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FAS2 encoding α subunit of fatty acid synthetase is one of essential genes in S. cerevisiae, and theFAS2‐G1250S mutation decreases carbon chain elongation activity during fatty acid synthesis, resulting in high productivity of ethyl caproate. Evaluation for mutant gene dosage using polyploids with isogenic background revealed the trade‐off relationship between ethyl caproate productivity and cell growth rate. We further demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains., Summary The yeast Saccharomyces cerevisiae, widely used for ethanol production, is one of the best‐understood biological systems. Diploid strains of S. cerevisiae are preferred for industrial use due to the better fermentation efficiency, in terms of vitality and endurance as compared to those of haploid strains. Whole‐genome duplications is known to promote adaptive mutations in microorganisms, and allelic variations considerably contribute to the product composition in ethanol fermentation. Although fermentation can be regulated using various strains of yeast, it is quite difficult to make fine adjustment of each component in final products. In this study, we demonstrate the use of polyploids with varying gene dosage (the number of copies of a particular gene present in a genome) in the regulation of ethanol fermentation. Ethyl caproate is one of the major flavouring agents in a Japanese alcoholic beverage called sake. A point mutation in FAS2 encoding the α subunit of fatty acid synthetase induces an increase in the amount of caproic acid, a precursor of ethyl caproate. Using the FAS2 as a model, we generated and evaluated yeast strains with varying mutant gene dosage. We demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains. Productivity of ethyl caproate gradually increased with mutant gene dosage among tetraploid strains. This approach can potentially be applied to a variety of yeast strain development via growth‐based screening.

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy., Unlike canonical macroautophagy, alternative autophagy does not require the factors Atg5 and Atg7. Here, the authors show that Wipi3 is essential for alternative autophagy, but not for canonical autophagy, and that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.

Various clinical and experimental findings have revealed the causal relationship between autophagy failure and oncogenesis, and several mechanisms have been suggested to explain this relationship. We recently proposed two additional mechanisms: centrosome number dysregulation and the failure of autophagic cell death. Here, we detail the mechanical relationship between autophagy failure and oncogenesis., Various clinical and experimental findings have revealed the causal relationship between autophagy failure and oncogenesis. Here, we detail their mechanical relationship.

Control-Flow Integrity (CFI) is a class of defensive techniques against control-flow attacks such as Return-Oriented Programming. We propose a light-weight CFI scheme for RTOS-based applications, TZmCFI, which utilizes TrustZone for Armv8-M, a hardware-assisted security feature for embedded systems with tight resource constraints. TZmCFI embodies several existing CFI techniques to provide a comprehensive protection. The traditional shadow stack technique is used to ensure stack integrity and validate function returns. To protect exception handlers, TZmCFI extends shadow exception stacks, which are a variant of the traditional shadow stack technique we proposed in our previous work, for RTOS integration and performance improvement. We conducted an experiment on Arm Versatile Express Cortex-M Prototyping System (V2M-MPS2+) to evaluate the run-time overhead of the proposed system.

Aircraft flight control usually requires driving flaps and ailerons. However, the air drags increase significantly due to the corners of flaps and aileron. Especially, the gap between mother wing and flap / aileron causes a drag increase. Therefore, studies are being conducted on morphing wings that smoothly and greatly deform the wing surface. For aircraft wing, it is needless to say that strength is important to sustain lift and drag for the aircraft during the flight. For morphing wings, in addition, actuators must be mounted inside the wing to enable the morphing deformation. Moreover, for the aircraft wing, weight is quite important. Therefore, carbon fiber reinforced plastic (CFRP) is currently most suitable for aircraft wing structural materials. However, it is difficult to mold CFRP so that it has sufficient strength and can be morphed. In this study, by using CFRP, the morphing wing structure was prototyped with targeting a small unmanned aerial vehicle (UAV) weighing 3 kg. The CFRP lattice structure that enables morphing deformation was designed and manufactured by applying the electrodeposition resin molding (ERM) method which was developed by the authors. In the ERM method, firstly, the carbon fiber was fixed with a jig according to the designed morphing wing structure, and immersed in the electrodeposition solution. Secondly, the epoxy polymer particle in the solution were electrophoresed and impregnated between carbon fibers. After thermal curing, the morphing wing structure was fabricated. Further, the loading-unloading torsion and bending tests of the morphing wing structure were carried out. Smooth morphing deformation and sufficient strength were confirmed.

Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.

Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.

Backbone circularization is an effective technique for protein stabilization. Here, we investigated the effect of a connector, an engineered segment that connects two protein termini, on the conformational stability of previously designed circularized variants of granulocyte colony-stimulating factor (G-CSF). Heat tolerance and chemical denaturation analyses revealed that aggregation resistance and thermodynamic stability of the circularized variants were superior to those of linear G-CSF. Crystal structure and molecular dynamics (MD) simulation of the most thermodynamically stable variant (C166) revealed a high number of intramolecular hydrogen bonds in both the connector region and Helix D adjacent to the connector region in the folded structure. MD simulations and theoretical calculations involving different force fields indicated a reduction in the main chain entropy of C166 in the unfolded state and increase in the intramolecular hydrogen bond energy of C166 in the folded structure. Although backbone circularization is usually considered to alter chain entropy of the unfolded state, the data indicated that it could also improve the conformational enthalpy of the folded state. Further structural examination of the connector region confirmed that protein design based on a statistical analysis of local structures is an effective approach for predicting an optimum connector length to improve the conformational stability of backbone-circularized proteins. Protein design using backbone circularization with an optimum connector length will be useful for the development of effective and safe protein therapeutics. DATABASE: Structural data are available in Protein Data Bank under the accession number 5ZO6.

Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on

Cellulose nanofibers (CNFs) have high strength. Therefore, many studies have been conducted to improve the mechanical properties of carbon fiber reinforced plastics (CFRP) by dispersing CNF in the matrix. However, since CNFs are hydrophilic, a hydrophobic treatment is required. Previously, authors showed the bending strength of CFRP could be enhanced by CNFs without a hydrophobic treatment. Applying the electro-activated deposition resin molding (ERM) method to fabricate CFRP, CNFs can be applied on the surface since the electro-activated deposition solution is essentially water-based and contains a polymer with epoxy groups. In this study, the CNF sheet was prepared by drying CNF dispersion in advance. Shortly after the ERM, the CNF sheet was applied to the surface of the CFRP. The mechanical properties of the CFRP were enhanced, the maximum elastic modulus and tensile strength were 2.3 times and 1.7 times of the CFRP specimen without the CNF sheet, respectively.

A multi-objective optimization method for the laminated composite fabricated by a tailored fiber placement machine that is an application of embroidering machine is presented. The mechanical properties of composite with curvilinear fibers including stiffness, volume fraction, and density are variable depending on curvatures of fibers. The present study first measures the relation between curvatures and mechanical properties. The measured results indicate that the stiffness of composite decreases linearly as the curvature increases. Then, the obtained relation is applied to the multi-objective optimization where the maximum principal strain and magnitude of curvature are employed as objective functions. Obtained Pareto optimum solutions are widely distributed ranging from the solutions with curvilinear fibers to those with straight fibers, and the curvilinear fiber has still advantages over straight fiber even its weakened stiffness.

Many researches on strengthening the matrix of Carbon Fiber Reinforced Plastic (CFRP) using cellulose nanofibers (CNFs) have been reported. However, hydrophobic treatment to modify the surface of C...

Aggregates of therapeutic proteins that can contaminate drug products during manufacture is a growing concern for the pharmaceutical industry because the aggregates are potentially immunogenic. Electron microscopy is a typical, indispensable method for imaging nanometer- to micrometer-sized structures. Nevertheless, it is not ideal because it must be performed with ex situ monitoring under high-vacuum conditions, where the samples could be altered by staining and drying. Here, we introduce a scanning electron-assisted dielectric microscopy (SE-ADM) technique for in-solution imaging of monoclonal immunoglobulin G (IgG) aggregates without staining and drying. Remarkably, SE-ADM allowed assessment of the size and morphology of the IgG aggregates in solution by completely excluding drying-induced artifacts. SE-ADM was also beneficial to study IgG aggregation caused by temporary acid exposure followed by neutralization, pH-shift stress. A box-counting analysis of the SE-ADM images provided fractal dimensions of the larger aggregates, which complemented the fractal dimensions of the smaller aggregates measured by light scattering. The scale-free or self-similarity nature of the fractal aggregates indicated that a common mechanism for antibody aggregation existed between the smaller and larger aggregates. Consequently, SE-ADM is a useful method for characterizing protein aggregates to bridge the gaps that occur among conventional analytical methods, such as those related to in situ/ ex situ techniques or size/morphology assessments.

In press processing, quality inspection of a product is often carried out for each lot in the post process stage. When a failure occurs, it may result in a large number of defective products due to the fast processing speed. In order to prevent this, it is ideal to immediately stop the processing just after the defect occurs. Therefore, confirming the processing state in-process is required. This study proposes a new quality inspection method for deep drawing processes by using the count rate of acoustic emission (AE) signals. To analyze the AE count, deep learning, which is a multilayered neural network, is employed to recognize defects during a deep drawing process. The material used was a ductile material of cold rolled steel plate and is relatively difficult to find cracks during the deep drawing process. Characteristics were clarified by analysis of AE counts at plastic deformation and brake of material, and performing the forming state recognition experiment by a multilayer neural network (deep learning) showed a maximum recognition rate of 97.3%. High recognition rate was obtained despite the small number of data used.

For the efficient manufacturing of carbon fiber reinforced plastics (CFRP), the electro-activated deposition resin molding (ERM) method is developed. The carbon fiber fabric is immersed in an electro-activated deposition solution containing polymer with epoxy groups. By energization , an epoxy resin is precipitated on the surface of the carbon fiber, and impregnation occurred in the solution. In this study, applying the ERM method, CFRP specimen with the hole was manufactured. Furthermore, the reinforcement by arranging the fiber circumferentially around the hole was installed. The carbon fiber fabric was sewn using the tailored fiber placement (TFP) machine. As a results, the effect of the reinforcement was confirmed. The tensile strength of CFRP with the hole and the reinforcement was the same as in the case without the hole. From the finite element analysis , it was confirmed that the reinforcement around the hole reduced the stress concentration.

Cellulose nanofiber (CNF) is high strength and lightweight, additionally, it is produced by sustainable natural resources such as wood. Therefore, it has been reported the mechanical properties of CFRP can be improved by dispersing CNF in the matrix. However, as CNF is hydrophilic, a hydrophobic treatment was applied in many previous studies. For efficient CFRP manufacturing, the authors have developed an electrochemical resin molding method by using aqueous electro-activated deposition which contains the polymer having an epoxy group. In this method, the carbon fiber fabric is immersed in the electro-activated deposition solution and energized, epoxy resin is precipitated around the carbon fiber and impregnated. In this study, shortly after electro-activated deposition, CNF without a hydrophobic treatment was applied to the surface of the resin-impregnated carbon fiber fabric. The mechanical properties of CFRP could be enhanced, and optimum weight fraction of CNF was revealed.

