Unsymmetry effect of hexa-<i>peri</i>-hexabenzocoronenes on columnar mesophase behaviour

<p>To address the effects of molecular symmetry on the phase of columnar liquid crystals, a series of unsymmetry (<i>C₁</i>) and symmetry (<i>D_3h</i>) hexa-<i>peri</i>-hexabenzocoronene derivatives was prepared. Compounds <b>3</b> and <b>4</b>, as well as a mixture of compounds <b>3</b>+<b>4</b>, all had a significantly lower melting temperature and clearing temperature than high-symmetry compounds (<i>D_6h</i>), and all results show a strong dependence on molecular symmetry.</p

Reactive Ground-State Pathways Are Not Ubiquitous in Red/Green Cyanobacteriochromes

Recent characterization of the red/green cyanobacteriochrome (CBCR) NpR6012g4 revealed a high quantum yield for its forward photoreaction [J. Am. Chem. Soc. 2012, 134, 130−133] that was ascribed to the activity of hidden, productive ground-state intermediates. The dynamics of the pathways involving these ground-state intermediates was resolved with femtosecond dispersed pump–dump–probe spectroscopy, the first such study reported for any CBCR. To address the ubiquity of such second-chance initiation dynamics (SCID) in CBCRs, we examined the closely related red/green CBCR NpF2164g6 from <i>Nostoc punctiforme</i>. Both NpF2164g6 and NpR6012g4 use phycocyanobilin as the chromophore precursor and exhibit similar excited-state dynamics. However, NpF2164g6 exhibits a lower quantum yield of 32% for the generation of the isomerized Lumi-R primary photoproduct, compared to 40% for NpR6012g4. This difference arises from significantly different ground-state dynamics between the two proteins, with the SCID mechanism deactivated in NpF2164g6. We present an integrated inhomogeneous target model that self-consistently fits the pump–probe and pump–dump–probe signals for both forward and reverse photoreactions in both proteins. This work demonstrates that reactive ground-state intermediates are not ubiquitous phenomena in CBCRs

Synaptotagmin I Regulates Patterned Spontaneous Activity in the Developing Rat Retina via Calcium Binding to the C2AB Domains

<div><h3>Background</h3><p>In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons (starburst amacrine cells, SACs) that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca²⁺-binding domains (C2A and C2B) which serve as Ca²⁺ sensors in neurotransmitter release. However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca²⁺ binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics.</p> <h3>Methodology/Principal Findings</h3><p>Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca²⁺ sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca²⁺-binding ability in C2A or C2B in SACs. Subsequent live Ca²⁺ imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca²⁺ transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca²⁺ transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca²⁺ transients.</p> <h3>Conclusions/Significance</h3><p>Through Ca²⁺ binding to C2A or C2B, the Ca²⁺ sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.</p> </div

Optically Guided Photoactivity: Coordinating Tautomerization, Photoisomerization, Inhomogeneity, and Reactive Intermediates within the RcaE Cyanobacteriochrome

The RcaE cyanobacteriochrome uses a linear tetrapyrrole chromophore to sense the ratio of green and red light to enable the <i>Fremyella diplosiphon</i> cyanobacterium to control the expression of the photosynthetic infrastructure for efficient utilization of incident light. The femtosecond photodynamics of the embedded phycocyanobilin chromophore within RcaE were characterized with dispersed femtosecond pump–dump–probe spectroscopy, which resolved a complex interplay of excited-state proton transfer, photoisomerization, multilayered inhomogeneity, and reactive intermediates. These reactions were integrated within a central model that incorporated a rapid (200 fs) excited-state Le Châtelier redistribution between parallel evolving populations ascribed to different tautomers. Three photoproducts were resolved and originates from four independent subpopulations, each with different dump-induced behavior: Lumi-G_o was depleted, Lumi-G_r was unaffected, and Lumi-G_f was enhanced. This suggests that RcaE may be engineered to act either as an <i>in vivo</i> fluorescent probe (after single-pump excitation) or as an <i>in vivo</i> optogenetic sample (after pump and dump excitation)

Comparison of wave characteristics following transfection.

<p>Retinal waves were measured from whole-mount retinal explant cultures after transfection with the DNA plasmid (pmGluR2-IRES2EGFP). Intact control was P0–P2 retinas in the same culture condition (DIV 3–4) without transfection. No significant differences were found among all groups. For wave frequency, <i>p</i> = 0.26 (Kruskal-Wallis method with Dunn <i>post-hoc</i> test); for wave duration, <i>p</i> = 0.58 (One-way ANOVA with Student-Newman <i>post-hoc</i> test); for wave amplitude, <i>p</i> = 0.87 (One-way ANOVA with Student-Newman <i>post-hoc</i> test).</p

Ca²⁺ transient frequency is reduced by weakened Ca²⁺ binding to the C2AB domains of Syt I.

<p>A. <i>Left,</i> The expression pattern of pmGluR2-IRES2EGFP in transfected whole-amount retinas. <i>Right,</i> The RGC layer labeled with the Ca²⁺ indicator fura-2 to measure the wave-associated Ca²⁺ transients after transfection. Scale bar for both panels, 25 µm. B. Spontaneous, correlated Ca²⁺ transients were monitored from randomly selected cells in the RGC layer. The example traces of fluorescent changes over time showed the Ca²⁺ transients in the nearby cells from one imaged region. Retinas were transfected with pmGluR2-IRES2EGFP (Control), pmGluR2-IRES2EGFP-Syt I (Syt I), pmGluR2-IRES2EGFP-Syt I-D230S (Syt I-C2A*), or pmGluR2-IRES2EGFP-Syt I-D363N (Syt I-C2B*). C. Summary of Ca²⁺ transient frequency after transfection. Data were from 23–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (**<i>p</i><0.01; ***<i>p</i><0.001; One-Way ANOVA with <i>post</i>-<i>hoc</i> Student-Newman-Keuls test.) D. Distributions of cumulative probability for Ca²⁺ transient frequency from individual cells. Data were from 1196–1489 cells out of the same data sets in C. E. Summary of fraction of cells with Ca²⁺ transients in one imaged region after transfection. Data were from 32–58 transfected regions out of the same data sets in C. (**<i>p</i><0.01; ***<i>p</i><0.001; Kruskal-Wallis method followed by <i>post</i>-<i>hoc</i> Dunn test.).</p

Syt I is strongly expressed in neonatal SACs and IPL.

<p>Ai–Aiii. Merged images of Syt I (green) and ChAT (red) staining in retinal cross-sections from P0–P2 rats. The colocalization of Syt I and ChAT labeling was found in the IPL (yellow). Scale bars, 25 µm. ChAT, choline acetyltransferase. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bi–Biii. Immunofluorescence labeling of Syt I (green) in the IPL of retinal cross-sections from P0–P2 rats. Ci–Ciii. Immunofluorescence staining of ChAT (red) labeling SACs and the IPL in retinal cross-sections from P0–P2 rats. Di–Diii. Merged images of Syt I and ChAT staining in the same retinal cross-sections. The Syt I and ChAT labeling was colocalized to the SAC somata (yellow) as indicated by arrows. Scale bars for B-D, 50 µm. Ei–Eiii. The high magnification for merged images of Syt I and ChAT staining in the boxes of Di–Diii. Scale bars for E, 5 µm.</p

Gene transfer into whole-mount retinas by the homemade electroporation device.

<p>A–B. Preparation of platinum (Pt) electrodes. Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15×15 mm with one extra 5×5 mm-overhanging square) was aligned to one side of the 7^th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8^th) slide was glued onto the (+) electrode base. Bi–ii. The arrangement of the (−) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) ×12 (width) ×3 (height) mm] on the (+) electrode. The (−) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (−) electrodes was adjusted by a micromanipulator that held the (−) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or α-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ∼65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments.</p

Pairwise correlation is not altered by weakened Ca²⁺ binding to the C2AB domain of Syt I.

<p>A. Representative pairwise cross-correlograms of 49 cells in one imaged region from the transfected retinas. Each event was a Ca²⁺ transient in an individual cell. Delay times were the time differences between Ca²⁺ transient peaks in cell pairs. Note that all correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were very similar with sharp peaks at t = 0 sec (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047465#pone-0047465-t002" target="_blank">Table 2</a> for statistics). The oscillatory behavior of the correlograms beyond | t | > ∼7.5 sec might reflect the bursting behavior of the distant cells. Bin widths for all correlograms were 1 sec. B. Pairwise C.I. values as a function of intercellular distance for control, Syt I, Syt I-C2A* and Syt I-C2B*. Data points were averages of medians within the 50 µm bins from 17–23 transfected retinas (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). The error bars were the standard errors of the C.I. values in each bin.</p

Comparison of wave correlograms for Syt I and its C2AB mutants.

<p>Retinal waves were measured from cultured whole-mount retinas expressing Syt I or its mutants (Syt I-C2A* and Syt I-C2B*) with the mGluR2 promoter. Control was transfected with pmGluR2-IRES2EGFP. Wave correlograms were constructed from 49 cell pairs in one imaged region and fitted to a Gaussian equation to yield the Gaussian mean and standard deviation (SD). No significant differences were found in Gaussian mean (0.00±0.00 sec) among all groups. For Gaussian SD, <i>p</i> = 0.6225 (One-Way ANOVA with Student-Newman <i>post-hoc</i> test).</p