N,N′,N′′-tris(2-pyridinyl)-1,3,5-benzenetricarboxamide, intermolecular N—H⋯O hydrogen bonds link the molecules via their amide groups into slanted ladder-like chains. Only two of the three amide groups in the molecule are involved in hydrogen bonding, which influences the degree of out-of-plane twisting at each amide group.In the first reported crystal structure involving the potential ligand  N,N′,N′′-tris(pyridin-2-yl)benzene-1,3,5-tricarboxamide, C24H18N6O3, intermolecular N—H⋯O hydrogen bonds link the molecules via their amide groups into slanted ladder-like chains, in which the uprights of the ladder are formed by the hydrogen-bonding interactions and the benzene ring cores of the molecules act as the rungs of the ladder. Only two of the three amide groups in the molecule are involved in hydrogen bonding and this influences the degree of out-of-plane twisting at each amide group, with the twist being more significant for those amide groups participating in hydrogen bonds.In the first reported crystal structure involving the potential ligand  N,N′,N′′-tris(n-pyridinyl)-1,3,5-benzenetricarboxamide , which has potential donor atoms on each pyridinyl ring and at the amide function.Branched coordinating ligands with potential donor atoms on each branch can be useful as spacers in the synthesis of coordination polymers and metal–organic frameworks. A frequently used starting material is benzene-1,3,5-tricarboxylic acid (trimesic acid), which can act as a three-way planar node-connector. A related, less frequently employed, ligand system is abIII coordination polymers. The crystal structure of the 2-pyridinyl derivative, N,N′,N′′-tris(2-pyridinyl)-1,3,5-benzenetricarboxamide, C24H18N6O3, (I)We are interested in constructing bismuth(III) coordination polymers . The 2-pyridinyl rings all lie with the ring N atom approximately cis to the amide N—H group.The asymmetric unit of (I)The bond lengths and angles in the molecule have normal values and the bond lengths around the amide groups Table 1 are not et al., 1995In the extended structure of (I)x, 1 − y, 1 − z, and with the 2-pyridinyl ring containing atom N6 in the centrosymmetrically related adjacent molecule on the other side at −x, 1 − y, 1 − z. These interactions reinforce the ladder structure, rather than linking adjacent ladders. For the interactions involving the 2-pyridinyl rings containing atoms N5 and N6, respectively, the distances between the centroids of the benzene and 2-pyridinyl rings are 3.8956 (6) and 3.8409 (6) Å, the perpendicular distances between the centroid of the benzene ring and the planes of the 2-pyridinyl rings are 3.4522 (5) and 3.4610 (4) Å, while the slippages of the centroids are 1.735 and 2.097 Å and the angles between the benzene and 2-pyridinyl ring planes are 3.11 (5) and 7.50 (5)°.The slanted stacking of the molecules as the rungs of the hydrogen-bonded ladder only allow weak π–π interactions, which occur between the central benzene ring and the 2-pyridinyl ring containing atom N5 in the centrosymmetrically related adjacent molecule on one side at 1 − et al., 2016et al., 1997Cc and Pbca Cc polymorph of the monohydrate, two of the 3-pyridinyl rings lie approximately trans to their adjacent amide N—H groups, while in the Pbca polymorph, all three of the 3-pyridinyl rings have the trans arrangement. Surprisingly, there are only three crystal structures reported for the tris(4-pyridinyl) analogue; the monohydrate  analogue acts as a ligand. In most of these, the ligand coordinates through the pyridinyl N atom, although the amide O atom is involved in a few examples. The tris(4-pyridinyl) analogue occurs as a ligand in six coordination complexes, all of which involve coordination through the pyridinyl N atom. Given the propensity of the pyridinyl N atom to act as the coordinating atom in these examples, the steric congestion between the 2-pyridinyl ring and the adjacent amide group of (I)via filtration through fluted filter paper and dried in air between filter papers. Crystals were grown by dissolving the product in warm methanol, filtering and allowing slow evaporation of solvent overnight. A small quantity of orange–yellow crystals was recovered from a yellow oil. 1H NMR δ: 11.03 , 8.76 , 8.44 , 8.28 , 7.90 , 7.22 ; ESI m/z: 438.14335 (predicted 438.14).A solution of 2-aminopyridine (0.96 g) in dichloromethane (DCM) (12 ml) and trimethylamine (TEA) (1.4 ml) was added dropwise to a solution of benzene-1,3,5-tricarboxylic acid trichloride in DCM (3.4 ml) at 273 K. A further 1.5 ml of TEA were added and reaction mixture stirred at room temperature for approximately 5 days until the dark-brown–red slurry turned yellow–orange. The reaction mixture was filtered under vacuum and washed with DCM. It proved difficult to isolate a purified product until the product was washed with copious amounts of water, then collected as a precipitate Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020005599/hb7909sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020005599/hb7909Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020005599/hb7909Isup3.cmlSupporting information file. DOI: 1998283CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The authors wish to make the following corrections to their paper : We haveco-acrylic acid)-graft-β-cyclodextrin Hydrogel for Thorium(IV) Adsorption”.1. The title of the original publication . The H–O–H bending vibrations of lattice water molecules in the complexes were expressed at 1635 cmAt the same time, we added a new Reference [23] to support our results in the second paragraph of page 3 and all the reference numbers after it have been changed accordingly. References [23–39] are now numbered [24–40].3. In the Section “2.3.2. Scanning Electron Microscopy Measurements” of the Materials and Methods:β-CD(AN-co-AA) resembled a cloud sheet, and the size of the gel sheet is 2 μm in length and 1 μm in width, mostly”. Instead, this statement should read, “The morphology of β-CD(AN-co-AA) has a loose flocculent structure”.On page 3, there are some mistakes in the second sentence in the last paragraph of the original publication incorrectly state, “The morphology of 4. In the Section “2.3.3. X-ray Diffraction Measurements” of the Materials and Methods:On page 4, the third line in the first paragraph, we have modified the powder X-ray diffractometer’s type information from “Rigaku, The Woodlands, TX, USA” to “Rigaku, Texas, USA”.β-CD has lost its crystalline characteristic.” To express the sentence more clearly, we would like to replace it with the following: “This further indicates that the product of copolymer is not a simply physical mixture.”The last sentence of this paragraph in the original publication is, “This further indicates that these three materials, combined by chemical bonds, have formed a hydrogel, and 5. In the Section “2.4. Adsorption Experiments” of the Materials and Methods:β-CD(AN-co-AA) hydrogel of Th(IV) was calculated by the changes between the initial and final equilibrium concentrations.”On page 4, we have added some detailed information about our experimentation. The last sentence of this paragraph should be corrected to, “Concentration of Th(IV) was analyzed with the Arsenazo-III spectro photometric method on a spectrophotometer . A quantity of 1 mL Th(IV)solution sample,1 mL 0.5 M HCl and 1.0 mL 0.1 % Arsenazo-III aqueous solution were added to a 25 mL glass flask, respectively, and the final solution volume was filled up to 25 mL by adding deionized water. After 15 min, the absorbance of the mixture liquid was measured at 662 nm, and the adsorption capacity of the 6. In order that readers understand our experiment more easily, we added detailed information about our experimental condition to some figures .Figure 4. Effect of equilibrium time on Th(IV) adsorption onto β-CD(AN-co-AA). ([Th(VI)]0 = 6.5334 × 10−4 M, pH = 2.95 ± 0.05, T = 298.15 ± 1.00 K, I = 0.50 M NaNO3).”On page 5, the new title and conditions of Figure 4 should be, “Figure 5. Test of pseudo-second-order adsorption kinetics plot for Th(IV). ([Th(VI)]0 = 6.5334 × 10−4 M, pH = 2.95 ± 0.05, T = 298.15 ± 1.00 K, I = 0.50 M NaNO3).”On page 6, the new title and conditions of Figure 5 should be, “Figure 6. Effect of the pH value of the solution on the adsorption of Th(IV) onto β-CD(AN-co-AA). ([Th(VI)]0 = 6.5334 × 10−4 M, m/V = 0.18 g/L, T = 298.15 ± 1.00 K, I = 0.50 M NaNO3).”On page 7, the new title and conditions of Figure 6 should be, “Figure 7. Effect of the ionic strength of the solution on the adsorption of Th(IV) onto β-CD(AN-co-AA). ([Th(VI)]0 = 6.5334 × 10−4 M, m/V = 0.18 g/L, pH = 2.95 ± 0.05, T = 298.15 ± 1.00 K).”On page 7, the new title and conditions of Figure 7 should be, “Figure 8. Effect of solid content on the adsorption of Th(IV) onto β-CD(AN-co-AA), squares show the value of qe (g/g) and dots show the value of “adsorption %” ([Th(VI)]0 = 6.5334 × 10−4 M, pH = 2.95 ± 0.05, T = 298.15 ± 1.00 K, I = 0.50 M NaNO3).”On page 8, the new title and conditions of Figure 8 should be, “Figure 9. Effect of the initial concentration of Th4+ on the adsorption behavior onto β-CD(AN-co-AA). .”On page 8, the new title and conditions of Figure 9 should be, “These changes have no material impact on the conclusions of our paper. The authors would like to apologize for any inconvenience caused to the readers by these changes.


