Recent findings suggest that memory allocation to specific neurons  in the amygdala is not random, but rather the transcription factor cAMP-response element binding protein (CREB) modulates this process, perhaps by regulating the transcription of channels that control neuronal excitability. Here, optogenetic studies in the mouse lateral amygdala (LA) were used to demonstrate that CREB and neuronal excitability regulate which neurons encode an emotional memory. To test the role of CREB in memory allocation, we overexpressed CREB in the lateral amygdala to recruit the encoding of an auditory-fear conditioning (AFC) memory to a subset of neurons. Then, post-training activation of these neurons with Channelrhodopsin-2 was sufficient to trigger recall of the memory for AFC, suggesting that CREB regulates memory allocation. To test the role of neuronal excitability in memory allocation, we used a step function opsin (SFO) to transiently increase neuronal excitability in a subset of LA neurons during AFC. Post-training activation of these neurons with Volvox Channelrhodopsin-1 was able to trigger recall of that memory. Importantly, our studies show that activation of the SFO did not affect AFC by either increasing anxiety or by strengthening the unconditioned stimulus. Our findings strongly support the hypothesis that CREB regulates memory allocation by modulating neuronal excitability. There is growing evidence for molecular and cellular mechanisms that regulate the allocation of memories to specific synapses and neurons in a neurocircuit . By direRecently, CREB has been proposed to regulate neuronal allocation by modulating neuronal excitability , 6, 9. IFig 1A) also included a ChR2 gene : 8 NaCl, 2 KH2PO4, 2 d-Glucose, 10 HEPES, 130 KMeSO4, 4 Mg-ATP, 7 Phosphocreatine Na, 0.3 GTP, 0.5 ADP. Patch pipettes were pulled with micropipette puller model P-97  from borosilicate glass capillary  with around 3 M resistance. Cells were visualized with an upright microscope using infrared or epifluorescent illumination, and whole-cell voltage-clamp recordings were made from cells with Multiclamp 700B. Responses were filtered at 2 kHz and digitized at 10 kHz. All data were acquired, stored and analyzed using pClamp 10 . These data are simply a replication of previous work : 135 NaCl, 5 KCl, 10 HEPES, 2 CaClous work , 17, 18.After the behavioral experiments, mice were sacrificed with sodium pentobarbital  and fixed with a transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer . Brains were sliced coronally (40 μm). The cannula tip locations were confirmed at the end of each experiment. Only those mice with validated bilateral cannula placements were included in the analysis. For cfos immunohistochemistry mice were sacrificed 90 minutes following behavior with sodium pentobarbital  and fixed with a transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer . Brains were sliced coronally (40 μm) and prepared for immunocytochemistry using either anti cFos (1:200) rabbit polyclonal antibodies  and an Alexa-568 conjugated anti-rabbit secondary antibody (1:250) , or anti GFP (1:1000) chicken polyclonal antibodies  and an Alexa-568 conjugated anti-chicken secondary (1:500) (A11041 Thermo Fisher Scientific) or anti mCherry (1:1000) mouse monoclonal antibody  and a Alexa-568 conjugated anti-mouse secondary (1:500) (A-11004 Thermo Fisher Scientific). For visualization of neurons an Alexa-455 conjugated Nissl NeuroTrace (Thermo Fisher Scientific) counter stain was used. Confocal microscopy was used to identify immunoreactive neurons. The lateral amygdala was anatomically defined according to the Paxinos and Franklin atlas . Immunor was designed to bilaterally channel 473 or 532 nm light through fiber optic cables designed to be inserted into the cannula implanted during surgery. The light source consisted of two DPSS lasers  operating at 473 nm and 532 nm delivering TEM00 mode laser beams. The beams are then coupled using a dichroic mirror to make both the beam co-linear and then expanded using a two lens telescope system. The expanded beam is then split in two arms at orthogonal directions using 50:50 beam splitter and then focused by two separate biconvex lenses on to a fiber coupling assembly with xyz adjustments. The optical fiber (200 um multi-mode) from both the arms are aligned using the XYZ adjustments to obtain an equal power output (+/- 2 mW) as measured by power meter held at the other end of the optical fiber. The laser source is modulated using TTL pulses generated from a signal generator . All the optical components including optical fiber are bought from Thorlabs, Newton, New Jersey. The end of the fiber to be inserted into the cannula was reinforced with a steel tube . A Plastics One injector and dummy were modified to allow the fiber to be positioned securely at and appropriate depth during behavior [An optogenetic setup behavior . entailed the placement of mice in a conditioning chamber, and 1 minute later, the presentation of a tone  that co-terminated with a shock . AFC was tested (Tone Test) 30 minutes after training. Mice were placed in a novel chamber, and 1 minute later, the tone was presented for 1 minute. The index of memory, freezing, was assessed via automated procedures . 15 minutes later ChR2 activation was achieved by illuminating the LA of Trained–CREB/ChR2 and Trained–ChR2 cohorts with a 5mW 473nm laser  1 minute after placement in a novel chamber. Activation continued for 1 minute (Optogenetic Activation) and the index of memory, freezing, was assessed via manual scoring procedures by researchers blind to the experimental conditions. The No-Training–CREB/ChR2 cohort was treated identically to the Trained–CREB/ChR2 and Trained–ChR2 cohorts expect no shock was administered during AFC. The Unpaired–CREB/ChR2 cohort was treated identically to the Trained–CREB/ChR2 and Trained–ChR2 cohorts except the shock preceded the tone by 1 minute. Any animal that had a loose or lost head cap, demonstrated signs of sickness or died, were excluded.AFC (Tone Training) for Trained–CREB/ChR2 and Trained–ChR2 cohorts  entailed the placement of mice in a conditioning chamber, and 1 minute later, the presentation of a tone  that co-terminated with a shock . Only the Step–SFO/VChR1 cohort received a 5 second pulse of 5mW 473nm blue light prior to the tone/shock presentation and, thereby, the depolarization of a subset of their neurons during the tone/shock association. The Reversed–SFO/VChR1 cohort received a 5 second pulse of 5mW 473nm blue light 55 seconds after the tone/shock presentation. All three cohorts were trained identically a second time 24 hours later. The mice received two training trials because the manipulations required for SFO activation  prior to AFC resulted in reduced AFC learning  for Step–SFO/VChR1, Ctrl–SFO/VChR1 and Reversed–SFO/VChR1 cohorts learning . Testing a cohort of SFO/VChR1 infused animals were placed on an elevated plus maze and allowed to explore for 3 minutes. Each animal then received a 5 second pulse of 5mW 473nm blue light and allowed to explore the elevated plus maze for another 3 minutes. The time spent in the open, center or closed portions of the maze for the minute prior to (Pre-Light) and following (Post-Light) blue light delivery was assessed with automated procedures.For the elevated plus maze  entailed the placement of SFO/VChR1 infused animals in a conditioning chamber, and 55 seconds later SFO was activated with a 5 second pulse of 5mW 473nm blue light. The presentation of a tone  immediately followed the SFO activation. Testing for AFC occurred 24 hours later. Mice were placed in a novel chamber, and 1 minute later, the tone was presented for 1 minute. Freezing was assessed with automated procedures. These animals were previously tested on the elevated plus maze.Pairing of step function opsin and tone  entailed the placement of SFO/VChR1 infused animals in a conditioning chamber, and 1 minute later, a tone  was presented that co-terminated with a foot-shock . Testing for AFC occurred 24 hours after training. Mice were placed in a novel chamber, and 1 minute later, the tone was presented for 1 minute (Pre-Pairing Tone Test). 24 hours later mice were placed in a novel chamber, and 55 seconds later SFO was activated with a 5 second pulse of 5mW 473nm blue light. SFO activation was immediately followed by the presentation of a tone . Testing for AFC occurred 24 hours later (Post-Pairing Tone Test). Mice were placed in a chamber, and 1 minute later, the tone was presented for 1 minute. Any animal that had a loose or lost head cap, demonstrated signs of sickness or died, were excluded.Pairing of step function opsin and tone on previously conditioned animals A priori knowledge regarding the (n) needed to observe a significant result  was used to determine the sample size. This was subsequently confirmed by observing the variance and significance of each individual experiment. Behavioral results over two standard deviations from the mean were excluded from analysis. One-sample two-sided t-tests against 0 were used to demonstrate statistically significant levels of auditory-fear learning. Unpaired two-sided t-tests are used to determine statistical differences between two cohorts. One-way (baseline subtracted) and two-way (raw data) analysis of variance (ANOVA) were used to determine statistical differences when three or more cohorts were present. Post hoc Tukey’s multiple comparison (one-way) or Bonferroni tests (two-way) were performed to further determine the relationships between cohorts if significance was demonstrated by ANOVA. In the caption of all figures we state which analysis has been used and that error bars are mean ± SEM, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.S1 Fig(a) Behavioral design for the four experimental cohorts: Trained—CREB/ChR2, Trained—ChR2, No-Training–CREB/ChR2 and Unpaired—CREB/ChR2. (b) A two-way ANOVA demonstrated a difference in freezing between cohorts  = 7.95, p < 0.001), baseline vs tone  = 23.11, p < 0.001) and an interaction between cohorts and baseline vs tone  = 3.04, p < 0.05) during the tone test. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs tone freezing for the Trained—CREB/ChR2 (p < 0.01) and the Trained—ChR2 (p < 0.001) cohorts whereas there was no difference between baseline vs tone for the No-Training–CREB/ChR2 (p > 0.05) and Unpaired—CREB/ChR2 (p > 0.05) cohorts. Mean freezing levels were (baseline / tone): Trained—CREB/ChR2 6.58 ± 2.09%, / 27.12 ± 6.90% n = 11; Trained—ChR2 7.47 ± 2.36% / 29.10 ± 4.93%, n = 17; No-Training—CREB/ChR2 1.21 ± 1.01% / 3.12 ± 2.11%, n = 12; and Unpaired—CREB/ChR2 3.06 ± 1.34% / 11.62 ± 4.89%, n = 11. (c) A two-way ANOVA demonstrated a difference in freezing between cohorts  = 11.39, p < 0.001), baseline vs light  = 30.24, p < 0.001) and an interaction between cohorts and baseline vs light  = 6.94, p < 0.001) during the optogenetic activation. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs light freezing for the Trained—CREB/ChR2 (p < 0.001) and the Trained—ChR2 (p < 0.01) cohorts whereas there was no difference between baseline vs light freezing for the No-Training–CREB/ChR2 (p > 0.05) and Unpaired—CREB/ChR2 (p > 0.05) cohorts. Importantly, Bonferroni’s post hoc test also determined that there was a significant difference in freezing to light between the Trained—CREB/ChR2 and the Trained—ChR2 cohorts (p < 0.001), the Trained—CREB/ChR2 and the No-Training–CREB/ChR2 cohorts (p < 0.001), and the Trained—CREB/ChR2 and the Unpaired—CREB/ChR2 cohorts (p < 0.001). Mean freezing levels were (baseline / light): Trained—CREB/ChR2 6.06 ± 3.91% / 48.79 ± 9.21%, n = 11; Trained—ChR2 1.27 ± 0.69% / 20.20 ± 5.87%, n = 17; No-Training—CREB/ChR2 1.81 ± 1.66% / 7.22 ± 3.73%, n = 12; and Unpaired—CREB/ChR2 0.00 ± 0.00% / 5.91 ± 5.09%, n = 11. Error bars are mean ± SEM, ** = p < 0.01, *** = p < 0.001, NS = not significant.(TIF)Click here for additional data file.S2 Fig(a) Behavioral design for the three experimental cohorts: Step—SFO/VChR1, Ctrl—SFO/VChR1 and Reversed—SFO/VChR1. (b) A two-way ANOVA demonstrated a difference in freezing between baseline vs tone  = 40.96, p < 0.001) but no difference in freezing between cohorts  = 0.87, p > 0.05) and no interaction between cohorts and baseline vs tone  = 1.89, p > 0.05) during the tone test. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs tone freezing for the Step—SFO/VChR1 (p < 0.001), Ctrl—SFO/VChR1 (p < 0.01) and Reversed—SFO/VChR1 (p < 0.01) cohorts. Mean freezing levels were (baseline / tone): Step—SFO/VChR1 33.92 ± 4.24%, / 69.13 ± 5.75% n = 21; Ctrl—SFO/VChR1 40.89 ± 7.59% / 73.67 ± 5.12%, n = 13; and Reversed—SFO/VChR1 35.60 ± 6.87% / 63.47 ± 6.48%, n = 17. (c) A two-way ANOVA demonstrated a difference in freezing between cohorts  = 5.56, p < 0.01) and baseline vs light  = 14.19, p < 0.001) but no interaction between cohorts and baseline vs light  = 2.03, p > 0.05) during the optogenetic activation. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs light freezing for the Step—SFO/VChR1 (p < 0.001) cohort whereas there was no difference between baseline vs light freezing for the Ctrl—SFO/VChR1 (p > 0.05) and Reversed—SFO/VChR1 (p > 0.05) cohorts. Importantly, Bonferroni’s post hoc test also determined that there was a significant difference in freezing to light between the Step—SFO/VChR1 and the Ctrl—SFO/VChR1 cohorts (p < 0.01), and the Step—SFO/VChR1 and the Reversed—SFO/VChR1 cohorts (p < 0.01). Mean freezing levels were (baseline / light): Step—SFO/VChR1 5.95 ± 1.43% / 22.14 ± 4.86%, n = 21; Ctrl—SFO/VChR1 3.21 ± 1.65% / 8.21 ± 2.75%, n = 13; Reversed—SFO/VChR1 1.76 ± 0.76% / 8.33 ± 2.49%, n = 17. Error bars are mean ± SEM, ** = p < 0.01, *** = p < 0.001, NS = not significant.(TIF)Click here for additional data file.S3 Fig(a) Behavioral design for the two experimental cohorts: Step–SFO/VChR1, and Ctrl–SFO/VChR1. (b) A two-way ANOVA demonstrated a difference in freezing between baseline vs tone  = 13.57, p < 0.001) but no difference in freezing between cohorts  = 0.59, p > 0.05) and no interaction between cohorts and baseline vs tone  = 0.02, p > 0.05) during the tone test. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs tone freezing for the Step—SFO/VChR1 (p < 0.01) and Ctrl—SFO/VChR1 (p < 0.05) cohorts. Mean freezing levels were (baseline / tone): Step—SFO/VChR1 9.99 ± 2.83%, / 24.68 ± 4.66% n = 23; Ctrl—SFO/VChR1 7.51 ± 2.62% / 21.25 ± 4.38%, n = 17. (c) A two-way ANOVA demonstrated a difference in freezing between baseline vs light  = 17.11, p < 0.001) but no difference in freezing between cohorts  = 0.08, p > 0.05) and no interaction between cohorts and baseline vs light  = 0.04, p > 0.05) during the 1st optogenetic activation. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs light freezing for the Step—SFO/VChR1 (p < 0.01) and Ctrl—SFO/VChR1 (p < 0.05) cohorts. Bonferroni’s post hoc test also determined that there was no significant difference in freezing to light between the Step—SFO/VChR1 and the Ctrl—SFO/VChR1 cohorts (p > 0.05). Mean freezing levels were (baseline / light): Step—SFO/VChR1 2.10 ± 0.59% / 9.85 ± 2.83%, n = 23; Ctrl—SFO/VChR1 1.18 ± 0.37% / 9.69 ± 2.34%, n = 17. (d) A two-way ANOVA demonstrated a difference in freezing between baseline vs light  = 34.89, p < 0.001) and a difference in freezing between cohorts  = 4.47, p < 0.05) and no interaction between cohorts and baseline vs light  = 2.63, p > 0.05) during the 2nd optogenetic activation. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs light freezing for the Step—SFO/VChR1 (p < 0.001) and Ctrl—SFO/VChR1 (p < 0.05) cohorts. Importantly, Bonferroni’s post hoc test also determined that there was a significant difference in freezing to light between the Step—SFO/VChR1 and the Ctrl—SFO/VChR1 cohorts (p < 0.05). Mean freezing levels were (baseline / light): Step—SFO/VChR1 6.52 ± 1.42% / 29.78 ± 4.41%, n = 23; Ctrl—SFO/VChR1 5.00 ± 1.46% / 18.23 ± 3.16%, n = 17. (e) Behavioral design for the two experimental cohorts: Step—SFO/VChR1, and Ctrl—SFO/VChR1. (f) A two-way ANOVA demonstrated a difference in freezing between baseline vs tone  = 27.86, p < 0.001) but no difference in freezing between cohorts  = 0.09, p > 0.05) and no interaction between cohorts and baseline vs tone  = 0.01, p > 0.05) during the tone test. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs tone freezing for the Step—SFO/VChR1 (p < 0.01) and Ctrl—SFO/VChR1 (p < 0.01) cohorts. Mean freezing levels were (baseline / tone): Step—SFO/VChR1 6.38 ± 4.75%, / 42.68 ± 9.69% n = 6; Ctrl—SFO/VChR1 9.06 ± 3.16% / 44.05 ± 6.25%, n = 5. (g) A two-way ANOVA demonstrated a difference in freezing between baseline vs light  = 5.45, p < 0.05) but no difference in freezing between cohorts  = 2.04, p > 0.05) and no interaction between cohorts and baseline vs light  = 4.13, p > 0.05) during optogenetic activation. Bonferroni’s post hoc test determined that there was a significant difference between baseline vs light freezing for the Step—SFO/VChR1 (p < 0.01) but not the Ctrl—SFO/VChR1 (p > 0.05) cohorts. Importantly, bonferroni’s post hoc test also determined that there was a significant difference in freezing to light between the Step—SFO/VChR1 and the Ctrl—SFO/VChR1 cohorts (p < 0.05). Mean freezing levels were (baseline / light): Step—SFO/VChR1 5.00 ± 2.30% / 19.44 ± 4.89%, n = 6; Ctrl—SFO/VChR1 7 ± 2.22% / 8.00 ± 2.77%, n = 5. Error bars are mean ± SEM, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = not significant.(TIF)Click here for additional data file.S4 Fig(a) Behavioral design for the elevated plus maze (EPM). (b) The exploration time in the open, center and closed portions of the EPM was monitored for the minute prior to (Pre-Light) and after (Post-Light) a step in excitability. Two-way ANOVA showed a significant effect of arm  = 109.6, p < 0.001) but no significant interaction  = 0.018, p > 0.05) or effect of light  = 3.7x10-7 p > 0.05). The mean exploration times: Pre-Light: Open; 5.96 ± 3.10%, Center; 4.95 ± 1.77%, Closed 49.09 ± 4.06%. Post-Light: Open; 6.43 ± 3.69%, Center; 5.21 ± 2.23%, Closed 48.35 ± 4.46%. (c) Behavioral design for the Step/Tone pairing. (d) There was no difference in freezing during the tone test between the baseline and tone  = -1.33, p > 0.05). The mean freezing levels (baseline / tone): 0.00 ± 0.00% / 2.35 ± 1.76%, n = 8 tone test. e) Behavioral design for the trained Step/Tone pairing. (f) A two-way ANOVA demonstrated no difference in freezing between baseline vs tone  = 2.68, p > 0.05), cohorts  = 0.946, p > 0.05) and no interaction between cohorts and baseline vs tone  = 1.10, p > 0.05) during the tone test. Bonferroni’s post hoc test determined that there was no significant difference between tone freezing for the Step—SFO/VChR1 and Ctrl—SFO/VChR1 (p > 0.05) cohorts. Mean freezing levels were (baseline / tone): Step—SFO/VChR1 13.22 ± 3.49%, / 22.06 ± 4.77% n = 15; Ctrl—SFO/VChR1 24.97 ± 5.12% / 24.64 ± 3.92%, n = 5. Error bars are mean ± SEM, NS = not significant.(TIF)Click here for additional data file.


In the title salt dihydrate, the conrotatory relationship between the carboxylate and nitro groups of the anion leads to a dihedral angle between them of 26.73 (15)°. Substantial charge-assisted water-O—H⋯O(carboxylate) hydrogen bonding leads to supramolecular zigzag chains. These are connected into a three-dimensional architecture by N—H⋯O and N—H⋯N hydrogen bonds. 5N2+·C7H5N2O4−·2H2O, the carboxylate and nitro groups lie out of the plane of the benzene ring to which they are bound , and as these groups are conrotatory, the dihedral angle between them is 26.73 (15)°. An intramolecular amino-N—H⋯O(carboxylate) hydrogen bond is noted. The main feature of the crystal packing is the formation of a supramolecular chain along the b axis, with a zigzag topology, sustained by charge-assisted water-O—H⋯O(carboxylate) hydrogen bonds and comprising alternating twelve-membered {⋯OCO⋯HOH}2 and eight-membered {⋯O⋯HOH}2 synthons. Each ammonium-N—H atom forms a charge-assisted hydrogen bond to a water molecule and, in addition, one of these forms a hydrogen bond with a nitro-O atom. The amine-N—H atoms form hydrogen bonds to carboxylate-O and water-O atoms, and the amine N atom accepts a hydrogen bond from an amino-H atom. The hydrogen bonds lead to a three-dimensional architecture. An analysis of the Hirshfeld surface highlights the major contribution of O⋯H/H⋯O hydrogen bonding to the overall surface, i.e. 46.8%, compared with H⋯H contacts (32.4%).In the anion of the title salt hydrate, H Beyond the structure determination of several polymorphs of the parent structure lead(II)]  and C7—O2 = 1.2746 (15) Å. While there is an intramolecular amino-N—H⋯O(carboxylate) hydrogen bond, Table 1The molecular structures of the constituents of (I)a. Each of the ammonium-N3—H atoms forms a charge-assisted hydrogen bond to a water molecule, with the HN4 atom also forming a hydrogen bond to a nitro-O4 atom indicating that the HN4 atom is bifurcated [i.e.: N—H⋯]. The amine-N4—H atoms form a hydrogen bond to a carboxylate-O1 atom and to a water molecule and at the same time accept a hydrogen bond from an amino-H atom, this being the only N—H⋯N hydrogen bond in the structure; the second amino-H atom forms an intramolecular hydrogen bond with the carboxylate-O1 atom, as mentioned above. Each of the water-H atoms forms a charge-assisted hydrogen bond with a carboxylate-O atom, leading to a zigzag supramolecular chain aligned along the b axis, as shown in Fig. 2b. The chain comprises alternating twelve-membered {⋯OCO⋯HOH}2 and eight-membered {⋯O⋯HOH}2 synthons. As shown in Fig. 2c, two of the ammonium-N3—H atoms bridge water molecules in the chain shown in Fig. 2b to form a non-symmetric, eight-membered {⋯HNH⋯OH⋯O⋯HO} synthon while the amine-H atoms provide a second bridge between water- and carboxylate-O atoms to form a ten-membered {⋯HNH⋯OH⋯O⋯HOH⋯O} synthon. Further hydrogen bonds to water molecules leads to the formation of additional synthons, i.e. ten-membered {⋯HNNH⋯O}2 and eight-membered {⋯HNH⋯O}2. A view of the unit-cell contents is shown in Fig. 2d. In addition to the above, π(phenyl)–π(phenyl) interactions are noted between inversion-related rings with the inter-centroid separation being 3.6190 (8) Å .As expected from the chemical composition of (I)et al., 2016dnorm in Fig. 4W and —H4W atoms and carboxylate-O1 and -O2 atoms are evident through the bright-red spots appearing near the respective donor and acceptor atoms, Fig. 4a. The donors of these interactions appear as light-blue spots near the water O—H atoms and the acceptors as red regions surrounding carboxylate-O1 and -O2 atoms on the Hirshfeld surface mapped over electrostatic potential in Fig. 3The Hirshfeld surface analysis of (I)W and -O2W atoms, and near the hydrazinium-H3N, H4N, H5N and H7N atoms in Fig. 4b are indicative of the hydrazinium-N—H⋯O(water) hydrogen bonds. In the same way, the amine-N4—H6N⋯O1 hydrogen bond is also viewed as a pair of bright-red spots near these atoms in Fig. 4b. The bifurcated ammonium-HN4 atom, forming comparatively weaker N—H⋯O hydrogen bonds compared to those just described, is viewed as the diminutive red spot in Fig. 4a. The presence of faint-red spots near the phenyl-C2–C4 atoms in Fig. 4b indicate their participation in edge-to-edge overlap with a symmetry-related phenyl ring, as seen in the short interatomic C⋯C contacts listed in Table 2b, two spots are noted in the vicinity of the O4 atom, one corresponding to the conventional hydrogen bond and the other (to the left) to the weak H2N⋯O4 interaction, Table 2dnorm- and shape-index mapped Hirshfeld surfaces highlighting the O—H⋯O and O—H⋯N hydrogen bonds, and short interatomic C⋯C, C⋯O/O⋯C and N⋯O/O⋯N contacts are illustrated in Fig. 5a and 5b, respectively.The two pairs of bright-red spots near each water-O1et al., 2007a–g, respectively; their relative contributions to the Hirshfeld surfaces are summarized in Table 3i.e. 46.8%, due to the involvement of all the acidic hydrogen atoms in hydrogen bonds, mainly to oxygen, many of which are charge-assisted. Reflecting this dominance, sharp spikes are evident in the fingerprint plot delineated into O⋯H/H⋯O contacts shown in Fig. 6b. The pair of green spikes have their tips at de + di ∼1.9 Å and extend linearly up to de + di ∼2.3 Å. The points merged within the plot up to de + di ∼2.7 Å indicate the presence of short interatomic O⋯H/H⋯O contacts, Table 2i.e. 32.4%, Fig. 6c, as relatively few hydrogen atoms are available to form interatomic contacts. The pair of tips at de + di ∼2.3 Å in the mirror-reflected saw-tooth distribution are due to short interatomic H⋯H contacts involving water- and hydrazinium-hydrogen atoms, Table 2d, represents two π–π stacking interactions. In the first of these, the symmetry-related phenyl rings have a face-to-face overlap to give the arrow-like distribution in lower  region at around de = di = 1.6 Å. This interaction is also seen as the flat region appearing about the phenyl ring on the Hirshfeld surface mapped over curvedness, shown in Fig. 7b and Table 2de = di = 1.8 Å, i.e. adjacent to first arrow-like distribution. Even though C⋯H/H⋯C contacts have a significant contribution to the Hirshfeld surface, i.e. 5.9%, as seen from the fingerprint plot in Fig. 6e, the interatomic separations are much greater than sum of their van der Waals radii and hence do not appear to have influence on the molecular packing. The presence of short interatomic C⋯O/O⋯C and N⋯O/O⋯N contacts in the crystal, Table 2f, and conical tips, Fig. 6g, at de + di ∼3.1 Å in their respective fingerprint plots. The small contributions from the other interatomic O⋯O, C⋯N/N⋯C, N⋯N and N⋯H/H⋯N contacts listed in Table 2The overall two-dimensional fingerprint plot for (I)Chemical context section above, it was indicated that in the crystallographic literature there are several ammonium salts of 2-amino-4-nitrobenzoate anions. The ammonium cations range from the simple ammonium cation 2O 2C=NH2]+, i.e. guanidinium : 3514(s), 3399(s), 3400–2500, 1680, 1553, 1526, 1425, 1359, 1276, 830, 733.Solutions of 2-amino-4-nitrobenzoic acid (1 mmol) in MeOH (10 ml) and hydrazine (1 mmol) in MeOH (15 ml) were mixed and heated under reflux for 30 min. The reaction mixture was left at room temperatures for three days and the red blocks that formed were collected. M.p. 375–377 K (dec.). IR (KBr: cmUiso(H) set to 1.2Ueq(C). The O- and N-bound H atoms were located from difference maps, but refined with O—H = 0.84±0.01 Å and Uiso(H) = 1.5Ueq(O), and with N—H = 0.86–0.88±0.01 Å and Uiso(H) = 1.2Ueq(N), respectively. Owing to poor agreement, two reflections, i.e. (202) and (212), were omitted from the final cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989017004352/hb7667sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017004352/hb7667Isup2.hklStructure factors: contains datablock(s) I. DOI: 1538892CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Genome organization changes during development as cells differentiate. Chromatin motion becomes increasingly constrained and heterochromatin clusters as cells become restricted in their developmental potential. These changes coincide with slowing of the cell cycle, which can also influence chromatin organization and dynamics. Terminal differentiation is often coupled with permanent exit from the cell cycle, and existing data suggest a close relationship between a repressive chromatin structure and silencing of the cell cycle in postmitotic cells. Heterochromatin clustering could also contribute to stable gene repression to maintain terminal differentiation or cell cycle exit, but whether clustering is initiated by differentiation, cell cycle changes, or both is unclear. Here we examine the relationship between chromatin organization, terminal differentiation and cell cycle exit.Drosophila wing, where epithelial cells transition from active proliferation to a postmitotic state in a temporally controlled manner. We find there are two stages of G0 in this tissue, a flexible G0 period where cells can be induced to reenter the cell cycle under specific genetic manipulations and a state we call “robust,” where cells become strongly refractory to cell cycle reentry. Compromising the flexible G0 by driving ectopic expression of cell cycle activators causes a global disruption of the clustering of heterochromatin-associated histone modifications such as H3K27 trimethylation and H3K9 trimethylation, as well as their associated repressors, Polycomb and heterochromatin protein 1 (HP1). However, this disruption is reversible. When cells enter a robust G0 state, even in the presence of ectopic cell cycle activity, clustering of heterochromatin-associated modifications is restored. If cell cycle exit is bypassed, cells in the wing continue to terminally differentiate, but heterochromatin clustering is severely disrupted. Heterochromatin-dependent gene silencing does not appear to be required for cell cycle exit, as compromising the H3K27 methyltransferase Enhancer of zeste, and/or HP1 cannot prevent the robust cell cycle exit, even in the face of normally oncogenic cell cycle activities.We focused our studies on the Heterochromatin clustering during terminal differentiation is a consequence of cell cycle exit, rather than differentiation. Compromising heterochromatin-dependent gene silencing does not disrupt cell cycle exit.The online version of this article (10.1186/s13072-017-0159-8) contains supplementary material, which is available to authorized users. Cellular differentiation is the acquisition of cell-type specific characteristics, driven by changes in gene expression. Changes in gene expression are largely controlled by transcription factors, which can be facilitated or impeded by chromatin modifications, binding site accessibility and chromatin organization. A reciprocal relationship exists between chromatin organization, modification and gene expression, and several studies have shown that chromatin organization and modifications can change during differentiation. For example, during neural differentiation silenced genes move to repressive compartments in the nucleus –3. In ce0 coordinated with terminal differentiation. Thus, the modification and organization of chromatin in the nucleus can be impacted by the differentiation process itself, but also by the changes in cell cycle dynamics during differentiation. For example, chromatin compacts and heterochromatin clusters as cells in the embryo cycle more slowly and become lineage restricted [Drosophila loci within constitutive heterochromatin show increased association in terminally differentiated postmitotic cells [There is also an intimate relationship between the cell cycle and chromatin organization and modifications. Chromatin in actively cycling cells is highly dynamic. During S-phase, new histones are incorporated onto nascent DNA requiring re-establishment of histone modifications . During stricted . In Drosic cells  and facuic cells . Methodsic cells , but theDrosophila wing [Here we directly address the relationship between heterochromatin organization, chromatin modification and cell cycle exit using the temporally controlled cell cycle exit in the ila wing –20. In