Aggregation of protein therapeutics can lead to immunogenicity and loss of function in vivo. Its effective prevention requires an understanding of the conformational and colloidal stability of protein and the improvement of both. Granulocyte colony-stimulating factor (G-CSF), which is one of the most widely used protein therapeutics, was previously shown to be conformationally stabilized by connecting its N- and C-termini with amide bonds (backbone circularization). In this study, we investigated whether circularization affects the colloidal stability of proteins. Colloidal stability was indirectly assessed by analyzing the aggregation behavior of G-CSF variants using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). Consequently, we found that the unfolded structure of circularized G-CSF was more compact than non-circularized G-CSF, and that backbone circularization improved its aggregation resistance against chemical denaturation by guanidine hydrochloride (GdnHCl). The improved aggregation resistance suggests that the expansion tolerance of circularized G-CSF in the unfolded state increased its colloidal stability. Thus, backbone circularization is an excellent method for enhancing the colloidal and the conformational stability of protein with minimal sequence changes. It is therefore expected to be effective in extending the storage stability of protein therapeutics, enhancing their biological stability.

The scope of embedded systems used by multi-core processors is widely applied to various products that require control, such as home appliances, industrial devices, transportation devices, and communication terminals. Model-Base Parallelization technology was proposed towards efficient parallelization to implement a control system in the introduction of multi-core processors. By applying MBP, it is possible to parallelization even details that people are not aware of, but the control system with numerous causal relationships still does not sufficiently utilize multi-core. Therefore, a method of inserting a time-delay is being studied as a method for improving the parallelization of multi-cores.In this paper, We describe the insertion location that improves parallelization and does not affect the control performance by inserting time-delay to implement the vector control system of PMSM on multi-core efficiently.

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy., Unlike canonical macroautophagy, alternative autophagy does not require the factors Atg5 and Atg7, but does require Ulk1. Here the authors show that phosphorylation of Ulk1 at Ser746 by RIPK3 is required for alternative autophagy initiation at the Golgi in response to genotoxic stress.

The I-shaped cross-sectional beam of CFRP (CFRP I-beam) is usually manufactured by the continuous protrusion method. Carbon fibers can only be arranged in the longitudinal direction. The CFRP I-bea...

The grafting of active peptides onto structurally stable scaffold proteins is effective for the generation of functional proteins. In this study, we aimed to develop a grafting method using ubiquitin as a scaffold protein. Ubiquitin is a small protein consisting of 76 amino acid residues that is highly stable against heat and pH stress, which are desirable characteristics for a scaffold protein. Moreover, its structure is maintained even if it is split or additional residues are inserted. Therefore, we assumed that grafting of an active peptide into ubiquitin would result in a functional protein. As a proof of concept, we developed the ubiquitin-based binder (UbB), into which the p53 (17-28) peptide was inserted between Ile36 and Pro37. The p53 (17-28) peptide, utilized as a model active peptide in this work, is known to bind to mouse double minute 2 homolog (Mdm2). Size exclusion chromatography and circular dichroism indicated that UbB maintained a similar structure to that of ubiquitin. The affinity for Mdm2 measured by surface plasmon resonance was 292 times greater than that of the p53 (17-28) peptide. These observations indicate that ubiquitin is a robust scaffold for peptide grafting. We hope that this study will aid further development of ubiquitin-based protein engineering.

The present study minimizes the number of layers of wing shaped plates fabricated by laminated composite (CFRP) under aeroelastic constraint which keeps stable flutter speed. The finite element analysis (FEA) for the aeroelastic characteristics requires considerable calculation effort. Therefore, the response surface method (RSM) is utilized to reduce the computational time for optimization where an approximate expression uses bending rigidity of composite plates as input and flutter speed as output. Then, distributed genetic algorithm (DGA) is employed as an optimizer and minimizes numbers of layers of composite plates by designing fiber orientation angles. The optimum wing model shows large amount of weight reduction and enough flutter speed in less computational time than without RSM.

A more efficient manufacturing process for CFRP is expected to save energy, time and cost for production. However, it is difficult to impregnate the resin efficiently and uniformly between the carbon fibers, micro void tend to remains and strength decreases. In this study, a novel manufacturing method of CFRP by using the electro-activated deposition resin molding was developed. The non-crimp carbon fiber fabric was immersed in the electro-activated deposition solution, that containing epoxy groups. And, the fabric was energized and heat cured. The electro-activated deposition conditions and tensile properties were investigated for the CFRPs. It was confirmed that resin could be impregnated for multiple plies of the non-crimp carbon fiber fabric without using autoclave and injecting viscous resin like VARTM. Comparing to the traditional manufacturing process, a step of previously mixing an epoxy resin prepolymer and a curing agent was not required. And, a process of vacuum suction could be eliminated, so, wrapping non-crimp carbon fiber fabric with vacuum bag was not necessary.

