runx3 and Th-Pok expression, i.e., via upregulation of Runx3 and downregulation of Th-Pok, although the detailed molecular mechanism by which TGF-β regulates these two transcriptional factors remains to be elucidated. Strikingly, we believe that the role of TGF-β in thymic γδT cells also includes facilitating those cells to maintain the potential to develop into CD8αα+ homodimers, instead of CD8αβ+ heterodimers, based on our data that CD8β on thymic γδT cells tends to be inhibited by TGF-β treatment. In splenic γδT cells, however, TGF-β is capable of driving both CD8α and CD8β upregulation, suggesting that splenic γδT cells are unlikely the precursors of TCRγδ+CD8αα+ IELs. For γδ IELs, TGF-β upregulates or maintains CD8α expression on TCRγδ+CD8αα+ IELs but does not induce it in TCRγδ+CD8α−β− IELs. This indicates that TGF-β promotes the development of TCRγδ+CD8αα+ IELs not through the conversion of TCRγδ+CD8α−β− IELs into TCRγδ+CD8αα+ IELs.However, the aforementioned function of TGF-β in the thymic γδT precursors clearly cannot explain the unique phenotype of TCRγδ−CD8−γδT cells in the thymus, spleen, and IEL have the distinct potential to be CD8α+ under stimulation of TGF-β1. In this regard, TGF-β1 increases CD8α but decreases CD8β in thymic γδT cells, upregulates both CD8α and CD8β in splenic γδT cells, and fails to induce CD8α and CD8β in TCRγδ+CD8α−β− IELs. Though the underlying mechanisms remain unknown, it could be due to the developing stage and tissue-specific imprint. Thymic γδT cells are less developed compared to splenic γδT cells or γδ IELs and, therefore, have a higher potential to be induced to express CD8α+. Splenic γδT cells, however, tend to express CD8αβ+ rather than CD8αα+ in response to sufficient TGF-β stimulation. The biological significance of this feature remains unknown, but it suggests that splenic γδT cells are unlikely to be the precursors of TCRγδ+CD8αα+ IELs. TCRγδ+CD8α−β− IELs should go through a similar developmental process to TCRγδ+CD8αα+ IELs but cannot express CD8αα, suggesting that they are stable in the CD8α−β− phenotype and might be terminally differentiated and, thus, less likely convert to be CD8α+ when encountering stimuli such as TGF-β. As expected39, we showed that the decrease in TCRγδ+CD8αα+ IELs in the absence of TGF-β signaling was not due to the suppression of proliferation but can be contributed by the increase in the apoptosis of the knockout TCRγδ+CD8αα+ IELs.Intriguingly, CD4+ phenotype that is distinct from γδT cells in other tissues4. As a crucial cytokine produced by IECs, TGF-β also indirectly modulates the maintenance and proliferation of TCRγδ+CD8αα+ IELs by regulating the expression of several genes on IECs, which are known to tightly influence the development of TCRγδ+CD8αα+ IELs40. Btnl is expressed on IECs and determines the maturation and expansion of Vγ7+ IELs extrathymically regardless of food antigen and microbiomes;19 IL-15 and IL-15Rα complexes are abundant in IECs and critical for proliferation and survival of TCRγδ+CD8αα+ IELs; MyD88-deficient mice also have less TCRγδ+CD8αα+ IELs due to IL-15 shortage42. We showed that the expression of Btnl, IL-15, and MyD88 on IECs are all downregulated in TGF-β signaling-deficient mice. Reduction of the expression of these genes led Smad3−/− IECs unable to maintain CD8αα+ on TCRγδ+CD8αα+ IELs in an IECs–IELs coculture system. However, TCRγδ+CD8α−β− IELs were rarely to be CD8α+ even co-cultured with WT IECs, further confirming their inability to become TCRγδ+CD8αα+ IELs. Both TCRγδ+CD8αα+ and TCRγδ+CD8α−β− IELs showed greater proliferation when co-cultured with Smad3−/− IECs, suggesting that factors promoting γδ IELs proliferation were released more from Smad3−/− IECs, although this remains to be fully understood. γδ IELs developed in an intestinal environment lacking Smad3 show a lower frequency of CD8αα+, Vγ7, and Vγ4 populations, indicating that the interaction between IECs and IELs is also important for the development and/or function of TCRγδ+CD8αα+ IELs.In addition, IECs provide an organ-specific environment for γδ IELs to obtain a special CD8αα20. As shown previously44, TGF-β signaling in γδ IELs is crucial for intestinal homeostasis and the control of DSS-induced IBD. Supporting this conclusion is the observation that