Development of a yeast cell surface display method using the SpyTag/SpyCatcher system

生体内タンパク質ライゲーションを用いた新規細胞表層ディスプレイ法の開発 --新しい手法によるタンパク質工学の進展--. 京都大学プレスリリース. 2021-05-28.Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution

A Clinical Trial of Kampo Formulae for the Treatment of Symptoms of Yusho, a Poisoning Caused by Dioxins and Related Organochlorine Compounds

The objective of this study was to evaluate the effectiveness of traditional herbal medicines (Kampo) on the symptoms of Yusho. Yusho is a mass food poisoning that was caused by ingestion of rice oil contaminated with dioxins and related organochlorines in 1968. Patients with Yusho suffer from skin symptoms (acneform eruptions, liability to suppuration and pigmentation), respiratory symptoms (cough and expectoration of sputum), neurological symptoms (numbness and paresthesia of extremities), arthralgia and general fatigue, and no effective treatment has yet been developed. In this clinical trial, four Kampo formulae (Bakumondo-to, Keigai-rengyo-to, Gosha-jinki-gan and Hochu-ekki-to) were administered to four representative Yusho symptoms (respiratory, skin, neurological symptoms and general fatigue), respectively. Twenty-seven Yusho patients were enrolled and two formulae were administered to each patient for half-a-year each. The effectiveness of Kampo formulae was estimated by changes in the intensity of symptoms measured by a visual analogue scale (VAS) of 100 mm recorded at baseline and after administration of each formula. The influence of Kampo formulae on patients' quality of life (QOL) was also assessed by the SF-36 (NBS). Twenty-five patients completed the treatment. Bakumondo-to significantly improved respiratory symptoms as well as patients' QOL in the context of vitality, compared with other formulae. In contrast, Hochu-ekki-to impaired patients' QOL in the context of physical functioning and vitality, compared with other formulae. This study demonstrated for the first time that a Kampo formula Bakumondo-to is useful for treating respiratory symptoms caused by dioxins

Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies

遊離型抗体の構造活性相関解析を迅速に評価可能とする新手法を開発. 京都大学プレスリリース. 2021-11-08.Optimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence–function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody–antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence–function relationships

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates

Paracrine IL-33 Stimulation Enhances Lipopolysaccharide-Mediated Macrophage Activation

BACKGROUND: IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation

Vibration testing based on impulse response excited by laser ablation

This paper proposes an innovative vibration testing method based on impulse response excited by laser ablation. In conventional vibration testing using an impulse hammer, high-frequency elements of over tens of kilohertz are barely present in the excitation force. A pulsed high-power YAG laser is used in this study for producing an ideal impulse force on a structural surface. Illuminating a point on a metal with the well-focused YAG laser, laser ablation is caused by generation of plasma on the metal. As a result, an ideal impulse excitation force generated by laser ablation is applied to the point on the structure. Therefore, it is possible to measure high-frequency FRFs due to the laser excitation. A water droplet overlay on the metal is used to adjust the force magnitude of laser excitation. An aluminum block that has nine natural frequencies below 40 kHz is employed as a test piece. The validity of the proposed method is verified by comparing the FRFs of the block obtained by the laser excitation, impulse hammer, and finite element analysis. Furthermore, the relationship between accuracy of FRF measurements and sensitivity of sensors is investigated

Frequency response function measurements of rotational degrees of freedom using a non-contact moment excitation based on nanosecond laser ablation

Frequency response functions (FRFs) are generally measured by investigating the input-output relationship of a target structure. An input is applied to the target structure and the output is measured. Typical vibration tests measure inputs as excitation forces using load cells or similar devices and outputs as velocities or accelerations using laser Doppler vibrometers or accelerometers. Each input or output has three translational degrees of freedom (DOF). Consequently, experimental models established based on such tests have three translational DOF. However, numerical models using finite element analysis (FEA) for existing structures have six DOF at each node (three translational and three rotational DOF). Therefore, numerical models using FEA and experimental models have gaps in their DOF, leading to diverse problems. This study realizes a non-contact measurement method for rotational DOF FRFs using an impulse excitation force generated by laser ablation (LA) where the input is a moment and the output is the velocity. A laser beam radiated from an Nd:YAG pulse laser emitter is divided using a half mirror, halving the laser pulse energy. Then, these laser beams are radiated on two points near a target structure's excitation point. The simultaneous occurrence of LA at these two points realizes a moment that acts on the target structure in a non-contact manner. This experiment uses a 5052 aluminum alloy plate where one end is fixed as a test piece. Both the auto FRFs and cross FRFs for the rotational DOF between two arbitrary points on the test piece are measured. The FRFs obtained by our method agree well with those obtained by FEA, demonstrating its efficacy. (C) 2019 The Authors. Published by Elsevier Ltd

Damage detection in pipes based on acoustic excitations using laser-induced plasma

A health-monitoring system is proposed to detect holes drilled in a pipe based on laser plasma acoustic excitations and acoustic measurements. In this system, an acoustic excitation is applied to a pipe via a laser-induced plasma in air generated by a high-power Nd:YAG pulse laser. Laser-induced plasmas can realize non-contact acoustic impulse excitations. A microphone is used to measure the time response of the acoustic pressure. In this study, we focus on the detection of a hole in the pipe. The reflection of the acoustic wave due to a hole drilled in the pipe induces a change in the time response of the acoustic pressure. Applying a continuous wavelet transform to the measured time response data with/ without the hole can locate the position of the hole. This study demonstrates the effectiveness of the present damage detection method based on an acoustic excitation using a laser-induced plasma

Damage detection in membrane structures using non-contact laser excitation and wavelet transformation

In this paper, a vibration testing and health monitoring system based on an impulse response excited by laser is proposed to detect damage in membrane structures. A high power Nd: YAG pulse laser is used to supply an ideal impulse to a membrane structure by generating shock waves via laser-induced breakdown in air. A health monitoring apparatus is developed with this vibration testing system and a damage detecting algorithm which only requires the vibration mode shape of the damaged membrane. Artificial damage is induced in membrane structure by cutting and tearing the membrane. The vibration mode shapes of the membrane structure extracted from vibration testing by using the laser-induced breakdown and laser Doppler vibrometer are then analyzed by 2-D continuous wavelet transformation. The location of damage is determined by the dominant peak of the wavelet coefficient which can be seen clearly by applying a boundary treatment and the concept of an iso-surface to the 2-D wavelet coefficient. The applicability of the present approach is verified by finite element analysis and experimental results, demonstrating the ability of the method to detect and identify the positions of damage induced on the membrane structure