Application of Homology Modeling by Enhanced Profile–Profile Alignment and Flexible-Fitting Simulation to Cryo-EM Based Structure Determination

Application of cryo-electron microscopy (cryo-EM) is crucially important for ascertaining the atomic structure of large biomolecules such as ribosomes and protein complexes in membranes. Advances in cryo-EM technology and software have made it possible to obtain data with near-atomic resolution, but the method is still often capable of producing only a density map with up to medium resolution, either partially or entirely. Therefore, bridging the gap separating the density map and the atomic model is necessary. Herein, we propose a methodology for constructing atomic structure models based on cryo-EM maps with low-to-medium resolution. The method is a combination of sensitive and accurate homology modeling using our profile–profile alignment method with a flexible-fitting method using molecular dynamics simulation. As described herein, this study used benchmark applications to evaluate the model constructions of human two-pore channel 2 (one target protein in CASP13 with its structure determined using cryo-EM data) and the overall structure of Enterococcus hirae V-ATPase complex.     

Cytoskeletal Protein 4.1G Is Essential for the Primary Ciliogenesis and Osteoblast Differentiation in Bone Formation

The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.     

A comparative study of the emotional assessment of automotive exterior colors in Asia

This article compares color assessments of automotive exteriors at four locations in China, Thailand and Japan. This study employed painted panels of twelve colors, thirty sensory words and fifteen pairs of emotion variables. It also used reference figures of nine cars that included four styling categories, namely sedans, compacts, sport utility vehicles and sports cars. The results of like-dislike tests revealed that basic automotive exterior colors, such as black, white and red, were highly preferred across locations. However, geographical differences are apparent in the preference for some non-basic colors. To elucidate these geographical differences, a Chi-square test and principal component analysis were performed on the data on emotion variables. The results revealed both similarities and differences in color preferences among the four locations. Color preferences were similar in Hong Kong and Shanghai when simply evaluated by color samples. However, similar preferences for automotive exterior colors were observed in Hong Kong, Kyoto and Bangkok, but not in Shanghai. These results suggest that there is fluctuation in the emotion evoked by a color when the color is considered in the context of automotive exteriors. Therefore, this study indicates that automotive sales can be secured in the global marketplace by creating a single palette of exterior colors based on similar color preferences across countries. However, these results also indicate that preparing market-specific automotive-color line-ups can be an effective marketing strategy.     

Melt-mixing by novel pitched-tip kneading disks in a co-rotating twin-screw extruder

Melt-mixing in twin-screw extruders is a key process in the development of polymer composites. Quantifying the mixing performance of kneading elements based on their internal physical processes is a challenging problem. We discuss melt-mixing by novel kneading elements called “pitched-tip kneading disk (ptKD)”. The disk-stagger angle and tip angle are the main geometric parameters of the ptKDs. We investigated four typical arrangements of the ptKDs, which are forward and backward disk-staggers combined with forward and backward tips. Numerical simulations under a certain feed rate and screw revolution speed were performed, and the mixing process was investigated using Lagrangian statistics. It was found that the four types had different mixing characteristics, and their mixing processes were explained by the coupling effect of drag flow with the disk staggering and pitched-tip and pressure flows, which are controlled by operational conditions. The use of a pitched-tip effectively controls the balance of the pressurization and mixing ability.     

Preparation of ultrafine polyurethane fiber web by laser‐electrospinning combined with air blowing

Thermoplastic polyurethane fiber webs were prepared using a laser‐heated electrospinning process combined with air blowing. The effect of spinning conditions such as air flow rate and air temperature on fiber diameter and molecular weight was investigated. Although the average fiber diameter decreased with increased air flow rate at each air temperature, the diameter increased when the air flow rate was >15 NL min^?1. In addition, the fiber was comparatively thicker with an increase in the air temperature. The variation in the fiber diameter tends to increase with the air flow rate, and a reduction in the molecular weight of the fiber by thermal degradation was suppressed. The thinnest and most uniform fiber with a diameter of 0.9 µm and a diameter coefficient variation of 15% was obtained at an air temperature of 25°C under an air flow rate of 15 NL min^?1. This fiber also had a minimum of decreased molecular weight. POLYM. ENG. SCI., 54:2605–2609, 2014. © 2013 Society of Plastics Engineers     

Structure and Nonlinear Optical Properties of Lanthanide Borate Glasses

The third–order nonlinear optical susceptibility, χ⁽³⁾, of lanthanide (lanthanum, praseodymium, neodymium, and samarium) borate glasses has been measured by the third harmonic generation method. The structure of the present glass system has been studied by infrared and Raman spectroscopic methods. The network structures of the present Ln₂O₃–B₂O₃ glasses have been confirmed to be basically similar to each other. Praseodymium, neodymium, and samarium borate glasses exhibit χ⁽³⁾ values that are larger than lanthanum borate glasses, because of the optical resonance effect, in accordance with the f – f transition. Especially, the χ⁽³⁾ value for 30Pr₂O₃·70B₂O₃ glass is 1.8 × 10⁻¹² esu, which is a factor of ∼60 larger than that of SiO₂ glass. This striking enhancement of χ⁽³⁾ is mainly attributed to the large transition moment to the first excitation state.     

Superior performance of non-activated multi-walled carbon nanotube polymer actuator containing ruthenium oxide over a single-walled carbon nanotube

The electrochemical and electromechanical properties of actuators developed using a non-activated multi-walled carbon nanotube (MWCNT)–ionic liquid (IL) gel electrode containing ruthenium oxide (RuO₂) were compared with only-MWCNT and only-single-walled carbon nanotube (SWCNT) based actuators. The double-layer capacitance of the non-activated MWCNT electrode containing RuO₂ was larger than that of the only-MWCNT electrode. The non-activated MWCNT polymer actuator containing RuO₂ surpassed the performance of the only-MWCNT and only-SWCNT actuators in terms of the strain and maximum generated stress. Both MWCNTs and RuO₂ were required to produce large strain and quick response actuators that surpassed the performance of the only-SWCNT polymer actuator and exhibited characteristics sufficient for practical applications (e.g. tactile display).     

A Comprehensive Study of the Interaction between Peptidoglycan Fragments and the Extracellular Domain of Mycobacterium tuberculosis Ser/Thr Kinase PknB

The Mycobacterium tuberculosis Ser/Thr kinase PknB is implicated in the regulation of bacterial cell growth and cell division. The intracellular kinase function of PknB is thought to be triggered by peptidoglycan (PGN) fragments that are recognized by the extracytoplasmic domain of PknB. The PGN in the cell wall of M. tuberculosis has several unusual modifications, including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues and amidation of d -Glu in the peptide chains. Using synthetic PGN fragments incorporating these diverse PGN structures, we analyzed their binding characters through biolayer interferometry (BLI), NMR spectroscopy, and native mass spectrometry (nMS) techniques. The results of BLI showed that muropeptides containing 1,6-anhydro-MurNAc and longer glycan chains exhibited higher binding potency and that the fourth amino acid of the peptide stem, d -Ala, was crucial for protein recognition. Saturation transfer difference (STD) NMR spectroscopy indicated the major involvement of the stem peptide region in the PASTA-PGN fragment binding. nMS suggested that the binding stoichiometry was 1:1. The data provide the first molecular basis for the specific interaction of PGN with PknB and firmly establish PGNs as the effective ligands of PknB.     

Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.