depletion of TGF-β signaling in γδT IELs leads to increased susceptibility to and exacerbation of DSS-induced colitis, as evidenced in both Smad3−/− mice and γδT cell-specific TGF-β receptor I-deficient mice, due to enhanced bacterial invasion and damage to the epithelial barrier resulting from the reduction of γδ IELs in these knockout mice. Furthermore, TGF-β-deficient TCRγδ+CD8αα+ IELs exhibit upregulation of pro-inflammation cytokines IFN-γ, IL-6, IL-21, IL-23α, and IL-12α, as well as downregulation of antimicrobial proteins such as RegIIIγ and RegIIIβ31. In addition to its importance in maintaining the integrity of the murine intestinal barrier, TGF-β has been shown to be effective in enhancing the cytotoxicity of expanded human Vδ2 cells in combination with IL-15, likely through the upregulation of CD103 and IL-9 expression in the presence of TGF-β46.Due to their special localization, rapid activation, and broad antigen recognition spectrum, γδ IELs serve as the first line of the intestinal immune system to detect bacteria invasion and maintain the integrity of the epithelial barrier+CD8αα+ IELs are CD8αα+Vγ7+ and CD8αα+Vγ1+, suggesting that TGF-β has the potential to alter the TCR repertoire and modulate the differentiation of γδ IELs. This hypothesis is further supported by the weaker ability of γδ IELs lacking TGF-β signaling to maintain the integrity and homeostasis of the intestinal barrier, indicating that the remaining γδ IELs without TGF-β exhibit distinct characteristics that may be caused by differences in TCR repertoire. However, we have yet to determine the detailed TCR repertoire specificity of γδ IELs lacking TGF-β signaling compared to cells from WT mice. We plan to investigate this issue in future studies.According to our results, the main diminished populations of TCRγδ+CD8αα+ IELs, which play a crucial role in maintaining immune homeostasis and resisting DSS-induced inflammation in the gut.In summary, we have elucidated a previously unrecognized mechanism in which TGF-β is a key factor in the formation and development of TCRγδRag1−/−, CD45.1, TCRδ ER Cre mice used in this study were purchased from The Jackson Laboratory. Tgfbr1f/fEsr1-cre, Tgfbr2f/fEsr1-cre, Smad2f/fER Cre, Smad3−/−, Smad2/Smad3 double KO mice , and Tgfbr1f/fTCRδERCre (Tgfbr1f/fcrossed with TCRδ ER Cre) mice were housed and bred in the animal facility in National Institute of Dental and Craniofacial Research. All mice were housed in specific pathogen-free conditions with adequate food and water supply, with 12 h light/dark cycle, 50% humidity, and room temperature between 25 to 27 °C, and the maximum number of mice in each cage is 5. All procedure of animal studies was performed under the National Institutes of Health guidelines for the use and care of live animals and were approved by the Animal Care & Use Committee (ACUC) of the National Institute of Dental and Craniofacial Research (NIDCR).6- to 8-week-old C57BL/6, TM Foxp3/Transcription Factor Staining Buffer Set (Cat# 00-5523-00) were from eBioscience. Mouse TCR γ/δ T cell Isolation Kit (130-092-125) was from Miltenyi Biotec.Anti-mouse CD45.1 (A20), anti-mouse CD45.2 (104), anti-mouse CD45 (30-F11), anti-mouse TCR γ/δ (GL3), anti-mouse TCR β (H57-597), anti-mouse CD8α (53-6.7), anti-mouse CD8β (YTS156.7.7), anti-mouse Vγ1.1 (2.22), anti-mouse Vγ1.2 (4B2.9), anti-mouse Vγ7 (F2.67), anti-mouse Ki67 (16 A8), anti-mouse CD25 (PC61), anti-mouse CD44 (IM7), anti-mouse CD45RB (C363-16A), anti-mouse CD103 (2E7), anti-mouse CCR9 (9B1), anti-mouse IFN-γ (XMG1.2), anti-mouse TNF-α (MP6-XT22), anti-mouse IL-17A (TC11-18H10.1), anti-mouse CD4 (GK1.5), and anti-mouse CD326 (G8.8) were purchased from Biolegend. Purified anti-mouse CD3 (145-2C11) was purchased from Bio X Cell. Recombinant mouse IL-2 (402-ML) and human TGF-β1 (240-B) were purchased from R&D Systems. SB431542 was obtained from Selleckchem (Cat# S1067). Cell Proliferation Dye eFluor™ 450 (Cat# 65-0842-90), eBioscience Cells were collected and adjusted into appropriate density for antibody staining. For cell surface marker staining, cells were incubated with antibodies for 20 min at 4 °C in the dark. Intracellular staining for cytokines detection, cells were treated with PMA (10 ng/mL), ionomycin (250 ng/mL), and Golgi-Plug (diluted by 1:1000) for 