A high-throughput sequence determination for one cyclic peptide immobilized on a single bead by the one-pot cyanidation followed by MALDI-TOF-MS/MS for discovery of interacting peptides.

One cyclic peptide immobilized on one gel-type bead has been employed for the discovery of both interacting peptides and/or medicinal medium-sized molecules. Although high-throughput characterization of recognized peptides has been a bottleneck, here, we describe direct liberation from beads by a one-pot reaction using 2-nitro-5-thiocyanatobenzoic acid followed by mass spectrometry to realize faster and routine sequencing of the peptide on the beads. This is useful for the investigation of protein-protein interactions as well as discovery of drug candidates.

Characterization of novel truncated apolipoprotein A-I in human high-density lipoprotein generated by sequential treatment with myeloperoxidase and chymase.

High-density lipoprotein (HDL) cholesterol is a well-known biomarker, which has been associated with reduction in the risk of cardiovascular diseases (CVD). However, some HDL anti-atherosclerotic functions may be impaired without altered HDL-cholesterol (HDL-C) level via its dysfunctional proteins or other physiological reactions in vivo. We previously showed that activated mast cell-derived chymase could modestly cleave apolipoprotein A-I (apoA-I) in HDL3, and further easily cleave lipid-free apoA-I. In contrast, myeloperoxidase (MPO) secreted by macrophages, the main cell type in atherosclerotic plaques, could oxidize HDL proteins, which might modify their tertiary structures, increasing their susceptibility to other enzymes. Here we focused on the co-modification and impact of chymase and MPO, usually secreted during inflammation from cells with possible co-existence in atheromas, on HDL. Only after sequential treatment with MPO and then chymase, two novel truncated apoA-I fragments were generated from HDL. One fragment was 16.5 kDa, and the cleavage site by chymase after MPO modification was the C-terminal of Tyr100 in apoA-I, cross-validated by three different mass spectrometry methods. This novel apoA-I fragment can be trapped in HDL particles to avoid kidney glomerular filtration and has a specific site for antibody generation for ELISA tests. As such, its quantification can be useful in predicting patients with CVD having normal HDL-C levels.

A novel cyclic peptide library immobilized on gel-type beads focusing on rapid construction and characterization for comprehensive drug discovery.

Medium sized molecules such as peptides and macrocycles have recently drawn much attention as potent sources of medicinal lead compounds, whereas the possibility of obtaining a practical drug from them remains limited. The present paper describes a concept of discovering novel medicinal targets or binding modes as well as lead compounds by the one-peptide-on-one-bead (OPOB) technology for comprehensive screening. The difficulty and problems in conventional drug discovery methods that generally deal with one predetermined target are considered. The building blocks used for the present libraries were selected based on previous results in development of peptidic drugs. Each constituent has the common structure of cyclic form prepared by disulfide of cysteinyl residues or thioether linkages, additionally a methionine residue was inserted for the site-specific rapid cleavage by cyanogen bromide to liberate the immobilized peptides allowing reliable characterization by MALDI-TOF-MS/MS without LC-purification. Thus, a high throughput construction method for cyclic peptide libraries as well as characterization of single bead are proposed for drug discovery.

Phospholipid synthesis inside phospholipid membrane vesicles.

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.

Linagliptin, A Xanthine-Based Dipeptidyl Peptidase-4 Inhibitor, Ameliorates Experimental Autoimmune Myocarditis.

This study sought to show the mechanism of how to ameliorate experimental autoimmune myocarditis (EAM) by administering dipeptidyl peptidase (DPP)-4 inhibitor linagliptin. The number of RAR-related orphan nuclear receptor gamma-positive Th17 cells infiltrated to the EAM myocardium was significantly attenuated by linagliptin treatment. Tandem mass spectrometry-based analysis demonstrated that DPP-4 binds to cathepsin G in EAM hearts, thereby protecting cathepsin G activity through inhibiting SerpinA3N activity. Linagliptin suppresses oxidative stress in EAM hearts as well. Thus, we found that DPP-4 plays a detrimental role in the progression of EAM by interacting with cathepsin G, which, in turn, suppresses SerpinA3N activity.

Electron inelastic mean free path in water.

Liquid phase transmission electron microscopy (LPTEM) is rapidly developing as a powerful tool for probing processes in liquid environments with close to atomic resolution. Knowledge of the water thickness is needed for reliable interpretation and modelling of analytical studies in LPTEM, and is particularly essential when using thin liquid layers, required for achieving the highest spatial resolutions. The log-ratio method in electron energy-loss spectroscopy (EELS) is often applied in TEM to quantify the sample thickness, which is measured relative to the inelastic mean free path (λIMFP). However, λIMFP itself is dependent on sample material, the electron energy, and the convergence and divergence angles of the microscope electronoptics. Here, we present a detailed quantitative analysis of the λIMFP of water as functions of the EELS collection angle (ß) at 120 keV and 300 keV in a novel nanochannel liquid cell. We observe good agreement with earlier studies conducted on ice, but find that the most widely used theoretical models significantly underestimate λIMFP of water. We determine an adjusted average energy-loss term Em, water, and characteristic scattering angle θE, water that improve the accuracy. The results provide a comprehensive knowledge of the λIMFP of water (or ice) for reliable interpretation and quantification of observations in LPTEM and cryo-TEM studies.

Mean Inner Potential of Liquid Water.

Improving our experimental and theoretical knowledge of electric potentials at liquid-solid boundaries is essential to achieve a deeper understanding of the driving forces behind interfacial processes. Electron holography has proved successful in probing solid-solid interfaces but requires knowledge of the materials' mean inner potential (MIP, V_{0}), which is a fundamental bulk material property. Combining off-axis electron holography with liquid phase transmission electron microscopy (LPTEM), we provide the first quantitative MIP determination of liquid water V_{0}=+4.48±0.19 V. This value is larger than most theoretical predictions, and to explain the disagreement we assess the dominant factors needed in quantum simulations of liquid water. A precise MIP lays the foundations for nanoscale holographic potential measurements in liquids, and provides a benchmark to improve quantum mechanical descriptions of aqueous systems and their interfaces in, e.g., electrochemistry, solvation processes, and spectroscopy.

Superior removal of selenite by periclase during transformation to brucite under high-pH conditions.

The sorption of selenite (Se(IV)) at trace (sub-ppm) to high concentrations on periclase (MgO) under high-pH conditions (pH > 10) was examined by macroscopic sorption experiments and nanoscale solid phase analyses via transmission electron microscopy and X-ray absorption spectroscopy. The maximum distribution coefficient (Kd) of Se(IV) on MgO was 100 L/g, the highest among any reported mineral sorbents at pH > 10. Since MgO is a metastable phase under ambient conditions, it transforms instantaneously to brucite (Mg(OH)2) in solution. Se(IV) was preferentially and homogeneously distributed onto the newly formed Mg(OH)2. The Mg(OH)2 formed thin flake-like platelets, which appeared to be aggregates of nanoscale Mg(OH)2 particles, the primary alteration product of MgO. The chemical form of Se(VI) adsorbed on nanoscale particles was outer-sphere complexes. Therefore, the outer-spherically adsorbed Se(IV) was occluded into the large flake-like Mg(OH)2 particles, resulting in its effective isolation from the solution.

TOPK is regulated by PP2A and BCR/ABL in leukemia and enhances cell proliferation.

Although treatment of chronic myeloid leukemia (CML) has improved with the development of tyrosine kinase inhibitors (TKIs), patients develop fatal blast crisis (BC) whilst receiving TKI treatment. Alternative treatments for cases resistant to TKIs are required. A serine/threonine protein kinase, T­lymphokine­activated killer cell­originated protein kinase (TOPK), is highly expressed in various malignant tumors. Binding of peptides to human leukocyte antigen was assessed via mass spectrometry in K562 CML cells. TOPK expression was assessed in various CML cell lines and in clinical samples obtained from patients with CML using reverse transcription­quantitative polymerase chain reaction and western blot assays. It was observed that TOPK was expressed abundantly in BCR/ABL­positive cell lines and at significantly higher levels in CML clinical samples compared with healthy donor samples. Overexpression of BCR/ABL or the presence of its inhibitor imatinib upregulated and downregulated TOPK expression, respectively, indicating that TOPK may be a target of BCR/ABL. TOPK inhibitor OTS514 suppressed proliferation of BCR/ABL­positive cell lines and colony formation of CD34­positive cells from patients with CML compared with lymphoma patients without bone marrow involvement. Furthermore, phosphorylation of TOPK was increased by protein phosphatase 2A (PP2A) inhibitor okadaic acid and was decreased in the presence of PP2A activator FTY720 compared with untreated samples. As constitutive BCR/ABL activity and inhibition of PP2A are key mechanisms of CML development, TOPK may be a crucial signaling molecule for this disease. Inhibition of TOPK may control disease status of CML, even in cases resistant to TKIs.

Nanowire Quantum Dots Tuned to Atomic Resonances.

Quantum dots tuned to atomic resonances represent an emerging field of hybrid quantum systems where the advantages of quantum dots and natural atoms can be combined. Embedding quantum dots in nanowires boosts these systems with a set of powerful possibilities, such as precise positioning of the emitters, excellent photon extraction efficiency and direct electrical contacting of quantum dots. Notably, nanowire structures can be grown on silicon substrates, allowing for a straightforward integration with silicon-based photonic devices. In this work we show controlled growth of nanowire-quantum-dot structures on silicon, frequency tuned to atomic transitions. We grow GaAs quantum dots in AlGaAs nanowires with a nearly pure crystal structure and excellent optical properties. We precisely control the dimensions of quantum dots and their position inside nanowires and demonstrate that the emission wavelength can be engineered over the range of at least 30 nm around 765 nm. By applying an external magnetic field, we are able to fine-tune the emission frequency of our nanowire quantum dots to the D2 transition of 87Rb. We use the Rb transitions to precisely measure the actual spectral line width of the photons emitted from a nanowire quantum dot to be 9.4 ± 0.7 µeV, under nonresonant excitation. Our work brings highly desirable functionalities to quantum technologies, enabling, for instance, a realization of a quantum network, based on an arbitrary number of nanowire single-photon sources, all operating at the same frequency of an atomic transition.

Mechanisms of Se(IV) Co-precipitation with Ferrihydrite at Acidic and Alkaline Conditions and Its Behavior during Aging.

Understanding the form of Se(IV) co-precipitated with ferrihydrite and its subsequent behavior during phase transformation is critical to predicting its long-term fate in a range of natural and engineered settings. In this work, Se(IV)-ferrihydrite co-precipitates formed at different pH were characterized with chemical extraction, transmission electron microscopy (TEM), and X-ray absorption spectroscopy (XAS) to determine how Se(IV) is associated with ferrihydrite. Results show that despite efficient removal, the mode and stability of Se(IV) retention in the co-precipitates varied with pH. At pH 5, Se(IV) was removed dominantly as a ferric selenite-like phase intimately associated with ferrihydrite, while at pH 10, it was mostly present as a surface species on ferrihydrite. Similarly, the behavior of Se(IV) and the extent of its retention during phase transformation varied with pH. At pH 5, Se(IV) remained completely associated with the solid phase despite the phase change, whereas it was partially released back into solution at pH 10. Regardless of this difference in behavior, TEM and XAS results show that Se(IV) was retained within the crystalline post-aging products and possibly occluded in nanopore and defect structures. These results demonstrate a potential long-term immobilization pathway for Se(IV) even after phase transformation. This work presents one of the first direct insights on Se(IV) co-precipitation and its behavior in response to iron phase transformations.

Probing the Chemistry of Adhesion between a 316L Substrate and Spin-on-Glass Coating.

Hydrogen silsesquioxane ([HSiO3/2] n)-based "spin-on-glass" has been deposited on a 316L substrate and cured in Ar/H2 gas atmosphere at 600 °C to form a continuous surface coating with submicrometer thickness. The coating functionality depends primarily on the adhesion to the substrate, which is largely affected by the chemical interaction at the interface between the coating and the substrate. We have investigated this interface by transmission electron microscopy and electron energy loss spectroscopy. The analysis identified a 5-10 nm thick interaction zone containing signals from O, Si, Cr, and Fe. Analysis of the energy loss near edge structure of the present elements identified predominantly signal from [SiO4]4- units together with Fe2+, Cr2+, and traces of Cr3+. High-resolution transmission electron microscopy images of the interface region confirm a crystalline Fe2SiO4 interfacial region. In agreement with computational thermodynamics, it is proposed that the spin-on-glass forms a chemically bonded silicate-rich interaction zone with the substrate. It was further suggested that this zone is composed of a corundum-type oxide at the substrate surface, followed by an olivine-structure intermediate phase and a spinel-type oxide in the outer regions of the interfacial zone.

Novel DHCR7 mutation in a case of Smith-Lemli-Opitz syndrome showing 46,XY disorder of sex development.

Smith-Lemli-Opitz syndrome is an autosomal recessive disease caused by mutations in 7-dehydrocholesterol reductase (DHCR7), which is rarely observed in Japan. We report a Japanese case with 46,XY disorder of sex development and Y-shaped 2-3 toe syndactyly. DHCR7 gene analysis revealed compound heterozygous mutations including the novel mutation H442R. Early diagnosis led to starting cholesterol treatment at an early age.

Effects of dissolved oxygen concentration and iron addition on immediate-early gene expression of Magnetospirillum gryphiswaldense MSR-1.

We report the effects of dissolved oxygen (DO) concentration and iron addition on gene expression of Magnetospirillum gryphiswaldense MSR-1 cells during fermentations, focusing on 0.25-24 h after iron addition. The DO was strictly controlled at 0.5% or 5% O2, and compared with aerobic condition. Uptake of iron (and formation of magnetosomes) was only observed in the 0.5% O2 condition where there was little difference in cell growth and carbon consumption compared to the 5% O2 condition. Quantitative reverse transcription PCR analysis showed a rapid (within 0.25 h) genetic response of MSR-1 cells after iron addition for all the genes studied, except for MgFnr (oxygen sensor gene) and fur (ferric uptake regulator family gene), and which in some cases was oxygen dependent. In particular, expression of sodB1 (superoxide dismutase gene) and feoB1 (ferrous transport protein B1 gene) was markedly reduced in cultures at 0.5% O2 compared to those at higher oxygen tensions. Moreover, expression of katG (catalase-peroxidase gene) and feoB2 (ferrous transport protein B2 gene) was reduced markedly by iron addition, regardless of oxygen conditions. These data provide a greater understanding of molecular response of MSR-1 cells to environmental conditions associated with oxygen and iron metabolisms, especially relevant to immediate-early stage of fermentation.

Stability of a Bifunctional Cu-Based Core@Zeolite Shell Catalyst for Dimethyl Ether Synthesis Under Redox Conditions Studied by Environmental Transmission Electron Microscopy and In Situ X-Ray Ptychography.

When using bifunctional core@shell catalysts, the stability of both the shell and core-shell interface is crucial for catalytic applications. In the present study, we elucidate the stability of a CuO/ZnO/Al2O3@ZSM-5 core@shell material, used for one-stage synthesis of dimethyl ether from synthesis gas. The catalyst stability was studied in a hierarchical manner by complementary environmental transmission electron microscopy (ETEM), scanning electron microscopy (SEM) and in situ hard X-ray ptychography with a specially designed in situ cell. Both reductive activation and reoxidation were applied. The core-shell interface was found to be stable during reducing and oxidizing treatment at 250°C as observed by ETEM and in situ X-ray ptychography, although strong changes occurred in the core on a 10 nm scale due to the reduction of copper oxide to metallic copper particles. At 350°C, in situ X-ray ptychography indicated the occurrence of structural changes also on the µm scale, i.e. the core material and parts of the shell undergo restructuring. Nevertheless, the crucial core-shell interface required for full bifunctionality appeared to remain stable. This study demonstrates the potential of these correlative in situ microscopy techniques for hierarchically designed catalysts.

The mevalonate pathway regulates primitive streak formation via protein farnesylation.

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.

High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages.

High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity.

High throughput sequencing of cyclic peptide immobilized on a gel-type single bead.

Relatively larger scale peptide libraries immobilized on a gel-type solid support consisting of 24 natural and non-natural amino acids by the "split and combine method" have been constructed to find interacting molecules. The diversity was ca. 200 millions of hexapeptides with cysteinyl residues forming cyclotide. Selected beads after screening can be sequenced by the conventional Edman degradation, although several restrictions and the problems are known. To resolve these, a novel combinatorial method involving partial acid hydrolysis followed by liquid chromatography with on-line mass spectrometric analyses has been established. Problems were uncovered in an early stage of the process. Uncertain assignment caused by byproducts derived from a cystine residue and other materials could be resolved by optimal hydrolysis conditions and derivatization before mass spectrometric analysis. Discrimination between Leu and Ile could be performed using high energy collision induced dissociation in the high resolution MALDI-TOF-MS/MS. The present optimized protocol is useful for discovery of sequences of interacting molecules and a second library construction.

Reagent-Free Synthesis and Plasmonic Antioxidation of Unique Nanostructured Metal-Metal Oxide Core-Shell Microfibers.

A photoresponsive inorganic microfiber with a plasmonic core-shell structure responds to visible light to achieve self-protection against oxidation in an open environment. The microfibers are synthesized via a newly developed reagent-free electrolytic method and have unique interfacial structures and high surface activity.