Pichia kudriavzevii (11 isolates), Candida ethanolica (1 isolate), Clavispora lusitaniae (2 isolates), Ogataea polymorpha (1 isolate), Hanseniaspora opuntiae (1 isolate), Lodderomyces elongisporus (1 isolate), Saccharomyces cerevisiae (2 isolates), C. tropicalis (5 isolates), Yarrowia lipolytica (2 isolates), Magnusiomyces ingens (1 isolate), and Magnusiomyces capitatus (3 isolates), Trichosporon insectorum (1 isolate), and Trichosporon asteroides (2 isolates). Seven strains, T. insectorum 4E-1D, M. capitatus 5E-1T and 5E-2D, T. asteroides 8E-1T and 8E-1D, and Y. lipolytica Fy-12 and Fy-13, showed high lipolytic activity ranged from 5.21 ± 0.09 to 45.68 ± 2.37 U/mL. Moreover, these seven strains exhibited good lipolytic activity after culturing in the medium containing palm oil (11.79 ± 0.67 to 28.19 ± 4.84 U/mL) and soy oil (9.14 ± 1.08 to 22.97 ± 0.69 U/mL) as lipase inducers. The result of this study suggests that the palm oil industrial wastes and Thai fermented foods could be promised as the invaluable sources of lipolytic yeasts.Thirty-three yeasts were isolated from palm oil industrial wastes and traditional fermented foods in Thailand. Based on the analysis of the sequences of the D1/D2 region of the large subunit ribosomal RNA gene (LSU rDNA) and their phenotypic characteristics, they were identified as  Lipases  are a class of hydrolase enzymes which can hydrolyse triglycerides at the lipid-water interface to free fatty acids, monoglycerides, diglycerides and glycerol , fermented pork (Mam-Moo), fermented pork sausage, fermented fish (Pla-Som) and fermented rice (Khao Kab), were collected from the markets in Bangkok, Thailand. One gram of each sample was enriched in a tube containing 10 mL lipolytic medium (0.3% (w/v) yeast extract, 0.3% (w/v) peptone, 0.01% (w/v) CaCl2 · 2H2O and 0.5% (v/v) Tween 20) supplemented with 200 mg·L−1 chloramphenicol  agar medium . The yeast isolates were maintained on YM agar slant at 4°C, freeze in YM broth supplemented with 10% (v/v) glycerol at −80°C, and lyophilised for long-term preservation.The palm oil industrial wastes including palm cake, palm shell and palm fibre were collected from 2 factories, Eastern Palm Oil Co., Ltd. and Suksomboon Co., Ltd. in Chonburi province and fermented food samples: fermented beef . The purified PCR product was sequenced at Macrogen (Korea). The sequences were compared with available sequence data using a BLASTN search on NCBI GenBank database  yeast extract, 0.5% (w/v) peptone, 0.2% (w/v) glucose, 0.01% (w/v) MgSO4 · 7H2O, 0.1% (w/v) K2HPO4 and 1% (v/v) Tween 20 as a lipase inducer, pH 7.0, incubated by shaking (180 rpm) at 30°C for 72 h. The culture was centrifuged at 14,000 rpm for 10 min and the supernatant was analysed for lipolytic activity.Thirty-three yeast isolates were cultured on YM agar medium at 30°C for 48 h. After cultivation, each isolate was standardised the turbidity with McFarland standard solution No. 2 to obtain approximately 1 × 106 CFU/mL in 0.85% NaCl and used as an inoculum. The inoculum (1%) was inoculated and cultivated in 100 mL of the production medium supplemented with 1% (v/v) of palm oil or soy oil under shaking condition at 30°C for 72 h. Cells were removed by centrifuging at 14,000 rpm for 10 min and the cultured supernatant were quantitatively determined for enzyme activity.The seven selected isolates, 4E-1D, 5E-1T, 5E-2D, 8E-1T, 8E-1D, Fy-12 and Fy-13, were grown at 30°C for 48 h in 500 mL Erlenmeyer flask containing 100 mL YM broth. Each isolate was standardised the turbidity with McFarland standard solution No. 2 to obtain approximately 1 × 10p-nitrophenyl butyrate (p-NPB) as substrate  into 10 mL of solution B  by using a volumetric flask, the final concentration of the substrate was 0.1 mM. The culture broth (20 μL) was added into 180 μL of the substrate and incubated for 60 minutes at 37°C. The release of p-nitrophenol was measured at 405 nm using a microplate reader . The production medium was used as a control. One Unit of lipase activity was defined as the amount of enzyme releasing 1 μmole fatty acid per minute at the assay condition.The lipolytic activity was determined spectrophotometrically using p ≤ 0.05) differences between the means.All experiments were conducted in triplicate and statistical analysis was performed by SPSS 21.0 software . Analysis of variance was carried out using the ANOVA procedure by the Duncan method to determine significant ; Candida ethanolica ; Clavispora lusitaniae ; Ogataea polymorpha ; Hanseniaspora opuntiae ; Saccharomyces cerevisiae ; Lodderomyces elongisporus ; Candida tropicalis ; Yarrowia lipolytica ; Magnusiomyces ingens  and Magnusiomyces capitatus . Two species in the basidiomycetes containing the isolate 4E-1D (Group XII) which was identified as Trichosporon insectorum and 2 isolates of Trichosporon asteroides .We found 11 species in the ascomycetes which were identified as P. kudriavzevii (33.4%), C. tropicalis (15.2%), M. capitatus (9.1%), Cl. lusitaniae (6.1%), S. cerevisiae (6.1%), Y. lipolytica (6.1%), T. asteroides (6.1%), C. ethanolica (3.0%), M. ingens (3.0%), H. opuntiae (3.0%), L. elongisporus (3.0%), O. polymorpha (3.0%) and T. insectorum (3.0%).The isolation rate of lipolytic yeasts in this study varied among different sources including foods, fruits, and wastes of palm oil factory which was founded in T. insectorum strain 4E-1D , Y. lipolytica strain Fy-12  and Y. lipolytica strain Fy-13 . The other selected isolates, 5E-1T, 5E-2D, 8E-1T and 8E-1D, also exhibited lipolytic activity varying from 11.79 to 16.33 U/mL in palm oil and from 9.14 to 11.84 U/mL in soy oil.The quantitative screening on Tween 20 liquid culture revealed varying levels of lipase activity. Most isolates showed lipolytic activity ranging from 1.05 to 3.12 U/mL . IsolateCandida tropicalis, Diutina rugosa, Moesziomyces antarcticus, Candida cylindracea, Candida parapsilosis, Cutaneotrichosporon curvatum, Yarrowia deformans, Rhodotorula glutinis, Rhodotorula pilimanae, Yarrowia lipolytica, Pichia bispora, Pichia xylosa, Pichia mexicana, Pichia silvicola, Pichia burtonii, Saccharomyces cerevisiae and Trichosporon asteroides were found to be the predominant terrestrial lipase-producing yeasts , olive oil (49 U/mL) and tributyrin (33 U/mL). This study suggested that Trichosporon, Magnusiomyces and Yarrowia strains could be further studied and used for lipase production.In addition, the two isolates of the genus o et al.  revealednd Revah  had studz et al.  reportedTrichosporon insectorum 4E-1D, Magnusiomyces capitatus 5E-1T and 5E-2D, Trichosporon asteroides 8E-1T and 8E-1D, and Yarrowia lipolytica Fy-12 and Fy-13, exhibited a significant level of lipolytic activity in Tween 20 liquid culture and were chosen for studying the lipolytic activity in palm and soy oils. Each selected strain showed varying levels of lipolytic activity in both palm and soy oils ranging from 9.14 to 28.19 U/mL. The further study focuses on the optimisation of lipase production by the best lipase producer, T. insectorum strain 4E-1D, as well as the characterisation and purification of lipase from the liquid medium which may provide for the commercial exploitation.The present study showed that Thai fermented foods and the palm oil industrial wastes could be employed as a valuable source for the isolation of lipolytic yeasts. Thirty-three isolates can be divided into 2 groups including ascomycetous (30 isolates) and basidiomycetous (3 isolates) yeasts. The seven isolates,


Heat energy bank: Accumulated heat energy is eternally preserved and extracted on demand by pressure. xTix3−O5). λ-ScxTix3−O5 not only accumulates heat energy from hot water but also could release the accumulated heat energy by the application of pressure. λ-ScxTix3−O5 has the potential to accumulate heat energy of hot water generated in thermal and nuclear power plants and to recycle the accumulated heat energy on demand by applying external pressure. Furthermore, it may be used to recycle waste heat in industrial factories and automobiles.In thermal and nuclear power plants, 70% of the generated thermal energy is lost as waste heat. The temperature of the waste heat is below the boiling temperature of water. Here, we show a long-term heat-storage material that absorbs heat energy at warm temperatures from 38°C (311 K) to 67°C (340 K). This unique series of material is composed of scandium-substituted lambda-trititanium-pentoxide (λ-Sc Generated thermal energy cannot be efficiently converted to electric power at thermal and nuclear power plants. Seventy percent of the generated thermal energy is discarded as waste heat (xMTi3O5). λ-Ti3O5 exhibits photo- and pressure-induced phase transitions . Here, we report the synthesis, crystal structure, and heat-storage properties of Sc-substituted λ-Ti3O5.Of these elements, only six have a stabilizing effect: Sc, Nb, Ta, Zr, Hf, and W . Thus, w3O5  measurements . We performed synchrotron x-ray diffraction (SXRD) measurements using beamline BL02B2 at SPring-8 to determine the crystal structure (C2/m) with lattice parameters of a = 9.84195 (4) Å, b = 3.79151 (1) Å, c = 9.98618 (4) Å, β = 91.1207 (3)°, and a unit cell volume of V = 372.572 (3) Å3 (fig. S3).We used an arc-melting technique to synthesize the Sc-substituted λ-Tical ball . The obt3O5. λ-Sc0.09Ti2.91O5 has a slightly larger unit cell volume than that of λ-Ti3O5 with a 0.4% expansion. In addition, the β phase is present as the minor phase. The β phase adopts a monoclinic crystal structure (space group C2/m) with lattice parameters of a = 9.7930 (4) Å, b = 3.8064 (14) Å, c = 9.4375 (4) Å, β = 91.5611 (3)°, and V = 351.66 (2) Å3. The scanning transmission electron microscopy (STEM) image shows stripe-like domains measuring approximately 100 nm × 200 nm . The as-prepared sample was compressed by pressures of 0.2 to 1.7 GPa with a hydraulic press. As the pressure increases, the λ-phase fraction decreases, while the β-phase fraction increases . The cro−1. The pressure- and heat-induced phase transitions were repeatedly observed (fig. S7).We measured the heat absorption mass of the sample after the pressure-induced phase transition by differential scanning calorimetry (DSC). We swept the sample compressed at 1.7 GPa with 22.7% of the λ phase and 77.3% of the β phase from 0°C (273 K) to 300°C (573 K). Heat absorption is observed with an absorption peak at 67°C (340 K) . ConsideCompared to the previous work (3O5 (Gsys) using a thermodynamic model based on the Slichter and Drickamer mean-field model (SD model) (Gsys) is described as Gsys= xΔH + γx(1 − x) + T{R[x ln x + (1 − x)ln(1 − x)] − xΔS}, with a cooperative interaction parameter (γ) between the λ phase and β phase due to the elastic interactions within the crystal. x is the ratio of λ phase, and R is the gas constant. From the result of the DSC measurement, the transition enthalpy (ΔH) is 75 kJ liter−1 (4.0 kJ mol−1), and the transition entropy (ΔS) is 0.22 kJ K−1 liter−1 (12 J K−1 mol−1). When the interaction parameters are set as a particular combination of values, the SD model calculation well reproduces the measurement data . Then, the thermally produced λ phase is maintained even at low temperatures in the cooling process  and 367 days (1 year) from the XRD measurement.According to previous reports on λ-Ti process . Thus, tFurthermore, we reproduced the pressure-induced phase transition from the λ phase to β phase. Applying pressure to the system causes the energy barrier to disappear and induces a phase transition from the λ phase to β phase . This pr3O5. Cooling water for a turbine in a power plant is pumped from a river or sea. As the water passes through the turbine, the water temperature increases due to heat exchange. The energy of hot water is transferred to Sc-substituted λ-Ti3O5 in tanks. Subsequently, water with a reduced thermal energy returns to the river or the sea. This system can mitigate the rise of river or sea water temperature. Energy-stored Sc-substituted λ-Ti3O5 can release its stored thermal energy by application of pressure, allowing energy to be used on demand. For example, the stored thermal energy can be supplied to buildings or industrial plants that are close to power plants, without using electricity. Moreover, taking advantage of the characteristic of holding the latent heat energy until pressure application, if energy-stored Sc-substituted λ-Ti3O5 is transported by truck, the heat energy can be used at a distant location. As for the efficiency, the transformation energy efficiency (e) value is evaluated on the basis of the temperature dependence of the enthalpy for the λ phase and β phase obtained by first-principles calculations and DSC measurements (fig. S8). For example, when the heat release temperature is 15°C (288 K) and the temperature increase is 1 K, the efficiency is 93%. When the temperature increase is 5 K, the efficiency is 77% (table S1).3O5, which absorb heat from hot water. After conducting first-principles calculations, we synthesize Sc-substituted λ-Ti3O5 ceramics with a heat absorption below 100°C (373 K). This heat absorption material below 100°C can recover the thermal energy from cooling water in power plant turbines, mitigating the rise in sea water temperatures. Moreover, the heat absorption temperature can be easily controlled by changing the Sc content in λ-Ti3O5 in accordance to the target application.In conclusion, we demonstrate heat-storage ceramics based on Sc-substituted λ-Ti0.105Ti2.895O5 with a heat absorption temperature at 45°C (318 K) and λ-Sc0.108Ti2.892O5 with a heat absorption temperature at 38°C . Sc-substituted λ-Ti3O5 will expand opportunities to use thermal energy as it can use thermal energy that is currently in the unused temperature range. In addition to electric power plants, other applications of the present material such as heat-storage usage to collect waste heat from factories, transportation vehicles, mobile phones, and electronic devices should be possible.These heat absorption temperature changes are attributed to the crossover temperature change of Gibbs energies. We successfully synthesize λ-Sc3+ and Ti4+ in λ-Ti3O5  code. The crystal structure of λ-Ti3O5 shown in , TiO2 (99.9% purity), and Sc2O3 (99.99% purity) in an Ar atmosphere at 0.05 MPa. Ti metal, TiO2, and Sc2O3 powders were mixed in a molar ratio of Ti:TiO2:Sc2O3 = 0.478:2.433:0.045. In this method, samples were melted and turned over three or four times on a copper cooling stage after solidification. These solidified samples were milled by hand before the measurements. The composition was confirmed by XRF, and the formula was determined to be Sc0.09Ti2.91O5: calcd: Ti, 62.4; O, 35.8; Sc, 1.8%; found: Ti, 62.0; O, 36.0; Sc, 2.0%, which is identical to the mixed ratio of the starting materials. Sc0.105Ti2.895O5 and Sc0.108Ti2.892O5 samples were also synthesized, and we measured the x-ray diffraction patterns (fig. S9). Moreover, Ti3O5 samples, substituted by 3 atomic % (at %) of Zr, Nb, Hf, Ta, and W, were synthesized and their x-ray diffraction patterns were measured (fig. S2). They mainly showed β-Ti3O5 patterns.Sc-substituted λ-Ti3O5 samples were determined by Rietveld analysis of the SXRD data collected in beamline BL02B2 at SPring-8  at a heating-cooling rate of 10 K/min and an air gas flow of 100 ml/min. Before DSC measurements, the samples containing both the λ phase and the β phase were compressed at 1.7 GPa to transform them from the λ phase to the β phase. In addition, the thermal properties of λ-Sc0.105Ti2.895O5 and λ-Sc0.108Ti2.892O5 samples were measured (fig. S9).The heat absorption properties of Sc-substituted Ti3O5 and β-Ti3O5 with supercells (1 × 3 × 1) and a k-mesh of 2 × 2 × 2 of the optimized structures were calculated using the Phonopy code in cooperation with the VASP code for the interatomic force constants calculations (0.09Ti2.91O5). That is, 1 of 36 Ti atoms was substituted by an Sc atom in the supercells. The differential energy of λ and β phase was calculated (ΔG = Gλ − Gβ). The calculated ΔG of Ti3O5 and Sc-substituted Ti3O5 are shown in 3O5 shows ΔG = 0 at 575°C (848 K), which is the crossover temperature of the calculated free energies corresponding to the phase transition temperature (3O5 showed ΔG = 0 at 341°C (614 K). The normalized temperature in 3O5. The crossover temperature of Sc-substituted Ti3O5 decreased about 27.6% from the temperature of Ti3O5. This temperature decrease ratio agreed well with the experimentally obtained decrease ratio of 27.7% calculated from the phase transition temperature of 470 K (197°C) reported in λ-Ti3O5  at 0 K were −2362.47 eV (λ-Ti3O5), −2376.44 eV (Sc-substituted λ-Ti3O5), −2372.45 eV (β-Ti3O5), and −2381.65 eV (Sc-substituted β-Ti3O5).To interpret the phase transition temperature, the Gibbs free energies of Sc-substituted λ-Tia + γbT + γcP). From the DSC measurement result, the ΔH value was 4.0 kJ mol−1, and the ΔS value was 11.7 J K−1 mol−1. When the parameters of γ were set as γa = 7 kJ mol−1, γb = −1.2 J K−1 mol−1, and γc = −0.37 J MPa−1 mol−1, the SD model calculations reproduced the long-term heat storage and pressure-induced phase transition, as shown in In the SD model calculations, the γ value depends on the temperature and pressure (i.e., γ = γ


The necessity of these activity bursts for sensorimotor control and their roles in specific phases of movement are unknown. Here we show that temporally-restricted optosilencing of spontaneous or sensory-evoked HON bursts disrupts locomotion initiation, but does not affect ongoing locomotion. Conversely, HON optostimulation initiates locomotion with subsecond delays in a frequency-dependent manner. Using 2-photon volumetric imaging of activity of >300 HONs during sensory stimulation and self-initiated locomotion, we identify several locomotion-related HON subtypes, which distinctly predict the probability of imminent locomotion initiation, display distinct sensory responses, and are differentially modulated by food deprivation. By causally linking HON bursts to locomotion initiation, these findings reveal the sensorimotor importance of rapid spontaneous and evoked fluctuations in HON ensemble activity.Appropriate motor control is critical for normal life, and requires hypothalamic hypocretin/orexin neurons (HONs). HONs are slowly regulated by nutrients, but also display rapid (subsecond) activity fluctuations  The importance of the neocortex, and other classical sensory and motor brain regions for subsecond sensorimotor transformations is well established . In contUnit recordings  and averHere, using cellular-resolution calcium imaging of >300 HONs during behavior, we were able to distinguish several HON types based on their natural activity profiles during self-initiated and sensory-evoked locomotor events in behaving mice. We quantified their distinct abilities and predicted future locomotion using machine learning. Using optogenetic manipulation of HONs time-locked to specific phases of locomotion or sensory stimulation, we found rapid functional coupling between HON activity and locomotion initiation.22.12+ sensor  indicating that many of the cells code for both speed and distance. Overall, speed was most strongly correlated with HON activity (statistics in figure legends). This correlation was evident across all behavior bouts irrespective of behavior class , 3.0 ± 2.0 s, 11; male mating (intromission), 13.8 ± 7.7 s, 15; female mating (intromission), 32.6 ± 29.4 s, 4; defecating, 6.7 ± 1.6 s, 5; urinating, 3.6 ± 1.3 s, 3; idle stationary , 22.5 ± 25.0 s, 19; conspecific approaches, 2.1 ± 2.8 s, 32; grooming, 38.4 ± 39.6 s, 19; feeding, 46.5 ± 38.8 s, 13; coprophagia, 19.4 s, 1; idle movement , 4.5 ± 2.9 s, 37; approaching conspecific, 3.0 ± 2.0 s, 43; approaching food, 5.2 ± 4.5 s, 22; chasing conspecific, 7.8 ± 7.1 s, 12; being chased (by conspecific), 6.8 ± 4.2 s, 21; escaping handler, 3.6 ± 3.8 s, 6). Across behavioral classes, activity of 33.3 % of HONs was strongly correlated (Pearson correlation coefficient >0.6) with the speed of locomotion during the behavior, 17.4 % with distance moved, and 1.9 % with duration I. There or class J, as welor class K, L.2.2ad libitum on a disk treadmill . In our highest quality recordings, the weighted average activity vector of these cells predicted movement onset of 75 % (9/12) of locomotion bouts 1.55 ± 0.36 s before they occurred T. To for2.3in vivo suggest that they do not normally fire above this frequency . This allowed HON firing to be controlled using green laser pulses A–C. Becarequency , we asserequency D, E. Therequency E, which requency . In addirequency F. Overalrequency Q. These To assess the role of natural HON activity in self-initiated locomotion, we next performed several loss-of-function experiments. First, we ablated HONs using a mouse line expressing the human diphtheria toxin receptor in HONs (O-DTR) . When thThe O-DTR method is not selective for HON activity during movement, but ablates all functions of HONs, including their slow indirect effects on movement endurance that may arise due to their role in sympathetic coordination of breathing . Therefo2.4−18; null −0.01 ± 0.01, visual 0.26 ± 0.02, tactile 0.14 ± 0.01; olfactory 0.38 ± 0.03), as was the average locomotor activity . The null responses also allowed us to delineate thresholds of detection throughout our analysis , whereas orexin population activity increased above baseline 2230 ± 440 ms before self-initiated locomotion onset J–M. Alth2.5t-test, n = 95 ON and up-down cells), while sensory-evoked movement was coupled with higher amplitudes than the activity profiles during self-initiated locomotion. ON cells in particular had significantly larger responses during sensory-evoked movement than during sensory responses while stationary  injected mice F, G. How2.6ad libitum for 2 h and recorded again  were generated as described before  and by Institutional Animal Welfare Ethical Review Panel. Animals of both sexes, aged 30–60 days at the beginning of the procedures were used and were housed in a controlled environment on a reversed 12 h light-dark cycle with food and water d before  and crosd before  and was d before H. O-DTR d before  were use4.2Two to three weeks prior to optical device implantation, WT mice were injected stereotactically with AAV1-hORX.GCaMP6s , AAV1-hORX.C1V1(t/s).mCherry or AAV1-hORX.ArchT.TdTomato . The GCaMP6s virus was generated as described before  using thUsing the orx.C1V1.mCherry virus, C1V1.mCherry was expressed with 96.0 ± 0.2 % specificity and 54.8 ± 3.4 % penetrance in HONs . A 0.8 mm diameter hole was drilled at the same position(s) as in virus injection surgery and the optical device was slowly (150 μm/min) lowered to a depth of 5.1 mm using an automated Luigs & Neumann micromanipulator controlled from Matlab. Lens placements in Fig. S2. For imaging experiments, the optical device was a 0.39 NA, 7.3 mm long, 0.6 mm diameter cylindrical graded refractive index (GRIN) lens (Grintech). For fiber photometry experiments in The mice received a single dose of 0.6 mg/kg dexamethasone and 5 mg/kg carprofen injections for two days as post-operative pain and anti-inflammatory medication. After at least two weeks of recovery, mice were trained for head-fixed awake experiments in 5–10 sessions of increasing length on the experimental running-wheel apparatus or for freely moving miniature endoscope recordings in a similar incremental regimen of wearing the head-mounted miniature microscope (∼2 g weight) in the test arena E.4.42+ imaging was performed with a head-mounted miniature microscope (Inscopix) which was attached to the animal’s head without anaesthesia to a magnetic baseplate that had previously been cemented at the correct focal height on top of the implant. The subject was then placed into the plexiglass-floored arena which it had already spent time in (during many hours of habituation sessions with the miniature microscope) and recording was begun after 10 min. A typical recording session lasted 30 min during which time a familiar opposite-sex conspecific was introduced by a gloved handler , and sometimes (rarely) the handler’s gloved hand would ‘chase’ the subject. Most of each recording was simply observation of the subject and conspecific in a familiar arena with wet food available ad libitum. The arena was lit with red LED lighting and was covered from view in a dark, quiet room and a silent fume extractor pipe was located 50 cm above the arena to eliminate background odors. Ca2+ imaging was performed at a frame rate of 10 frames/s. Behavior tracking was done with a CCD camera (Lumenera Infinity) below the arena acquiring at 60 frames/s and synchronized with Calcium imaging using an LED mounted next to the arena and facing the behavior camera. The LED was driven to flash on every acquired Calcium imaging frame with a TTL pulse from the microscope DAC board. Behavior analysis was done in ImageJ by manually classifying behaviors and measuring distance and time travelled during each behavior bout.Freely moving Ca4.5ad libitum. Each mouse was recorded in 2–8 sessions to obtain the full datasets presented here, and cell numbers from each mouse (26–85) were stable across recording sessions . Imaging was performed either in a dark room with red background lighting  and a femtosecond-pulsed mode-locked Ti:sapphire laser (Spectra-physics Mai Tai HP Deepsee 2) at 950 nm through a 20 ×  air-IR objective at 31frames/s with 512 × 512 pixel resolution using custom Labview software. With 5-plane imaging using the electro-tunable lens volume rate was 5.1 volumes/s. Images were obtained with a 510/80 nm band-pass emission filter.Visual stimuli were delivered with a blue LED positioned at eye level 10 cm away and visible to both eyes. Tactile stimuli were delivered with a fast TTL driven valve device (picospritzer) through a through a fan shaped nozzle  so that the geometry of the airpuff impinging on the animal was insensitive to small postural changes. Tactile stimuli were either 0.2 s pulses of 30 psi pressure delivered to the left side lower abdomen from the side as a vertical fan pattern  or 2 s pThe presentation of sensory stimuli was synchronized with image acquisition using custom routines in Matlab to generate timing triggers. Within Matlab, a National Instruments USB-6008 DAQ board was used to output TTL triggers and count imaging frames. Stimuli were presented in blocks consisting of a visual, tactile, auditory and 1–3 odors presented for 0.2 s  or tacti4.6GCaMP6s was excited with a blue LED  for 2-photon data or Mosaic (Inscopix) for miniature endoscope data. Imaging data were down-sampled spatially by 2*2 (2-photon) or 3*3 (miniature endoscope) binning to reduce file size. Cell outlines were drawn manually, and, in head-fixed 3-D 5-plane imaging, outlines containing the same cell were tracked across planes. Mean intensity within each region of interest (ROI) was used to generate rawF and npF with a 3 frame moving average, then average activity for each cell specific signal was calculated from bouts aligned to the onset of locomotion  were used to deliver light through shielded 0.2 mm diameter optic fibers to the bilateral implants. Light intensity out of the fiber was set to 3 mW for ArchT stimulation  and 20 mW for C1V1 stimulation (10 ms pulses for 2.5 s at indicated frequency). HONs in O-DTR mice were deleted by two injections of 150 ng diphtheria toxin (Sigma D0564) diluted to 1 μg/ml in saline, administered i.p. on day 0 and day 2. Recordings for DT + conditions were started on day 12. WT control mice were treated identically in parallel. In experiments in 4.92 and 5% CO2), icy cutting solution containing (in mM): 90 N-methyl-d-glucamine, 20 HEPES, 110 HCl, 3 KCl, 10 MgCl2, 0.5 CaCl2, 1.1 NaH2PO4, 25 NaHCO3, 3 pyruvic acid, 10 ascorbic acid and 25 d-glucose. 350 μm thick coronal brain slices were cut on a vibratome (Campden) and allowed to recover for 5–15 min at 37 °C in cutting solution, followed by 45–55 min at 22 °C in artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 3 KCl, 2 MgSO4, 2 CaCl2, 1.1 NaH2PO4, 26 NaHCO3, 0.1 pyruvic acid, 0.5 l-glutamine, 0.4 ascorbic acid and 25 d-glucose, continuously gassed with 95 % O2 and 5% CO2.Coronal brain slices from P60-180 animals were prepared after instant cervical dislocation and decapitation. The brain was rapidly dissected and cooled in continuously gassed (95 % O4.102 and 5% CO2. 3–6 MOhm patch pipettes were filled with intracellular solution containing (in mM): 130 K-gluconate, 5 NaCl, 2 MgSO2, 10 HEPES, 0.1 EGTA, 4 Mg-ATP, 0.4 Na-GTP, 2 pyruvic acid, 0.1 Alexa-594, 0.1 % biocytin, and ∼10 mM KOH (to set pH to 7.3). Whole cell recordings were not analysed if the access resistance was above 25 MOhm. Voltage recordings were sampled at 10 kHz and low-pass filtered at 3 kHz with HEKA EPC10 usb amplifiers and acquired with HEKA patchmaster software. ArchT and C1V1 were stimulated with green light from a xenon lamp (Sutter lambda 4DG controlled from HEKA patchmaster) through a TRITC-filter. Concurrent GCaMP6s fluorescence recordings were performed using the miniature endoscope with a GRIN lens implant recovered from a successful in vivo recording. This assembly was mounted on a micromanipulator and moved into focus at about 350 μm away from the patch clamped cell. Patch clamp data were analyzed in Matlab.Patch clamp recordings were performed in a submerged chamber with 3−5 ml/min superfusion with ACSF, continuously gassed with 95 % O4.11Mice were overdosed with ketamine/xylazine (100 mg/kg and 10 mg/kg respectively) and transcardially perfused with 4% PFA. Brains were post-fixed for 24 h. Coronal brain slices were sectioned at 50 μm using a cryostat or a vibratome. Sections were blocked in PBS with 0.3 % Triton X-100 and 1% bovine serum albumin (blocking solution) for 1 h, incubated with goat anti-orexin  over-night, washed, incubated with Alexa 647 conjugated donkey anti-goat  for 3.5 h, washed and mounted. Antibodies were applied blocking solution. Confocal micrographs were acquired on an Olympus FV1000 and merged in imageJ.4.12t-test or Wilcoxon rank sum test as stated. All statistics were performed using statistical functions in Matlab. Sigmoid fits in 50 is half-maximal response and IC50 half-maximal decrease. Fitting was done using custom routines in Matlab.All data are shown as mean ± SEM unless stated otherwise. Statistical significance was determined by paired Student EC50h+Fh C or P=PmIC50h+Fh E, where Mahesh M. Karnani: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Cornelia Schöne: Formal analysis, Investigation, Methodology, Writing - review & editing. Edward F. Bracey: Investigation, Methodology, Writing - review & editing. J. Antonio González: Investigation, Methodology, Writing - review & editing. Paulius Viskaitis: Investigation, Methodology. Han-Tao Li: Investigation, Methodology. Antoine Adamantidis: Conceptualization, Methodology, Writing - review & editing. Denis Burdakov: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Writing - original draft, Writing - review & editing.The authors declare no competing interests.


Pipers griffithii leaves known as zeylenone has been determined.The crystal structure 5-[(benzoyloxy)methyl]-5,6-dihydroxy-4-oxocyclohex-2-en-1-yl benzoate a natural product from  21H18O7, was confirmed by single-crystal X-ray diffraction. The crystal structure has three chiral centers at positions C1, C5 and C6 of the cyclohexanone ring, but the absolute configuration could not be determined reliably. The methyl benzoate and benzoyloxy substituents at positions C1 and C5 of the cyclohexenone ring are on the same side of the ring with the dihedral angle between their mean planes being 16.25 (10)°. These rings are almost perpendicular to the cyclohexenone ring. The benzoate groups and two hydroxyl groups on the cyclohexenone ring form strong hydrogen bonds to consolidate the crystal structure. In addition, weak C—H⋯O hydrogen bonds also contribute to the packing of the structure.The crystal structure of the natural product zeylenone, C Piperaceae and Annonaceae. The biological activity of zeylenone was reported as inducing apoptosis in the mitochondria of gastric cancer cells  could not be deduced from the X-ray data because of the large standard deviation of the Flack parameter [0.0 (3)]. The two main substituents are methyl benzoate and benzoyloxy at positions C1 and C5, and positioned at the same side of the cyclohexenone ring. The dihedral angle between the methyl benzoate and benzoyloxy mean planes is 16.24 (10)°, indicating that the rings are almost coplanar. The dihedral angle between the cyclohexenone ring and the methyl benzoate and benzoyloxy rings are 74.92 (9) and 69.23 (10)°, respectively, indicating that the aromatic and cyclohexenone rings are almost perpendicular. The conformation of the cyclohexenone ring, the core structure of (−)-zeylenone, is described as a half-chair based on the torsion angles H4—C4—C3—C2  and C5—C6—C1—C2  and the puckering parameters .The molecular structure of the title compound (I)i and O1—H1⋯O5ii, Fig. 2a, Table 1bc plane . In addition, the crystal packing features weak C—H⋯O hydrogen-bonding interactions  calculations at the M062X/6-31G(d) level using Discovery Studio visualizer  of the experimental and optimized structures are given in Table 2The DFT-optimized geometry was compared with the geometry obtained from the crystal structure using the molecular overlay module based on 50% steric and 50% electrostatic similarities in the EHOMO − ELUMO) is 7.61 eV. The large energy gap indicates the stability of the title compound.Finally, the molecular orbitals of zeylenone were calculated. The HOMO and LUMO plots are shown in Fig. 5R, 5S, 6R. A circular dichroism study of the synthesized product gave (+)-zeylenone  was macerated at room temperature in hexane for a week and then filtered. This was repeated with the remaining Piper powder using ethyl acetate. The filtrate was evaporated to yield about 2.60 g crude extract from ethyl acetate, which was dissolved again in ethyl acetate and mixed with silica gel. The mixture was evaporated by rotary evaporator, loaded on the column and eluted by gradient elution using 20–50% EtOAc in hexane. The fractions were collected and combined, monitoring with thin layer chromatography, to provide eleven fractions. The sixth fraction was separated by column chromatography using MeOH:EtOAc:Hexane (1:4:5) as eluents, yielding a pale-yellow solid (0.60 g), which was recrystallized from dichloromethane and hexane (1:1), giving colourless in CIF crystals, m.p. 423–424 K.1H NMR : δ 3.22 , 4.11 , 4.38 , 4.60 , 4.85 , 5.96 , 6.34 , 6.96 , 7.38–7.44 , 7.56 , 7.94 , 8.02 . 13C NMR (CDCl3): δ 65.4, 69.2, 71.6, 77.2, 128.4, 128.5, 128.6, 128.7, 129.1, 129.7, 129.78, 133.4, 133.7, 142.6, 165.3, 166.1, 196.2. Mass spectroscopy m/z 383.1125 (M + 1)+. IR : 712 cm−1 ; 1103 cm−1 ; 1277 cm−1 ; 1593 cm−1 ; 1705 cm−1  ; 2933 cm−1 ; 3423 cm−1 .Uiso(H) = 1.5Ueq(C) or 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020007793/vm2234sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020007793/vm2234Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020007793/vm2234Isup3.cmlSupporting information file. DOI: 2008756CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


By contrast to other tissues however, the uterine compartment also included non-Vγ6+, IFN-γ-producing cells; was strikingly enriched in young mice; expressed genes hitherto associated with the uterus, including the progesterone receptor; and did not require microbes for development and/or maintenance. This notwithstanding, γδ T-cell deficiency severely impaired resistance to reproductive tract infection by Candida albicans, associated with decreased responses of IL-17-dependent neutrophils. These findings emphasise tissue-specific complexities of different mucosal γδ cell compartments, and their evident importance in lymphoid stress-surveillance against barrier infection.This most comprehensive analysis to date of γδ T cells in the murine uterus reveals them to compose a unique local T-cell compartment. Consistent with earlier reports, most cells expressed a canonical Vγ6Vδ1 TCR, and produced interleukin (IL)-17A upon stimulation. Nonetheless, contrasting with earlier reports, uterine γδ T cells were not obviously intraepithelial, being more akin to sub-epithelial Vγ6Vδ1 Clear counterparts of these cells were recently identified in human gut.3Many tissues harbour two categories of lymphocytes which are largely noncirculating.+ dendritic epidermal T cells (DETC) and small intestinal intraepithelial lymphocytes (IEL) are defined by their respective expressions of the Vγ5 and Vγ7 gene segments, which are developmentally selected by epithelial butyrophilin-like (Btnl) molecules, Skint1 and Btnl1, respectively.5 Selection is independent of microbial colonisation,6 and occurs within discrete developmental windows: prenatal for DETC,8 and postnatal day 17–35 for Vγ7+ cells.3 It induces strong responsiveness to innate stimuli,9 such as stress-induced ligands for the activating NK receptor, NKG2D,10 while suppressing Rorc and Sox13 expression, thereby diminishing the cells’ potential to produce interleukin (IL)-17A, in favour of IFN-γ, TNF, IL-13, and granzymes that contribute to the cells’ cytolytic potentials.11 Seemingly reflective of these effector capabilities, γδ+ T cells are associated with limiting skin and intestinal carcinogenesis.13Murine TCRγδ14While some properties of tissue-associated γδ T cells are shared across anatomical sites, others seem site-specific, as was recently considered for γδ T cells in the gingiva.RM. In this regard, we have focused on the murine female reproductive tract (FRT). A TCRγδ+ uterine IEL compartment was described many years ago, that was limited to use of a quasi-monomorphic Vγ6Vδ1 TCR.15 Interestingly, cells with the same TCR were described in the lung, tongue, gut lamina propria, and dermis,16 although those cells are predominantly sub-epithelial, with potentially unique relationships with specific tissues.17Thus, it is clearly important to better characterise each tissue-associated γδ T-cell compartment, particularly in the cases of organs housing T+ cells have been considered to be microbe-dependent,19 and those cells populating the gut lamina propria only expanded into a prevalent subset following oral infection, e.g. with Listeria monocytogenes.20 Apparently consistent with this, mucosal Vγ6Vδ1+ cells were shown to provide anti-microbial protection, particularly against re-challenge, and this was primarily ascribed to IL-17A production.23 Likewise, so-called γδ17 cells have been strongly implicated in inflammatory immunopathologies, including psoriasis, neuroinflammation, and cancer,26 although some have also been ascribed amphiregulin-dependent contributions to tissue homoeostasis.14Most commonly, mucosal Vγ6Vδ1+ thymocytes have been reported to apoptose upon TCR cross-linking,27 although there are counter-arguments in favour of the cells’ selection.28 Conceivably, the lack of endogenous selecting elements might be compensated for by the development of γδ17 cells being driven by the microbiome, perhaps related to the cells’ provision of anti-microbial protection.The cells’ production of IL-17A is considered to reflect a lack of developmental selection events akin to those shaping IEL compartments. Indeed, Vγ6Vδ1+ population. In contrast to earlier reports,15 uterine cells were largely stromal, evoking sub-epithelial γδ T cells in the dermis and gut. Like many of those cells, most uterine TCRγδ+ cells expressed a canonical Vγ6Vδ1 TCR and produced IL-17A upon stimulation. Nonetheless, the cells were heterogeneous in including a minor subset of non-Vγ6+ cells producing IFN-γ. Furthermore, uterine γδ cells phenocopied epidermal and intestinal IEL in that their development and/or maintenance was regulated by a distinct time window in early life, and was independent of microbes. This notwithstanding, γδ cells provided non-redundant protection against vaginal Candida albicans infection of adult mice.In this study, a comprehensive analysis of murine uterine T lymphocytes has revealed a unique TCRγδ+ cells accounted for over half the T cells in the uterus of mice aged 4 weeks old or younger  showed that the cells’ morphology was more lymphoid than dendritic, akin to intestinal IEL rather than DETC Fig. . Immunohial Fig. . Indeed,30  or Vγ5 (expressed by DETC) Fig. . Instead30 Fig. .Fig. 2Su−4−5− cells, ~80% of productive rearrangements corresponded to the canonical rearrangement of Vγ6 to Jγ115  to Jδ2.15 In addition, there were some productive Vδ1 rearrangements to Jδ1, such that all in all, Vδ1 rearrangements accounted for ~99% of productive sequences 3. The cells’ expression of ICOS, PD-1, and CD69 evoked several types of tissue-resident populations, including TCRαβ+ TRM cells.1 Conversely, the relatively modest expression of CD127, which encodes the IL7Rα chain, contrasted with its high expression on other γδ17 cells.32The uterine γδ T-cell phenotype from mice aged 2–3 weeks Fig. , red waslls Fig. , blue. U+ cells in left panel, middle row; Fig. + and were all Vγ6−CD44lo, whereas IL-17A-producers were predominantly Vγ6+ and all CD45RB−CD44+ ; CD44+ mature Vγ1,4,5− TCRγδ+ thymocytes ; and Vγ1,4,5− cells from the uterus  and from lungs   to interrogate the representation of each tissue-specific signature in uterine and lung γδ cells, respectively, relative to mature Vγ6+ thymocytes, as shown in Fig. In sum, we could conclude that lung and uterine Vγ1,4,5p value for the uterus signature, whereas lung γδ T cells displayed the highest enrichment score and lowest p value for the lung signature, as reflected in the graphs in Fig. + γδ thymocytes versus γδ T cells from uterus  and those regulating surfactant protein maturation (Lamp3) , and the gene, Pgr, encoding progesterone receptor  Fig. . This wator Fig. . To prector Fig. . Given tone Fig. . Thus, V20 we next asked whether the cells required microbial colonisation for their development and/or maintenance. In fact, signature CD45RB−, CD44+ uterine γδ T cells displayed comparable frequencies, phenotypes and cytokine production profiles in 4-week-old and 7–8-week-old germ-free (GF) versus conventional specific pathogen-free (SPF) mice, albeit that the fluorescence intensity of CD3 was slightly increased in GF mice  versus wild-type mice. Moreover, we examined whether any potential phenotypes might be exaggerated if mice were outbred, as would be the case in the wild. Thus, wild-type or Tcrd−/− FVB females were time-mated to C57BL/6J males and implantation rates evaluated following intravenous delivery of Evans blue dye at E5.5 . We therefore interrogated breeding in γδ T-cell-deficient 35 Fig. . This re35 Fig. , and a vles Fig. . While tble Fig. . Moreove 36 Fig. .37 we asked whether γδ T-cell deficiency might negatively impact host-protective responses to intravaginal infection by C. albicans. Indeed, Tcrd−/− mice showed highly significant, two-log fold increases in fungal growth within the FRT : A microbially enriched mouse housing model was designed at NMBU. Indoor pens built of galvanised steel (1.1 m × 2.4 m × 1.2 m) were prepared with woodchip bedding and enriched with soil, straw, and faecal content from farm animals. Three-week-old female and male C57BL/6JRJ mice were acclimatised for 1 week under conventional, pathogen-free conditions in individually ventilated cages (IVCs), before being distributed into different environments. Animals were primed in their respective housing environment for 4 weeks. For breeding, two pen-housed females and one male were brought together in IVCs enriched with pen material. After 10 days, the females returned to the pens to deliver. Female controls were mated under conventional, pathogen-free conditions in IVCs.All experiments were performed according to the UK, German and Norwegian (FOTS-18012) animal protection laws.+ cells, cell suspensions were incubated with anti-Vγ6 (1C10-1F7), followed by anti-mouse IgG1-APC (BioLegend).Uteri, vaginas, and lungs from mice were collected, minced, and digested using the Multi Tissue Dissociator kit-1 (Miltenyi). Samples were transferred into GentleMACS C tubes (Miltenyi) containing 2.5 mL digestion mix and incubated at 37 C for 40 min. Tissues were homogenised using GentleMACS program C and filtered through 70-μm strainers. Single-cell suspensions were stained with Live/Dead Aqua (Invitrogen), Fc-blocked (BD Biosciences) and stained with specific antibodies (TCRβ-BV605 (H57-597-BD Biosciences), TCRδ-BV421 (GL3-BioLegend), CD45-eV605 (30-F11-eBioscience), Vγ1-FITC (2.11-BioLegend), Vγ4-APC (UC3-10A6-BioLegend), Vγ5-PE , CD3-APCCy7 (17A2-BioLegend), CD27-FITC (LG.7F9-eBioscience), CD44-PECy7 (IM7-BioLegend), CD45RB-BV650 (16A-BD Biosciences), CD5-BV605 (UCHT2-BD Biosciences), ICOS-BV605 (7E.17G9-BD Biosciences), PD-1-BV605 (29F.1A12-BioLegend), CD127-BV711 (SB/199-BD Biosciences), CD69-PECy7 (H1.2F3-BioLegend), CD11b-FITC (M1/70-eBioscience), and Ly6G-PerCPCy5.5 (RB6-8C5-eBioscience). For identification of Vγ62-AF647 (BioLegend). Cells were acquired with a BD X20 or Symphony and analysed with FlowJo (TreeStar).For intracellular staining, cell suspensions were stimulated for 3 h with PMA 50 ng/mL, ionomycin 1 µg/mL, and brefeldin A 10 µg/mL. After surface staining, cells were treated with Foxp3-Fix/Perm buffer (BioLegend), followed by staining with anti-IL-17A-PE (TC11-18H10.1-BioLegend) and anti-IFN-γ-BV421 (XMG1.2-BioLegend). Alternatively, permeabilised cells were stained with anti-Pgr (Alpha PR6-Abcam) or anti-Esr (E115-Abcam), followed by goat-anti-mouse-IgG-PE (Thermo) or goat-anti-rabbit-F(ab′)+TCRδ− cells relative to the epithelium, images were 3D rendered and the minimal distance between each TCRδ+ or CD3+TCRδ− cell and EpCAM+ regions assessed via Definiens. For high-resolution analysis, samples were scanned using an Instant Structured Illumination Microscope  using a ×100 1.45 NA objective and analysed with Nikon or Fiji (NIH).For whole mount staining, uteri were fixed in Zamboni, blocked with 5% BSA and stained with TCRδ-AF647 (Biolegend-118134), CD3-FITC (BD-553061), and EpCAM (Biolegend-118201), followed by anti-rat-AF568 (Invitrogen-A-11077). Z-Sections were acquired on a Leica SP5 microscope using a ×40 1.25 NA objective and processed and analysed with Fiji (NIH). To determine the position of γδ T cells and CD3Alternatively, mouse uteri were fixed in 10% NBF for 24 h at RT and paraffin-embedded. For immunohistochemistry, following antigen retrieval, peroxidase blocking, and incubation with 1% BSA, 3-μm sections were incubated with anti-CD3 (ab134096-Abcam) or EpCAM (14-5791-EBioscience). Antibodies were detected with goat-anti-rabbit-Biotin (BA-1000-Vector) or rabbit-anti-rat-Biotin (BA-4001-Vector), followed by detection with DAB (Vector). Slides were counterstained with hematoxilin, dehydrated, cleared, and mounted.For confocal microscopy, slides were probed with anti-rabbit-AF647 and anti-rat-AF488, incubated with DAPI and mounted with Prolong Gold Antifade (Invitrogen).+TCRδ+Vγ1−4−5− cells were sorted from the uterus from C57BL/6J females into RLT Plus buffer. RNA was extracted with RNeasy micro kit Plus (Qiagen). TCR sequencing was performed with mouse Immunoverse TCRα/β/δ/γ kit (Archer Immunoverse).CD3+TCRδ+Vγ1−4−5− cells were sorted from C57BL/6J females, and thymic CD24+ (immature) or CD44+ (mature) CD3+TCRδ+Vγ1−4−5− cells were sorted from E18.5 embryos, into RLT Plus buffer. RNA was extracted with RNeasy micro kit Plus (Qiagen). cDNA was prepared using the NuGEN Ovation RNA-Seq System, followed by library preparation with the NuGEN Ovation UltraLow system. Sequencing was carried out on a HiSeq-4000 (Illumina), with read lengths of 75 bp.Uterus and lung CD3p value below 0.05 were deemed significant. Normalisation and variance-stabilising transformation (VST) were applied before performing euclidean distance-based clustering.Raw reads were quality and adaptor trimmed using cutadapt-1.9.1. Reads were aligned and quantified using RSEM-1.3.0/STAR-2.5.2 against mouse genome GRCm38 and annotation 86 (Ensembl). Differential gene expression analysis was performed in R-3.6.0 using DESeq2 (version 1.24.0). Genes with an adjusted 34GSEA (version 3.0) was performed using Preranked analysis with the classic scoring scheme. Gene lists were generated with the results of differential expression analysis, and ranked using the Wald statistic. Uterus and lung genesets were generated with signatures from Supplementary Table 7 of ref. Z-scores. For heatmaps of uterus and lung signatures, genes were ranked based on the differences mean(Uterus) − mean(Lung) and mean(Lung) − mean(Uterus), respectively.Heatmaps were made in R-3.6.0 using ComplexHeatmap (version 2.0.0). VST was applied on raw counts. Then, for each experimental condition mean VST was computed across samples and used to compute 57 Uterine or lung cell suspensions were cultured at a 1 × 106 cells/mL in phenol red-free RPMI containing 10% charcoal-treated FCS, glutamine, Pen/Strep, 1 mM pyruvate, β-mercaptoethanol, and 1X NEAA (complete medium), supplemented with 5 ng/mL rIL-23 (eBioscience), 5 ng/mL rIL-1β (R&D Systems), 10 μg/mL anti-IFN-γ (eBioscience), and 1 μg/mL indomethacin (Sigma-Aldrich), for 3 days in 96-well plates coated with 1 μg/mL anti-TCRδ (eBioscience). Cells were harvested and re-plated for 3 days in the same medium, without TCR stimulation. Finally, cells were harvested and incubated for 3 days in complete medium with 20 ng/mL rIL-7 (Peprotech). Throughout the culture protocol, 1 μM progesterone (Sigma-Aldrich) or DMSO were added.Uterine and lung γδ17 cells were expanded using a protocol adapted from McKenzie et al.Tcrd−/− females were timed mated with C57BL/6J males. For analysis of implantation, females were injected i.v. with 1% Evans Blue 5 days post coitum. Uteri were harvested after 3 min, and implantation sites detected as areas of Evans Blue accumulation. In addition, females were also culled at E14.5 or E15.5. Following uterus excision, embryos were enumerated, weighed and the CR and OF length measured. Placentas were harvested and fixed in NBF for histological analysis. FFPE placental sections were stained with H&E, and endomucin and cytokeratin-8 immunohistochemistry were performed.WT FVB or 6 cfu of C. albicans 529 L in 10 µL of PBS. On day 7 post infection, mice were culled and lavaged with 100 µL of PBS for fungal burden assessment. Uterine lysates were homogenised in GentleMACS tubes, programme E. The burden was determined by plating serial dilutions of vaginal and uterine samples in YEPD + chloramphenicol plates.Three days prior to infection, 100 µg of estradiol-17-valerate (Sigma) in sesame oil were administered subcutaneously. For infection, mice were anesthetised and inoculated intravaginally with 5 × 10t test. When multiple experimental groups were present, one-way ANOVA with Sidak’s post-hoc test was applied. To establish statistical significance of changes over time, we used one-way ANOVA with a post-hoc test for linear trend.Differences between experimental groups were analysed using two-tailed Student’s Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Tables


Gamma-delta (γδ) T cells are a subset of T cells that promote the inflammatory responses of lymphoid and myeloid lineages, and are especially vital to the initial inflammatory and immune responses. Given the capability to connect crux inflammations of adaptive and innate immunity, γδ T cells are responsive to multiple molecular cues and can acquire the capacity to induce various cytokines, such as GM-CSF, IL-4, IL-17, IL-21, IL-22, and IFN-γ. Nevertheless, the exact mechanisms responsible for γδ T cell proinflammatory functions remain poorly understood, particularly in the context of the central nervous system (CNS) diseases. CNS disease, usually leading to irreversible cognitive and physical disability, is becoming a worldwide public health problem. Here, we offer a review of the neuro-inflammatory and immune functions of γδ T cells, intending to understand their roles in CNS diseases, which may be crucial for the development of novel clinical applications. Together, gamma-delta (γδ) and alpha-beta (αβ) T cells represent two different T cell lineages that have been defined by their expression of αβ or γδ T cell receptors (TCRs) . AlthougUnlike αβ T cells, γδ T cells are a relatively minor subset of T lymphocytes in the peripheral blood (PB), comprising only 1–5% of lymphocytes circulating . However+ γδ T cells are present in the skin, Vγ7+ γδ T cells lie in the intestinal, Vγ6+ γδ T cells localize to the reproductive mucosa, and Vγ1+ or Vγ4+ γδ T cells are found in secondary lymphoid organs  receptors such as NKG2D  -4-hydroxy-3methyl-but-2-enyl pyrophosphate] and host-derived  phosphorylated metabolites originating from the isoprenoid metabolic mevalonate and non-mevalonate pathways, through association with butyrophilin 3A1 (BTN3A1) and BTN3A2 –16. Moreas NKG2D –20. It ias NKG2D –24. Alth T cells , 22. Besolecules , 26. Furns (HSP) –29.Recently, some discrete population of T cells that coexpressed αβ-γδ TCRs and Vγ-Cβ TCRs have been identified , 31. Amovia caspase-1 and inflammasome complex. γδ T cells secrete IL-17 in response to IL-1β, IL-18, and IL-23 in the absence of TCR (+ (Foxp3) -expressing Treg cells conversion, as well as promoting effector T (Te) cells response (Activation and development of γδ T cells promoting CNS inflammation are chiefly mediated by dendritic cells (DCs). The immunostimulatory component induces IL-1β, IL-6, IL-18, and IL-23 by DCs e of TCR –40. Durie of TCR –44. Moree of TCR . Also, Iresponse , 47. Theresponse .Activated DCs also promotes the induction of other proinflammatory cytokines from γδ T cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-21, and IL-22 , 40 Fi–51; howe+ Treg or Th17 cells  signals alone, in the absence of primary (TCR) and secondary (costimulation) signals, IL-17-producing T helper (Th17) cells require primary, secondary, and cytokine (IL-6 and TGF-β) signals to generate IL-17  , a murine MS model, is used to research the proinflammatory mechanism underlying CNS . Before elopment –77. Besielopment . Togetheelopment . Nonethe−/− and IL-23p19−/− mouse strains are both resistant to EAE (+ T cells (IL-17 and IFN-γ double-positive) is observed at the peak of EAE  reconstitute γδ T celln in EAE . These fThe main consequence of ischemic brain injury is manifested as the CNS tissue necrosis, due to the loss of nutrition. The tissue necrosis leads to a secondary inflammation, involving the accumulation of specific immune cells, especially neutrophils, macrophages, and T cells, which is a critical factor to the entire pathophysiology , 97.−/− mice illustrated a diminishment in infarct extent only 1 day after the ischemic injury, whereas IL-17 deficiency led to reduced infarct size after 4 days. Long-term, deficiencies of IL-17 and IL23 demonstrated obviously diminished CNS injury, relative to WT, or even IFN-γ−/−, mice  protectiveness and subsequent tissue inflammation; however, inflammation is also crucial to CNS immunity, as reduced γδ T cell expansion leads to increased host vulnerability to viral infection , 108. Fo−/− brain abscess mice  were detected elevated IL-17, and γδ T cells were the source of IL-17  researches involving the function of γδ T cells in models of CNS bacterial infection . For exaof IL-17 –114. Simof IL-17 . Also, Iof IL-17 . An incrof IL-17 . High leof IL-17 . NeverthImmune responses and neuroinflammation involving γδ T cells are also critically involved in CNS traumatic diseases. Diseases resulting from CNS trauma usually involve irreversible sensory, motor, and autonomic impairments . Periphe+ γδ T cells, exert a detrimental role in SCI, probably by providing an important origin of IFN-γ, which induces macrophages to adopt the M1 phenotype, with increased secretion of inflammatory cytokines, such as TNF-α , the Natural Science Foundation of Guangdong Province , and the Science and Technology Program of Guangzhou (No. 201803010001).CS was employed by Lunan Pharmaceutical Group Co. Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Crystals were obtained by slow evaporation of an acetonitrile solution of the title compound, which crystallizes in the triclinic space group PA new dithiolene derivative has been synthesized from [TBA] 2[Zn(dmit)2] and 3-chloro-2,4-pentanedione yielded single crystals of the title compound, -3,3′-[bis(sulfanediyl)]bis(4-hydroxypent-3-en-2-one), C13H14O4S5, after solvent evaporation. The title compound crystallizes in the triclinic space group PThe reaction between [TBA] The electrophilic nature of the acetylacetone (acac) motif and the high electron density on the sulfur atoms drive the nucleophilic substitution to completion. The title compound is a double β-dicarbonyl compound that contains two acetylacetone moieties, which are found in their enolic form in the solid state. Concerning the reactivity of the title compound, it is able to undergo acid or base-catalyzed α-hydrogen substitution reactions, in which the rate-determining step is the formation of the enol or enolate anion PThe title compound crystallizes in the triclinic space group it Fig. 1. Its chec axis and extend in a sheet-like fashion, forming a network that propagates along the axis with all rings following the same orientation. The sheets are linked by out-of-plane C13—H13B⋯S1 short contacts, generating stacks along the a axis with S⋯S short contacts between adjacent molecules [S5⋯S5iv = 3.5688 (6) Å]. In addition, the nucleophilic atom S3 is oriented towards the electrophilic C5, leading to an S3⋯C5iii [3.471 (2) Å] contact, further contributing to the extension of the network along the c-axis direction. Molecules of the title compound also associate with neighboring molecules above and below the thiocarbonyl ring planes through the acac backbone by C4—H4⋯S1 and C9—H9⋯S1 contacts. The acac backbone lies nearly perpendicular to the rings, and there are several key interactions between the carbonyl oxygen atoms  and neighboring methyl hydrogen atoms (H8A and H8C) with lengths in the range 2.56-2.66 Å. However, atom O4 is not involved in any interactions with hydrogen atoms, and instead makes short contacts with both C12 and C13.The title compound exhibits numerous intermolecular interactions, namely four C—H⋯O, three C—H⋯S, three C⋯O, one S⋯C, and one S⋯S interaction Fig. 2 and 2 ▸.dnorm is shown in Fig. 3et al., 2007CrystalExplorer17 , H⋯H , O⋯H/H⋯O , C⋯H/H⋯C , S⋯C/C⋯S , S⋯O/O⋯S , S⋯S , O⋯C/C⋯O , O⋯O , and C⋯C  interactions. Thus, the Hirshfeld surface analysis indicates that the most significant contributions arise from S⋯H and H⋯H contacts.The Hirshfeld surface 2] and 4 eq. of 3-chloro-2,4-pentanedione in 100 mL of acetonitrile under argon for 1 h, after which it was cooled and left under stirring overnight. Activated carbon was added and stirring continued for 1 h. The resulting mixture was filtered and washed with cold acetonitrile. The solvent was evaporated under reduced pressure, and ethyl acetate was added to precipitate ZnCl2. The remaining solution was filtered, followed by evaporation of the solvent, giving a yellow crystalline solid (67.3% yield). NMR analyses were performed on a Bruker AV-700 spectrometer using chloroform-d (CDCl3) as solvent. The solvent signals at 7.26 and 77.00 ppm were used as internal standards for proton and carbon, respectively. 1H NMR  δ 15.40  5.11, 2.48 . 13C NMR  δ 24.92, 102.30, 131.40, 197.78, 209.18.The synthesis of the title compound was carried out by refluxing 1 eq. of [TBA]et al., 1976via resonance of the conjugated π-system and intramolecular hydrogen bonding. Furthermore, the enol is the less polar of the two tautomers because the intramolecular hydrogen bond reduces the dipole–dipole repulsion of the two carbonyls in the keto form. The equilibrium of β-dicarbonyl compounds has been studied extensively and it has been shown that tautomeric interconversion between the diketo and enol forms is relatively slow and can be observed by NMR. Under normal conditions, the enolic form predominates in equilibrium  =1.5Ueq and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020010695/dx2025sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020010695/dx2025Isup2.hklStructure factors: contains datablock(s) I. DOI: 1984599CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Dear Editor,c-erbAα and c-erbAβ genes. Their human homologs are designated THRA and THRB. The c-erbAα gene located on chromosome 17 encodes two different TRα isoforms. One is functional TH-binding TRα1 and the other is a dominant-negative splice variant, TRα2, lacking TH binding activity [In Dr. Magdalena Szaryńska’s recent letter, she expressed one confusing aspect she met in activity . T3 inteactivity . TRα2 is


The dicarboxylic acid was reduced to 1,1′-ferrocenedimethanol using LiAlH4 and subsequently converted to 1,1′-bis(azidomethyl)ferrocene in the presence of NaN3. The diazide was treated with 2-ethynylpyridine under ‘click’ conditions to give the title compound in 75% yield. The FeII center lies on an inversion center in the crystal. The two pyridyltriazole wings are oriented in an anti conformation and positioned exo from the FeII center. In the solid state, the molecules interact by C—H⋯N, C—H⋯π, and π–π interactions. The complexation of the ligand with [Cu(CH3CN)4](PF6) gives a tetranuclear dimeric complex.The title compound, [Fe(C The CCp—Csp3 and CCp—N bond lengths involving C6 are 1.4910 (19) and 1.4700 (18) Å, respectively. The pyridyltriazole moiety is oriented exo from the FeII center, with the least-squares planes of the Cp and triazole rings forming a dihedral angle of 65.68 (5)°. The nitrogen donor atoms of the pyridyltriazole units adopt an anti conformation, as is often observed in this type of chelating ligand  and the Cp carbon atom C5 forms a C—H⋯N interaction with a C⋯N distance of 3.4240 (19) Å to triazole N2 . These two contacts form a ring with graph-set motif et al., 1990x, y + 1, z). Thus, the C—H⋯N contacts form a two-dimensional network normal to [001]. The pyridyltriazole moieties stack in an anti-parallel fashion about inversion centers. The pyridyl moiety of one molecule has a π–π interaction with the triazole moiety of another molecule with a dihedral angle of 11.27 (10)° and centroid–centroid distance of 3.790 Å . In addition, there are also C—H⋯π interactions between the hydrogen atom of the pyridyl moiety with the cyclopentadienyl ring . These two interactions thus form centrosymmetric dimers, illustrated in Fig. 4The crystal structure of the title compound is consolidated by intermolecular C—H⋯N Table 1, C—H⋯π, et al., 2016et al., 2010m-xylylene, VAJVIN 4]PF6 was added to a suspension of the ligand in DMF in a 1:1 ratio, the mixture was completely soluble, indicating the formation of a complex. To avoid oxidation of the complex, the resultant solution was diffused with diethyl ether vapor under nitrogen for 3 d. Under these conditions, a bright-yellow microcrystalline solid was formed. At room temperature, the 1H NMR spectrum of the complex showed a simple pattern containing the same set of signals for the ligand, indicative of the presence of one single species in solution. Compared to the spectrum of the free ligand, the proton signals of the complex, especially for the pyridyltriazole coordination pocket, are shifted downfield  ligands and their AgI complexes + with a similar isotopic pattern as the theoretical simulation ferrocene, 2. To a stirred solution of 1,1′-ferrocenedicarboxylic acid  in dry THF (400.0 mL), 1.0 M LiAlH4  was added dropwise at room temperature under N2. The reaction vigorously produced hydrogen gas. The reaction mixture was refluxed for 2 h, by which time the starting compound was consumed, as evidenced by TLC. The reaction was again cooled to room temperature, and ethyl acetate (5 mL) and water (10 mL) were added in sequence with constant stirring. The product was extracted with ethyl acetate (4 × 150 mL). The combined organic layer was dried with anhydrous MgSO4 and volatiles removed in vacuo to give 2  as a brown solid. The analytically pure product was obtained by recrystallization from toluene upon cooling. 1H NMR : δ 4.07 , 4.13 , 4.19 , 4.30 . 13C NMR : δ 67.4, 67.7, 69.6, 89.7.Synthesis of 1,1′-bis(azidomethyl)ferrocene, 3. Caution! Organic azides with low C/N ratio are potentially dangerous. However, we did not encounter any problem during the synthesis of diazide and its subsequent derivatization. To a stirred solution of 1,1′-hydroxymethylferrocene  in glacial acetic acid (7.5 mL), sodium azide  was added. The reaction was stirred for 3 h at 323 K under nitrogen. The reaction mixture was neutralized with a saturated solution of sodium bicarbonate. The product was extracted with chloroform (2 × 50 mL). The organic phase was dried with anhydrous MgSO4 and the volatiles removed in vacuo to give 3  as a viscous liquid. IR : 2092 (s), 1733 (w), 1240 (m). 1H NMR : δ 4.10 , 4.19 , 4.22 .Synthesis of 1,1′-bis(pyridyltriazolylmethyl)ferrocene, 4. To a stirred solution of 1,1′-bis(azidomethyl)ferrocene  in a mixture of DMF and water (4:1) (20 mL), Na2CO3 , CuSO4·5H2O , ascorbic acid , and 2-ethynylpyridine  were added in sequence. The reaction mixture was stirred for 20 h at room temperature, and then poured into an NH3/EDTA solution  and the mixture extracted with chloroform (3 x 100 mL). The organic layer was collected, dried over MgSO4, and evaporated to dryness. The crude product was purified by trituration with cold diethyl ether to give 4  as a light-brown solid. X-ray quality crystals of the compound were obtained by vapor diffusion of diethyl ether into its solution in chloroform, m.p.: decomposes above 463 K. 1H NMR : δ 4.24 , 4.32 , 5.33 ,7.21, 7.75 , 8.05 , 8.15 , 8.53  13C NMR : δ 49.8, 69.8, 70.2, 81.9, 120.3, 121.5, 122.9, 137.0, 148.4, 149.4, 150.2. HRESI–MS: m/z = 501.1416 [4+H]+ , 523.1238 [4+Na]+ .I complex of 1,1′-bis(pyridyltriazolylmethyl)ferrocene, 5.Synthesis of Cu To a nitrogen-purged stirred suspension of 4  in DMF (10 mL), [Cu(CH3CN)4](PF6)  was added. The reaction produced a clear yellow solution, which was stirred for 2 h at room temperature. The reaction mixture was diffused with nitrogen-purged diethyl ether using a cannula for 3 d. The solution was decanted and the product was washed with diethyl ether and dried under a slow stream of nitrogen to give 5  as a yellow microcrystalline solid. A 1H NMR sample was prepared by dissolving the compound in DMSO-d6 and transferring the solution into an NMR tube under nitrogen. 1H NMR : δ 4.21 , 4.29 , 5.43 , 7.90 , 8.04 , 8.43 , 9.05 . HRESI–MS: m/z = 1273.0915 (Cu242](PF6)+ , 563.0650 [Cu4](PF6)+ .2) and with Uiso(H) =1.2Ueq for the attached C atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020011901/pk2645sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020011901/pk2645Isup2.hklStructure factors: contains datablock(s) I. DOI: 2026000CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of 64Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs 64Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for 64Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for 64Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted 64Cu-AB1-HLys-DOTA group than in the 64Cu-HLys-DOTA group and 2‐3 times higher than in the PBS control group at 1, 4, and 24 h post injection. 64Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that 64Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria.Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N′,N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with  Infectious diseases are one of the top ten leading causes of death in the world and the number one cause of death in low-income developing countries. Current diagnostic methods for bacterial infections are time-consuming as they require samples from the patient to be cultured for hours to weeks before enough bacteria are isolated to run destructive diagnostic tests , 2. As p99mTc-methylene diphosphonate, 67Ga-citrate, and 18F-FDG citrate PET-CT of bone and joint infection offered lower radiation dose, as well as earlier and shorter imaging times, and its sensitivity and specificity was found to be lower than [67Ga ]citrate TF) has been used to image Staphylococcus aureus (SA) and found to detect the infected lesions better than [68Ga]Cl3; additionally, [68Ga]TF was also found to detect the Gram-negative bacteria, Proteus mirabilis [68Ga and used to distinguish inflamed muscle from SA-infected muscle [Another radioisotope of interest is ]citrate . In othepathways . 68Ga-apirabilis . In othed muscle .64Cu radioisotope for imaging purposes has increased over the years, and several studies have utilized this isotope to label compounds for detecting regions of infection ATSM DTPA-prothrombin was used to noninvasively detect infection lesions as compared to the bacteria-free control mice, which had no accumulation at the site of endothelial trauma ATSM . In micel trauma .Staphylococcus aureus (SA) even in the presence of serum proteins, while bypassing mammalian cells and Gram-negative bacteria fluoro-d-glucose ([18F]FDG) was purchased from PETNET . All solvents and reagents were obtained from Sigma-Aldrich Company unless otherwise specified. Staphylococcus aureus (SA), ATCC 6538, and Pseudomonas aeruginosa (PA), ATCC 47085, were purchased from ATCC , and Whatman 3 M silica gel thin-layer chromatography (TLC) plates were purchased from Fisher Scientific . Diethylenetriaminepentaacetic acid (DTPA) was purchased from Sigma . 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) was purchased from Macrocyclics . C18 SepPak cartridges were obtained from Waters Corporation (Milford. MA). Radiometric instant thin-layer chromatography measurements were accomplished using a TLC scanner (Bioscan AR-2000 Scanner). Reverse-phase chromatography analysis was performed on a C18 column (PA) with an infinity diode-array UV detector  and a PMT/NaI remote radioactive detector . Laura radiochromatography software  was used to quantify chromatograms by integration. Radioactive samples were counted using a 2480 Wizard II automatic gamma counter . Imaging studies were done by using GNEXT PET-CT (Sofie BioSciences) . Acquired PET/CT images were analyzed with Inveon™ Research Workplace (IRW) (Siemens).HLys-DOTA and AB1-HLys-DOTA were synthesized by following a previously described protocol for solid-phase peptide synthesis , and the64Cu  was produced in-house at the University of Alabama at Birmingham Cyclotron facility via the 64Ni64Cu nuclear reaction. Production and purification of 64Cu was conducted using modified methods from literature FDG imaging was also performed in infected animal models as described in the PET imaging section below to further confirm the existence of infection in the right thigh and the absence thereof in the left thigh.Mice for infection studies were anesthetized prior to the intramuscular infection of SA or PA. Animals were immobilized with isoflurane. Bacteria cells concentrated in sterile PBS were injected into the thigh muscle and the site of injection pinched to minimize bleeding. SA or PA suspension (50 μCi) of 64Cu-HLys-DOTA or 64Cu-AB1-HLys-DOTA was injected into prewarmed tail vein of the animals under anesthesia. Animals were euthanized at 1 and 4 h after radiotracer administration (n = 4 for each tracer at each time point). At 24 h after small animal PET imaging, the mice were also euthanized for a post-PET biodistribution. In a similar set of experiment, excess of the unlabeled peptide, 150 nmol, was coinjected with the radiolabeled peptides per mouse. The organs of interest were collected, weighed, and measured for radioactivity content using a gamma counter. The data were background-corrected and decay-corrected. The biodistribution data were expressed as percentage of injected radioactive dose per gram of tissue (%ID/g) for selected organs as the mean value of four mice.For biodistribution studies, 3.3–3.7 MBq (90–100 μCi), was administrated via tail vein injection of prewarmed mice under anesthesia. The inflammatory response caused by the infection was confirmed by intravenous injection of [18F]FDG, 3.7 MBq (100 μCi), into mice infected on the right thigh muscle with SA or PA and on the left with PBS as control. For this small animal PET imaging, 1 and 4 h post-injection static scans were collected. Following imaging studies, the mice were sacrificed for biodistribution. Mice were allowed to recover from anesthesia until the time of imaging. Mice were anesthetized with 2‐3% isoflurane/oxygen and imaged on the small animal PET/CT scanner. Static images were collected at 1, 4, and 24 h for 15, 20, and 30 min, respectively. PET images were coregistered with CT image for anatomical colocalization. Regions of interest (ROI) were manually drawn over organs of interest with CT anatomical guidelines, and the associated radioactivity was measured using Inveon Research Workstation software. Standard uptake values (SUV) were calculated as nCi/cc × animal weight/injected dose, and comparisons in pharmacokinetics of radiolabeled peptides were assessed.Small animal PET/CT imaging studies were conducted in SA-infected and PA-infected animal models. Each radiolabeled peptide, 3.3–3.7 MBq (90–100 p < 0.05) were considered statistically significant.Statistical calculations were carried out using Prism 7 (GraphPad Software) and expressed as mean ± SD. One-way analysis of standard deviation at 95% confidence level , respectively, at 56°C. Incubations at lower temperature resulted in lower yields  was significantly higher than that of 64Cu-HLys-DOTA at all time points: 0.08, 0.67, 1, 4, and 24 h; 98.5 ± 3.5% vs 39.1 ± 3.3% at 24 h, Pseudomonas aeruginosa (PA), a Gram-negative bacterium. At 5 min after incubation, the uptake of 64Cu-AB1-HLys-DOTA and 64Cu-HLys-DOTA showed 9.8 ± 4.4% and 1.9 ± 0.4% binding to PA, respectively, which increased slightly to 13.3 ± 1.1% and 5.2 ± 0.3%, respectively, at 24 h after incubation. This indicates a 5–10 times lower uptake of the peptides in PA as compared to SA confirming the specificity of AB1 for Gram-positive bacteria. Specific bacterial cell uptake was confirmed by the low uptake (9.4 ± 2.3%) of 64Cu-AB1-HLys-DOTA in mammalian SKBR3 breast cancer cells at 24 h post incubation.Excellent stability of 6 cfu/g of bacteria was found and only one PBS-control muscle was positive with colony at 1.8 × 105 cfu/g cfu. [18F]FDG in vivo characterization of the infection site verified high uptake in the SA-infected muscle versus the control muscle at 1 and 4 h post injection, p < 0.005.The development of infection was evident by presence of a palpable mass filled with abscess observed during dissection and harvesting. The infected muscle and control muscle were harvested, homogenized, and the CFU of bacteria counted. In the infected muscle, 4.8 ± 2.5 × 1064Cu-AB1-HLys-DOTA and 64Cu-HLys-DOTA were assessed in mice intramuscularly infected with S. aureus on the right thigh muscle and PBS injected in the left thigh muscle as control. The biodistribution showed that the uptake of both peptides in the infected muscle was significantly higher than the PBS-injected control muscle of the same mouse at all time points: 1, 4, and 24 h post injection, as shown in 64Cu-AB1-HLys-DOTA conjugate in the infected thigh was 2‐3 times higher than the control muscle at later time points, although no significant difference was observed at 1 h (4.8 ± 2.5 %ID/g vs. 2.5 ± 0.5 %ID/g). At 4 h, the dose uptake of 64Cu-AB1-HLys-DOTA in the infected muscle was 3.6 ± 0.5 %ID/g vs. 1.1 ± 0.4 %ID/g in the control muscle, p < 0.0005. Similarly, at 24 h, the uptake of 64Cu-AB1-HLys-DOTA in the infected muscle was 2.6 ± 0.6 %ID/g vs. 0.8 ± 0.1 %ID/g in the control muscle, p < 0.005. In contrast, the dose accumulation of 64Cu-HLys-DOTA in the infected muscle was only 1- or 2-fold higher than the control muscle in the same mouse, except at 24 h where no significant difference was observed between them. At 1 and 4 h, the uptake of 64Cu-HLys-DOTA in the infected muscle vs. control muscle was 1.3 ± 0.5 %ID/g vs. 0.6 ± 0.1 %ID/g, p < 0.05, and 0.8 ± 0.1 %ID/g vs. 0.4 ± 0.1 %ID/g, p < 0.005, respectively. We also observed that uptake in the infected muscle was 4 times higher in the targeted 64Cu-AB1-HLys-DOTA group compared to that in the 64Cu-HLys-DOTA group, as shown in 64Cu-AB1-HLys-DOTA in the infected muscle was 4.8 ± 2.5, 3.6 ± 0.5, and 2.6 ± 0.6 %ID/g vs. 1.3 ± 0.5, 0.8 ± 0.1, and 0.6 ± 0.1 %ID/g for 64Cu-HLys-DOTA, respectively, at 1, 4, and 24 h post injection, p < 0.005 at 4 and 24 h. The highest accumulation of the radiolabeled peptides was observed in the liver and kidney with 64Cu-AB1-HLys-DOTA also showing some uptake in the lungs. 64Cu-AB1-HLys-DOTA also showed higher retention in the blood and heart than 64Cu-HLys-DOTA at 1 and 4 h post injection but at 24 h, both peptides had similar activity in the blood. A similar experiment using an excess of the unlabeled peptides, 150 nmol, did not show a statistical difference (data not shown) in the uptake of the peptides at the infection site. This may indicate that the binding sites at the bacterial surface cannot be saturated.The biodistribution and pharmacokinetics of the peptides 64Cu-AB1-HLys-DOTA in the infected muscle was visibly observed via small animal PET/CT imaging as early as 1 and 4 h post injection, 64Cu-AB1-HLys-DOTA than in the images of 64Cu-HLys-DOTA at 1 and 4 h post injection in agreement with the biodistribution data. SUV analysis of the PET images showed 2.4 to 4-fold increase in accumulation of 64Cu-AB1-HLys-DOTA vs. 64Cu-HLys-DOTA at the infection. The mean SUV of 64Cu-AB1-HLys-DOTA and 64Cu-HLys-DOTA in the infected muscle was 0.70 ± 0.08 vs. 0.17 ± 0.06, p < 0.0001, 0.49 ± 0.04 vs. 0.16 ± 0.02, p < 0.0005, and 0.31 ± 0.01 vs. 0.13 ± 0.01, p < 0.0001 at 1, 4, and 24 h post injection, respectively. There was no statistical difference between mean SUV uptake of 64Cu-HLys-DOTA in the infected muscle and 64Cu-AB1-HLys-DOTA uptake in the control muscles at all time points. This indicates 64Cu-HLys-DOTA uptake in the infected muscle is as low as the background uptake of 64Cu-AB1-HLys-DOTA observed in the control muscle. Mean SUV analysis of the heart supports the trend in the biodistribution study with blood retention of 64Cu-AB1-HLys-DOTA at 0.65 ± 0.08 vs. 0.25 ± 0.05 for 64Cu-HLys-DOTA, p < 0.0005, at 1 h. At 24 h post injection, both peptides had similar dose retention in the heart: 0.26 ± 0.04 and 0.21 ± 0.04 for 64Cu-AB1-HLys-DOTA and 64Cu-HLys-DOTA, respectively. The images also showed high signal in the liver and kidney possibly due to the fast clearance of the cationic peptides through these organs.The uptake of 64Cu-AB1-HLys-DOTA peptide for Gram-positive bacteria was confirmed by low uptake in the Gram-negative bacteria Pseudomonas aeruginosa (PA) as observed in the PET images and the 4 h post-PET biodistribution, shown in Supporting Information 64Cu-AB1-HLys-DOTA uptake in the SA-infected muscle at 1 and 4 h was significantly higher than that in the PA-infected muscle: 0.70 ± 0.08 vs. 0.28 ± 0.10, p=0.0052, and 0.49 ± 0.04 vs. 0.19 ± 0.10, p=0.0288, respectively. A similar trend was observed in the 24 h post-PET biodistribution with percent uptake of 64Cu-AB1-HLys-DOTA in the SA-infected muscle at 2.64 ± 0.57 %ID/g vs. 0.49 ± 0.11 %ID/g in the PA-infected muscle, p=0.0039.Specificity of 18F]FDG uptake in the SA-infected muscle was significantly higher than that in the control muscle at 4 h post injection with mean SUV of 1.96 ± 0.19 vs. 0.41 ± 0.09, p < 0.0001, which confirmed the presence of the SA infection [18F]FDG in the SA-infected muscles was also confirmed with post-PET imaging biodistribution data at 4 h , as shown in FPTMP showed high uptake at the infection and low background signal in normal tissues and other noninfection inflammation sites but did not differentiate between Gram-negative and Gram-positive strains [nfection . Althoug strains . Thus, f64Cu-AB1-HLys-DOTA showed higher uptake in S. aureus bacteria cells in vitro and improved accumulation at the infection site of SA-inoculated mice compared with the noncovalently targeting 64Cu-HLys-DOTA. In the small-animal PET images, the dose uptake of 64Cu-AB1-HLys-DOTA at the infected site was distinguishable as early as 1 h after administration, indicating its potential for fast detection of infection. These results illustrate that the 64Cu-labeled AB1-HLys-DOTA peptide could be used as imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacterial infection.The purpose of this study was to investigate the potential of AB1-HLys-DOTA peptide to image and distinguish infection due to Gram-positive bacteria from Gram-negative bacteria with higher specificity than HLys-DOTA peptide.


Three salts containing the 4-(4-fluorophenyl)piperazin-1-ium cation have been prepared and structurally characterized. 10H14FN2+·C7H3N2O7−, (I), the anion contains an intramolecular O—H⋯O hydrogen bond, and it has a structure similar to that of the picrate ion. The cations and anions are linked into [001] chains of rings by a combination of two three-centre N—H⋯(O)2 hydrogen bonds. The anion in 4-(4-fluorophenyl)piperazin-1-ium hydrogen oxalate, C10H14FN2+·C2HO4−, (II), is planar, and the cations and anions are linked into (100) sheets by multiple hydrogen bonds including two-centre N—H⋯O, three-centre N—H⋯(O)2, O—H⋯O, C—H⋯O and C—H⋯π(arene) types. In 4-(4-fluorophenyl)piperazin-1-ium hydrogen -tartrate monohydrate, C10H14FN2+·C4H5O6−·H2O, (III), the anion exhibits an approximate non-crystallographic twofold rotation symmetry with antiperiplanar carboxyl groups. A combination of eight hydrogen bonds, encompassing two- and three-centre N—H⋯O systems, O—H⋯O and C—H⋯π(arene) types, link the independent components into a three-dimensional framework. Comparisons are made with some related structures.Three salts containing the 4-(4-fluorophenyl)piperazin-1-ium cation have been prepared and structurally characterized. In 4-(4-fluorophenyl)piperazin-1-ium 2-hydroxy-3,5-dinitrobenzoate, C N-(4-fluorophenyl)piperazine (4-FPP) is a major metabolite piperazin-1-yl]butan-2-yl}pyridine-3-carboxamide), used in the treatment of autistic disorders piperazine (MeOPP), also used as a recreational drug -hydrogentartrate, which crystallizes from ethyl acetate as a monohydrate (III)We have recently reported the structure of MeOPP and those of a number of salts derived from it  form, was used in the synthesis of (III)P212121.Compounds (I)–(III) are all 1:1 salts Figs. 1–3 ▸ ▸ iIn each of (I)–(III), the piperazine ring adopts an almost perfect chair conformation, with the 4-fluorophenyl substituent occupying an equatorial site. The value of the ring-puckering angle θ –(III), the location of the remaining acidic H atom was initially deduced from difference-Fourier maps, and then confirmed by refinement of the atomic coordinates, reinforced by inspection of the final difference-Fourier map and of the relevant C—O bond lengths, where the single and double bonds have distances entirely typical of their types (Allen cf. Scheme), as has been observed in picrate  anions By contrast, the anion in compound (II)S(6) motif 1] Fig. 4. There i1] Fig. 4, which l1 screw axis along , are linked by a combination of an asymmetric three-centre N—H⋯(O)2 hydrogen bond and a two-centre O—H⋯O hydrogen bond  1 − x, 1 − y, 2 − z] of 3.0108 (14) Å, but it is unclear whether this has any structural significance.The component ions in compound (II)d Table 2 to form d Table 2. This chon Fig. 6. The comet al., 1998abet al., 2000z < 1.0 if Fig. 3. The int.0 Fig. 7. A secons Table 3. Finallys Table 3, therebyet al., 2019P21/c, their unit-cell dimensions are very different, as is the manner of their supramolecular assembly. Thus, in the structure of (IV), a combination of N—H⋯O and C—H⋯O hydrogen bonds links the component ions into a chain of centrosymmetric rings in which rings of et al., 2019et al., 2012aet al., 2012bIt is of interest briefly to compare the structures reported here with those of some closely related compounds. An obvious comparison is between compound (I)N-(4-fluorophenyl)piperazine  was dissolved in methanol (10 ml) and a solution of the appropriate acid (0.55 mmol) in methanol (10 ml)  was then added; the mixtures were briefly stirred at 323 K before being set aside at ambient temperature to crystallize. After two days, the resulting solid products were collected by filtration and dried in air. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature and in the presence of air, of solutions in ethyl acetate for (I)All starting materials were obtained commercially, and all were used as received. For the preparation of compounds (I)–(III), 2), or 0.98 Å  and with Uiso(H) = 1.2Ueq(C). The H atoms bonded to N or O atoms were refined with Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O), giving the N—H and O—H distances shown in Tables 1x parameter  − (I−)]/[(I+) + (I−)], was −1.5 (7). Since a single enantiomer, the  form, of tartaric acid was used in the preparation of compound (III)Crystal data, data collection and refinement details are summarized in Table 410.1107/S2056989020006398/wm5557sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989020006398/wm5557Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020006398/wm5557IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020006398/wm5557IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989020006398/wm5557Isup5.cmlSupporting information file. DOI: 2003726, 2003725, 2003724CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: