Long-read sequencing identifies the pathogenic nucleotide repeat expansion in RFC1 in a Japanese case of CANVAS.

Recently, a recessively inherited intronic repeat expansion in replication factor C1 (RFC1) was identified in cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Here, we describe a Japanese case of genetically confirmed CANVAS with autonomic failure and auditory hallucination. The case showed impaired uptake of iodine-123-metaiodobenzylguanidine and 123I-ioflupane in the cardiac sympathetic nerve and dopaminergic neurons, respectively, by single-photon emission computed tomography. Long-read sequencing identified biallelic pathogenic (AAGGG)n nucleotide repeat expansion in RFC1 and heterozygous benign (TAAAA)n and (TAGAA)n expansions in brain expressed, associated with NEDD4 (BEAN1). Enrichment of the repeat regions in RFC1 and BEAN1 using a Cas9-mediated system clearly distinguished between pathogenic and benign repeat expansions. The haplotype around RFC1 indicated that the (AAGGG)n expansion in our case was on the same ancestral allele as that of European cases. Thus, long-read sequencing facilitates precise genetic diagnosis of diseases with complex repeat structures and various expansions.

Direct phase-locking of a Ti:Sapphire optical frequency comb to a remote optical frequency standard.

We report on an ultralow noise optical frequency transfer from a remotely located Sr optical lattice clock laser to a Ti:Sapphire optical frequency comb through telecom-wavelength optical fiber networks. The inherent narrow linewidth of the Ti:Sapphire optical frequency comb eliminates the need for a local reference high-finesse cavity. The relative fractional frequency instability of the optical frequency comb with respect to the remote optical reference was 6.7(1) × 10-18 at 1 s and 1.05(3) × 10-19 at 1,000 s including a 2.9 km-long fiber network. This ensured the optical frequency comb had the same precision as the optical standard. Our result paves the way for ultrahigh-precision spectroscopy and conversion of the highly precise optical frequency to radio frequencies in a simpler setup.

Origins of Water Molecules in the Photosystem II Crystal Structure.

The cyanobacterial photosystem II (PSII) crystal structure includes more than 1300 water molecules in each monomer unit; however, their precise roles in water oxidation are unclear. To understand the origins of water molecules in the PSII crystal structure, the accessibility of bulk water molecules to channel inner spaces in PSII was investigated using the water-removed PSII structure and molecular dynamics (MD) simulations. The inner space of the channel that proceeds toward the D1-Glu65/D2-Glu312 pair (E65/E312 channel) was entirely filled with water molecules from the bulk region. In the same channel, a diamond-shaped cluster of water molecules formed near redox-active TyrZ in MD simulations. Reorientation of the D2-Leu352 side chain resulted in formation of a hexagonal water network at the Cl-2 binding site. Water molecules could not enter the main region of the O4-water chain, which proceeds from the O4 site of the Mn4CaO5 cluster. However, in the O4-water chain, the two water binding sites that are most distant from the protein bulk surface were occupied by water molecules that approached along the E65/E312 channel, one of which formed an H-bond with the O4 site. These findings provide key insights into the significance of the channel ends, which may utilize water molecules during the PSII photocycle.

Structurally conserved channels in cyanobacterial and plant photosystem II.

In the cyanobacterial photosystem II (PSII), the O4-water chain in the D1 and CP43 proteins, a chain of water molecules that are directly H-bonded to O4 of the Mn4Ca cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4-PsbU channel). The cyanobacterial PSII structure also shows that the O1 site of the Mn4Ca cluster has a chain of H-bonded water molecules, which is linked with the channel that proceeds toward the bulk surface via PsbU and PsbV (O1-PsbU/V channel). Membrane-extrinsic protein subunits PsbU and PsbV in cyanobacterial PSII are replaced with PsbP and PsbQ in plant PSII. However, these four proteins have no structural similarity. It remains unknown whether the corresponding channels also exist in plant PSII, because water molecules are not identified in the plant PSII cryo-electron microscopy (cryo-EM) structure. Using the cyanobacterial and plant PSII structures, we analyzed the channels that proceed from the Mn4Ca cluster. The cyanobacterial O4-PsbU and O1-PsbU/V channels were structurally conserved as the channel that proceeds along PsbP toward the protein bulk surface in the plant PSII (O4-PsbP and O1-PsbP channels, respectively). Calculated protonation states indicated that in contrast to the original geometry of the plant cryo-EM structure, protonated PsbP-Lys166 may form a salt-bridge with ionized D1-Glu329 and protonated PsbP-Lys173 may form a salt-bridge with ionized PsbQ-Asp28 near the O1-PsbP channel. The existence of these channels might explain the molecular mechanism of how PsbP can interact with the Mn4Ca cluster.