4 h at 37 °C incubator. After being stained with surface markers, cells were fixed with the fixation/permeabilization buffer solution according to the manufacturer’s instructions. Apoptosis of cells was investigated by staining Annexin V and 7-AAD solution diluted with Annexin binding buffer for 30 min at room temperature. Intranuclear staining were performed by using fixation/permeabilization buffer solution according to the manufacturer’s instructions.15. Small intestines were removed from Peyer patches, opened, and washed with 1× PBS several times to clean gut content, then cut into four to six pieces and incubated in IEL buffer  for 20 min in 37 °C incubator with shaking. Suspensions were filtered by 70 μm and 40 μm strainer, centrifuged on a 44 and 70% percoll density gradient at 1800 rpm for 20 min with brake 0. IELs can be obtained on the layer between 44% and 70% percoll.IELs from mouse small intestines were obtained as described previously19. Mouse small intestines were opened and washed with cold PBS to remove bacteria and gut content. Clean intestines were cut into small pieces and incubated in DMEM supplemented with 5% FBS, 5 mM EDTA and 0.145 mg/mL dithiothreitol for 20 min on a turning wheel. Tissue pieces were transferred into a new tube with 5–10 mL medium, with a vortex of 15 s three times to get more epithelial cells; all the media were collected into a container with suspensions, filtered by 70 and 40 μm strainer, centrifuged 1000 rpm for 10 min at 4 °C; cells were suspended by lysis buffer for 3 min and washed once by PBS. Stained by flow antibodies for FACS sorting, CD45−CD326+ cells from above were gated and collected for experiments.IECs were isolated in a way as described in the previous publication+Tgfbr1f/fEsr1-cre or Tgfbr2f/fEsr1-cre mice (treated with tamoxifen for 5 days) and CD45.1+ C57BL/6 mice. CD45.2+ and CD45.1+ BM were mixed with a ratio of 1:6, then injected into irradiated (450 rads) Rag1−/− mice intravenously. Four to five weeks later, mice were sacrificed and cell populations were isolated for staining. Age-matched littermates of Smad3+/+ or Smad3−/− recipients were irradiated (450 rads) 6 h before BM cell transfer. BM cells were isolated from C56BL/6 J mice and injected intravenously into Smad3+/+ or Smad3−/− recipients. One month after transfer, IELs were isolated and analyzed by flow cytometry.BM cells were isolated from CD45.2+CD45+TCRγδ+ cells, and cultured in 96 round plate in the RPMI-1640 complete medium, supplemented with anti-CD3 , IL-2 (10 ng/mL), with or without TGF-β1 , and SB431542 (5 uM). TCRγδ+CD8αα+ IELs and TCRγδ+CD8α−β− IELs were sorted from IELs by FACSAria Sorter with gating live+CD45+TCRγδ+CD8α+β− or live+CD45+TCRγδ+CD8α−β− specifically and cultured in RPMI-1640 complete medium supplemented with anti-CD3, IL-2 (100 U/mL), IL-3 (10 ng/mL), IL-4 (10 ng/mL), and IL-15 (10 ng/mL) as previously described in ref. 19, treated with or without TGF-β1 , or SB431542 (5 uM).Pure mouse γδT cells were sorted from mouse splenocytes or thymocytes by FACSAria Sorter with staining live+CD8αα+ IELs or TCRγδ+CD8α−β− IELs were sorted from IELs by FACSAria Sorter with gating live+CD45+TCRγδ+CD8α+β− or live+CD45+TCRγδ+CD8α−β−, and were mixed with pure intestinal epithelial cells (sorted by gating live+CD45+CD326+ from IECs) as a ratio 1:10 (IELs:IECs), which is the ratio of IELs to IECs in mice gut according to the published studies1. Cells were cultured in 96 round plates in RPMI-1640 medium, which was supplemented with anti-CD3, IL-2, IL-3, and IL-4 for 3 days before being prepared for flow cytometry.TCRγδTgfbr1f/fTCRδ ER Cre mice at 7–12 days of age were cut and incubated in lysis buffer at 50 °C overnight, then diluted with dH2O for PCR cocktail preparation. The primers used for Tgfbr1f/f mouse strain are F: TTCTGCTAATCCTGCAGTAAAC; R: ACCCTCTCACTCTTCCTGAGT. And the primers for the TCRδ ER Cre mouse strain are WT R: GCTTCCAAAACACTTGCACA; Common F: GGAGAGTTTTCCTAGCAGCA; Mutant R: ACACCGGCCTTATTCCAAG. PCR products were separated on 2% agarose gel with EtBr staining to distinguish bands of genes.Tails of Hprt (Mm00446968_m1), CD8α (Mm01182197_g1), CD8β1 (Mm00438116m1), Runx3 (Mm00490666), Zbtb7b (Mm00784709_s1), Btnl1 (Mm01281669_m1), Il-15 (Mm00434210), MyD88 (Mm00440338), CCL25 (Mm00436443), or Cdh1 (Mm01247357).Total RNA of isolated IECs or 18-h cultured γδT cells were extracted by using RNeasy Mini Kit (QIAGEN) according to the instruction of the manufacturer and reversed transcribed by High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR was performed using TaqMan assays with primers of Smad3−/− or age- and gender-matched Smad3+/+ mice were given 3% DSS drinking water continuously for 7 days; body weight and disease activity index were monitored during treatment. Tgfbr1f/fTCRδ ER Cre and Tgfbr1+/+TCRδ ER Cre littermates were treated with tamoxifen (10 mg/mL) five times to deplete TβR1 specifically on γδT cells before 3% DSS water treatment. Body weight and disease activity index were monitored during treatment.47. Small intestinal tissues were taken from mice, fixed, and embedded into paraffin before being cut into 5-μm sections; then they were deparaffinized in two changes of xylene, rehydrated in 95% and 90% ethanol for 10 min respectively; 16 S rRNA bacteria probe: (AminoC6 + Alexa488) GCTGCCTCCCGTAGGAGT (Eurofins MWG Operon) was diluted to the concentrations of 100 nM to 1 μM by hybridization buffer and incubated at 50 °C for 3 h. DAPI was stained for microscope detection.FISH experiments for bacteria invasion in the intestinal barrier were conducted based on the procedures described in the previous publicationt-test was used for the comparison of two independent experimental groups; the comparison of more than two groups was conducted by one-way ANOVA. RNA-seq data were analyzed by DeSeq2 for gene expression and normalization. P value threshold for the statistical difference was 0.05.Statistical analysis was conducted by GraphPad Prism 9 and shown in figure legends. Unpaired two-tailed Student’s Supplementary InformationSupplementary Dataset S1


The title compound is a phthalimide-protected polyamine with a protonated central nitrogen atom. The crystal packing features a hydrogen-bond network, a two-coordinated chloride ion, and off-set π–π stacking. 20H18N3O4+·Cl−·2H2O, is a phthalimide-protected polyamine that was synthesized by a previous method. It was characterized by ESI–MS, 1H NMR, and FT–IR. Crystals were grown from a solution of H2O and 0.1 M HCl. The central nitrogen atom is protonated and forms hydrogen bonds with the chloride ion and a water molecule. The two phthalimide units make a dihedral angle of 22.07 (3)°. The crystal packing features a hydrogen-bond network, two-coordinated chloride, and off-set π–π stacking.The title compound {systematic name: bis[2-ethyl]azanium chloride dihydrate}, C These units point in opposite directions to each other from the perspective of the central nitrogen atom. The central tetrahedral nitrogen atom (NH2) forms hydrogen bonds with a water molecule and the chloride ion.The molecular structure of the title compound is shown in Fig. 13.b-axis direction ⋯Cg (N3/C13–C20) centroid–centroid distance is 4.0143 (7) Å. A hydrogen-bond network (Table 1A), a water molecule (O1W), and a second water molecule (O2W). Both water molecules  also form hydrogen bonds with phthalimide oxygen atoms . The chloride ions form two hydrogen bonds with the protonated amine and a water molecule.The crystal structure features off-set π–π stacking between phthalimide groups running along the on Fig. 2. The Cg k Table 1 exists b4.et al., 20162CH2NH2)2 resulted in 1707 hits, while the structure with protonated amines +HN(CH2CH2NH3+)2 resulted in 182 hits. One of these structures is the triprotonated diethylenetriamine trichloride , 386.1 (M + Na+). 1H NMR  δ ppm 7.75 , 3.75 , 3.0 , 1.60 . FTIR (cm−1) = 3326 ν(N—H), 1698 ν(C=O). Crystals suitable for X-ray crystallography were grown by evaporation, with the compound dissolved in a solution of H2O and 0.1 M HCl.Following a previous protocol (Utz 6.Uiso(H) = 1.2Ueq(N). C-bound and water H atoms were positioned geometrically  and refined as riding with Uiso(H) = 1.2–1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023004565/ex2070sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023004565/ex2070Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023004565/ex2070Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023004565/ex2070Isup4.cmlSupporting information file. DOI: 2264952CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: