Quantitative evaluation of temporal partial coherence using 3D Fourier transforms of through-focus TEM images

AbstractWe evaluate the temporal partial coherence of transmission electron microscopy (TEM) using the three-dimensional (3D) Fourier transform (FT) of through-focus images. Young's fringe method often indicates the unexpected high-frequency information due to non-linear imaging terms. We have already used the 3D FT of axial (non-tilted) through-focus images to reduce the effect of non-linear terms on the linear imaging term, and demonstrated the improvement of monochromated lower-voltage TEM performance [Kimoto et al., Ultramicroscopy 121 (2012) 31–39]. Here we apply the 3D FT method with intentionally tilted incidence to normalize various factors associated with a TEM specimen and an imaging device. The temporal partial coherence of two microscopes operated at 30, 60 and 80kV is evaluated. Our method is applicable to such cases where the non-linear terms become more significant in lower acceleration voltage or aberration-corrected high spatial resolution TEM

Detection of Arc/Arg3.1 oligomers in rat brain: constitutive and synaptic activity-evoked dimer expression in vivo

The immediate early gene product activity-regulated cytoskeleton-associated protein (Arc or Arg3.1) is a major regulator of long-term synaptic plasticity with critical roles in postnatal cortical development and memory formation. However, the molecular basis of Arc function is undefined. Arc is a hub protein with interaction partners in the postsynaptic neuronal compartment and nucleus. Previous in vitro biochemical and biophysical analysis of purified recombinant Arc showed formation of low-order oligomers and larger particles including retrovirus-like capsids. Here, we provide evidence for naturally occurring Arc oligomers in the mammalian brain. Using in situ protein crosslinking to trap weak Arc–Arc interactions, we identified in various preparations a prominent Arc immunoreactive band on SDS-PAGE of molecular mass corresponding to a dimer. While putative trimers, tetramers and heavier Arc species were detected, they were of lower abundance. Stimulus-evoked induction of Arc expression and dimer formation was first demonstrated in SH-SY5Y neuroblastoma cells treated with the muscarinic cholinergic agonist, carbachol, and in primary cortical neuronal cultures treated with brain-derived neurotrophic factor (BDNF). In the dentate gyrus (DG) of adult anesthetized rats, induction of long-term potentiation (LTP) by high-frequency stimulation (HFS) of medial perforant synapses or by brief intrahippocampal infusion of BDNF led to a massive increase in Arc dimer expression. Arc immunoprecipitation of crosslinked DG tissue showed enhanced dimer expression during 4 h of LTP maintenance. Mass spectrometric proteomic analysis of immunoprecipitated, gel-excised bands corroborated detection of Arc dimer. Furthermore, Arc dimer was constitutively expressed in naïve cortical, hippocampal and DG tissue, with the lowest levels in the DG. Taken together the results implicate Arc dimer as the predominant low-oligomeric form in mammalian brain, exhibiting regional differences in its constitutive expression and enhanced synaptic activity-evoked expression in LTP.publishedVersio

Development and Validation of Arc Nanobodies: New Tools for Probing Arc Dynamics and Function

Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions.publishedVersio

Relationship of magnetic ordering and crystal structure in lanthanide ferromagnets Gd, Tb, Dy, and Ho at high pressures

The pressure dependence of the magnetic ordering temperatures for the lanthanide ferromagnets Gd, Tb, Dy, and Ho has been investigated in the pressure region up to 18 GPa by two types of magnetic measurements using a superconducting quantum interference device (SQUID). The present magnetic measurements enabled us to investigate the pressure dependence of the magnetization intensity at low magnetic fields as well as the magnetic ordering temperatures. Their results are interpreted in the light of such previous experiments as magnetic susceptibility, magnetization, electrical resistance, neutron diffraction, and Mössbauer spectroscopy measurements. All of the magnetic orderings in the above four elements were suppressed down to less than the detection level, being related to the structural transition. The ferromagnetic ordering in Gd, Tb, Dy, and Ho is stabilized in the hcp structure. The magnetic anomalies due to the helimagnetic ordering of Tb and Dy disappear at the Sm-to-dhcp transition and the hcp-to-Sm transition, respectively, while that of Ho disappears in the Sm-type phase near the Sm-to-dhcp transition

Ring-fused porphyrins: extension of π-conjugation significantly affects the aromaticity and optical properties of the porphyrin π-systems and the Lewis acidity of the central metal ions

Here, we report the effects of ring fusion, which causes expansion of the π-conjugation circuits of the porphyrin derivatives to the fused meso-aryl groups, on the aromaticity and the magnetic properties of porphyrin derivatives. These studies revealed the facts that the ring fusion with five-membered rings causes not only the remarkable red shifts of the absorption bands and narrowed HOMO–LUMO gaps, but also the contribution of anti-aromatic resonance forms to the magnetic properties as observed in the 1H NMR spectra. The optical absorption and magnetic circular dichroism (MCD) spectroscopies indicate that the increase in the number of the fused rings causes stabilization of the LUMO level of the porphyrin derivatives and as a result induces the loosening of the LUMO degeneracy that is generally observed for porphyrins. The electronic structure of a quadruply fused porphyrin derivative was experimentally clarified by the ESR studies on the 1e−-oxidized and 1e−-reduced species in THF. Furthermore, we revealed the substituent effects of the fused meso-aryl groups of quadruply fused porphyrins (QFPs) on the crystal structures, absorption spectra and redox potentials; the sensitiveness of the substituent effects shows that the π-conjugation circuits extended to the fused meso-aryl groups. Additionally, the elongation of the bond lengths between the pyrrolic nitrogen and the central metal ions in QFP–metal complexes causes a remarkable increase of the Lewis acidity of the central metal ions

A simple DMSO-based method for cryopreservation of primary hippocampal and cortical neurons

Background Primary neuronal cultures are widely used to elucidate fundamental aspects of neuronal anatomy, physiology, cell biology, and neuronal dysfunction in animal models of disease. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it is often difficult to produce high-quality cultures consistently in a single laboratory, and to compare results between laboratories. To overcome these issues, cryopreservation can be used to obtain more standardized, high-quality banks of neuronal cultures. New Method In this study, we present a simplified cryopreservation method for rodent primary hippocampal and cortical neurons from embryonic day 18.5 fetuses, using DMSO-containing traditional cell freezing medium. Results Cryopreserved neurons stored for more than 1 year in liquid nitrogen were assessed by cell imaging, as well as biochemical signaling transduction and gene expression in response to pharmacological treatments. Cryopreserved neuronal cultures were comparable to freshly prepared cultures in terms of: (1) neuronal viability, (2) neuronal morphology and maturation, (3) functional synapse formation, (4) stimulus responsiveness. These results indicate that DMSO-cryopreserved neurons are equivalent to freshly prepared neurons both developmentally and functionally. Comparison with Existing Methods Our method is simple and does not require special reagents or equipment. Conclusions Introduction of the cryopreserved neurons as a standard laboratory practice has the potential to increase the robustness and reproducibility of findings between laboratories and reduce the number of animals used in research

A simple DMSO-based method for cryopreservation of primary hippocampal and cortical neurons

Background Primary neuronal cultures are widely used to elucidate fundamental aspects of neuronal anatomy, physiology, cell biology, and neuronal dysfunction in animal models of disease. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it is often difficult to produce high-quality cultures consistently in a single laboratory, and to compare results between laboratories. To overcome these issues, cryopreservation can be used to obtain more standardized, high-quality banks of neuronal cultures. New Method In this study, we present a simplified cryopreservation method for rodent primary hippocampal and cortical neurons from embryonic day 18.5 fetuses, using DMSO-containing traditional cell freezing medium. Results Cryopreserved neurons stored for more than 1 year in liquid nitrogen were assessed by cell imaging, as well as biochemical signaling transduction and gene expression in response to pharmacological treatments. Cryopreserved neuronal cultures were comparable to freshly prepared cultures in terms of: (1) neuronal viability, (2) neuronal morphology and maturation, (3) functional synapse formation, (4) stimulus responsiveness. These results indicate that DMSO-cryopreserved neurons are equivalent to freshly prepared neurons both developmentally and functionally. Comparison with Existing Methods Our method is simple and does not require special reagents or equipment. Conclusions Introduction of the cryopreserved neurons as a standard laboratory practice has the potential to increase the robustness and reproducibility of findings between laboratories and reduce the number of animals used in research

Discovery and biological characterization of a novel scaffold for potent inhibitors of peripheral serotonin synthesis

Aim: Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation. Results & methodology: Screening using a thermal shift assay for TPH1 binders yielded Compound 1 (2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one), which showed high potency (50% inhibition at 98 ± 30 nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure–activity relationships studies revealed several analogs of 1 showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of 1, with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed. Conclusion: This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin

Substituent Effects at the β‑Positions of the Nonfused Pyrroles in a Quadruply Fused Porphyrin on the Structure and Optical and Electrochemical Properties

We have synthesized <b>2</b>, a derivative of zinc­(II) quadruply fused porphyrinato (Zn^IIQFP) that is tetrabrominated at the β-positions of the two nonfused pyrroles, by treatment of Zn^IIQFP with <i>N</i>-bromosuccinimide. X-ray diffraction analysis of a single crystal obtained from a THF solution of <b>2</b> by vapor diffusion of ethanol (EtOH) revealed that <b>2</b> formed an unprecedented dimeric structure, (<b>2</b>)₂-L (L = EtOH), in which one of the brominated QFP ligands acts as a bridging ligand in an unprecedented μ-η³:η¹ coordination mode. In the dimeric structure, the two QFP ligands showed a unique η³ coordination mode for both Zn^II centers. In (<b>2</b>)₂-EtOH, one of the pyrrolic nitrogen atoms of the two nonfused pyrroles dissociates from the Zn^II center, and the dissociated pyrrolic nitrogen atom coordinates to the Zn^II center of the other molecule in the dimer. The Zn^II center having the μ-η³:η¹-QFP ligand is coordinated by an EtOH molecule, and the other Zn^II center is coordinated by the η³-QFP ligand and one nitrogen atom of the bridging QFP ligand. The dimeric structure is stable and maintained even in a solution of noncoordinating solvents such as dichloromethane. The bromo groups of <b>2</b> can be substituted with phenyl groups under Suzuki coupling conditions to afford the tetraphenyl derivative, <b>3</b>. Furthermore, the effects of the substituents at the β-positions on the optical and electrochemical properties and Lewis acidity of the Zn^II centers have been investigated. The redox potentials were positively shifted by introduction of electron-withdrawing groups at the β-positions, and the shift widths exhibited a linear correlation to the Hammett parameters of the substituents

Periodontal Healing by Implantation of Collagen Hydrogel-sponge Composite in One-wall Infrabony Defects in Beagle Dogs

The purpose of this study was to evaluate the effect of implanted collagen hydrogel-sponge composite on periodontal wound healing. One-wall infrabony defects (depth: 5 mm; width: 3 mm) were surgically created in three beagle dogs. The exposed root surface was planed and demineralized with EDTA. In the experimental group, the defects were filled with collagen hydrogel-sponge composite. Conversely, no collagen hydrogel-sponge composite was applied to defects in the control group. Histomorphometric parameters were evaluated four weeks after surgery. In the experimental group, regeneration of alveolar bone and cementum was frequently observed. Periodontal ligament tissue was reestablished between the alveolar bone and cementum. New bone height, new bone area, new cementum and new periodontal ligament in the experimental group were significantly greater than those in the control group (p < 0.05). These findings suggest that implanted collagen hydrogel-sponge composite facilitates periodontal wound healing in one-wall infrabony defects in beagle dogs