Non-Covalent Loading of Anti-Cancer Doxorubicin by Modularizable Peptide Self-Assemblies for a Nanoscale Drug Carrier.

We prepared nanoscale, modularizable, self-assembled peptide nanoarchitectures with diameters less of than 20 nm by combining ß-sheet-forming peptides tethering a cell-penetrating peptide or a nuclear localization signal sequence. We also found that doxorubicin (Dox), an anti-cancer drug, was non-covalently accommodated by the assemblies at a ratio of one Dox molecule per ten peptides. The Dox-loaded peptide assemblies facilitated cellular uptake and subsequent nuclear localization in HeLa cells, and induced cell death even at low Dox concentrations. This peptide nanocarrier motif is a promising platform for a biocompatible drug delivery system by altering the targeting head groups of the carrier peptides.

The Existence of an Isolated Hydronium Ion in the Interior of Proteins.

Neutron diffraction analysis studies reported an isolated hydronium ion (H3 O+ ) in the interior of d-xylose isomerase (XI) and phycocyanobilin-ferredoxin oxidoreductase (PcyA). H3 O+ forms hydrogen bonds (H-bonds) with two histidine side-chains and a backbone carbonyl group in PcyA, whereas H3 O+ forms H-bonds with three acidic residues in XI. Using a quantum mechanical/molecular mechanical (QM/MM) approach, we analyzed stabilization of H3 O+ by the protein environment. QM/MM calculations indicated that H3 O+ was unstable in the PcyA crystal structure, releasing a proton to an H-bond partner His88, producing H2 O and protonated His88. On the other hand, H3 O+ was stable in the XI crystal structure. H-bond partners of isolated H3 O+ would be practically limited to acidic residues such as aspartic and glutamic acids in the protein environment.

Crystal structures of copper-depleted and copper-bound fungal pro-tyrosinase: insights into endogenous cysteine-dependent copper incorporation.

Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe(513) on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193-201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19-26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys(92), which is covalently bound to His(94) via an unusual thioether linkage in the holo-form, and Cys(522) and Cys(525) of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information.

Synthesis and SAR studies of benzyl ether derivatives as potent orally active S1P1 agonists.

We report herein the synthesis and structure-activity relationships (SAR) of a series of benzyl ether compounds as an S1P1 receptor modulator. From our SAR studies, the installation of substituents onto the central benzene ring of 2a was revealed to potently influence the S1P1 and S1P3 agonistic activities, in particular, an ethyl group on the 2-position afforded satisfactory S1P1/S1P3 selectivity. These changes of the S1P1 and S1P3 agonistic activities caused by the alteration of substituents on the 2-position were reasonably explained by a docking study using an S1P1 X-ray crystal structure and S1P3 homology modeling. We found that compounds 2b and 2e had a potent in vivo immunosuppressive efficacy along with acceptable S1P1/S1P3 selectivity, and confirmed that these compounds had less in vivo bradycardia risk through the evaluation of heart rate change after oral administration of the compounds (30 mg/kg, p.o.) in rats.

Biphenyl-derived phosphepines as chiral nucleophilic catalysts: enantioselective [4+1] annulations to form functionalized cyclopentenes.

Because of the frequent occurrence of cyclopentane subunits in bioactive compounds, the development of efficient catalytic asymmetric methods for their synthesis is an important objective. Introduced herein is a new family of chiral nucleophilic catalysts, biphenyl-derived phosphepines, and we apply them to an enantioselective variant of a useful [4+1] annulation. A range of one-carbon coupling partners can be employed, thereby generating cyclopentenes which bear a fully substituted stereocenter [either all-carbon or heteroatom-substituted (sulfur and phosphorus)]. Stereocenters at the other four positions of the cyclopentane ring can also be introduced with good stereoselectivity. An initial mechanistic study indicates that phosphine addition to the electrophilic four-carbon coupling partner is not the turnover-limiting step of the catalytic cycle.

Design of a dynamic polymer interface for chiral discrimination.

Enantioselective wetting of a chiral polymer film was demonstrated. The contact angle of chiral liquids on the film was strongly dependent on their chirality although their physical properties including surface tension were identical. Such wetting behavior resulted from the enantioselective surface reorganization involving local conformational change of the polymer chains at the liquid interface. The concept of "dynamic interface for chiral discrimination" has possible potential for the development of materials capable of chiral sensing, optical resolution, and asymmetric synthesis.

Activation mechanism of melB tyrosinase from Aspergillus oryzae by acidic treatment.

The pro form of recombinant tyrosinase from Aspergillus oryzae (melB) shows no catalytic activity, but acid treatment (around pH 3.5) of protyrosinase activates it to induce tyrosinase activity. Circular dichroism spectra, gel filtration analysis, and colorimetric assay have indicated that acid treatment around pH 3.5 induced the disruption of the conformation of the C-terminal domain covering the enzyme active site. These structural changes induced by the acid treatment may open the entrance to the enzyme active site for substrate incorporation. To compare the mechanism of hydroxylation by the acid-treated tyrosinase with that by trypsin-treated tyrosinase, a detailed steady-state kinetic analysis of the phenolase activity was performed by monitoring the O(2)-consumption rate using a Clark-type oxygen electrode. The results clearly show that the phenolase activity (phenol hydroxylation) of the activated tyrosinase involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase (Yamazaki and Itoh in J. Am. Chem. Soc. 125:13034-13035, 2003) and activated hemocyanin with urea (Morioka et al. in J. Am. Chem. Soc. 128:6788-6789, 2006).

Lateral olfactory tract usher substance (LOTUS), an endogenous Nogo receptor antagonist, ameliorates disease progression in amyotrophic lateral sclerosis model mice.

Nogo-Nogo receptor 1 (NgR1) signaling is significantly implicated in neurodegeneration in amyotrophic lateral sclerosis (ALS). We previously showed that lateral olfactory tract usher substance (LOTUS) is an endogenous antagonist of NgR1 that prevents all myelin-associated inhibitors (MAIs), including Nogo, from binding to NgR1. Here we investigated the role of LOTUS in ALS pathogenesis by analyzing G93A-mutated human superoxide dismutase 1 (SOD1) transgenic (Tg) mice, as an ALS model, as well as newly generated LOTUS-overexpressing SOD1 Tg mice. We examined expression profiles of LOTUS and MAIs and compared motor functions and survival periods in these mice. We also investigated motor neuron survival, glial proliferation in the lumbar spinal cord, and neuromuscular junction (NMJ) morphology. We analyzed downstream molecules of NgR1 signaling such as ROCK2, LIMK1, cofilin, and ataxin-2, and also neurotrophins. In addition, we investigated LOTUS protein levels in the ventral horn of ALS patients. We found significantly decreased LOTUS expression in both SOD1 Tg mice and ALS patients. LOTUS overexpression in SOD1 Tg mice increased lifespan and improved motor function, in association with prevention of motor neuron loss, reduced gliosis, increased NMJ innervation, maintenance of cofilin phosphorylation dynamics, decreased levels of ataxin-2, and increased levels of brain-derived neurotrophic factor (BDNF). Reduced LOTUS expression may enhance neurodegeneration in SOD1 Tg mice and ALS patients by activating NgR1 signaling, and in this study LOTUS overexpression significantly ameliorated ALS pathogenesis. LOTUS might serve as a promising therapeutic target for ALS.

Multifunctions of MelB, a fungal tyrosinase from Aspergillus oryzae.

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2)OH (reductant), the proenzyme bound dioxygen to give a stable (µ-η(2):η(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.

Efficacy of specimen pasting after cold snare polypectomy for pathological evaluation of horizontal margins.

Background and study aims Several studies have reported that pathological horizontal margin evaluation cannot be diagnosed in cold snare polypectomy (CSP) specimens. We conducted a prospective randomized controlled trial to determine the efficacy of pasting CSP specimens on paper for pathological horizontal margins. Patients and methods This was a single-center, prospective study conducted at Osaka Saiseikai Nakatsu Hospital. In this study, the indications for CSP were adenomas ≤ 10 mm. Colorectal polyps resected by CSP were randomized to the pasting and non-pasting groups after exclusion of fragmented specimens, and the extended CSP specimens pasted on paper were formalin-fixed in the pasting group. The primary endpoint was rate of unclear horizontal margins after CSP. Results A total of 216 CSP specimens were analyzed. The rate of unclear horizontal margins was significantly lower in the pasting group than in the non-pasting group (15.1 % vs 33.6 %, P  = 0.002). CSP specimen pasting significantly reduced the rate of unclear horizontal margins. On multivariate analysis, non-pasting group (odds ratio [OR], 2.69; 95 % confidence interval [CI], 1.38-5.41; P  = 0.003) and right colon (OR, 1.98; 95 %CI, 1.01-4.01; P  = 0.047) were independent risk factors for unclear horizontal margins in CSP specimens. Conclusions Pasting the extended specimen is important for accurate pathological examination after CSP.

Anchored Palladium Complex-Generated Clusters on Zirconia: Efficiency in Reductive N-Alkylation of Amines with Carbonyl Compounds under Hydrogen Atmosphere.

Carbon-nitrogen bond formation is an important method on both laboratory and industrial scales because it realizes the production of valuable pharmaceuticals, agrochemicals, and fine chemicals. Direct reductive N-alkylation of amines with carbonyl compounds via intermediary imine compounds, especially under catalytic hydrogenation conditions, is one of the most convenient, economical, and environmentally friendly methods for this process. Here we report a novel palladium species on zirconia having specific activity towards hydrogenation of imines but other carbonyl groups remaining intact. The present catalytic property offers a practical synthetic method of functionalized secondary amines by reductive N-alkylation under mild conditions with high atom-efficiency. Mechanistic studies revealed that the catalytically active species is the palladium cluster, which is generated in situ from molecular palladium complexes on the support by exposure to atmospheric hydrogen. These fundamental findings are expected to progress in developing novel cluster catalysts for chemical processes directed towards a sustainable society.

Pulmonary actinomycosis mimicking lung cancer on 18F-fluorodeoxyglucose positron emission tomography: a case report.

BACKGROUND: Pulmonary actinomycosis is a chronic disease characterized by abscess formation, draining sinuses, fistulae, and tissue fibrosis. It can mimic other conditions, particularly malignant and granulomatous diseases, and is perhaps extremely challenging to diagnose. CASE PRESENTATION: A 64-year-old Japanese man presented with 6-week history of a painful solid lump in the chest wall. Chest computed tomography scan revealed a mass-like consolidation in the left upper lobe, with rib erosion and direct extension into the anterior chest wall. 18F-fluorodeoxyglucose positron emission tomography scan showed increased metabolic activity in the mass, which is indicative of primary lung cancer. The bronchoscopy and computed tomography scan-guided transthoracic biopsy results were considered nondiagnostic. Finally, the patient was diagnosed with pulmonary actinomycosis via surgical resection. He completed an 8-week course of antibiotic therapy and experienced no recurrence. CONCLUSIONS: There is no difference in positron emission tomography/computed tomography scan findings between actinomycosis and malignancy. Therefore, pulmonary actinomycosis should be considered in the differential diagnosis of cases involving intensive activity on 18F-fluorodeoxyglucose positron emission tomography scan.

Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76.

Background: Some strains of Streptococcus mitis exhibit ß-hemolysis due to the ß-hemolytic activity of cholesterol-dependent cytolysin (CDC). Recently, a gene encoding an atypical lectinolysin-related CDC was found in S. mitis strain Nm-76. However, the product of this gene remains uncharacterized. We aimed to characterize this atypical CDC and its molecular functions and contribution to the pathogenicity of S. mitis strain Nm-76. Methods: Phylogenetic analysis of the CDC gene was conducted based on the web-deposited information. The molecular characteristics of CDC were investigated using a gene-deletion mutant strain and recombinant proteins expressed in Escherichia coli. Results: The gene encoding CDC found in Nm-76 and its homolog are distributed among many S. mitis strains. This CDC is phylogenetically different from other previously characterized CDCs, such as S. mitis-derived human platelet aggregation factor (Sm-hPAF)/lectinolysin and mitilysin. Because this CDC possesses an additional N-terminal domain, including a discoidin motif, it was termed discoidinolysin (DLY). In addition to the preferential lysis of human cells, DLY displayed N-terminal domain-dependent facilitation of human erythrocyte aggregation and intercellular associations between human cells. Conclusion: DLY functions as a hemolysin/cytolysin and erythrocyte aggregation/intercellular association molecule. This dual-function DLY could be an additional virulence factor in S. mitis.

Inhibition of Crmp1 Phosphorylation at Ser522 Ameliorates Motor Function and Neuronal Pathology in Amyotrophic Lateral Sclerosis Model Mice.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder that affects upper and lower motor neurons; however, its pathomechanism has not been fully elucidated. Using a comprehensive phosphoproteomic approach, we have identified elevated phosphorylation of Collapsin response mediator protein 1 (Crmp1) at serine 522 in the lumbar spinal cord of ALS model mice overexpressing a human superoxide dismutase mutant (SOD1G93A). We investigated the effects of Crmp1 phosphorylation and depletion in SOD1G93A mice using Crmp1S522A (Ser522→Ala) knock-in (Crmp1ki/ki ) mice in which the S522 phosphorylation site was abolished and Crmp1 knock-out (Crmp1-/-) mice, respectively. Crmp1ki/ki /SOD1G93A mice showed longer latency to fall in a rotarod test while Crmp1-/-/SOD1G93A mice showed shorter latency compared with SOD1G93A mice. Survival was prolonged in Crmp1ki/ki /SOD1G93A mice but not in Crmp1-/-/SOD1G93A mice. In agreement with these phenotypic findings, residual motor neurons and innervated neuromuscular junctions (NMJs) were comparatively well-preserved in Crmp1ki/ki /SOD1G93A mice without affecting microglial and astroglial pathology. Pathway analysis of proteome alterations showed that the sirtuin signaling pathway had opposite effects in Crmp1ki/ki /SOD1G93A and Crmp1-/-/SOD1G93A mice. Our study indicates that modifying CRMP1 phosphorylation is a potential therapeutic strategy for ALS.

Post-translational His-Cys cross-linkage formation in tyrosinase induced by copper(II)-peroxo species.

Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (µ-η(2):η(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.

Proton transfer pathway from the oxygen-evolving complex in photosystem II substantiated by extensive mutagenesis.

We report a structure-based biological approach to identify the proton-transfer pathway in photosystem II. First, molecular dynamics (MD) simulations were conducted to analyze the H-bond network that may serve as a Grotthuss-like proton conduit. MD simulations show that D1-Asp61, the H-bond acceptor of H2O at the Mn4CaO5 cluster (W1), forms an H-bond via one water molecule with D1-Glu65 but not with D2-Glu312. Then, D1-Asp61, D1-Glu65, D2-Glu312, and the adjacent residues, D1-Arg334, D2-Glu302, and D2-Glu323, were thoroughly mutated to the other 19 residues, i.e., 114 Chlamydomonas chloroplast mutant cells were generated. Mutation of D1-Asp61 was most crucial. Only the D61E and D61C cells grew photoautotrophically and exhibit O2-evolving activity. Mutations of D2-Glu312 were less crucial to photosynthetic growth than mutations of D1-Glu65. Quantum mechanical/molecular mechanical calculations indicated that in the PSII crystal structure, the proton is predominantly localized at D1-Glu65 along the H-bond with D2-Glu312, i.e., pKa(D1-Glu65) > pKa(D2-Glu312). The potential-energy profile shows that the release of the proton from D1-Glu65 leads to the formation of the two short H-bonds between D1-Asp61 and D1-Glu65, which facilitates downhill proton transfer along the Grotthuss-like proton conduit in the S2 to S3 transition. It seems possible that D1-Glu65 is involved in the dominant pathway that proceeds from W1 via D1-Asp61 toward the thylakoid lumen, whereas D2-Glu312 and D1-Arg334 may be involved in alternative pathways in some mutants.