An accurate and straightforward analytical method is proposed for the free vibration of a laminated composite rectangular plate with blended layers. The plate with blended layers is defined here as a laminated plate where some of the outer layers are composed of multiple subareas with different orientation angles of straight fibers. A finite element code is developed as well to verify accuracy of the proposed method. In numerical comparison, the natural frequencies calculated by the present method agree well with those from the FEM code for rectangular plates with two, three, four and five subareas. Subsequently, the use of this analytical method is tested in vibration optimization, i.e. design for maximization of fundamental frequencies. This frequency optimization is formulated by changing the fiber orientation angles in multiple subareas simultaneously, and is carried out by using permutation in simple cases and by Genetic Algorithm (GA) for a larger number of frequency calculations. It is observed that introduction of blended layers is very effective approach to design the dynamic characteristics of laminated composite plates .

Recently, theoretical and experimental methods were proposed for organizing carbon fibers in straight and curvilinear arrangements to obtain a uniform stress distribution in the design of three-dimensional (3D) carbon fiber reinforced thermosetting plastic (CFRP). In this study, to establish an efficient CFRP manufacturing method and realize the curvilinear arrangement of carbon fibers, we develop an electrodeposition resin molding technique. That is, immersing a noncrimp fabric in the electrodeposition solution and energizing electricity, the fabric is impregnated by the resin; thus, a CFRP with curvilinear carbon fibers can be efficiently produced without autoclaving and vacuum packing. We then confirm the optimal electrodeposition conditions for maximizing the tensile strength of the CFRP, and obtain the 3D shape of the CFRP by keeping the noncrimp fabric in solution along the mold.

A cycle of dropping and shaking a vial containing antibody solution was reported to induce aggregation. In this study, antibody solutions in glass prefillable syringes with or without silicone oil lubrication were subjected to the combined stresses of dropping and shaking, using a friability testing apparatus. Larger numbers of subvisible particles were generated, regardless of silicone oil lubrication, upon combination stress exposure than that with shaking stress alone. Nucleation of antibody molecules upon perturbation by an impact of dropping and adsorption of antibody molecules to the syringe surface followed by film formation and antibody film desorption were considered key steps in the particle formation promoted by combination stress. A larger number of silicone oil droplets was released when silicone oil-lubricated glass syringes containing phosphate buffer saline were exposed to combination stress than that observed with shaking stress alone. Polysorbate 20, a non-ionic surfactant, effectively reduced the number of protein particles, but failed to prevent silicone oil release upon combination stress exposure. This study indicates that stress-stability assays using the friability testing apparatus are effective for assessing the stability of biopharmaceuticals under the combined stresses of dropping and shaking, which have not been tested in conventional stress-stability assays.

Backbone circularization of protein is a powerful method to improve its structural stability. In this paper, we presumed that a tight connection leads to much higher stability. Therefore, we designed circularized variants of a granulocyte-colony stimulating factor (G-CSF) with a structurally optimized terminal connection. To estimate the appropriate length of the connection, we surveyed the Protein Data Bank to find local structures as a model for the connecting segment. We set the library of local structures composed of “helix–loop–helix,” subsequently selected entries similar to the G-CSF terminus, and finally sorted the hit structures according to the loop length. Two, five, or nine loop residues were frequently observed; thus, three circularized variants (C163, C166, and C170) were constructed, prepared, and evaluated. All circularized variants demonstrated a higher thermal stability than linear G-CSF (L175). In particular, C166 that retained five connecting residues demonstrated apparent Tm values of...