ow/y, w, hs-FLP; en-GAL4, UAS-GFP/UAS-E2F1, UAS-DP; tub-gal80TS/+w/y, w, hs-FLP; en-GAL4, UAS-GFP/UAS-CycD, UAS-Cdk4; tub-gal80TS/UAS-E2F1, UAS-DPw/y, w, hs-FLP; en-GAL4, UAS-GFP/+; tub-gal80TS/UAS-CycE, UAS-Cdk2w/y, w, hs-FLP; en-GAL4, UAS-GFP/UAS-CycD, UAS-Cdk4; tub-gal80TS/+w/y, w, hs-FLP; tub > CD2 > GAL4, UAS-GFP/UAS-CycD, UAS-Cdk4; tub-gal80TS/UAS-E2F1, UAS-DPw/y, v; en-GAL4, UAS-GFP/+; tub-gal80TS/UAS-E(z)RNAi (Bloomington 33659)w/y, v; en-GAL4, UAS-GFP/+; tub-gal80TS/UAS-Su(var)205RNAi (Bloomington 33400)w/w, Y10C; en-Gal4, UAS-RFP/+; +/UAS-Su(var)205RNAi (Bloomington 33400)TS were maintained in 18 °C to suppress Gal4 in early development. To disrupt G0 with cell cycle regulators, white prepupae were collected and shifted to 28 °C to indicated time points. For E(z) knockdown experiments, L3 larva were shifted from 18 to 28 °C to induce E(z) RNAi. For HP1 knockdown, crosses were kept in 28 °C after egg laying (AEL). For clonal expression of cell cycle regulators, animals were heat-shocked in 37 °C for 8 min during 48–72 h AEL and then kept in 18 °C. White prepupae were collected and shifted to 28 °C to indicated time points. All timings are adjusted according to the equivalent development at 25 °C as described previously [All the crosses containing gal80Imaginal disks or pupal wings were dissected in 1 × PBS and fixed in 4% paraformaldehyde/1 × PBS for 30 minutes. Samples were washed twice in 1 × PBS, 0.1% Triton X, 10 min each and incubated in PAT  for 10 min for larval tissues and 3 × 20 min for pupal tissues. Samples were then incubated with primary antibodies for 4 h or 4 °C overnight followed by three washes and secondary antibodies at room temperature for 4 h or 4 °C overnight. Primary antibodies used in this study include: Anti-phospho-Ser10 histone H3, 1:2000 rabbit (Millipore #06-570) or mouse ; Anti-GFP, 1:1000 chicken (Life Technologies A10262) or 1:1000 rabbit (Life Technologies A11122); Anti-pH2Av, 1:100 mouse ; Anti-H3K27me3, 1:500 rabbit (Millipore #07-449); Anti-HP1, 1:250 mouse ; Anti-H2Av, 1:500 rabbit (Active Motif #39715); Anti-H3, 1:500 mouse ; Anti-H3ac, 1:500 rabbit (Millipore #06-599); Anti-H3K4me3, 1:500 rabbit (Millipore #07-473); Anti-H3K9me3, 1:500 rabbit (Millipore #07-523) or (Active Motif #39161); Anti-H3K27ac, 1:500 rabbit (Abcam ab4729); Anti-H4ac, 1:500 rabbit (Millipore #06-866); Anti-H4K16ac, 1:500 rabbit (Millipore #07-329); Anti-H4K20me3, 1:500 mouse (Abcam ab78517); Anti-E2F, 1:500 guinea pig (kindly provided by Dr. Terry L. Orr-Weaver); Anti-Ubx, 1:250 mouse ; Anti-D1, 1:200 guinea pig (kindly provided by Dr. Yukiko Yamashita). DNA was labeled by 1 μg/ml DAPI in 1 × PBS, 0.1% Triton X for 10 min. F-actin was stained using 1:100 rhodamine–phalloidin  in 1 × PBS for 4 h.Images were taken with a 100 × oil objective on a Leica SP5 confocal with a system optimized z-section of 0.13 μm. Three-dimensional reconstructions were performed using the “3D viewer” function in Leica LAS AF software. Images of whole pupal wings in Figs. For integrated intensity quantifications, we used maximum projections of 12 continuous z-sections of confocal images. We developed a toolkit in MATLAB (Release 2015b) that automatically segments nuclei and foci within nuclei and integrates the pixel intensities with the help of the Advocacy and Research Support, U. Michigan LSA-IT. To identify nuclei, images were smoothed using a circular averaging filter through the fspecial and imfilter function of MATLAB. Next, a watershed algorithm was applied to segment nuclei from the background, and nuclei were masked using local maxima with an h-maxima transform. Thresholds were manually set and checked for each image to accurately delineate nuclei. GFP positive versus negative was established using an intensity threshold for the GFP channel. Integrated intensities for all nuclei were exported to Excel. Segmentation and measurement of foci followed a similar process for foci within the defined nuclear regions. In brief, foci were segmented using a watershed algorithm and then further measured for pixel intensity and number, which was used for foci area and intensity measurements.Alexa-488 probes against the rDNA internal transcribed spacer (ITS) region and Cy3 probes against AACAC repetitive satellite sequences were kindly provided by Dr. Yukiko Yamashita. For FISH, fixed tissues were treated with 2 mg/ml Rnase A in 1 × PBS, 0.1% Triton X at 37 °C for 10 min, and rinsed in 2 × SSC/1 mM EDTA/0.2% Tween 20. Then tissues were incubated in 2 × SSC/1 mM EDTA/0.2% Tween 20 solution with increasing formamide concentration from 20, 40 to 50% for 15 min to 30 min. Finally, tissues were incubated in 100 μl hybridization solution with 50 μl formamide, 20 μl 50% dextran sulfate, 20 μl 2 × SSC/1 mM EDTA/0.2% Tween 20 and 10 μl of 10 μm probe for 15 min at 91 °C and left at 37 °C overnight. Quantification of size for rDNA loci area was carried out using our customized MATLAB toolkit. For the quantification of AACAC satellite to the chromocenter, we used a single 0.13-μm z-section with the strongest FISH signal and measured the relative distance of the center of the FISH signal to the brightest Dapi-stained region and corrected for the total nuclear radius using the Leica LAS AF software.FACS was performed on dissociated wings to measure DNA content on an Attune Cytometer (Life Technologies) as described .T7-Wee-fwd, TAATACGACTCACTATAGGGATGACTTTGACAAGGACAC;T7-Wee-rev, TAATACGACTCACTATAGGATCTAGTCGATTGACGCATT;T7-Myt1-fwd, TAATACGACTCACTATAGGAATTGCACGACGACAAACAC;T7-Myt1-rev, TAATACGACTCACTATAGGTGTCCAGATGGATGAGATTC;T7-Myt1-fwd2, TAATACGACTCACTATAGGACAACAATCTGAACCGAAGC;T7-Myt1-rev2, TAATACGACTCACTATAGGTGGAGCCATATACCTCGAAT;T7-GFP-fwd, TAATACGACTCACTATAGGCATGTGGTCTCTCTTTTCGTT7-GFP-rev, TAATACGACTCACTATAGGGGGCACAAATTTTCTGTCAGT7-CycB-fwd TAATACGACTCACTATAGGGGCGTTTTTGCGTTCGAATTT7-CycB-rev, TAATACGACTCACTATAGGCAATTGCAAGTACGTGCGTT.Kc167 cells were kindly provided by Dr. K. Cadigan and cultured as described . For RNAWestern blots were performed on staged fly wings using Bio-Rad TGX precast 4–20% gels and high-sensitivity ECL reagents (Thermo) to detect HRP-conjugated secondary antibodies . Mouse aDrosophila is unclear. To examine this, we immunostained for various chromatin marks and chromatin binding proteins in wild-type Drosophila wings at three stages with distinct proliferation parameters. We examined quickly proliferating second instar larval (L2) wings, slowly proliferative wandering third instar larva (L3) and postmitotic 28 h pupal wings  that uses DAPI staining to mask individual nuclei followed by foci segmentation and measurement. We measured clustering of heterochromatin foci as a function of the integrated intensity for each focus  , 31. ThiDrosophila cell culture. In Drosophila Kc cells, the overall cell doubling time is controlled by the negative and positive regulators of the G2/M transition Wee/Myt and Cyclin B, respectively [cyclin B. Slowing the cell cycle increased the clustering and intensity of H3K27Me3 and H3K9Me3 compared to controls exposed to RNAi to GFP  using probes against the internal transcribed spacer region between the 18S RNA and 28S rDNA loci, which are tandemly repeated on the X, and measured the total rDNA area before and after cell cycle exit in the wing , 33. In An increase in chromatin clustering could be correlated with a global reduction in gene expression when cells become postmitotic . To testE(z) and/or the H3K9Me3 binding protein HP1. As E(z) and HP1 perform many functions during development, we turned to an inducible system with RNAi to alter gene function after embryogenesis. We used the engrailed-Gal4 driver with a temperature sensitive Gal80  to turn on UAS-driven expression of dsRNAs to E(z) and HP1 in the posterior wing from the early L1 and L3 stages, respectively. We then dissected wings at 24–28 h APF and stained for the mitotic marker phosphorylated phospho-Ser-10 histone H3 (PH3) to determine whether cells in the posterior wing delayed or bypassed cell cycle exit. We saw no effect of E(z) or HP1 reduction on cell cycle exit despite a clear loss of H3K27Me3 and HP1 in the posterior wing  [Pc by RNAi, but observed no effect on cell cycle exit despite de-repression of UBX in the wing  . We therE(z) and/or HP1 to examine whether loss of heterochromatin-dependent gene silencing can further delay cell cycle exit in the presence of high E2F activity. However, inhibition of E(z), HP1 or E(z) + HP1 together did not further compromise cell cycle exit in the presence of high E2F activity , can cause 1–2 extra cell cycles in the pupa wing between 24 and 36 h APF followed by a delayed entry into G0 at 36 APF , esc, Su(z)12) and Su(var)3-9) as well as several other histone modifying enzymes  and Su(var)3-9, which may indicate an expansion of heterochromatin in differentiating cells that enter a robust G0. Consistent with this idea we also observe an increase in the H2A variant H2Av in E2F-expressing cells in robust G0  was expressed in the posterior wing from the L3 stage and postmitotic wings at 26–28 h were examined for mitoses as indicated by PH3 and effective knockdown of PRC1 function by de-repression of the PRC1 target gene Ubx. Flow cytometry was also performed to measure cells that enter S and G2 phases. Green trace indicates cells from the posterior wing expressing the indicated transgenes. Black trace: control non-expressing anterior wing cells. Compromising PRC1 activity does not delay cell cycle exit. Scale bars = 100 μm.Additional file 2: Figure S3. Two stages of G0 in differentiating wings. E2F was expressed in the posterior wing to delay cell cycle exit. 28 h and 42 h APF pupal tissues were dissected and immunostained for PH3 (to label mitoses) and E2F1. The anterior/posterior boundary is specified by the white line. Overexpression of E2F delays entry into G0 until 36 h. At 42 h cells expressing high E2F1 are postmitotic (in robust G0). CycD/Cdk4 + E2F expression in the posterior wing is able to bypass the robust G0 to promote continued cycling, as shown by abundant mitoses (PH3) at 42 h. Bar = 50 μm.Additional file 3: Figure S1. Global levels of histone modifications do not dramatically change at cell cycle exit. (A-D) Quantitative western blots were performed on wings of the indicated stages to assess the levels of modified or total histone H3 or HP1. Control (Ctrl) and E2F samples are from 28 h postmitotic wings overexpressing GFP or E2F respectively. Total H3K9Me3, H3K27Me3, and HP1 levels do not dramatically change with cell cycle exit, however they increase with E2F expression. Modifications associated with active chromatin, H3K4Me3 and H3K27Ac also do not dramatically change with cell cycle exit, but increase upon E2F expression.Additional file 4: Table S1. Chromatin modifiers/organizers/remodelers that are upregulated upon E2F1/DP expression in pupal wings.Additional file 5: Figure S4. Clustering of heterochromatin can be disrupted within one cell cycle. E2F was overexpressed in the posterior wing from 10 h APF. 12 h later (within approximately one cell cycle) tissues were immunostained for indicated histone modifications. The posterior region is labeled by the expression of GFP and the anterior/posterior boundary is specified by the white line. The distribution of staining intensity in 1112–1339 nuclei, binned into three ranges, is shown at bottom. E2F disrupts heterochromatin clustering within one cell cycle. P values were determined by an unpaired t test; ****< 0.0001.Additional file 6: Figure S5. Delaying cell cycle exit disrupts heterochromatin. (A) CycE/Cdk2 or CycD/Cdk4 complexes were overexpressed in the posterior wing from 0 h APF. The anterior/posterior boundary is indicated by the white line. At 28 h (flexible G0) or 42 h APF (robust G0) pupal tissues were dissected and immunostained for the indicated histone modifications. (B) The distribution of staining intensity from 492 to 976 nuclei, binned into three ranges, is shown. Wings expressing E2F or CycD/Cdk4 to delay cell cycle exit were stained for mitoses (PH3) and the mitotic index at 27 h was quantified for the posterior compartment (C-D). The degree of heterochromatin disruption correlates with the number of cells cycling. P-values were determined by an unpaired t test; ****P value < 0.0001.Additional file 7: Table S2. Genes associated with senescence that are upregulated during robust G0 in the presence of ectopic E2F1/DP.


Each metal atom adopts a distorted pentagonal–bipyramidal SnC E)-2-[(2-hydroxyphenylimino)methyl]quinolin-8-ol derivative that reacted with di-n-butyltin oxide with release of H2O to yield the chelate title complex, [Sn2(C4H9)4(C16H10N2O2)2]. The compound crystallizes in the triclinic space group P-1, with two independent centrosymmetric dimers in the unit cell. Each features a typical pincer-type structure where the dianionic ligand is tetradentate, coordinating to the central tin atom through both phenolate oxygen atoms, as well as through the quinoline and imine N atoms. Each metal atom adopts a distorted pentagonal–bipyramidal SnC2N2O3 coordination arising from the N,N′,O,O′-tetradentate deprotonated Schiff base, one bridging phenolate O atom of the neighbouring ligand and two butyl groups in the axial sites.Condensation of 8-hydroxyquinoline-2-carbaldehyde with 2-aminophenol gave the ( One of the aims of our research is the structural analysis, particularly their coordination modes which has influence on their biological effects. The title compound (I)n-Bu2SnL units [molecule 1: Sn1 to C126 and molecule 2: Sn2 to C226 ] comprise the asymmetric unit. The dimerization of these monomeric units occurs through the quinolin-8-olate group oxygen atoms, leading to a central four membered (SnO)2 ring with a metal–metal separations Sn1⋯ Sn1 3.9593 (5) Å and Sn2⋯ Sn2 = 4.0132 (5) Å.The molecular structures of the two independent molecules in the compound (I)E)-2-(oxidophenylimino)methyl)quinolin-8-olate ligands are essentially planar and act as N,N′,O,O′-tetradentate ligands, forming a lozenge-shaped plane with the two n-butyl groups bonded to the same Sn atom on opposite sides. These n-butyl groups in molecule 1 display an ordered extended conformation while those in molecule 2 display mainly disordered gauche conformations, probably due to packing considerations -2-[(oxidophenylimino)methyl]quinolin-8-olate ligands and n-butyl groups occupying axial positions [C123—Sn1—C119 = 171.11 (12)° and C223—Sn2—C219 = 171.72 (13)° ° Fig. 1. There an-butyl groups attached to the Sn atoms and the aromatic H atoms of the ligand (Table 1a). Such interactions generate a two-dimensional supramolecular structure parallel to the bc plane .The lozenge planes of the two independent molecules are nearly perpendicular [angle between planes = 84.20 (3)°], giving rise to several weak C—H⋯π interactions between the d Table 1. These id Table 1a. Such ne Fig. 2b.2-quinolin-8-olato) ligands  compounds with bis(μn-butyltin(IV) oxide and solvents were purchased from Aldrich and used without further purification. Elemental analysis were performed using an Eager 300 analyzer. The infrared spectra were recorded on Perkin Elmer 1600 FT spectrometer in the 4000–400 cm−1 range. Melting points were measured on a Fisher–Johns melting-point apparatus and are uncorrected. 119Sn spectra were recorded with a Bruker AVANCE-II, 300 MHz NMR spectrometer operating at 111.81 MHz and using a 4mm CP-MAS probe. NMR 119Sn chemical shift referencing is toward tetramethyltin.3-Hydroxyquinoline-2-carboxaldehyde, 2-aminophenol, di-N-(2-hydroxyphenyl)carboximidoyl]quinolin-8-ol (II) (0.378 mmol) and di-n-butyltin oxide (0.378 mmol) were dissolved in toluene in a 100 ml flask equipped with a Dean–Stark funnel. This mixture was refluxed for 1.5 h. After refluxing, the solvent was distilled. The red crystalline product was recrystallized from a mixture of 2,3-dichlorobutane/hexane 3/1. M.p. 468–471 K. Yield 69.4% The dark-red crystalline product was characterized by elemental analysis, calculated for C24H28N2O2Sn 0.3H2O, C 57.55, H 5.61, N 5.59. Found C 57.30, H 5.32, N 5.51. RMN 119Sn solid state: −462.57 ppm. IR  3074w, 2954s, 2922s, 2867m, 2854m, 1591m, 1581m, 1523m, 1504m, 1470s, 1446m, 1429m, 1377w, 1342m, 1329s, 1308m, 1286m, 1188w, 1142m, 1135m, 1094w, 907w, 881w, 839w, 752m, 742m, 549w, 468w.Compound (I)Ueq value of the parent atom. The n-butyl groups in molecule 2 display some degree of orientational disorder, which was modeled into two orientations using geometrical  and ADP  restraints.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016018867/bg2598sup1.cifCrystal structure: contains datablock(s) I, Compound_I. DOI: 10.1107/S2056989016018867/bg2598Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016018867/bg2598Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016018867/bg2598Isup4.cdxSupporting information file. DOI: 1519148CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


PIK3CD encoding p110δ cause a human immunodeficiency known as activated PI3K delta syndrome. Currently, idelalisib is the only selective p110δ inhibitor that has been FDA approved to treat certain B cell malignancies. p110δ inhibitors can suppress autoantibody production in mouse models, but limited clinical trials in human autoimmunity have been performed with PI3K inhibitors to date. Thus, there is a need for additional tools to understand the effect of pharmacological inhibition of PI3K isoforms in lymphocytes. In this study, we tested the effects of a potent and selective p110δ inhibitor, IPI-3063, in assays of B cell function. We found that IPI-3063 potently reduced mouse B cell proliferation, survival, and plasmablast differentiation while increasing antibody class switching to IgG1, almost to the same degree as a pan-PI3K inhibitor. Similarly, IPI-3063 potently inhibited human B cell proliferation in vitro. The p110γ isoform has partially overlapping roles with p110δ in B cell development, but little is known about its role in B cell function. We found that the p110γ inhibitor AS-252424 had no significant impact on B cell responses. A novel dual p110δ/γ inhibitor, IPI-443, had comparable effects to p110δ inhibition alone. These findings show that p110δ is the dominant isoform mediating B cell responses and establish that IPI-3063 is a highly potent molecule useful for studying p110δ function in immune cells.The class I phosphoinoside-3-kinases (PI3Ks) are important enzymes that relay signals from cell surface receptors to downstream mediators driving cellular functions. Elevated PI3K signaling is found in B cell malignancies and lymphocytes of patients with autoimmune disease. The p110δ catalytic isoform of PI3K is a rational target since it is critical for B lymphocyte development, survival, activation, and differentiation. In addition, activating mutations in  Class IA PI3Ks are heterodimers composed of two subunits, a regulatory subunit (p85) and one of three catalytic subunits . The class IB PI3K is composed of unique regulatory subunits (p101 or p84) with the catalytic subunit p110γ. Whereas p110α and p110β are ubiquitously expressed, p110δ and p110γ expression is mainly restricted to leukocytes. The importance of PI3K activation in various cancers has led to development of many small molecule PI3K inhibitors targeting individual isoforms or subgroups (Phosphoinoside-3-kinase (PI3K) enzymes are lipid kinases that produce 3′-phosphorylated phosphoinositides, which act as second messengers to relay signals from cell-surface receptors to downstream mediators. The class I PI3Ks produce phosphatidylinositol-3,4,5-triphosphate (PIPPIK3CD encoding p110δ cause a human immunodeficiency known as activated PI3K delta syndrome (APDS), which is associated with chronically activated lymphocytes that undergo apoptosis or senescence . IPor p110γ . We asseTo assess survival, purified mouse B cells were incubated for 48 h in either B-cell activating factor (BAFF) or interleukin-4 (IL-4) with various concentrations of IPI-3063 and IPI-443. The results showed that both IPI-3063 and IPI-443 reduced BAFF-dependent survival in a dose-dependent manner, approaching the effect of GDC-0941 Figure . The selin vitro. In addition, selective p110γ inhibition alone had no effect on B cell survival or proliferation.Next, we evaluated the effects of PI3K inhibitors on B cell proliferation. We stimulated CFSE-stained cells with αIgM + IL-4 or with LPS for 72 h, or with αCD40 + IL-4 or LPS + IL-4 for 96 h. Cells were treated with vehicle or GDC-0941, IPI-3063, IPI-443, or AS-252424 at various concentrations. Histograms of CFSE fluorescence in the cells show that IPI-3063 blocked proliferation in α-IgM + IL-4 stimulated B cells at the lowest concentration tested (1 nM)  and decreases differentiation into plasmablasts that secrete low-affinity IgM , 19. Act1 Figure B. IPI-441 Figure B. In cel+ Figure C.+B220lo population. IPI-443 also caused a dose-dependent decrease starting at 1 nM. In cells treated with IPI-3063 or IPI-443, the highest concentrations inhibited plasmablast differentiation to the same degree as GDC-0941. Measuring IgM secretion by ELISA showed similar trends  followed by addition of ATP/diC8-PtdInsP2 mixture to give final concentrations of 3 mM ATP and 500 µM diC8-PtdInsP2. Reactions were incubated at room temperature for 2 h, with PI3K activity assessed via the Promega ADP-Glo Max assay kit (cat. no. V7002) according to the manufacturer’s instruction. Plates were read on Envision plate reader in luminescence mode.Human recombinant PI3K-α (cat. no. 14-602-K), -β (cat. no. 14-603-K), -δ (cat. no. 14-604-K), and -γ (cat. no. 14-558-K) were purchased from Millipore. Phosphatidylinositol 4,5 bis phosphate P2) was purchased from Avanti Polar Lipids, Inc. PI3K-α, β, and δ are heterodimers consisting of full length p110α, p110β, or p110δ catalytic subunit and the p85α regulatory subunit. PI3K-γ is a monomer of the p110γ catalytic subunit. Samples of kinase  were incubated with inhibitor for 30 min at room temperature in reaction buffer  for 30 min and Raw264.7 cells with 25 nM C5a (RnD Systems) for 3 min in the presence of inhibitor. SKOV3 and 786.0 cells were not stimulated. Medium was then aspirated and 50 µL/well of ice-cold lysis buffer was added. pAKT level was determined according to the manufacturer’s instruction.Phospho-Akt1 (S473) sandwich ELISA antibody kit  was utilized to analyze pAKT signal in cells as described previously by Winkler et al.  for dat. BrieflyC57BL/6 mice were bred at the University of California, Irvine, and used at between 6 and 12 weeks of age. All animals were studied in compliance with protocols approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. The p110δ-selective PI3K inhibitor IPI-3063 and p110δ/γ PI3K inhibitor IPI-443 were synthesized at Infinity Pharmaceuticals. These compounds and the pan-PI3K class I inhibitor GDC-0941 (LC laboratories) were dissolved in DMSO. The p110γ PI3K inhibitor AS-252424 (Chemdea) was dissolved in ethanol. Inhibitors were included throughout the indicated cell treatment periods.6 cells/mL. For plasmablast differentiation, B cells were stimulated with 5 µg/mL LPS (Sigma) for 72 h, and for IgG1 CSR, B cells were stimulated 5 µg/mL anti-CD40 (HM40-3) agonistic antibody (eBioscience), or 5 µg/mL LPS (Sigma), together with 5 ng/mL mIL-4 (R&D Systems) for 96 h. All B cells were cultured in RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol.Mouse splenic B cells were purified by negative selection (eBioscience Magnisort Mouse B cell enrichment kit). B-cell purity was >95% as measured by FACS analysis  using anti-B220 antibody (BioLegend). Purified B cells were seeded at a final concentration of 0.5 or 0.25 × 10Analysis was performed on western blots using ImageJ to measure mean fluorescence intensities of each band. Phospho-AKT S473 and phospho-ERK1/2 signal was normalized with actin measurements and fold change was calculated using the stimulated/no drug control.6 cells/mL with a concentration of 2.5 µM CFSE. Flow cytometric data were analyzed using FlowJo software (TreeStar).Before cell surface staining, cells were incubated with TruStain fcX in FACS buffer (0.5% BSA + 0.02% NaN3 in 1× HBSS) to block Fc receptors for 10 min on ice. Staining with antibodies was subsequently performed, also with FACS buffer and on ice for 20 min. Flow cytometry antibodies and other reagents used were as follows: B220 (RA3-6B2), IgG1 (A85-1), CD138 (281-2), and 7-Aminoactinomycin D. CFSE labeling of B cells to track proliferation was performed by resuspending cells to a concentration of 10 × 10For cell culture ELISA to measure total IgM, supernatants from purified B cells stimulated with LPS were collected after 3 days and diluted 1:1,000 in 2% (wt/vol) BSA in PBS. Nunc Maxisorp plates (Thermo Fisher) were coated with anti-mouse IgM  at 10 µg/mL in 50 µL of total sample in PBS and allowed to incubate overnight at 4°C. Diluted supernatant samples were incubated on coated plates for 1 h at 37°C. HRP-conjugated rabbit anti-mouse IgM secondary antibody (Zymed) was used.6 cells/mL and cultured with 2 µg/mL human CD40L (eBioscience) + 5 µg/mL anti-human IgM/IgG (eBioscience) + 100 µg/mL hIL-2 (R&D Systems) + 100 µg/mL hIL-21 (R&D Systems). All B cells were cultured in RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol.Peripheral blood from normal volunteers was obtained through an Institutional Review Board-approved protocol. Peripheral blood mononuclear cells (PBMCs) were first purified from blood by density gradient centrifugation using Ficoll-Paque. Human B cells were then purified from PBMCs by negative selection (eBioscience Magnisort Human B cell enrichment kit). B-cell purity was increased from 4% to >70% as measured by FACS analysis  using anti-CD19 PE conjugated antibody (eBioscience). Purified B cells were seeded at a final concentration of 0.1 × 10Live animals (mice) were studied in compliance with protocols approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.DF designed research and wrote the manuscript. HC designed and performed research and wrote the manuscript. SM, PN, AM, LJ, JD, and EB designed and performed research. DW, KM, and JK designed research and wrote the manuscript.DW, JD, EB, KM, and JK were employees and shareholders at Infinity Pharmaceuticals, Inc., at the time of these studies. DF previously served as a consultant for Infinity Pharmaceuticals. The other 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.


Type 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ.1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation.Phosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the βIn cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified. •PP2A subunit B56δ is phosphorylated on β-adrenergic stimulation of cardiomyocytes.•Phosphorylation occurs at Ser573 and increases B56δ-PP2A catalytic activity.•1-adrenoceptor subtype and protein kinase A.Response is mediated by the β•Phosphorylated B56δ abundance is increased in pathological cardiac hypertrophy. PP2A holoenzymes comprise a 65-kDa scaffold  is localized in chromosome region 6p21.1 and gives rise to three splice variants that differ at the N-terminus Little information is available regarding B56δ expression, regulation and function in the heart. Mice lacking B56δ were the first mice with targeted disruption of a B56 subunit to be studied; however, their cardiac phenotype was not studied 1AR and is mediated by PKA, and (iii) B56δ phosphorylation at S573 is necessary for βAR-mediated stimulation of PP2A activity.In view of the importance of the Gs-adenylate cyclase-cAMP-PKA pathway in mediating cardiomyocyte responses to β-adrenergic receptor (βAR) stimulation 22.1C, PLB and phospho-S/T kinase substrate antibodies were from Cell Signaling Technology . GAPDH, H2B and B56γ antibodies were from Abcam . B56α and B56δ antibodies were from BD Biosciences (610615) and Bethyl (A301-100A), respectively. PP2AA and B55α antibodies were from Santa Cruz . GFP and α-Actinin antibodies were from Roche (11814460001) and Sigma (A7732), respectively. pS16 PLB and pS282 cMyBP-C antibodies were from Badrilla (A010-12AP) and Enzo Life Sciences (ALX-215-057-R050), respectively. The cMyBP-C antibody was a kind gift from Professor Mathias Gautel 6-benzoyl cAMP (BNZ), and okadaic acid were from Calbiochem . The pEGFP-C1 vector encoding PPP2R5D and cardiac tissue from littermate B56δ knock out (KO) and WT mice were kind gifts from Professor Veerle Janssens cTnI, pS23/24 cTnI, PP2A2.2PPP2R5D in a pEGFP-C1 vector using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene). The adenoviral vectors expressing wild type (WT) B56δ (AdV.GFP-B56δ-WT) and mutated (S573A) B56δ (AdV.GFP-B56δ-S573A) were constructed using the AdEasy system To replace S573 with alanine, a single point mutation was introduced into human 2.3l-creatine, 5 mM carnitine, 5 mM taurine and 100 IU/ml penicillin/streptomycin, and were cultured in plastic 6-well plates pre-coated with laminin. Cells were maintained in a humidified incubator  for 2 h after which, the medium was replaced with fresh medium and cells were incubated overnight. Where indicated, cells were transduced with adenoviruses 2 h post-plating. AdV.GFP was used at MOI 30. AdV.GFP-B56δ-WT and AdV.GFP-B56δ-S573A were both used at MOI 100.ARVM were isolated from the hearts of adult male Wistar rats by collagenase-based enzymatic digestion, as previously described 2.4Unless otherwise stated, ARVM were incubated with vehicle or 10 nM ISO for 10 min. PRO (100 nM), CGP (100 nM), ICI (100 nM) or vehicle was added to the culture medium 10 min before ISO stimulation. H89 (10 μM), PKI (10 μM) or vehicle was added 30 min before ISO stimulation. Cells were exposed to BNZ (500 μM) or vehicle for 30 min, and to OA (0.1 or 1 μM) or vehicle for 60 min. Experiments were performed at 37 °C.2.5g for 30 min at 4 °C. Proteins in the soluble fraction (supernatant) were denatured in 3X Laemmli sample buffer. Proteins in the insoluble fraction (pellet) were resuspended in 1X Laemmli sample buffer.The subcellular fractionation method was adapted from methods described in previous publications 2.62. Prior to transfer of proteins to PVDF membranes, gels were incubated 15 min in transfer buffer containing 1 mM EDTA then 15 min in transfer buffer alone.Heat-denatured protein samples were resolved on Tris-glycine SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) and 5% (w/v) non-fat milk. Incubation with primary antibodies was performed overnight at 4 °C and incubation with secondary antibodies was performed for 2 h at room temperature. Protein bands were visualized on chemiluminescence film following incubation of the membranes with ECL Western Blotting Detection Reagents . Signal intensity was quantified on a calibrated GS-800 densitometer (Bio-Rad). Phosphate affinity (PhosTag) SDS-PAGE was performed on Tris-glycine gels containing 50 μM acrylamide-pendent PhosTag™ and 100 μM MnCl2.7ARVM were prepared for imaging on a confocal florescence microscope as previously described 2.8GFP, GFP-B56δ-WT and GFP-B56δ-S573A were immunoprecipitated from ARVM lysates using a GFP antibody. Cells were harvested in ice-cold lysis buffer containing: 50 mM Tris (pH 8), 138 mM NaCl, 27 mM KCl, 1% (v/v) Triton-X100 and complete mini protease inhibitor. The cleared lysate containing 500 μg of protein was incubated with anti-GFP (4 μg) over-night at 4 °C. Immunocomplexes were collected using Protein G beads. After three washes in lysis buffer, these were resuspended in Laemmli buffer.2.9pNPP Protein Phosphatase Assay kit (AnaSpec), as recommended by the manufacturer. Cells expressing heterologous proteins were washed in phosphate-buffered saline and harvested in 1X lysis buffer with added 1% (v/v) Triton-X100 and protease inhibitors. Master mixes of whole cell lysates in assay buffer (40 mM Tris (pH 8.4), 34 mM MgCl2, 4 mM EDTA and 4 mM DTT) were prepared on ice. Incubation with 5 nM okadaic acid or DMSO was performed for 10 min. Assays were performed in triplicates, in wells of 96-well plates. Individual wells contained 10 μg of protein in 50 μl of master mix and 50 μl of pNPP solution. Absorbance at 405 nm was determined in a plate reader pre-heated to 30 °C. Measurements were recorded at 5 min intervals, for a total of 30 min. The amount of pNP produced was determined using the equation c = A ÷ (ɛ × l), where c is the concentration of pNP, A is the optical absorbance of pNP at 405 nm, ɛ is the molar extinction coefficient of pNP  and l is the path length.Protein phosphatase activity was determined using the SensoLyte 2.102 for subsequent analysis.Transverse aortic constriction (TAC) surgery was performed in adult mice by Professor Brian Cooley . Male C57Bl/6 mice at 9 weeks of age were anaesthetised by intraperitoneal injection of 100 mg/kg ketamine and 15 mg/ml xylazine. Constriction of the aortic arch was achieved by ligation of the aorta with 6-0 silk, using a 27.5-gauge spacer. Sham animals were subjected to the same surgical procedure, with no constrictive suture. Hearts were harvested 7 days after the surgical procedure and frozen in liquid N2.11t-test, or One-Way ANOVA followed by Tukey's multiple comparisons test. P < 0.05 was considered significant. In figure legends, n indicates the number of independent experiments.Quantitative data are presented as mean ± SEM. Differences between groups were analysed by unpaired 33.119 mouse heart .To explore the effect of βAR stimulation on B56δ phosphorylation in cardiomyocytes, cell lysates from unstimulated and ISO-stimulated ARVM were resolved by standard- and PhosTag SDS-PAGE. Immunoblot analysis was performed with a polyclonal antibody raised against a peptide sequence at the C-terminus (aa 552-602) of human B56δ, which is conserved in the rat and mouse protein A. When p3.21 or β2ARs, respectively. ISO-induced B56δ phosphorylation at S573 was abolished by CGP and partially attenuated by ICI (1ARs 6-benzoyl-cAMP (BNZ) We next investigated the signaling mechanisms that underlie ISO-induced B56δ phosphorylation at S573. The response was blocked by propranolol, a non-selective βAR antagonist, confirming a βAR-mediated mechanism A. To detd by ICI A. ReflecICI 1ARs A. Guided3.3We have shown previously that ISO stimulation in ARVM induces a change in the subcellular distribution of a different B56 isoform, B56α 3.4To study the functional importance of B56δ phosphorylation at S573, we constructed adenoviral vectors for heterologous expression of GFP-tagged WT or non-phosphorylatable (S573A) B56δ  in ARVM. Control cells were transduced with an adenovirus that expresses GFP alone (AdV.GFP). Comparable GFP protein expression was achieved following 18 h incubation with AdV.GFP at multiplicity of infection (MOI) 30 and AdV. GFP-B56δ-WT or AdV.GFP-B56δ-S573A at MOI 100 . Immunoblot analysis of the same samples with the B56δ antibody confirmed comparable expression of GFP-B56δ-WT and GFP-B56δ-S573A, and showed that, relative to cells expressing GFP alone, expression of endogenous B56δ was unchanged . To further characterize the adenoviral vectors, we determined protein levels of endogenous PP2A subunits in transduced cells. Expression of GFP-B56δ-WT and GFP-B56δ-S573A was associated with increased expression of C subunits, but no change in the expression of A subunits. Expression of other B subunit isoforms  was also unchanged A. To detS/T) residues in the RRXS/T motif. This antibody detected basal S/T phosphorylation in GFP-B56δ-WT and the signal was markedly increased following ISO stimulation . In contrast, the antibody did not detect GFP-B56δ-S573A in the absence or presence of ISO stimulation , indicating that in ARVM S573 is the only B56δ site that is phosphorylated.In the HEK293 cell line, PKA activation has been reported to induce B56δ phosphorylation not just at S573 but also at S88, albeit to a much smaller extent To explore whether ablation of the S573 phosphorylation site impacts on B56δ subcellular localization, we determined the distribution of GFP-B56δ-WT and GFP-B56δ-S573A in soluble and insoluble compartments of fractionated cells. As in earlier experiments B, the puStudies in vitro and in non-cardiac cells have shown that B56δ phosphorylation at S573 increases the activity of its associated C subunit 3.5S/T motif detected phosphorylation of GFP-B56δ-WT but not GFP-B56δ-S573A , confirming the ability of the method to reveal differential phosphorylation of S/T residues in abundantly expressed proteins. In complementary experiments, we determined the phosphorylation status of cTnI, cardiac myosin binding protein-C (cMyBP-C) and phospholamban (PLB), which are phosphorylated by PKA following βAR stimulation and play important roles in mediating inotropic and lusitropic responses. PhosTag SDS/PAGE-immunoblot analysis of cTnI showed that the abundance of non-phosphorylated (0P), mono-phosphorylated (1P) and bis-phosphorylated (2P) cTnI was comparable in cells expressing GFP-B56δ-WT and those expressing GFP-B56δ-S573A, in the absence or presence of ISO stimulation  that selectively inhibits PP2A 3.6Finally, to explore whether the phosphorylation and/or the expression of cardiac B56δ is altered in a pathophysiological setting, we examined these in a mouse model of pressure overload-induced cardiac hypertrophy. Seven days post-surgery, heart weight/tibia length was 10.1 ± 0.8 mg/mm in mice subjected to TAC and 7.7 ± 0.2 mg/mm in mice subjected to sham operation , reflecting cardiac hypertrophy in the former. The abundance of both pS573 B56δ and total B56δ was increased in the hearts of mice subjected to TAC relative to mice subjected to sham operation . These f41AR and activation of PKA, and (iii) βAR/PKA-mediated phosphorylation of B56δ at S573 increases PP2A activity.The present study has revealed for the first time in a cardiac context that (i) acute simulation of βARs induces a rapid and sustained phosphorylation of the PP2A regulatory subunit B56δ selectively at S573, (ii) this phosphorylation occurs principally through stimulation of the β1AR-mediated activation of protein kinases 1AR-mediated regulation of Ser/Thr phosphatases that serve an opposing role is restricted largely to type 1 phosphatase (PP1), whose activity is regulated through PKA-mediated phosphorylation of the PP1-specific inhibitor of protein phosphatase-1 (I-1) In healthy myocardium, catecholamines regulate a variety of cellular processes primarily by β1AR antagonist) and ICI (a selective β2AR antagonist) indicate that the ISO response is mediated primarily by the β1AR subtype (1AR is comparable to that of ICI for the β2AR 1AR) 1AR signaling by the former antagonist. On the basis of this observation and evidence that in ARVM the relative β1AR:β2AR abundance is 65:35 2AR activation and that its relative ineffectiveness in inhibiting ISO-induced B56δ phosphorylation arose from a minor role for this βAR subtype in mediating that response.Here, we provide robust new evidence that in ARVM the non-selective βAR agonist ISO induces phosphorylation of the PP2A regulatory subunit isoform B56δ selectively at S573, in a rapid and sustained manner . Our dat subtype A. In celThe data that we have obtained from complementary studies, in which we explored the role of PKA in mediating B56δ phosphorylation at S573, indicate that activation of this kinase is both necessary for the ISO-induced response and sufficient per se to induce such phosphorylation. Thus, ISO-induced B56δ phosphorylation was significantly attenuated by pre-treatment of ARVM with either H89 or PKI, which inhibit PKA through distinct mechanisms We have shown previously that βAR stimulation by ISO in ARVM alters the subcellular localisation of another PP2A regulatory subunit isoform, B56α C protein that accompanied the heterologous expression of GFP-tagged B56δ in either form  revealed the presence of PP2AA and PP2AC proteins in comparable abundance (C subunits) was increased in ARVM expressing GFP-B56δ-WT but not those expressing GFP-B56δ-S573A physiological role(s) of B56δ phosphorylation, our data from an exploratory analysis of mouse hearts with pressure overload-induced cardiac hypertrophy show that, while the relative phosphorylation of B56δ was unaltered, the absolute abundance of pS573 B56δ was increased significantly in that setting. This change may be a compensatory response to pressure overload-induced cardiac hypertrophy, or play a mechanistic role in its pathogenesis. Notwithstanding, the dynamic regulation of B56δ phosphorylation and expression in cardiac hypertrophy warrants investigation of the functional consequences of their targeted manipulation in appropriate models. In that context, it is interesting to note also that B56δ expression has been reported to be increased in dog hearts following myocardial infarction and in the setting of tachypacing-induced non-ischemic heart failure In conclusion, the present study has identified and characterised a novel mechanism that is likely to regulate a specific pool of PP2A activity in cardiomyocytes in response to βAR stimulation, through PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573. The functional importance of this novel mechanism now requires the establishment of a model system that allows replacement of endogenous B56δ with a non-phosphorylatable (S573A) mutant (e.g. a knock-in mouse) and the application of broad-spectrum phosphoproteomics analyses, as well the examination of cardiac responses to (patho)physiological stimuli, in such a model.None.


Z,5Z)-5-(2-methoxy-2-oxoethylidene)-2-{(E)-[2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enylidene]hydrazinylidene}-4-oxothiazolidin-3-yl)fumarate displays a conformational disorder which inverts the configuration of the chiral C atom within the cyclohexylidene ring.The crystal structure of the title compound, dimethyl 2-((2 22H27N3O7S, were determined from the synthetic pathway and by X-ray analysis. This compound is a new 4-thiazolidinone derivative prepared and isolated as pure product from thiosemicarbazone carvone. The molecule is built up from an oxothiazolidine ring tetrasubstituted by a methoxy–oxoethylidene, a maleate, an oxygen and a cyclohexylidene–hydrazone. The cyclohexylidene ring is statistically disordered over two positions, resulting in an inversion of configuration for the substituted carbon.The crystal structure and the conformation of the title compound, C The resulting product 2In recent years, the synthesis of heterocyclic systems containing nitrogen and sulfur has attracted great interest because of their broad spectrum of pharmacological activities. The thiazol nucleus is found in a large number of natural products -5-(2-methoxy-2-oxoethylidene)-2-{(E)-[2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enylidene]hydrazinylidene}-4-oxothiazolidin-3-yl)fumarate.The structure of A = S1/C2/N3/C4/C5/N2/C7/O4/C10) are roughly coplanar with the largest deviation from the mean plane being 0.085 (2) Å for C10. The butadiene fragment (C1′/C2′/C3′/C4′A/C4′B) of the cyclohexylidene ring is twisted slightly with respect to this plane, making a dihedral angle of 8.3 (2)°. The methoxycarbonyl group (C11/O11/O12/C12) is also twisted slightly with respect to plane A, with a dihedral angle of 8.2 (2)°. The methoxycarbonyl groups (C6/O61/O62/C14 and C9/O91/O92/C13) of the fumarate group make dihedral angles of 70.06 (7) and 75.59 (9)°, respectively, with the thiazolidine ring.The title molecule is built up from an oxothiazolidine ring tetrasubstituted by a methoxy-oxoethylidene, a fumarate, an oxygen and a cyclohexylidene-hydrazone Fig. 1. As expeR C5′A and S C5′B) and so the crystal might be considered as a racemate. Could the crystal be considered as a co-crystal built up from the combination of R and S configurations? It is difficult to answer this question.The most striking feature of this structure is the conformational statistical disorder which affects the cyclohexylidene ring: atoms C6′ and C5′ are split over two positions, each of half occupancy, with respect to the mean plane of the butadiene (C1′–C4′) fragment . Such disorder inverts the configuration at C5 -thiosemicarbazone carvone 1 and dimethyl acetylenedicarboxylate (1.25 eq) in anhydrous MeCN (50 mL), was heated under reflux for 30 min. After the completion of the reaction (the progress of the reaction was monitored by TLC), the solvent was evaporated to dryness. The crude product was purified by silica gel chromatography (230–400 mesh) using hexane/ethyl acetate (95:5) as eluent. The pure thiazolic product 2 was obtained in 65% yield. Slow evaporation from an ethanolic solution of the title compound gave crystals of 2 suitable for crystallographic analysis.A solution of  = 1.5Ueq(C) for methyl H atoms and 1.2Ueq for all other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017001190/xu5897sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017001190/xu5897Isup2.hklStructure factors: contains datablock(s) I. DOI: 1529291CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


To the Editor:Borrelia miyamotoi causes a recently recognized tickborne zoonosis in Eurasia and North America  were sufficient to identify the I. pacificus–borne spirochete as a sister taxon to B. miyamotoi from elsewhere , except for these modifications: clpA (53°C); clpX forward 5′-CCGTTGCTATTTGTTTTGAATGCTCT-3′ (55°C); pepX forward 5′-TTAAAACTTGATGATAAATGGTCATTA-3′ and reverse 5′-TTAAAACTTGATGATAAATGGTCATTA-3′ (52°C); pyrG forward 5′-CTTTTAGTAATTGAGATTGGTGGT-3′ and reverse 5′-CAGCATCAAGTATTCCACAAAC-3′ (55°C); recG forward 5′-CTAGCATTCCTTTAGTTGAGGC-3′ and reverse 5′-TTSTGTTAAAGGTTCCTTATAAAG-3′ (52°C); rplB forward 5′-ATTAAAACTTATAGGCCAAAAAC-3′ and reverse 5′-GGCTGACCCCAAGGAGAT-3′ (55°C); and uvrA forward 5′-GCTTAAATTTTTAATTGATGTTGGA-3′ and reverse 5′-CAAGGAACAAAAATRTCAGGC-3′ (52°C). On a Bio-Rad T100 thermal cycler  and with Apex Master mix , PCR extension at 72°C was 1.5 min for clpX and 1.0 min for others; final elongation was for 5 min at 72°C. Products were sequenced over both strands at GENEWIZ  by the Sanger method either directly or after cloning into a plasmid vector. Resultant sequences were aligned with homologous sequences (http://doua.prabi.fr/software/seaview). The equal length MLST sequences, as specified  and identical to the IGS of other B. miyamotoi in I. pacificus ticks . As observed previously (Ixodes species.We determined a rooted neighbor-joining phylogram of 16S ribosomal RNA gene sequences of onestari , panel A strains , panel BB. miyamotoi in I. pacificus ticks and organism strains associated with other Ixodes species. However, we found a close phylogenetic relationship between organisms from the far-western and the northeastern United States.In conclusion, we identified differences in several genetic loci between


II ion in the title compound shows a distorted square-pyramidal coordination geometry with two N and one O atoms of the mpmapOH ligand and two Cl anions. In the crystal, molecules are connected by hydrogen bonds and π–π interactions, forming a strong supramolecular network along the a-axis direction.The Cu 2(C10H16N2O)], has been synthesized and characterized by synchrotron single-crystal X-ray diffraction and FT–IR spectroscopy. The 2-methyl-2-[(pyridin-2-ylmethyl)amino]propan-1-ol (mpmapOH) ligand, including pyridine, amine and hydroxy groups, was synthesized by the reaction of 2-amino-2-methylpropan-1-ol with pyridine-2-carbaldehyde and was characterized by NMR spectroscopy. In its CuII complex, the metal ion has a distorted square-pyramidal coordination geometry with two N and one O atom of the mpmapOH ligand and one chloride anion in the equatorial plane, and the second chloride in an axial position. The bond lengths involving the CuII ion range from 1.9881 (10) to 2.0409 (9) for the Cu—N and Cu—O bonds, and from 2.2448 (5) to 2.5014 (6) Å for the equatorial and axial Cu—Cl bonds, respectively. Intermolecular hydrogen bonds (N—H⋯Cl and O—H⋯Cl) and face-to-face π–π interactions stabilize the molecular structure and give rise to a two-dimensional supramolecular structure extending parallel to (101).The title compound, [CuCl The Cu—Cl bond lengths are 2.2448 (5), and 2.5014 (6) Å, respectively, with the larger value corresponding to the axial chloride ligand. The equatorial atom Cl1 lies 0.332 (1) Å above the equatorial plane, away from the axial chloride anion Cl2. The bite angles of the five-membered chelate rings involving C5, C6 and C7, C10 atoms are 82.92 (4) and 82.97 (4)°, respectively. The bond angles around the copper ion range from 82.92 (4) to 161.51 (4)°.In the title compound (I) Fig. 1, the copb axis  was prepared as follows. 2-Amino-2-methyl-1-propanol  was dissolved in MeOH (30 mL) followed by the addition of 2-pyridinecarboxaldehyde  under a nitrogen atmosphere. The resulting mixture was strirred at room temperature for three hours, and then NaBH4  was added slowly. The mixture was again stirred at room temperature overnight. The yellow solution was evaporated to dryness under reduced pressure. The residue was dissolved in CH2Cl2 and the undissolved solids were filtered off. The solution was washed with H2O and dried over MgSO4. After removal of the drying agent and solvent, the mpmapOH ligand was obtained as a yellow oil. Yield: 6.67 g (74%). 1H NMR : δ = 0.98 2–CH2), 3.22 2–2CH–OH), 3.75 , 7.21 , 7.42 , 7.71 , 8.45 . To an MeOH solution (10 mL) of CuCl2·H2O  was added dropwise an MeOH solution (10 mL) of mpmapOH ; the color became dark blue, and the solution was stirred for 30 min at room temperature. Blue crystals of (I) were obtained by diffusion of diethyl ether into the dark-blue solution for several days, and were collected by filtration and washed with diethyl ether and dried in air. Yield: 247 mg (67%). FT–IR : 3217, 3172, 3072, 2968, 1609, 1569, 1444, 1382, 1280, 1165, 1044, 984.The title compound (Uiso(H) were set to 1.2 or 1.5Ueq of their respective parent atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016013773/zl2674sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016013773/zl2674Isup2.hklStructure factors: contains datablock(s) I. DOI: 1501276CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Assembled structures of dyes have great influence on their coloring function. For example, metal ions added in the dyeing process are known to prevent fading of color. Thus, we have investigated the influence of an addition of copper(II) ion on the surface structure of alkyl-derivatized indigo. Scanning tunneling microscope (STM) analysis revealed that the copper(II) complexes of indigo formed orderly lamellar structures on a HOPG substrate. These lamellar structures of the complexes are found to be more stable than those of alkyl-derivatized indigos alone. Furthermore, 2D chirality was observed. Dyes are colored substances capable of absorbing certain wavelengths of light intensively. Many of the dyes have a large π-conjugated system, and the color of dyes is highly influenced by the electronic state of the system. In consequence, assembled structural changes often induce the change in the electronic state of the π-conjugated system and result in color variations. Color changes via polymorphism, phase transition, etc. have been reported elsewhere ,2,3. ThunIND, n = alkyl chains length) shown in nIND formed characteristic monolayer structures on HOPG [nIND)2], the copper(II) complex of CnIND, shown in nIND)2] using STM. This investigation was carried out to understand the influence of the addition of metal ions, which often stabilizes the adsorption of dyes on the substrate. This phenomenon is known as mordanting. Mordanting [nIND and those of [Cu(CnIND)2], the mechanism of stabilization via mordanting may be found. In addition, [Cu(CnIND)2] exhibited a unique structure exhibiting 2D and 3D chirality, which is described later.Indigo dye is an organic dye exhibiting a distinct blue color, used for dyeing jeans blue. The indigo molecule has a planar structure due to the presence of a π-conjugated system and thus tends to assemble on a HOPG or metallic substrate. In our previous study, the surface structures of alkyl-derivatized indigo In the previous study, the self-assembled structures on HOPG of C16IND were analyzed via STM . C16IND 2] formed an orderly structure, and the dislocation was not observed observed . The ord“2D chirality” of the adsorbed molecules on the substrate has been intensively studied ,19,20. W2] on the HOPG surface exhibits distinguishable 2D chirality, as shown in [Cu(C16IND)2] and [Cu(C20IND)2] with longer alkyl chains than [Cu(C16IND)2]. As a result, [Cu(C18IND)2] and [Cu(C20IND)2] molecules were found to form basically similar structures to [Cu(C16IND)2]. These molecules formed a similar lamellar structure, and alkyl chains were interdigitated as shown in 2] seem to be aligned along the HOPG substrate lattice, suggesting that the surface structure matches well with the HOPG lattice and becomes stable.Alkyl chains lengths are known to influence the self-assembled structures of alkyl-derivatives ,24,25,262] also exhibited a different surface structure 2] appears as shown in [Cu(C20IND)software  2] induced a structural difference.Compared with the type 1 structure of  was obtained as blue crystals in ca. 25% yield. Elemental analysis, Calc: C, 74.28; H, 7.99; N, 5.41%, Found: C, 73.92; H, 8.29; N, 5.35%; IR (KBr): ν 1694 cm−1 (C=C), 1702 (C=O), 2848–2950 (C-H); UV-vis λmax (ε): 299 nm (5.7 × 104 L·mol−1·cm−1), 350 nm (2.1 × 104 L·mol−1·cm−1), 651 nm (2.1 × 104 L·mol−1·cm−1), 752 nm .us study . A total2] and [Cu(C20IND)2] with the same scheme as that with which [Cu(C16IND)2] was synthesized described above, using C18IND and C20IND respectively instead of C16IND. [Cu(C18IND)2]: Elemental analysis, Calc: C, 74.86; H, 8.31; N, 5.14%, Found: C, 74.84; H, 8.17; N, 5.39%; IR (KBr): ν 1695 cm−1 (C=C), 1702 (C=O), 2847–2960 (C-H); UV-vis λmax (ε): 299 nm (5.5 × 104 L·mol−1·cm−1), 348 nm (2.1 × 104 L·mol−1·cm−1), 650 nm (2.2 × 104 L·mol−1·cm−1), 752 nm . [Cu(C20IND)2]: Elemental analysis, Calc: C, 75.39; H, 8.61; N, 4.88%, Found: C, 75.58; H, 8.74; N, 4.71%; IR (KBr): ν 1695 cm−1 (C=C), 1702 (C=O), 2847–2948 (C-H); UV-vis λmax (ε): 299 nm (5.7 × 104 L·mol−1·cm−1), 349 nm (2.2 × 104 L·mol−1·cm−1), 650 nm (2.2 × 104 L·mol−1·cm−1), 752 nm .We synthesized [Cu(C18IND)2], [Cu(C18IND)2], and [Cu(C20IND)2] on HOPG were observed via STM . STM scans were performed at the o-dichlorobenzene/HOPG interface, at room temperature and under ambient pressure. STM tips were prepared via electrochemical etching from Tungsten wire . The details of the measuring conditions are as follows: each solution of [Cu(C16IND)2], [Cu(C18IND)2], and [Cu(C20IND)2] in o-dichlorobenzene was prepared in a concentration of 1.0 mmol·L−1. A drop of this o-dichlorobenzene solution was applied onto a freshly cleaved surface of HOPG . Then, STM scans were performed at the solution/HOPG interface in constant current mode. The STM images were corrected and analyzed via WSxM 5.0 software [Surface structures formed by [Cu(C16IND)software  (NanotecnIND)2]). STM images revealed that the assembled structures of the complexes were more stable than those of alkyl-derivatized indigos alone, suggesting the effect of mordanting. [Cu(CnIND)2] formed orderly lamellar structures on a HOPG substrate, and their alkyl chains were interdigitated. In the STM study of [Cu(C16IND)2], 2D chirality was observed; λ-structure and δ-structure were distinguished by molecular orientation. In the STM study of [Cu(C20IND)2], two types of structures with different lengths of b-axis were observed. The difference between these structures was found to derive from the 3D chirality of [Cu(C20IND)2]. In summary, [Cu(CnIND)2] formed orderly surface structures involving 2D chirality, and these surface structures were altered by 3D chirality.We investigated the two-dimensional self-assembled structures formed by copper(II) complexes of alkyl-derivatized indigo ([Cu(C


II cation is coordinated by three N atoms of three different 1,3,5-tris(imidazol-1-ylmethyl)benzene ligands, one O atom of an ethane-1,2-diol molecule, by a sulfate anion and a water molecule, forming a distorted octahedral NiN3O3 coordination geometry. The tripodal 1,3,5-tris(imidazol-1-ylmethyl)benzene ligands link the NiII cations to generate a metal–organic chain running along [100].The Ni II coordination polymer of 1,3,5-tris(imidazol-1-ylmethyl)benzene, namely catena-polynickel(II)] ethane-1,2-diol monosolvate monohydrate], {[Ni(SO4)(C18H18N6)(C2H6O2)0.5(H2O)]·C2H6O2·H2O}n, was synthesized and characterized by elemental analysis, IR spectroscopy and single-crystal X-ray diffraction. The NiII cation is coordinated by three N atoms of three different 1,3,5-tris(imidazol-1-ylmethyl)benzene ligands, one O atom of an ethane-1,2-diol molecule, by a sulfate anion and a water molecule, forming a distorted octahedral NiN3O3 coordination geometry. The tripodal 1,3,5-tris(imidazol-1-ylmethyl)benzene ligands link the NiII cations, generating metal–organic chains running along the [100] direction. Adjacent chains are further connected by O—H⋯O hydrogen bonds, resulting in a two-dimensional supermolecular architecture running parallel to the (001) plane. Another water molecule and a second ethane-1,2-diol molecule are non-coordinating and are linked to the coordinating sulfate ions via O—H⋯O hydrogen bonds.A new one-dimensional Ni In the present work, we carried out the solvothermal reaction between NiSOPII ion, one sulfate anion, one timb ligand, half a coordinating ethane-1,2-diol molecule, one coordinating water molecules, one additional lattice water molecule and one non-coordinating ethane-1,2-diol solvent molecule. As shown in Fig. 1II cation exhibits an irregular octahedral NiN3O3 coordination geometry and is coordinated by three N atoms  from three different tripodal timb ligands and three O atoms  from a coordinating water molecule, a sulfate anion and a coordinating ethane-1,2-diol molecule, respectively (H2O)2(SO4)]·EtOH·H2O}n (ClO4)2 -1,3,5-triazine; Ni—N = 2.097 (5)–2.151 (4) Å; Wan et al., 2004The title complex (I)II atom is coordinated to three individual timb ligands and each timb ligand in turn connects three nickel(II) atoms to generate an infinite laddered chain along the [010] direction , as observed in the Ni compound reported by Xu et al. (2009i), 8.7577 (4) Å (Ni1⋯Ni1ii) and 11.7296 (6) Å (Ni1i⋯Niii)  and one 24-membered macrocyclic ring (B) exist in the above-mentioned chain 2(SO4)]·EtOH·(H2O)}n in which A and B are 24-membered macrocyclic rings , giving rise to a two-dimensional supermolecular structure running parallel to (001) plane , 1,3,5-tris(imidazol-1-ylmethyl)benzene (0.1 mmol), water (6 ml) and ethane-1,2-diol (2 ml) were mixed and placed in a thick Pyrex tube, which was sealed and heated to 413 K for 72 h. After cooling to room temperature, blue block-shaped crystals  suitable for X-ray analysis were obtained. Elemental analysis calculated for C21H31N6NiO9S: C 41.86, H 5.15, N 13.95%; found: C 41.90, H 5.12, N 13.86%. IR : 3378 (s), 1612 (m), 1521 (s), 1445 (m), 1400 (w), 1283 (w), 1234 (m), 1119 (s), 1055 (s), 963 (w), 830 (m), 750 (s), 661 (s), 637 (m).NiSOUiso(H) = 1.2Ueq(C). O-bound H atoms of the water and ethane-1,2-diol molecules were either located in difference Fourier maps or placed in calculated positions so as to form a reasonable hydrogen-bonding network, as far as possible. Initially, their positions were refined with tight restraints on the O—H and H⋯H distances  in order to ensure a reasonable geometry. They were then constrained to ride on their parent O atoms, with Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017000470/zl2688sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017000470/zl2688Isup2.hklStructure factors: contains datablock(s) I. DOI: 1470095CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: