N = 288 recordings) to investigate whether it is possible to reliably identify cell type  based on their electrophysiological characteristics. We quantified 15 electrophysiological variables in each recorded cell. Individually, none of the variables could reliably distinguish the cell types. We therefore constructed a logistic regression model that included all quantified variables, to determine whether they could together identify cell type. The model identified cell type with 94% accuracy. This model was applied to a dataset of cells recorded from hyperglycaemic βV59M mice; it correctly identified cell type in all cells and was able to distinguish cells that co-expressed insulin and glucagon. Based on this revised functional identification, we were able to improve conductance-based models of the electrical activity in α-cells and generate a model of δ-cell electrical activity. These new models could faithfully emulate α- and δ-cell electrical activity recorded experimentally.The α-, β- and δ-cells of the pancreatic islet exhibit different electrophysiological features. We used a large dataset of whole-cell patch-clamp recordings from cells in intact mouse islets ( All tThe whole-cell patch-clamp technique, applied to intact islets, is the perfect experimental paradigm for understanding the electrophysiological properties of islet cells. However, within a mouse islet, the different cell types are not present in equal proportions; β-cells are the most abundant , with α-cells 15–20%) and δ-cells (5–10%) being relatively sparse –20% and –18, progThis has motivated the development of strategies to improve identification of islet cell type. β-cells can be separated from non-β-cells by autofluorescence-activated cell sorting . Althoug+ currents and a voltage-gated Na+ current that inactivates at very hyperpolarized potentials [+ current [+ current with depolarized inactivation properties relative to the β-cell [2+ channels [Currently, electrophysiological identification of cell type  relies on two criteria. The first is that β-cells are larger than non-β-cells ,34–44. Ttentials ,39,45. I current ,38,46–48e β-cell ,49,50 anchannels .Here we reviewed the electrophysiological fingerprints of mouse α-, β- and δ-cells. We recorded and analysed a large dataset of whole-cell voltage-clamp recordings (288 recordings) made from cells in intact mouse islets, whose cell type was subsequently unequivocally determined by immunocytochemistry. We used these data to investigate the validity of these properties for cell identification and to produce a mathematical model for identifying islet cell type. We show that this model can reliably identify islet cell type and can be successfully used to monitor transdifferentiation of cells in a diabetic mouse model (βV59M) . Our fin2.2.1.+ (KATP) channel in β-cells (βV59M mice). This dataset consisted of 13 recordings from βV59M mice, and 15 from littermate controls.Recordings from 288 cells in islets from five different strains of mouse with a normoglycaemic phenotype were used in this study. The mouse strains were NMRI, C57BL/6, EPAC2-KO , GYY 3232 and SS2.2.Mice were killed by cervical dislocation, and islets isolated by liberase digestion (schedule 1 procedure). Islets were used for acute experiments and were not maintained in tissue culture for less than 16 h. A new islet was used for each cell recording.2.3.ulse software . Currents were filtered at 2.9 kHz and digitized at more than 10 kHz. Currents were compensated for capacitive transients and leak current subtraction was conducted. The extracellular medium consisted of (mM) 118 NaCl, 20 tetraethylammonium-Cl (TEA-Cl), 5.6 KCl, 1.2 MgCl2, 5 HEPES (pH 7.4 with NaOH), 2.6 CaCl2 and 1 d-glucose. Two intracellular (pipette) solutions were used (solution 1 and solution 2). Solution 1 contained (mM) 125 K-glut, 10 KCl, 10 NaCl, 1 MgCl2, 5 HEPES, 3 MgATP and 0.05 EGTA (KOH buffered). Solution 2 contained 15 Cs-glut, 10 CsCl, 10 NaCl, 1 MgCl2, 5 HEPES, 3 MgATP, 0.05 EGTA (CsOH buffered). All chemicals were from Sigma-Aldrich. Only recordings with an access resistance of less than 50 MΩ were used for analysis.Whole-cell currents were recorded in intact islets using the standard whole-cell configuration. Measurements were performed using an EPC-10 patch-clamp amplifier and P2.4.−1) was included in the intracellular solution to allow identification of the cell recorded from. Following voltage-clamp experiments, islets were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) overnight and permeabilized with 0.3% Triton X-100. Non-specific binding was blocked by pre-treatment for 2 h with 5% normal goat serum before incubating with the different primary antibodies for 4–12 h , sheep anti-glucagon  and rabbit anti-somatostatin ). After washing with PBS, the islet was incubated for 1 h in secondary antibodies (Alexa 633 goat anti-guinea pig (insulin), Alexa 405 goat anti-mouse (glucagon) and Alexa 543 goat anti-rabbit (somatostatin)). Biocytin labelling was visualized by using Alexa Fluor 488 conjugated streptavidin . Islets were then washed and imaged on a confocal microscope .In all recordings, cell identity  was subsequently established by immunocytochemistry. Biocytin  and validating  the model. A description of this model and the modelling process is given in appendix B.A multinomial logistic regression model was constructed. For a given set of electrophysiological measures from a specific cell recording, this model can be used to predict the cell type. The model process requires a dataset for constructing . The toolbox uses the multinomial logistic regression model presented to predict cell type, given a set of user-defined inputs . We have also made available on GitHub the entire dataset of 288 cell recordings that can be tested with the multinomial regression model.The multinomial logistic regression model was constructed in SPSS . The model developed was coded into a freely available Matlab toolbox for predicting cell type. The toolbox and SPSS files are available from GitHub . If the data passed normality criteria , a parametric test was conducted with the appropriate post hoc test (Tukey). If the normality criteria were not met, a Kruskal–Wallis test with Dunn's multiple comparison test was conducted.All recorded variables were compared across cell types using one-way ANOVA (Pχ2) will test whether there is an association between this variable and cell type. For post hoc tests, we adopted the approach described by Sharpe [Some of the variables used to identify cell type, such as the presence/absence of an outward transient current, are categorical . A contiy Sharpe ; contingy Sharpe .2.8.−10. The models are described in appendix C and can be obtained from GitHub (https://github.com/IsletCellType).Conductance-based (Hodgkin–Huxley-like) models were used to simulate electrical activity in a model of an α-cell and a δ-cell. All conductance-based models were solved numerically in the software package XPPAUT  using thIn what follows, it will be clear from the context whether we are referring to either (i) a conductance-based model of α- or δ-cell electrical activity or (ii) a multinomial logistic regression model for predicting islet cell type.3.We analysed the electrophysiological variables of 288 cells in intact islets from mice with a normoglycaemic phenotype.3.1.Ccell) in β-cells  was significantly larger than that seen in α-cells  and δ-cells . α-Cells and δ-cells did not differ in their cell size (p = 0.556). Given that Ccell is frequently used to identify cell type [Ccell alone can identify cell type (equation (B 2) and Ccell alone is an inadequate indicator of cell type.Cell capacitance  was larger in β-cells  than in α-cells  or δ-cells . There was no difference in G between α-cells and δ-cells (p = 0.215).The whole-cell conductance  in α-cells  was statistically lower than in β-cells . G density in δ-cells  was no different from that in β-cells (p = 0.184) or α-cells (p = 0.536).3.3.+ current  evoked in α-cells  was significantly smaller than that in β-cells  and δ-cells . There was no difference in Imax between δ- and β-cells (p = 0.14). We explored whether Imax could be used to predict cell type in a multinomial logistic regression model (equation (B 2) and Imax alone cannot reliably identify cell type.The maximum amplitude of the Nant Imax; a evoked 3.4.+ current differed between cell types (c–e). Inactivation in α-cells was half-maximal (V2 h) at −38.4 ± 1.4 mV (N = 141), as observed in pancreatic slices [N = 91; p = 0.187). In contrast, V2 h was significantly more hyperpolarized in β-cells  than in either α-cells (p < 0.001) or δ-cells (p < 0.001). There was no difference in V2 h between α- and δ-cells (p = 0.22).The voltage dependence of steady-state inactivation of the Nall types c–e. Inacc slices . This vaV2 h is often used to distinguish β-cells from non-β-cells [V2 h alone could be used to distinguish cell type. We first did this by investigating whether a simple criterion could enrich the β-cell population; the number of cells with V2 h < a fixed cut-off were counted. The cut-off ranged from −25 to −100 mV in 1 mV increments. For each cut-off, the numbers of α-, β- and δ-cells that pass this criterion were counted. The percentage of these cells that were β-cells  and the percentage of β-cells that pass this criterion (figure 2g) were then calculated. As the cut-off became more hyperpolarized, β-cell enrichment increased. However, the percentage of β-cells that passed this criterion also decreased. Therefore, attempting to enrich β-cells with a criterion based on V2 h comes with a cost—a drastic decrease in sample size. We further demonstrated that V2 h cannot reliably identify cell type by constructing a multinomial logistic regression model of cell type, with one independent variable ). The model was unable to identify δ-cells (0% correct) and correctly identified cell type with an overall accuracy of 54% only  than in α-cells  and δ-cells . The slope factor was also significantly smaller in α-cells than in δ-cells (p < 0.001).The slope factor of steady-state inactivation was greater in β-cells . The average time constant of decay in δ-cells  was significantly greater than that in α-cells  and β-cells . Slow tail currents were present in 0/141 α-cells, 4/56 (7%) β-cells and 59/91 (65%) δ-cells (c). The presence of a slow tail current in δ-cells was statistically different from that in α-cells (p < 0.001) and β-cells (p < 0.001). This contrasts with previous studies which have used the presence of a slow tail current to identify α-cells [We next analysed slow tail currents in all cells a,b. The  δ-cells c. The pr α-cells ,31,38,463.6.+ current) to define an α-cell [b). The presence of a transient outward current was statistically different between δ-cells and α-cells (p = 0.0029). When only recordings with intracellular solution 1 (K-glut) were considered, its prevalence in δ-cells (67%) was also greater than that in α-cells .Many groups have used the presence of a transient TEA-resistant outward current  were counted. The percentage of cells that passed the criterion that were β-cells  and the percentage of all β-cells that pass this criterion (figure 5b) were then calculated. For example, 41 cells passed the criterion Ccell > 6 pF; 12 α-cells, 21 β-cells and eight δ-cells. Therefore, this rule only enriched β-cells in the sample to 51%. Moreover, 35 (56−21) β-cells did not pass this criterion; a 63% reduction in potential sample size. The results were still poor when we applied a stricter criterion; only four β-cells passed the criterion Ccell > 9.4 pF (100% enrichment), but this came with a 92% reduction in sample size (4/56 β-cells). We conclude that using Ccell alone to distinguish β-cells from non-β-cells is inadequate.Electrophysiological criteria have been employed in many studies to distinguish β-cells from non-β-cells. For example, islet cells with ll > 5pF  and Ccelll > 6pF  have beec–f). The electrophysiological variables significantly predicting cell type (β-cells from non-β-cells) included Ccell and Imax. The model was able to distinguish β-cells from non-β-cells with 91% accuracy in the model construction dataset; 32/40 β-cells were correctly assigned as β-cells, and 127/135 non-β-cells were assigned as non-β-cells . When the model was applied to the model validation dataset it again could identify β-cells from non-β-cells with 97% accuracy . We conclude that, when taken together, the electrophysiological variables quantified can distinguish β-cells from non-β-cells with a high degree of accuracy.We therefore used the model construction dataset to construct a binary logistic regression model, to determine whether the electrophysiological variables could collectively distinguish β-cells from non-β-cells c–f. The -β-cells c,d. Whenaccuracy e,f. We c3.8.a and Ileak, Raccess, Ccell, kh, Imax, Rinput, transient current, ratio current, V2 h and tail current. In what follows, this model is used to predict islet cell type.A multinomial logistic regression model was developed to investigate whether the electrophysiological variables could be used together to identify all three cell types, rather than just distinguish β-cells from non-β-cells. The model construction dataset was used for fitting the model parameters . The modfigure 6N = 113; figure 6b,c). These data demonstrate that the model is applicable to other datasets, as it can predict islet cell type in the model validation dataset with an overall accuracy of 94%.The model was applied to the model validation dataset N = 113;  to see hV2 h and Ccell—variables typically used to distinguish β-cells from non-β-cells [figure 6e(i)). The most important variable for distinguishing δ-cells from α-cells was the presence of a slow Ca2+ tail current (figure 6e(ii)). The presence of an A-current—which has frequently been employed to distinguish these two cell types [Imax, indicating that a large Na+ current is an important distinguishing feature of δ-cells from α-cells. These findings do not conform to standard practice for identifying cell type and therefore highlight the importance of using our multinomial logistic regression model to identify cell type.To rank the variables in the model by their importance for identifying islet cell type, standardized coefficients were calculated as described by Menard . V2 h an-β-cells ,44–47,50ll types ,38,46–483.9.V2 h was significantly hyperpolarized (−64.1 ± 7.1) compared with correctly assigned α-cells . Moreover, it did not differ from that of β-cells that were correctly identified by the model ). Furthermore, 64% of the incorrectly identified α-cells (9/14) exhibited a double sigmoidal h∞, compared with 2.5% of cells in the correctly labelled α-cell population (figure 7a(ii)). These incorrectly identified α-cells therefore have ‘β-cell-like’ Na+ channel properties. The model supported this idea; the probability that the incorrectly identified α-cells were β-cells (Pβ = 0.52 ± 0.1) was significantly larger than for correctly identified α-cells . In particular, the model revealed that these α-cells have ‘β-cell-like’ properties.We characterized the cells whose cell type was incorrectly identified by the model . The mod3.10.Pβ = 0.95 ± 0.04) and 10 α-cells (Pα = 0.86 ± 0.05) correctly. In βV59M mice, the model correctly identified all three β-cells (Pβ = 0.91 ± 0.04) and four α-cells (Pα = 0.94 ± 0.02). The remaining six recordings from βV59M mice were revealed  to be from cells co-expressing insulin and glucagon (ins+/glu+). The model identified all of these cells as β-cells (Pβ = 0.67 ± 0.05). The probability that these cells were α-cells (Pα = 0.37 ± 0.05), as predicted by the model, was significantly larger than the probability that β-cells from WT  or βV59M  mice were identified as α-cells by the model. Furthermore, the model was less certain that these βV59M ins+/glu+ cells were β-cells; Pβ in βV59M ins+/glu+ cells was smaller than in WT (p = 0.019) and βV59M (p = 0.04) β-cells. Therefore, although the model predicted these six βV59M ins+/glu+ cells to be β-cells, it also revealed that they had ‘α-cell-like’ electrophysiological properties.To investigate whether the model could accurately identify islet cell type from mice with a hyperglycaemic phenotype, an additional 13 cell recordings were made in βV59M mice and 15 in wild-type control mice WT; . The mod4.Here we have quantified numerous electrophysiological variables in α-, β- and δ-cells from intact mouse islets. Our study highlights the perils of using a single electrophysiological variable to distinguish cell type and demonstrates that some established methods for functional identifying cell type are misleading figures –4. We sh4.1.When recording membrane potential in the perforated patch-clamp configuration, the electrical activity of the cell in response to application of glucose is one method by which cell identity can be alluded to. The aim of this study was to provide a tool for accurately identifying cell type when membrane potential recording is not required or cannot be used (namely in voltage-clamp experiments under the standard whole-cell configuration).a,b). Criteria based on cell capacitance alone may moderately enrich the cell type of interest, but will significantly reduce the sample size. Thus, even if subsequent criteria are applied , the da4.2.+ current variables, the input resistance (1/G) and cell capacitance are significant predictors of cell type, when important experimental confounders (e.g. access resistance and leak current) are controlled for. Interestingly, the model revealed that the leak current—an experimental confounder—is a significant predictor of cell type . This is supported by transcriptome data from DiGruccio et al. [+ channels in both α- and δ-cells (figure 4d) [KCNA4 and KCND2 are preferentially expressed in δ-cells, and KCND1 and KCND3 exhibit similar levels of expression in α- and δ-cells. Similarly, Adriaenssens et al. [The presence/absence of an A-current has been used in many studies as an identifying feature for α-cells/δ-cells, respectively ,38,46–48figure 4o et al.  that repgure 4d) . KCNA4 as et al.  recentlys et al. . In conc+ channels [+ channels that inactivate; it blocks both slowly inactivating and non-inactivating K+ currents of delayed rectifier type, including Shaker family members Kv1.1 [Shab-related Kv2.1 and Shaw-related Kv3.1 [How do we reconcile this fact with the observation that 4-aminopyridine (4-AP) reduces glucagon secretion in mouse islets ? First, channels , it is nrs Kv1.1 , Kv1.2 [rs Kv1.1 , Kv1.3 [rs Kv1.1 , Kv1.5 [rs Kv1.1 ,68 and Krs Kv1.1 , as welled Kv3.1 . Secondled Kv3.1 .4.4.To demonstrate the importance of our improved characterization of the electrophysiological properties of α- and δ-cells, we used our findings to develop models of the electrical activity in these cell types figures  and 10.4.5.+ current which we demonstrate is present in 20% of α-cells . Furthermore, the cell capacitance reported in previously published models was 5 pF [Ccell for α-cells reported here  or previously [b,c), a feature not seen experimentally [Ccell to 4 pF and modified the Na+ current parameters in the model to fit our experimental data, the similarity between the model and the experimental data under simulated high-glucose improved. In particular, in low-glucose conditions, the spike height of the model action potential overshot 0 mV and had an amplitude of more than 50 mV, as seen experimentally [Conductance-based mathematical models of the electrical activity of α-cells have provided us with invaluable insights into the mechanisms regulating glucagon secretion –75. Howe α-cells . It also dataset a. Furthewas 5 pF –74, whiceviously ,45. Thesmentally . We thermentally ,52. Morementally . These r4.6.et al. [+ current kinetics in the model were fitted to experimental data from a δ-cell recording by the improved parameter estimation method proposed by Willms et al. [2+ tail currents . We also changed Ccell in the model (from 5 to 4 pF) to fit the experimental data for δ-cells . When the model was simulated under current clamp conditions, it produced large-amplitude spikes that overshot 0 mV and had large after-hyperpolarizations (figure 10b). Similar spikes were seen experimentally using the perforated patch-clamp configuration (figure 10c).A conductance-based model of δ-cell electrical activity calibrated against experimental data does not exist. Recently, Watts et al.  generateet al. . We thers et al. . Given tcurrents  and A-cucurrents , T-type 4.7.+ current density are altered in dispersed islet cells [We have focused our model on characterizing islet cell type from recordings made from intact islets. Some studies, however, use dispersed islet cells. Our model was not tested against recordings from dispersed cells for two reasons. First, cell identification by immunocytochemistry is straightforward in dispersed cells. Second, there is evidence that both cell size and Naet cells .α-, β- and δ-cells in human islets possess very distinct electrophysiological features compared with their mouse counterparts –80. UnliThe modelling process outlined in this study would also be helpful in the study of human islets. Such a model could be used to determine the key electrophysiological variables that identify cell type, making comprehensible the defining electrophysiological properties of these heterogeneous cells. It could also be used to predict the disease state (non-diabetic/diabetic) of a donor given a set of electrophysiological variables. Such a modelling procedure would illuminate which electrophysiological properties differ in diabetes, while correctly controlling for experimental confounders.In conclusion, we have conducted a comprehensive analysis of the electrophysiological properties of islet cells traditionally used for identifying cell type, in a large population of recordings. We used this dataset to reveal which electrophysiological fingerprints were reliable for identifying cell type, and then constructed a logistic regression model that can be used to predict islet cell type with 94% accuracy. These data were successfully used to not only predict cell type in diabetic mouse models, but also improve conductance-based models of α- and δ-cells.


The asymmetric unit of the title complex comprises a supramolecular dimer composed of the Δ(−) and Λ(−) optical isomers, which are linked by strong hydrogen-bonds, two nitrate anions and one water molecule. In the crystal, the dimers are joined by numerous hydrogen bonds, forming a three-dimensional framework. L) which was used in the preparation of the mononuclear title complex, [Ni(C13H12N3O)(C13H13N3O)]NO3·0.5H2O. As a result of the presence of HL and L in the [{Ni(HL)(L)}]+ unit, the complex appears to be a supramolecular dimer composed of the Δ(−) and Λ(−) optical isomers, which are linked by strong hydrogen-bonds. As well as the dimer generated by two mononuclear [{Ni(HL)(L)}]+ cations, the asymmetric unit also contains two nitrate anions and one water molecule. Each Ni atom is coordinated to two ligand molecules by a nitrogen atom of the pyridine ring, an imine nitrogen atom and a phenolic oxygen atom of one of the ligand molecules and a phenolate oxygen atom of the other organic molecules. The environment around the cation is a distorted octahedron. The basal planes are defined by the two nitrogen atoms of the pyridine rings and the two phenolic oxygen atoms of the ligand, the apical positions being occupied by the azomethine atoms. The O atoms of one of the nitrate ions are disordered over two sets of sites in a 0.745 (9):0.255 (9) ratio. In the crystal, the dimers are linked by numerous hydrogen bonds, forming a three-dimensional framework.The 2-hydrazinopyridine precursor has been widely used to prepare ligands of various kinds by condensation with carbonyl compounds. These types of ligands are suitable for synthesizing novel transition metal (II) complexes with interesting magnetic properties. In this context we have synthesized the ligand 1-(2-hydroxyphenyl-2-ethylidene)-2-(pyridin-2-yl)hydrazine (H We combined 2-hydroxyacetophenone and 2-hydrazino pyridine to prepare a ligand with four potential donor sites  that acts as a tridentate ligand. In trying to coordinate the 1-(2-hydroxyphenyl-2-ethylidene)-2-(pyridin-2-yl)hydrazine ligand to the first series of transition metals in ethanol, we obtained a nickel(II) complex.Organic ligands derived from salicylaldehyde containing N and O donor atoms are widely used in coordination chemistry (Wang L)(L)}]+ cations, which are strongly hydrogen bonded, two nitrate anions and one water molecule. The O atoms of one of the nitrate ions are disordered over two sets of sites in a 0.745 (9):0.255 (9) ratio. As a result of the presence of HL and L in the [{Ni(HL)(L)}]+ unit, the complex is chiral. The dimer is formed by the Δ(−) and Λ(−) optical isomers because of the clockwise and anti-clockwise arrangement of the ligands around the Ni2+ ion. The two optical isomers of the dimer are linked by strong O—H⋯O hydrogen bonds between the phenoxo oxygen atoms and the phenolic hydrogen atoms (O1—H1O⋯O4 and O3—H3O⋯O2) with a mean H⋯A distances of 1.64 Å.Fig. 12+ ion is hexacoordinated in an octahedral environment. Each Ni2+ ion is bonded to a ligand molecule, whose phenolic function is deprotonated and to a second neutral ligand molecule. The basal plane of the octahedron around each Ni2+ ion is occupied by two nitrogen atoms from the pyridine moieties, a phenolic oxygen atom and a phenolate oxygen atom. The apical positions are occupied by the nitrogen atoms of the imine functions. The angles (Table 1L)2] where HL is 2-[(piperidin-2-ylmethylimino)methyl]phenol  and Λ(−) optical isomers, which are linked by strong hydrogen bonds Table 2. The dimps Fig. 2. These i13H13N3O (HL), yield 60%, m.p. 388 K. Calculated: C, 68.70; H, 5.77; N, 18.49. Found: C, 68.72; H, 5.76; N, 18.46%. IR (cm−1): 3289 (ν O—H), 3051 (ν N—H), 1514 (ν C=N), 1576, 1507, 1493, 1247 (ν C—O), 1145, 1043 (ν N—N), 756. 1H NMR: δ (ppm): 2.3 , 6.79–6.85 , 8.7 , 12.9 . 13C NMR: δ(ppm): 12, 107, 116, 117, 118, 119, 120, 127, 130, 138, 149, 156, 158. A mixture of NiCl2·6H2O (1 mmol) in ethanol (10 mL) was added to a solution of HL (2 mol) in 10 mL of ethanol. The mixture was stirred for 60 min and the resulting greenish solution was filtered. The filtrate was kept at 298 K and after six days, green crystals suitable for X-ray analysis appeared and were collected by filtration. [C26H26N7NiO5.5], yield 40%. Calculated: C, 53.54; H, 4.49; N, 16.81. Found: C, 53.50; H, 4.52; N, 16.76%. μeff (mB): 1.8. ΛM (S cm2 mol−1): 5. IR (cm−1): 3289, 3051, 3289, 1614, 1576, 1375, 1229, 1015.A mixture of 2-hydrazinopyridine (1 mmol) and 2-hydroxyacetophenone (1 mmol) in ethanol (10 mL) was stirred under reflux for 60 min. On cooling, a yellow precipitate was obtained. After filtration, the resulting solid was dried in a desiccator. C2 groups were located in difference-Fourier maps and refined using a riding model with Uiso(H) = 1.5Ueq(O). Other H atoms  were geometrically optimized  and refined as riding with Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for all other H atoms. High thermal motion for the O atoms of one of the nitrate group was noted, indicating some disorder in their positions. Each of these O atoms was distributed over two sites with a refined occupancy ratio of 0.745 (9):0.255 (9).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018005261/tx2004sup1.cifCrystal structure: contains datablock(s) I. DOI: 1834549CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II atoms, forming a ladder-like polymeric complex.Both the 2,5-dicarboxybenzene-1,4-dicarboxylate dianions and pyridyl-terpyridine ligands bridge the Zn II complex, [Zn2(C10H4O8)Cl2(C20H14N4)2]n, the ZnII cations are bridged by both 2,5-dicarboxybenzene-1,4-dicarboxylate dianions and 4′-(pyridin-3-yl)-4,2′:6′,4′′-terpyridine ligands, forming ladder-like polymeric chains propagating along [1-10]. The Cl− anion further coordinates the ZnII cation to complete a distorted tetrahedral environment. In the 4′-(pyridin-3-yl)-4,2′:6′,4′′-terpyridine ligand, the three sideward pyridine rings are twisted with respect to the central pyridine ring by 39.27 (12), 14.89 (13) and 3.36 (13)°, respectively. In the crystal, classical O—H⋯N hydrogen bonds and weak C—H⋯O and C—H⋯Cl hydrogen bonds link the chains into a three-dimensional supramolecular architecture. π–π stacking is observed between the pyridine and benzene rings of neighbouring polymeric chains, with a centroid-to-centroid distance of 3.7280 (14) Å.In the title polymeric Zn The bond lengths around Zn1 are similar to those reported by Wang et al.  angles range from 97.21 (8) to 115.73 (8)° and the tetrahedron edge lengths range from 2.992 (3) to 3.591 (2) Å. Each 344-pytpy ligand act as 2-connecting node, linking two Zn atoms by the outer N-terminal atoms , the central and another outer pyridine N atom  are free. The H2bta2− anion is located on an inversion center, and bridges two ZnII atoms through the two carboxylate groups. In this way, chains propagating along [1As shown in Fig. 1 al. 2009. The X—Zed Fig. 2.In the crystal, classical O—H⋯N hydrogen bonds, weak C—H⋯O and C—H⋯Cl hydrogen bonds Table 1 link theet al., 20142  and 1,2,4,5-benzenetetracarbonic acid  were adequately dispersed in 10 mL of distilled water, and then the mixture was sealed and heated to 453 K for three days under hydrothermal conditions. The vial was then allowed to cool to room temperature. Colorless block-shaped crystals were collected .4′-(3-Pyridyl)-4,2′:6′,4′′-terpyridine was synthesized according to a literature method (Yang Uiso(H) = 1.2Ueq(C) and 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016016285/xu5893sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016016285/xu5893Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016016285/xu5893Isup3.cdxSupporting information file. DOI: 1509671CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title complex shows selective sensitivity to detecting nitrobenzene in DMF media due to the fluorescent quenching. 2(C6H6N4)]n, the central CdII atom is coordinated by four chloride ligands and two N atoms from a chelating 2,2′-bi-1H-imidazole molecule, leading to a distorted octahedral Cl4N2 coordination set. As a result of the μ2-bridging character of the Cl ligands, chains parallel to the c axis are formed, with the chelating 2,2′-bi-1H-imidazole ligands decorated on both sides of the chain. The luminescence properties of the complex dispersed in dimethylformamide shows that the emission intensities are significantly quenched by nitrobenzene.In the polymeric title compound, [CdCl Its crystal structure and luminescence sensing of solvent molecules are reported in this communication.We have explored the self-assembly of CdClII atom is coordinated by four chloride ligands and two nitrogen atoms from a chelating 2,2′-bi-1H-imidazole ligand, forming a distorted Cl4N2 octahedral coordination set °. The μ2-bridging character of the four Cl ligands leads to the formation of a chain expanding parallel to the c axis  and 3.9452 (14) Å, respectively. In addition, there are intermolecular hydrogen bonds between the imidazole N atoms and coordinating Cl atoms of neighboring chains Br2]n has a very similar composition to the title compound and also shows an arrangement of polymeric chains constructed from the bridging behaviour of the Br ligand .A search of the Cambridge Structure Database , 2,2-dimethylsuccinic acid , 2,2′-bi-1H-imidazole  in water (8 ml) was stirred vigorously for 1 h at 333 K. Slow evaporation of the clear solution resulted in the separation of block-like colorless crystals as a pure phase. The crystals were washed with ethanol, and dried at room temperature. Calculated: C, 22.70; H, 1.90; N, 17.65; found: C, 22.51; H, 2.58; N, 17.49%.A mixture of CdClUiso(H) = 1.2Ueq(C). H atoms attached to the N atoms were found from difference maps but constrained with N—H = 0.86 Å and Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016013736/wm5319sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016013736/wm5319Isup2.hklStructure factors: contains datablock(s) I. DOI: 1501229CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We found selective augmentation of Vγ4+ γδ27− cells with higher IL-17 production in KLF10-deficient mice. Surprisingly, KLF10-deficient CD127hi Vγ4+ γδ27−-17 cells expressed higher levels of CD5 than their wild-type counterparts, with hyper-responsiveness to cytokine, but not T-cell receptor, stimuli. Thymic maturation of Vγ4+ γδ27− cells was enhanced in newborn mice deficient in KLF10. Finally, a mixed bone marrow chimera study indicates that intrinsic KLF10 signaling is requisite to limit Vγ4+ γδ27−-17 cells. Collectively, these findings demonstrate that KLF10 regulates thymic development of Vγ4+ γδ27− cells and their peripheral homeostasis at steady state.γδ T cells, known to be an important source of innate IL-17 in mice, provide critical contributions to host immune responses. Development and function of γδ T cells are directed by networks of diverse transcription factors (TFs). Here, we examine the role of the zinc finger TFs, Kruppel-like factor 10 (KLF10), in the regulation of IL-17-committed CD27 Therefore, KLF10-deficient CD4+ T cells that are hyper-activated by TCR stimuli are less differentiated into Treg cells than wild-type (WT) controls , a transcription factor (TF) containing zinc finger DNA-binding domains, revealed its role in the induction of and balance between Foxp3controls , while T− γδ T (γδ27−-17) cells are present in peripheral lymph nodes (pLN) as well as regional tissues, including dermis, lung, and peritoneal cavity  (hiCD62LloCD127hi)  cells  , 9, most cells , 23.27−-17 cells and, thus, IL-17-producing capacity is considered to emerge by default from uncommitted early thymocytes  and/or intrinsic pre-programming of a gene regulatory network of diverse TFs –26. It iymocytes , 11, 27.ymocytes , leaving pathway , 9, 22; + γδ27−-17 cells. We found selective enlargement of IL-17-committed Vγ4+ γδ27− cells, but not of other IL-17-producing αβ T cells, in KLF10-deficient mice. TCR or cytokine (IL-7 or IL-1β plus IL-23) stimulation on γδ T cells could induce KLF10, which in turn differently regulates γδ T-cell responsiveness to these stimuli. Moreover, KLF10 deficiency affected the expression level of CD5, a stable indicator of TCR strength, on mature Vγ4+ γδ27−-17 cells within the neonatal thymus. These results suggest that the biology of Vγ4+ γδ27−-17 cells is dependent on transcriptional control by KLF10, which is differentially associated with TCR and cytokine signaling.Here, we identify KLF10 as a novel TF that negatively regulates the development and homeostasis of Vγ4KLF10-deficient mice with C57Bl/6 (B6) background were kindly provided by Dr. Woon Kyu Lee  . B6.Rag1Mouse peripheral lymph nodes , mesenteric lymph node, spleen, thymus, and lung were homogenized by mechanical disaggregation, strained through a 70-µm strainer (BD Biosciences), and washed in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum (FBS). Peritoneal cells were obtained from peritoneal lavage in cold phosphate-buffered saline (PBS) containing 5% FBS.2+ mobilization, cells from WT and knockout (KO) mice were first surface-stained with anti-CD45.2-PerCP-Cy5.5 and anti-CD45.2-Alexa 700, respectively, for subsequent identification of the two strains. Cells from both strains were mixed together at a ratio of 1:1 and loaded for 45 min at 37°C with the membrane-permeable fluorescent Ca2+ indicator dye, Indo-1 AM (Invitrogen), at a concentration of 4 µM in RPMI medium plus 5% (vol/vol) FBS. Cells were then stained for surface markers and were kept on ice. Before stimulation, cell aliquots were allowed to equilibrate to 37°C for 5 min and analyzed by flow cytometry. After acquisition of background intracellular Ca2+ concentrations for 30 s, cells were stimulated with biotinylated anti-CD3ε antibody and crosslinked by the addition of streptavidin. Samples were acquired on Canto II or Aria II (BD Biosciences) and data were analyzed with FlowJo software (TreeStar). Antibodies used for flow cytometric analyses were fluorochrome-labeled mAbs against mouse γδTCR (GL3), CD3ε (145-2C11), CD27 (LG.3A10 or LG.7F9), CD25 (PC61), CD69 (H1.2F3), CD5 (53-7.3), CD24 (M1/69), CD103 (2E7), CD122 (TM-b1), CD132 (TUGm2), CD127 (eBioSB/199), Ly6C (HK1.4), CD28 (E18), CD44 (IM7), CD62L (MEL-14), CCR6 (29-2L17), NK1.1 (PK136), CD45.1 (A20), CD45.2 (104), Vγ1 (2.11), Vγ4 (UC3-10A6), IL-17 (TC11-18H10), IFN-γ (XMG1.2), CD4 (RM4-5), CD8α (53-6.7), TCRβ (H57-597), pErk1/2 (D13.14.4E), pZap70 (n3kobu5), pSTAT3 (D3A7), and pSTAT5 (47/Stat5) .Single-cell suspensions were first blocked with anti-CD16/32 antibody  and then stained with antibodies at 4°C for 20 min in staining buffer . For intracellular cytokine staining, the cells were stimulated for 4 h with 50 ng/ml phorbol 12-myristate 13-acetate  and 750 ng/ml ionomycin (Sigma-Aldrich) in the presence of brefeldin A (BD Biosciences). The cells were then fixed, permeabilized with a BD Cytofix/Cytoperm Kit according to the manufacturer’s instructions (BD Biosciences), and stained for IL-17 and IFN-γ. Intracellular phosphorylated protein staining was performed as described  with mod− γδ27+, Vγ4+ γδ27+, Vγ4− γδ27−, and Vγ4+ γδ27−) were sorted using an Aria II with a purity of at least 98%. Alternatively, total γδ cells were manually isolated by negative selection as described previously , and anti-CD11b (M1/70) antibodies together with MagniSort™ Streptavidin Negative Selection Beads according to the manufacturer’s instructions (eBioscience). The negatively selected cells were then stained with fluorochrome-labeled anti-γδTCR, anti-CD27, and/or anti-Vγ4 antibodies. γδ subsets (Vγ4eviously  with modCells were cultured in RPMI 1640 containing 10% (vol/vol) FBS, 50 µM 2-mercaptoethanol, 1% (vol/vol) non-essential amino acids, 10 mM HEPES, and 1% (vol/vol) antibiotics/antimycotic solution . For TCR stimulation, the cells were incubated for the indicated times with plate-bound anti-CD3ε mAb  only or in combination with plate-bound anti-CD28 mAb . For cytokine stimulation, IL-7 , TGF-β , IL-6 , IL-1β , and IL-23  were added to the medium.6 cells) from B6.CD45.2 wild-type (WT) or B6.CD45.2 KLF10-deficient mice were intravenously transferred to B6.CD45.1 mice, which were irradiated at 900 cGy. For mixed BM chimeras, B6.CD45.1/2 mice were lethally irradiated at 900 cGy and intravenously administered 5 × 106 B6.CD45.1 WT cells mixed 1:1 with 5 × 106 B6.CD45.2 KLF10-deficient BM cells. Recipient mice were sacrificed and analyzed for reconstituted γδ T cells after at least 12 weeks.Bone marrow cells  mice were stained with Cell Trace Violet  and injected intravenously with 2 × 10Eef1a1  via the ΔΔCt method. The following primers were used: Efa1 forward, 5′-TCCACCGAGCCACCATACA-3′, reverse, 5′-CCAACCAGAAATTGGCACAA-3′; Klf10 forward, 5′-ACCCAGGGTGTGGCAAGAC-3′, reverse, 5′-AGCGAGCAAACCTCCTTTCA-3′; Rorc forward, 5′-TCAGCGCCCTGTGTTTTTCT-3′, reverse, 5′-CAAATTGTATTGCAGATGTTCCA-3′; Sox13 forward, 5′-CTGCCACCTGGGTTACTTTGA-3′, reverse, 5′-GAGTGGCGTGATGAACATGTG-3′.RNA was purified from cells using a Qiagen RNeasy kit. After reverse transcription into cDNA, PCR was performed with a StepOnePlus real-time PCR system (Applied Biosystems) and iTaq SYBR Green Supermix (Applied Biosystems) and relative expression was displayed in arbitrary units or as a percent of maximum expression, normalized to t-test was used for BM chimeras. The two-tailed, unpaired t-test or two-way ANOVA followed by a Bonferroni post hoc test were used for all other data sets. Mean fluorescence intensity (MFI) indicates geometric MFI. Robust coefficient of variation (CV) defines 100 × 1/2 [intensity (at 84.13 percentile) − intensity (at 15.87 percentile)/Median] and was determined by FlowJo software. The robust CV is a normalized SD not as skewed by outlying values as the CV.Prism software (GraphPad) was used for all statistical analyses. All quantitative data are shown as mean ±  standard deviation (SD) unless otherwise indicated. The two-tailed, paired + and CD8+ αβ T cells, were significantly increased in pLN and lung of KO mice . Analysis of discrete LNs  confirmed the higher frequencies of γδ T cells, with the exception of mesenteric LN (mLN). Interestingly, the increase in γδ T cells was largely attributable to γδ27− cell augmentation , in which innate-like γδ-17 cells are reported to be absent  γδ T cells in the analyzed organs . Intriguingly, the frequencies of Tregs  and Th17 cells  among CD4+ T cells were unchanged in KO mice relative to those in WT mice. In summary, these data suggested that the generation of γδ27− cells was enhanced in KO mice.CD27 expression discriminated γδ subsets with CD44, CD62L, NK1.1, and CCR6; pLN γδpe cells , 32. Con27− cell augmentation in KO mice, we investigated whether KLF10 deficiency affected homeostatic proliferation of γδ27− cells. Considering that the absolute number of KO pLN γδ27− cells was about twice higher than that in WT pLN cells . The homeostatic expansion of KO γδ27− cells was superior to that of WT γδ27− cells, with a ratio as great as threefold , which demonstrated that the change in these surface molecules on γδ27− cells   in pLN of KO mice, considerably more IL-17+ cells were observed in γδ27− cells from KO pLN cells after stimulation with PMA plus ionomycin  , we exam+ γδ27− cells. Under IL-7 treatment the absolute number of Vγ4+ and Vγ1−Vγ4− γδ27− cells from KO mice was considerably increased compared with their WT counterparts . These data suggested that STAT3 activation might be involved in the hyper-responsiveness of KO γδ27− cells to IL-7. On the other hand, contrary to the aforementioned in vivo Vγ4+ γδ27−-17 cell-specific regulation of KLF10 , which indicated that KLF10 also inhibited the activation of γδ27− cells triggered by inflammatory stimuli. Of note, Klf10 expression was increased in γδ27− cells stimulated with IL-1β plus IL-23, as well as those stimulated with IL-7 . Together, these data suggested that homeostatic and inflammatory cytokine signaling could induce KLF10, which in turn, as a negative feedback signaling factor, might impair γδ27− cell responsiveness to these innate stimuli.We next examined whether KLF10 is also involved in the reactivity of γδ T cells . Consist27+ cells could readily expand under TCR/CD28 stimuli, whereas γδ27− cells showed different patterns of proliferation and, especially, Vγ4+ γδ27− cells hardly expanded . Therefore, KLF10 has a minor role in TCR-triggered activation of peripheral γδ subsets. To further investigate whether the level of engagement by the antigen or costimulatory receptor influenced KLF10 involvement in the TCR response, we treated the cells with different doses of agonist antibodies. Vγ4+ γδ27− cells from both strains responded similarly to a high level of antigen engagement even in the absence of costimulatory signaling, but still showed an impaired proliferative response compared to that of γδ27+ cells ; similar results were found with costimulatory stimuli. Therefore, neither the strong antigen receptor nor costimulatory signaling caused a substantial discrepancy in TCR response of peripheral γδ subsets between the two strains.Next, we sought to determine whether KLF10 might be involved in TCR-triggered activation of peripheral γδ T cells , 32. γδ2ignaling . Althougs Figure , a rapid27− cells than in γδ27+ cells , meaning that they are a heterogeneous group receiving discrete TCR-signal strengths. Such a two-peak pattern was also observed for CD127 , one of the markers identifying innate-like γδ-17 cells , 1327− cs Figure B and furs Figure C.hiCD127lo γδ27− cells closely resembled γδ27+ cells rather than CD5loCD127hi γδ27− cells in terms of surface protein expression  in young mice (2 weeks old) but greatly increased (45.5%) in the adult (8 weeks old)  γδ27− cells of the thymus  and pLN  γδ27− cells of the adult thymus , in accordance with the previous report on sequential Vγ waves with age  was genuinely augmented by KLF10 deletion , effector (CD44loCD62Llo), and memory  phenotypes of CD4+ or CD8+ T cells (data not shown). Because CD5 is positively associated with the strength of TCR signaling that T cells received during their selection within the thymus (+ γδ27−-17 cell maturation actively occurred (loCD44hi) thymic Vγ4+ γδ27− cells under conditions of KLF10 deficiency, whereas the difference in CD5 was equivocal on the immature (CD24hiCD44lo) cells  cells among Vγ4+ γδ27− cells and their absolute number  stage  but, interestingly, closely similar to CD4+CD8+ double-positive (DP) CD69+ cells of the αβ lineage, based on low expression of genes involved in metabolism and energy production (www.immgen.org) reported that Klf10 in γδ T cells was closely correlated with genes encoding metabolic molecules, which showed lower expression in the immature Vγ4+ subset than in other Vγ subsets (data not shown) (hiCD69–) stage  , 37. Whe+ γδ27−-17 cells. We reconstituted irradiated CD45.2+ WT or KO mice with congenic WT BM cells and analyzed CD45.1+ γδ27− cells after at least 12 wks . Reconstitution of Vγ4+ γδ27− cells and CD5loCD127hi γδ27− cells in KO recipients was achieved at a level comparable to that in WT recipients, excluding the hematopoietic system-extrinsic effect of KLF10 on the homeostasis of Vγ4+ γδ27− 17 cells  showed a higher proportion of KO BM-derived cells among total CD3ε+ cells (data not shown). This suggested that KLF10-deficient BM cells outcompeted their WT counterparts during reconstitution of hematopoietic-derived cells, with the fact that KLF10 transcripts are at high level in long- and short-term repopulating hematopoietic stem cells (www.immgen.org).Finally, we examined whether KLF10 extrinsically or intrinsically controlled homeostasis of Vγ4s Figure A. By con+ γδ27− cells. In the mixed BM chimera setting, a proportion of Vγ4+ γδ27− cells in KO BM-derived γδ T cells was relatively higher than that of their WT counterpart . Indeed, KO BM-derived γδ27− cells contained higher proportions of Vγ4+ cells and CD5loCD127hi cells than their WT BM-derived counterparts , indicating that KLF10 function in γδ-17 cells was irrelevant to TGF-β/Smad signaling for γδ-17 cell development  and iNKT-17 (IL-17+CD1d-tet+TCRβ+) cells, as well as Treg cells, suggesting a novel function of KLF10 unique to innate-like γδ-17 cells. Meanwhile, the slight increase of IFN-γ+ γδ27+ cells in pLN of KO mice might demand further investigation.Early studies revealed that KLF10-deficient mice had defective Treg cell generation under inflammatory conditions, emphasizing the role of KLF10 as a TF in the balance between Treg and Th17 cell differentiation . Althougelopment . Intrigu27− cells to overcome the competitive inhibition by αβ T cells, reflecting the increased sensitivity of IL-7 signaling in KO γδ27− cells. This was confirmed by the hyper-responsiveness of KO γδ27− cells to exogenous IL-7 stimulation with increased STAT3 activation. KO γδ27− cells also hyper-responded to IL-1β plus IL-23 stimuli. Of note, KLF10 transcription in γδ27− cells was dramatically induced by IL-6 (data not shown), IL-7, or IL-1β plus IL-23, which are known to trigger STAT3 activation to induce RoRγt expression , which is different from the Vγ4+ subset-specific γδ27− cell enrichment observed in KO mice. IL-7Rα expression was not only similar between Vγ4+ and Vγ1−Vγ4− γδ27− subsets, both of which are main producers of innate IL-17 among γδ T cells, but was also unchanged by KLF10 deficiency (data not shown). These data suggest that Vγ4− γδ27− cells, presumably Vγ1−Vγ4− γδ27− subsets, are under homeostatic control by certain factors counteracting the effect of an active IL-7R-KLF10 signal axis  γδ27− subset , suggesting that Sox13 may not be associated with the selective effect of KLF10 deficiency on Vγ4+ γδ27− cell development .C-HY conceived the idea. C-HY and GK designed the experiments and wrote the manuscript. GK, MG, SK, KK, Y-CK, and CK performed the experiments. GK interpreted the data. J-HC, W-KL, K-DS, HC, Y-MP, and SH provided critical comments. All authors contributed to discussion of the results followed by writing and reviewing the manuscript.The 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.


AD), the leading cause of dementia, is pathologically characterized by β‐amyloid plaques and tau tangles. However, there is also evidence of lipid dyshomeostasis‐mediated AD pathology. Given the structural diversity of lipids, mass spectrometry is a useful tool for studying lipid changes in AD. Although there have been a few studies investigating lipid changes in the human hippocampus in particular, there are few reports on how lipids change in each hippocampal subfield . Since each subfield has its own function, we postulated that there could be lipid changes that are unique to each.Alzheimer's disease , using an analysis workflow developed in‐house. Hematoxylin, eosin, and luxol fast blue staining were used to precisely delineate each anatomical hippocampal subfield. Putative lipid identities, which were consistent with published data, were assigned using MS/MS.We used matrix‐assisted laser desorption/ionization‐imaging mass spectrometry to investigate specific lipid changes in each subfield in AD tissue. While the distribution pattern of lipids did not change in AD, the abundance of some lipids changed, consistent with trends that have been previously reported. However, our results indicated that the majority of these lipid changes specifically occur in the CA1 region. Additionally, there were many lipid changes that were specific to the DG.Both positively and negatively charged lipid ion species were abundantly detected in normal and AD pathogenesis.Matrix‐assisted laser desorption/ionization‐imaging mass spectrometry and our analysis workflow provide a novel method to investigate specific lipid changes in hippocampal subfields. Future work will focus on elucidating the role that specific lipid differences in each subfield play in  Unless otherwise stated, all other reagents were purchased from Sigma‐Aldrich.1,5‐Diaminonaphthalene, red phosphorous, and ammonium formate were purchased from Sigma‐Aldrich . Tissue‐Tek2.2Fresh, frozen postmortem human brain tissue, from the hippocampus block , and thaw‐mounted on to pre‐cooled indium‐tin oxide‐coated MALDI glass slides . The slides were then dried for an hour in a dry‐seal, grease‐free desiccator , under vacuum, and then washed with ammonium formate as previously outlined by Angel, Spraggins, Baldwin, and Caprioli  to reduc2.4m/z 400–2,000. MALDI‐IMS data for each age‐ and gender‐matched normal and AD case were acquired in the same dataset. One section from each age‐ and gender‐matched normal and AD case was used to acquire data in negative ion mode, using 75 laser shots per spectrum, and another section from each case was used to acquire data in positive ion mode using 100 laser shots per spectrum. Since lipid distributions were found to be highly reproducible between sections from the same case (data not shown), one dataset from each matched pair was used for subsequent data analysis. Following data acquisition, DAN matrix was removed by 70% ethanol, and tissue sections were stained with hematoxylin and eosin (H&E) and luxol fast blue (LFB) for histological analysis.Mass spectrometric analyses were performed at an accelerating voltage of +20 kV or −20 kV on a Bruker UltrafleXtreme MALDI‐TOF/TOF mass spectrometer , equipped with a 2 kHz Smartbeam II™ UV MALDI laser and 60–70 μm laser beam size, operating in reflector mode. Delayed extraction parameters (120 ns) were optimized for signal intensity and mass resolution, and red phosphorous  was used as an external calibrant before data collection. The distance between raster points was set to 100 μm. Data were collected in the range of 2.5m/z list based on previous publications on the mammalian brain lipidome , based on their histological appearance. A receiver‐operator characteristic (ROC) analysis was done on each ROI of each age‐ and gender‐matched pair, to statistically analyze m/z values that were either increased or decreased in AD. The ROC analysis results were then evaluated to find m/z values that were consistently statistically increased or decreased, in each ROI, across all six datasets. Distribution maps of these selected m/z values were generated using SCiLS lab 2015b , with automatic hotspot removal and edge‐preserving weak image denoising. The relative intensity change in these selected m/z values in AD was calculated as a percentage (%) change from normal, and the graphs showing this change for each region were generated using GraphPad Prism version 6 for Windows .Data were analyzed using the workflow outlined in Fig. 2.6m/z range in positive ion mode could not be identified accurately using the LIPID MAPS database (RRID:SCR_003817) as the library is not yet complete and is being continuously updated  and analyzed using the LIPID MAPS database , 778.6 (PE 39:5‐H−/PPE 40:4‐H−), 779.6 (PG 37:6‐H−), 791.6 (PE 40:6‐H−), 794.6 (PE 40:4‐H−), and 795.7 (PG 38:5‐H−/PA 44:10‐H−). In contrast, we detected a relative increase in m/z 739.2, 786.6 (PS 36:2‐H−), 810.6 (PS 38:4‐H−), and 843.7.There were ten lipids that were differentially expressed in AD across the whole hippocampus, of which the majority showed a relative decrease Fig. B. These 3.2.2m/z 797.7 (PG 38:4‐H−), 917.7, and 918.7 (SF 26:0 (0H)‐H−), were increased in white matter in AD, m/z 778.6 (PE 39:5‐H−/PPE 40:4‐H−) was the only lipid species that was decreased in this region , 789.6 (SM 36:1‐H−), 797.7 (PG 38:4‐H−), 810.6 (PS 38:4‐H−), 813.7, and 843.7. In contrast, m/z 767.7 (PE 38:4‐H−), 774.6 (PE 39:7‐H−/PPE 40:6‐H−), 778.6 (PE 39:5‐H−/PPE 40:4‐H−), 779.6 (PG 37:6‐H−), 791.6 (PE 40:4‐H−), 794.6 (PE 40:4‐H−), and 795.7 (PG 38:5‐H−/PA 44:10‐H−) were decreased.In contrast to the four lipids that were differentially expressed in white matter, 14 lipids were differentially expressed in gray matter, of which half were increased and half were decreased Fig. D. The li3.2.4m/z 600.5 (Cer 39:4‐H−), 767.6 (PE 38:4‐H−), 778.6 (PE 39:5‐H−/PPE 40:4‐H−), 779.6 (PG 37:6‐H−), 791.6 (PE 40:6‐H−), 794.6 (PE 40:4‐H−), 795.7 (PG 38:5‐H−/PA 44:10‐H−), 796.6 (PE 40:3‐H−). There were six lipids that were increased in the CA1 region, again reflecting the change seen in gray matter. These were m/z 739.2, 786.6 (PS 36:2‐H−), 789.6 (SM 36:1‐H−), 810.6 (PS 38:4‐H−), 813.7, and 843.7. There was only one lipid that was differentially expressed in the CA2/3 region in AD, and this was m/z 843.7, which was increased , 789.6 (SM 36:1‐H−), and 843.7.Of the CA1, CA2/3, and CA4 regions, the CA1 region had the most changes in lipid expression in AD. There were eight lipids that were decreased in the CA1 region, of which six reflected the change seen in gray matter , 646.5 (Cer N24:1‐H−), 647.5 (PA 32:0‐H−), 719.6, 786.6 (PS 36:2‐H−), 813.7, 843.7, 885.6 (PI 38:4‐H−), and 886.7. The seven lipids that were decreased were m/z 791.6 (PE 40:6‐H−), 794.6 (PE 40:4‐H−), 795.7 (PG 38:5‐H−/PA 44:10‐H−), 905.7, 906.7 (SF 24:0 (0H)‐H−), 918.7 (SF 26:0 (0H)‐H−), and 1574.0 (GM1 d20:1/18:0).There were 16 lipids that were differentially expressed in the DG in AD, of which eight were specifically only changed in this region Fig. H. There m/z values that were detected by our analysis. Overall, when they were differentially expressed, generally PE and PG showed a decrease, while PS, PI, SF, and SM, were increased. Cer was increased in the DG, while the only GM was decreased in the same region.The differential expression of these lipids in AD, in the subfields of the hippocampus, has been summarized in Table m/z 600.5 (Cer 39:4‐H−), 794.6 (PE 40:4‐H‐), and 885.6 (PI 38:4‐H−) were expressed with a higher abundance in gray matter. However, even when expressed in gray matter, they did not show a uniform distribution. Many lipids, such as m/z 778.6 (PE 39:5‐H−/PPE 40:4‐H−), 794.6 6 (PE 40:4‐H−), and 885.6 (PI 38:4‐H−) were highly expressed in the CA1 region. Lipids that were abundantly expressed in the CA1 region were also usually expressed in the DG and CA4 region. However, others such as m/z 646.5 (Cer N24:1‐H−), 719.6, and 786.6 6 (PS 36:2‐H−), were highly expressed in the DG region alone, at least in AD. Six out of the 26 lipids detected in this analysis showed an inverse pattern of distribution, in that they were highly expressed in white matter. These were m/z 797.7 (PG 38:4‐H−), 813.7, 905.7, 906.7 (SF 24:0 (0H)‐H−), 917.7, and 918.7 (SF 26:0 (0H)‐H−).The distribution images clearly show the region‐specific differential expression of these lipids and their relative increase or decrease in AD Fig. . The maj3.3Analysis of the positive ion mode datasets yielded 17 lipids that were differentially expressed, in at least one of the subfields of the hippocampus Fig. A in AD. 3.3.1m/z 496.5 , 720.6 (PE 34:0 + H+), 721.6, 730.5, 753.7 (SM 36:1 + Na+), 1463.1, 1478.1, 1479.2, 1481.1, and 1483.1.The only lipid that was decreased across the whole hippocampus was 3.3.2m/z 522.4 was the only lipid that was decreased in white matter in AD, there were six lipids that were increased , 720.6 (PE 34:0 + H+), 730.5, 1463.1, 1479.2, and 1481.1.While 3.3.3m/z 496.5 was the only one that showed a decrease. In contrast, m/z 718.6 (PE 34:1 + H+), 720.6 (PE 34:0 + H+), 721.6, 730.5, 1463.1, 1464.2, 1467.2, 1478.1, 1479.2, 1481.1, 1483.1, 1495.1, and 1507.3 were increased in gray matter in AD.There were 14 lipids that were differentially expressed in AD in gray matter Fig. D. The li3.3.4m/z 496.5 was the only one that showed a decrease , 720.6 (PE 34:0 + H+), 721.6, 730.5, 1463.1, 1464.2, 1467.2, 1478.1, 1479.2, 1481.1, and 1483.1.Of the 12 lipids that were differentially expressed in the CA1 region in AD, m/z 718.6 (PE 34:1 + H+), 720.6 (PE 34:0 + H+), 721.6, 730.5, 1463.1, 1464.2, 1467.2, 1478.1, 1479.2, 1481.1, and 1483.1.All eight lipids that were differentially expressed in the CA2/3 region were increased in AD Fig. F. These m/z 718.6 (PE 34:1 + H+), 1463.1, 1464.2, 1478.1, 1479.2, 1481.1, 1483.1, and 1507.3.The CA4 region showed a similar pattern, with an increase in eight lipids in AD Fig. G. The li3.3.5m/z 706.5 (PC 30:0 + H+), 718.6 (PE 34:1 + H+), 720.6 (PE 34:0 + H+), 721.6, 730.5, 1463.1, 1464.2, 1467.2, 1478.1, 1479.2, 1481.1, 1483.1, 1495.1, and 1507.3. Table m/z 496.5, 1467.2, and 1495.1, or in white matter, for example, m/z 1463.1, 1464.2, and 1507.3. Others, such as m/z 721.6, 1478.1, 1481.1, and 1483.1 showed a more homogenous expression through the hippocampus, with some hotspots in AD.There were nine lipids that were increased in the DG in AD Fig. H. These 706.5 PC 0:0 + H+,4m/z 795.7 [PG 38:5‐H−/PA 44:10‐H−]), and may contain more than one lipid species. To confirm the identities of each lipid detected in our study, high mass resolution IMS equipment would be required. Nonetheless, this discussion focuses on the lipid assignments that we were able to putatively make using MS/MS and previously published results on the mammalian lipidome.This study demonstrates the anatomical compartments in which lipids were differentially expressed in the human hippocampus in AD. Due to the mass resolution limitations of the MALDI‐TOF, peaks could not always be assigned a single lipid identity ‐H− are the only lipid species that show a departure from this trend, as the former was decreased in the CA1 region, but increased in the CA4 and DG, while the latter was decreased in the DG but increased in white matter. It is however, important to note, that the extent to which the same lipid species was changed, which is indicated by the percentage change in Tables With the exception of PC 30:0 + HOf the 43 lipids that were changed in at least one hippocampus subfield in AD, only approximately half were detected when the hippocampus was analyzed as a whole. This highlights the merit in analyzing various subfields of a region of interest separately, using MALDI‐IMS. Additionally, with the exception of some lipids that were detected in positive ion mode that showed a homogenous distribution, most lipids were abundantly expressed either in gray matter or in white matter Figs . There im/z 1,400–1,500 region in positive ion mode, given their proximity to expected m/z values, we suspect that some of these might be GMs. There is evidence that GM1 aggregation into clusters is accelerated in a cholesterol‐rich environment, as with aging and apolipoprotein E4 (apoE4) expression ‐H−, and Cer N24:1‐H− have been reported previously , consequently promoting Aβ biogenesis  and University of Auckland (lmen018 PReSS Account).None declared. Click here for additional data file.


The layers are linked across [100] through amine N—H⋯O hydrogen bonds to arsonate and water O-atom acceptors, giving overall three-dimensional network structures. p-arsanilic acid) manifest an isotypic series with the general formula [M2(C6H7AsNO3)2(H2O)3], with M = K {poly[di-μ3-4-aminophenylarsonato-tri-μ2-aqua-dipotassium], [K2(C6H7AsNO3)2(H2O)3], (I)}, Rb {poly[di-μ3-4-aminophenylarsonato-tri-μ2-aqua-dirubidium], [Rb2(C6H7AsNO3)2(H2O)3], (II)}, and Cs {poly[di-μ3-4-aminophenylarsonato-tri-μ2-aqua-dirubidium], [Cs2(C6H7AsNO3)2(H2O)3], (III)}, in which the repeating structural units lie across crystallographic mirror planes containing two independent and different metal cations and a bridging water molecule, with the two hydrogen p-arsanilate ligands and the second water molecule lying outside the mirror plane. The bonding about the two metal cations in all complexes is similar, one five-coordinate, the other progressing from five-coordinate in (I) to eight-coordinate in both (II) and (III), with overall M—O bond-length ranges of 2.694 (5)–3.009 (7) (K), 2.818 (4)–3.246 (4) (Rb) and 2.961 (9)–3.400 (10) Å (Cs). The additional three bonds in (II) and (III) are the result of inter-metal bridging through the water ligands. Two-dimensional coordination polymeric structures with the layers lying parallel to (100) are generated through a number of bridging bonds involving the water molecules (including hydrogen-bonding interactions), as well as through the arsanilate O atoms. These layers are linked across [100] through amine N—H⋯O hydrogen bonds to arsonate and water O-atom acceptors, giving overall three-dimensional network structures.The structures of the alkali metal  complex salts with 4-aminophenylarsonic acid ( Compounds (I)M = K), (II)In an attempt to complete the structures of the alkali metal series of M1 and M2), which lie on crystallographic mirror planes that also contain one of the coordinating water molecules (O2W), with the hydrogen p-arsanilate ligands and the second water molecules   lying across the mirror plane M—O bond length ranges are 2.694 (5)–3.009 (7) Å (K)  (Tables 1M2O5 polyhedron in (I)ii donors, the μ2-O2Wi , 4.2500 (11) Å [for (II)] and 4.3498 (15) Å [for (III)]. There are also slightly shorter M1⋯M1i separations in all structures: 4.079 (3) Å (I)M2⋯As1 separations are present within the repeat unit in all three structures: 3.6369 (19) Å (I)With (II) Tables 2 and 3 ▸.W provides hydrogen-bonding links across the mirror plane to arsonate O13 acceptors . Fig. 4 Tables 4 and 6 ▸.W acceptors  x, −y + 2, z + p-arsanilate O atom (O12) forms an intra-layer hydrogen bond with an O11 acceptor, giving overall three-dimensional network structures in all cases –(III) and in those examples which have been previously mentioned in the Chemical context section of this article, are worthy of noting here. Mixed-metal-ligand examples ], Rb2CO3 [for (II)] or Cs2CO3 [for (III)], in 20 ml of 50% ethanol/water (v/v). Room temperature evaporation of the solutions gave colourless crystal plates of the title compounds from which specimens were cleaved for the X-ray analyses.Compounds (I)–(III) were synthesized by heating together for 5 min, 1 mmol quantities of 4-aminophenylarsonic acid and 0.5 mmol of either KUiso(H) = 1.2Ueq(N) or 1.5Ueq(O). Other H atoms were included in the refinement at calculated positions, C—H = 0.95 Å, and treated as riding with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989017000445/wm5350sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989017000445/wm5350Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017000445/wm5350IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017000445/wm5350IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 1526425, 1526424, 1526423CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S(6) ring motif. The conformation about the C=N bond is E, and the aromatic ring of the julolidine moiety is inclined to the benzene ring by 3.74 (14)°.In the title compound, the hydroxy group forms a intramolecular hydrogen bond to the imine N atom and generates an  23H29N3O, was synthesized from the condensation reaction of 8-hydroxyjulolidine-9-carbaldehyde and N,N-diethyl-p-phenylenediamine. The hydroxy group forms a intramolecular hydrogen bond to the imine N atom and generates an S(6) ring motif. The conformation about the C=N bond is E, and the aromatic ring of the julolidine moiety is inclined to the benzene ring by 3.74 (14)°. One of the fused non-aromatic rings of the julolidine moiety adopts an envelope conformation and the other has a screw-boat conformation. In the crystal, molecules are linked by C—H⋯π interactions involving the aromatic julolidine ring, forming slabs parallel to the bc plane. The tricyclic fragment of the julolidine ring and the azomethine C=N bond are disordered over two sets of sites with a refined occupancy ratio of 0.773 (3):0.227 (3).The title compound, C The molecule is non-planar, with the dihedral angle between benzene ring (C1–C6) and the aromatic ring (C12–C17) of the julolidine moiety being 3.74 (14)°.The molecular structure of the title compound is illustrated in Fig. 1et al., 2012viz. 5-diethylamino-2-[(E)-iminomethyl]phenol and 8-{(E)-[(4-chlorophenyl)imino]methyl}-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyridoquinolin-9-ol imino]methyl}-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H,5H-pyridoquinolin-8-ol  in absolute ethanol (3 ml). Two drops of HCl were added to the reaction solution and it was stirred for 30 min at room temperature. The resulting yellow precipitate was recovered by filtration, washed several times with a small portions of ice EtOH and then with diethyl ether to give 130 mg (78%) of the title compound. Colourless block-like crystals, suitable for X-ray diffraction analysis, were obtained within three days by slow evaporation of a solution in methanol.An ethanolic solution of 8-hydroxyjulolidine-9-carboxaldehyde  was added to Uiso(H) = 1.5Ueq and 1.2Ueq(C) for other H atoms. The tricyclic fragment of the julolidine ring and the azomethine C=N bond are disordered over two sets of sites with a refined occupancy ratio of 0.773 (3):0.227 (3). The non-hydrogen atoms of the major fraction were refined anisotropically while those of the minor fraction were refined isotropically, and one disordered atom, C21A, is probably further disordered, but this was not corrected for. The bond lengths C1—N2 and C1—N2A were refined with distance restraints of 1.40 (2) Å.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016019733/su5338sup1.cifCrystal structure: contains datablock(s) I, global. DOI: Click here for additional data file.10.1107/S2056989016019733/su5338Isup2.cmlSupporting information file. DOI: 1521905CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


J13-2-12Y, together with six biosynthetically related compounds (4−9). All structures were determined by extensive spectroscopic analysis, chemical derivatization, and ECD experiments. A plausible biosynthetic pathway of 1−3 was proposed. In addition, the antimicrobial activities of all isolated compounds were evaluated against Staphylococcus aureus 209P (bacterium) and Candida albicans FIM709 (fungus).Phyllomeroterpenoids A−C ( In general, meroterpenoids originate from a dual-biosynthetic pathway7. This is composed of a non-terpenoid pathway and a terpenoid pathway, such as the polyketide/terpenoid and shikimate/terpenoid pathways.Meroterpenoids, such as fumagillinAcorus tatarinowii is a common and important medicinal plant, and its dried rhizomes have a long history of being used as traditional Chinese medicine (TCM) as Shi Chang Pu (Acori Tatarinowii Rhizoma) to treat many diseases, such as nervous ailments, dysentery, bronchitis, intermittent fevers ect8. A. tatarinowii is rich with asarones, which show antimicrobial activity9. The micro-environment of A. tatarinowii is special due to the existence of abundant antimicrobial asarones, and the microorganisms living in this habitat should be distinctive. During our recent search for bioactive compounds from microorganisms14, chemical investigation on a TCM endophytic fungal strain of Phyllosticta sp. J13-2-12Y from the leaves of A. tatarinowii was carried out. Through this investigation, three unusual meroterpenoids, phyllomeroterpenoids A–C (1−3), were isolated, along with six biosynthetically related compounds (4−9)  are multi-biosynthetic pathway derived meroterpenoids, whose structures are composed of one guignardianone unit from the amino acid pathway, one C7 unit from the pentose phosphate pathway, and one monoterpene unit from the terpenoid pathway. The guignardianone unit is the skeleton of the guignardianone derivative, while the C7 unit and the monoterpene unit compose the guignardone-type meroterpenoids. In this study, we report the isolation and structural elucidation of 1−9 as well as their antimicrobial activities. In addition, a plausible biogenetic pathway of 1−3 is proposed.4−6) and three guignardone-type meroterpenoids (7−9) were identified as -guignardianone C (4)16, -botryosphaerin B (5)17, -phenguignardic acid methyl ester (6)16, −17-hydroxylated guignardone A (7)18, -guignardone B (8)19, and −12-hydroxylated guignardone A (9)18 by comparisons of their NMR (recorded in CDCl3) and ECD data with references. In addition, the NMR data of 4 in CD3OD  was obtained as a yellowish oil. The positive ion at m/z 551.2277 [M + H]+  from HRESIMS indicated the molecular formula of C31H34O9 (index of hydrogen deficiency = 15). In the1H NMR spectrum of 1, the characteristic signals of five aromatic protons , three olefinic protons , one O-methine [δH 4.54 ], two O-methylenes , and three methyls  were observed. Among them, the five aromatic protons indicated the existence of a mono-substituted benzene ring moiety in 1. Combined with the DEPT-135 spectrum, 31 signals were observed in the13C NMR spectrum, which can be assigned to eight sp2 quaternary carbons (including one ketone carbonyl and two ester carbons), six sp2 methine carbons, one sp2 methylene carbon, three sp3O-quaternary carbons, four sp3 methine carbons (including one O-methine carbon), six sp3 methylene carbons (including two O-methylene carbons), and three methyl carbons. Based on the analysis of1H−1H COSY experiment, four subunits  were revealed as shown in Fig. 1H−1H COSY, the HMBC correlations -guignardianone C (4)16 and the above analysis of1H−1H COSY. Based on the molecular formula and the key HMBC correlation from H2-17 to C-10′, these two moieties can be combined, and the planar structure was established as shown in Fig. Phyllomeroterpenoid A  was obta3-13′/H3-14′ indicated that the configuration of the double bond of ∆2′ as Z. Furthermore, the13C NMR data of guignardianone unit in 1 were quite similar to those of -guignardianone C (4)16, which confirmed the above deduction. In addition, the alkaline hydrolysis of 1 give a major reaction product (1a) that was identified as −17-hydroxylated guignardone A (7)18 by HPLC, NMR data, and electronic circular dichroism (ECD) comparisons  was obtained as a yellowish oil. The positive ion at m/z 553.2455 [M + H]+  from HRESIMS indicated the molecular formula of C31H36O9 (index of hydrogen deficiency = 14). The detailed 2D NMR analysis -guignardianone C (4) -guignardone B (8) -guignardianone C (4)  Table  revealed8) Table  showed a4) Table , the key4) Table  between  Figures –S6 and t3) was obtained as a yellowish oil, and its molecular formula was the same as that of 1 (C31H34O9) as determined by HRESIMS. Based on comparison of the NMR data with -guignardianone C (4)16 and detailed NMR analyses -guignardianone C (4)16, the key NOESY correlations  and3JH-12, Ha-13 (6.9 Hz) in 3 were the same as those in 9  and Candida albicans FIM709 (fungus). All compounds exhibited different antimicrobial activities , and three known guignardone-type meroterpenoids (7–9) were isolated from the TCM endophytic fungal strain of Phyllosticta sp. J13-2-12Y. In addition, unusual meroterpenoids (1–3), the heterozygotes of guignardianone and guignardone-type meroterpenoid were also obtained. Phyllomeroterpenoids A–C (1–3) are composed of one guignardianone unit, one C7 unit, and one monoterpene unit, and they are multi-biosynthetic pathway derived meroterpenoids from the amino acid/pentose phosphate/terpenoid pathways. They could originate from phenyalanine30, 2-epi−5-epi-valiolone (EEV)33, and a monoterpenoid as shown in Fig. Guignardone-type meroterpenoids are a rare kind of meroterpenoidThe detail of instruments applied in this work are provided in supporting information.A. tatarinowii collected from Guangxi Medicinal Botanical Garden, Guangxi Province, People’s Republic of China. The details of isolation and identification can be found in supporting information.The strain numbered as J13-2-12Y was isolated from the leaves of 2O  to yield 4 fractions (W1–W4). Fraction W2 (3.2 g) was further separated by MPLC on ODS CC (4 × 45 cm) with a elution system of MeOH-H2O  for 420 min at 20 mL/min to afford fractions W2-1–W2-6. Fraction W2-6 (0.4 g) was separated by silica gel CC using a elution system of cyclohexane- EtOAc  to yield 7 fractions (W2-6-1–W2-6-7). 1  was isolated from fraction W2-6-2 (20.0 mg) by preparative HPLC using MeCN–H2O  at 3 mL/min. 2  was obtained from fraction W2-6-4 (15.0 mg) by preparative HPLC with MeCN–H2O  at 3 mL/min. 3  was separated from fraction W2-6-7 (19.0 mg) by preparative HPLC using MeCN–H2O  at 3 mL/min. 5  was isolated from fraction W2-2 (0.6 g) by preparative HPLC using MeOH–H2O  at 3 mL/min. 4  was isolated from fraction W2–4 (0.6 g)by preparative HPLC using MeOH–H2O  at 3 mL/min. 6  was isolated from fraction W2–5 (0.4 g) by preparative HPLC using MeCN–H2O  at 3 mL/min. Fraction W1 (4.6 g) was further separated by MPLC on ODS CC (4 × 45 cm) eluted with MeOH-H2O  for 400 min at 20 mL/min to afford 6 fractions (W1-1–W1–6). Fraction W1-2 (0.7 g) was subjected to silica gel CC with a elution system of cyclohexane-EtOAc  to obtain 4 fractions (W1-2-1–W1-2-4). 8  was isolated from fraction W1-2-3 (122.0 mg) by preparative HPLC using MeCN–H2O  at 3 mL/min to yield. 7  and 9  were isolated from fraction W1-2-2 (87.0 mg) by preparative HPLC using MeCN–H2O  at 3 mL/min.The fermented material was extracted with EtOAc for three times, and the solvent was evaporated to dryness under vacuum to obtain a crude extract (42.8 g). Then the crude extract was separated by silica gel CC (4 × 15 cm) with a elution system of cyclohexane-MeOH  to yield a cyclohexane portion  and a MeOH portion . The MeOH portion  was subjected to ODS CC (4 × 30 cm) eluting with MeOH–H1): yellowish oil; [α]27D –38.3 ; UV (MeOH) λmax (log ε) 204 (3.67), 222 (3.44), 269 (3.66), 295 (3.68), 308 (3.60); IR (KBr) vmax 3441, 2938, 1796, 1755, 1658, 1616, 1450, 1364, 1256, 1178, 1029, 977, 905, 758, 689 cm−1; ECD λnm (∆ε)  222 (+9.53), 255 (+12.77), 292 (–14.64) nm; ESI-MS (positive): m/z 1123 [2M + Na]+, 573 [M + Na]+; HRESIMS (positive): m/z 551.2277 [M + H]+ .Phyllomeroterpenoid A (2): yellowish oil; [α]27D –18.7 . UV (MeOH) λmax (log ε) 204 (3.67), 222 (3.46), 267 (3.66), 295 (3.61), 309 (3.55). IR (KBr) vmax 3447, 2979, 2935, 1799, 1746, 1655, 1619, 1450, 1370, 1299, 1249, 1181, 1124, 1036, 977, 758, 693 cm−1; ECD λnm (∆ε)  221 (+10.02), 258 (+11.33), 295 (–9.88) nm; ESI-MS (positive): m/z 575 [M + Na]+, 553 [M + H]+; HRESIMS (positive): m/z 553.2455 [M + H]+ .Phyllomeroterpenoid B (3): yellowish oil; [α]27D –45.0 ; UV (MeOH) λmax (log ε) 204 (3.82), 222 (3.62), 263 (3.93), 294 (3.69), 308 (3.62); IR (KBr) vmax 3435, 2924, 1796, 1755, 1655, 1613, 1447, 1382, 1246, 1038, 891, 684 cm−1; ECD λnm (∆ε)  221 (+14.16), 260 (+24.27), 293 (–18.84) nm; ESI-MS (positive): m/z 1123 [2M + Na]+, 573 [M + Na]+; HRESIMS (positive): m/z 551.2285 [M + H]+ .Phyllomeroterpenoid C (1 (1 mg) was treated with 2 N KOH (200 μL), THF (200 μL), and CH3OH (200 μL), and stirred at 25 °C for 4 h. After neutralizing with 10% HCOOH and extracting with EtOAc, the EtOAc layer was evaporated to dryness and dissolved in MeOH. Then, 1a (0.4 mg) was isolated from the mixture by analytical HPLC  with MeOH-H2O  at 1 mL/min, and its 1H NMR spectrum and ECD spectrum were identical with those of 7  was treated with 2 N KOH (200 μL), THF (200 μL), and CH3OH (200 μL), and stirred at 25 °C for 4 h. After neutralizing with 10% HCOOH and extracting with EtOAc, the EtOAc layer was evaporated to dryness and dissolved in MeOH. Then, 2a (0.4 mg) was isolated from the mixture by analytical HPLC  with MeOH-H2O  at 1 mL/min, and its 1H NMR spectrum and ECD spectrum were identical with those of 8 (Figures A sample of  Figures  and S6.S. aureus 209P and C. albicans FIM709 were measured in sterile 96-well plates using the broth microdilution method35, and the detail can be found in supporting information.The antimicrobial activities against Supporting information


N′-(pyridin-2-ylmethylene)picolinohydrazide. In both compounds, the Cd2+ ion is ligated by one O atom and two N atoms of the tridentate ligand, and by two halide ions. Both have fivefold coordination spheres with a distorted square-pyramidal geometry.The title compounds are cadmium bromide and cadmium iodide complexes of the ligand  2(C12H10N4O)]·CH3OH, (I), and [CdI2(C12H10N4O)], (II), are cadmium bromide and cadmium iodide complexes of the ligand (E)-N′-(pyridin-2-ylmethylene)picolinohydrazide. Complex (I) crystallizes as the methanol monosolvate. In both compounds, the Cd2+ cation is ligated by one O atom and two N atoms of the tridentate ligand, and by two bromide anions forming a Br2N2O pentacoordination sphere for (I), and by two iodide anions forming an I2N2O pentacoordination sphere for (II), both with a distorted square-pyramidal geometry. In the crystal of complex (I), molecules are linked by pairs of N—H⋯O and O—H⋯Br hydrogen bonds, involving the solvent molecule, forming dimeric units, which are linked by C—H⋯Br hydrogen bonds forming layers parallel to (101). In the crystal of complex (II), molecules are linked by N—H⋯I hydrogen bonds, forming chains propagating along [010]. In complex (II), measured at room temperature, the two iodide anions are each disordered over two sites; the refined occupancy ratio is 0.75 (2):0.25 (2).The title compounds, [CdBr Hydrazone ligands are one of the most important classes of flexible and versatile polydentate ligands and show very high efficiency in chelating transition metal ions , and have the potential to form mono- and multinuclear structures -N′-(pyridin-2-ylmethylene)picolinohydrazide, obtained by condensation of an equimolar mixture of 2-pyridinecarbaldehyde and picolinic acid hydrazide in methanol.The cadmium(II) ion, has a 2+ cation has a fivefold Br2N2O coordination sphere with a distorted shape and a τ5 value of 0.33 A/I2A:I1B/I2B. Considering the major components only, the Cd1—I1A and Cd1—I2A bond lengths are 2.736 (3) and 2.7128 (19) Å, respectively, and the Cd1—N2 bond length is 2.336 (3) Å. Atom Cd1 is ligated by one O atom (O1) and two N atoms (N1 and N2) of the tridentate ligand, and by two iodide anions. Atom Cd1 has a fivefold I2N2O coordination sphere with a distorted shape and a τ5 value of 0.28.In compound (II)In the crystal of compound (I)N′-[(E)-4-hydroxybenzylidene]pyridine-4-carbohydrazide-κN1}diiodidocadmium methanol disolvate ethylidene]picolinohydrazide-κ2N′,O}cadmium methylidene]pyridine-2-carbohydrazide-κ2N′,O}cadmium) cadmium -4-(2-hydroxybenzylideneamino)benzoato-κ2O,O′]cadmium in which layers are built from strong O—H⋯O hydrogen bonds picolinohydrazide  in 30 ml of methanol was treated with a methanolic solution of the appropriate cadmium(II) salt (0.5 mmol); CdBr2 for complex (I)2 for (II)4O10 in vacuum.A solution of the ligand Uiso(H) = 1.5Ueq(O) and 1.2Ueq for other H atoms. Owing to poor agreement, two reflections, (iUso(H) = 1.2Ueq(C). The N-bound H atoms were located in a difference-Fourier map but were refined with a distance restraint of N—H = 0.86 (4) Å with Uiso(H) = 1.2Ueq(N). In complex (II)Crystal data, data collection and structure refinement details for compounds (I)10.1107/S2056989017005308/su5361sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989017005308/su5361Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017005308/su5361IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1543006, 1543005CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, the benzoic acid molecules are linked via O—H⋯O hydrogen bonds, forming inversion dimers with R22(8) motifs. The dimers are linked through weak C—H⋯O hydrogen bonds into a chain structure along [-101]. The chains stack along the a axis through S⋯S and S⋯C short contacts, forming layers parallel to the ac plane.In the title compound (systematic name: 4,4′-{dithiolo[4,5- The dihedral angles between planes A and B and A and C are 31.24 (4) and 26.83 (6)°, respectively. Plane B makes dihedral angles of 85.88 (11) and 82.03 (15)°, respectively, with the benzene C9–C14 and C16–C21 rings. These benzene rings are approximately parallel, subtending a dihedral angle of 11.82 (14)°. All bond lengths and angles in the TTF fragment are within the range of the values for a neutral TTF molecule  Å and S1⋯C20v = 3.456 (5) Å; symmetry codes: (iv) x − 1, y, z; (v) −x + 1, −y, −z + 1], forming a layer parallel to the ac plane  reported by Mézière  al. 2000 has a si3,  and 4,5-bis(thiolmethylbenzoate)-1,3-dithiole-2-thion, 4,  were synthesized by the literature method  and 4 (465 mg 0.1 mmol) were mixed in triethylphosphite (5 ml) and heated at 393 K for 6 h. P(OEt)3 was then removed under reduced pressure and the red residue was purified by column chromatography on silica gel (DCM) to give 310 mg of a red powder of 2 (yield = 53%). 1H NMR : 8.01 , 7.41 , 4.28 , 3.94 .Compounds 1 was obtained by hydrolysis reaction of compound 2: A 50 ml flask was charged with compound 2  under an N2 atmosphere. Degassed methanol (6 ml) and THF (6 ml) were added to generate a suspension. In a separate flask, sodium hydroxide  was dissolved in degassed water (4 ml). The sodium hydroxide solution was added to compound 2 and the reaction was heated to reflux for 8 h. The reaction was then cooled to room temperature and the volatiles were removed in vacuo. Hydrochloric acid  was added to afford a maroon precipitate, which was collected by filtration and washed with water (50 ml). The product was collected and dried under high vacuum for 12 h to afford 1 as a maroon solid . 1H NMR : 8.02 ,7.43 , 4.28 . Elemental analysis calculated for C22H14O6S6: C 46.62, H 2.49%; found: C 46.67, H 2.51%. Red crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of an ethyl acetate solution of the title compound. Elemental analysis calculated for C22H14O6S6·0.25H2O: C 46.26, H 2.56%; found: C 46.29, H 2.58%.Finally, compound Uiso(H) = 1.2Ueq(O). H atoms bonded to C and O(water) atoms were positioned geometrically and included in the refinement in the riding-model approximation  with Uiso(H) = 1.2Ueq(C or O). In the refinement, the occupancy of the lattice water molecule was fixed at 0.25, which was estimated from the results of element analysis and gave acceptable displacement parameters for the water O atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017011070/is5477sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017011070/is5477Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017011070/is5477Isup3.cmlSupporting information file. DOI: 1548509CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The two molecular components are held together through an O—H⋯O hydrogen bond.The asymmetric unit comprises one 1,3-bis°. There are two intramolecular O—H⋯N hydrogen bonds, each generating an S(6) ring motif. In the crystal, classical O—H⋯O hydrogen bonds connect the 1,3-diazinane and water molecules into columns extending along the b axis. The crystal structure was refined as a two-component twin with a fractional contribution to the minor domain of 0.0922 (18).In the title hydrate, C Current research of our group is directed toward the synthesis of cyclic aminals with conformational interest, which may have the structural requirement for hydrogen-bonded interactions. Obvious targets are the 5-hydroxy-1,3-diazinanes because a hydroxyl group in the six-membered 1,3-diazacyclic ring may alter the conformational preferences resulting from the interactions of the hydroxyl group and the endocyclic nitrogen atoms -1,3-diazinan-5-ol monohydrate (II) and this study has shown that the hydroxyl substituent on the 1,3-diazinane ring is disordered over two positions, namely one component equatorial and the other axial , 1,3-bis(3-tert-butyl-2-hydroxy-5-methylbenzyl)-1,3-diazinan-5-ol monohydrate. The X-ray study again reveals that compound crystallizes with a solvent water molecule that links to the organic molecule through an O—H⋯O hydrogen bond. Furthermore, the hydroxyl group in the pyrimidine ring is also disordered over two positions .We have previously reported the synthesis and crystal structure of 1,3-bis(3-tert-butyl-2-hydroxy-5-methylbenzyl)-1,3-diazinan-5-ol molecule and a water molecule. These components are connected by an O3—H3⋯O1W hydrogen bond (Table 1Q = 0.588 (2) Å, θ = 176.9 (5) and φ = 245 (9)°. Atoms N1 and N2 are essentially tetrahedral (bond-angle sums are 331.5° for N1 and 331.6° for N2), with their benzyl substituents in equatorial positions and the lone pairs axial. The aromatic rings of these substituents are roughly parallel, with a dihedral angle between the two benzene rings of 19.7 (4)°. Intramolecular O—H⋯N hydrogen bonds form between the pyrimidine N atoms and the OH groups of the benzyl substituents and the pyrimidine N atoms, each with an S(6) graph-set motif (Table 1II)   Å is slightly longer than the typical value for an N—C bond [1.469 Å]. The remaining C—N bonds in the molecule are also typical and compare well with those found in the the related structure  set to 1.2Ueq(O). The H atoms of the water molecule were fixed in their found locations with Uiso(H) set to 1.5Ueq(O). C-bound H atoms were fixed geometrically (C—-H = 0.95 or 0.99 Å) and refined using a riding-model approximation, with Uiso(H) set to 1.2Ueq of the parent atom. The crystal was a two-component twin with a fractional contribution to the minor domain of 0.0922 (18).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016013645/sj5503sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016013645/sj5503Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016013645/sj5503Isup3.cmlSupporting information file. DOI: 1500903CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The ReI atom resides in an octahedral C3ClN2 coordination sphere. The Re—C bond trans to the chloride ligand is noticeably longer compared to the other two Re—C distances. Weak C—H⋯Cl hydrogen-bonding interactions consoldiate the packing of the molecules. In this design, the pyAm ligand was employed due to its well-known pharmacokinetic properties.The title compound, [ReCl(pyAm)(CO) In this design of pyAm ligand, the adamantyl moiety has been included beacuse of its well-known pharmacokinetic properties  complexes make them invaluable for a range of applications, such as light-emitting devices, nonlinear optical materials, radiopharmaceuticals, reagents for CO-reduction chemistry and photopolymerization °. The sixth site is occupied by a chloride ligand. The equatorial plane composed of atoms N1, N2, C2 and C3 is satisfactorily planar, with a mean deviation of 0.034 Å. In this complex, the chelate ring composed of atoms Re1, N1, C8, C9 and N2 is almost planar, with a mean deviation of 0.007 Å. The Re—Cl bond is considerably longer [1.963 (4) Å] compared to the other two Re—C bonds [1.918 (4) and 1.920 (3) Å], which can be attributed to the trans-labilizing effect arising from the chloride ligand across this bond.The molecular structure of the title complex is shown in Fig. 1l Table 1. The pyAc axis is shown in Fig. 3The crystal packing of the title complex reveals few nonclassical hydrogen-bonding interactions of the C—H⋯Cl type Table 2, leadinget al., 2016R)(CO)3], where R represents substituted or unsubstituted aromatic amines. The complex [ReCl(2-PP)(CO)3] [where 2-PP = N-(pyridin-2-ylmethylidene)aniline] has space-group symmetry P21/n (CO)3] {where L1 = 4-[(pyridin-2-ylmethylidene)amino]phenol} has P21/n space-group symmetry, with unit-cell dimensions close to those of [ReCl(2-PP)(CO)3] (CO)3]  were structurally characterized (CO)3], was synthesized  were added in a mixture of 15 ml of methanol and 5 ml of chloroform and allowed to reflux for 24 h. After this time, the reaction mixture was allowed to cool to room temperature, whereupon an orange precipitate was observed. The orange solid was collected by filtration and dried under vacuum to obtain 44.2 mg (55%) of the title complex. Single crystals were obtained by layering hexanes over a dichloromethane solution.A slurry of 50 mg (0.138 mmol) of [ReCl(CO)Uiso(H) = 1.2Ueq(C). One reflection  I. DOI: 10.1107/S205698901601255X/wm5310Isup2.hklStructure factors: contains datablock(s) I. DOI: 1497515CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information: