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Glassin is a water-soluble protein from the siliceous skeleton of a marine sponge that adsorbs tightly to silica at pH 7.0-9.0 and accelerates silica particle formation from silicic acid. Glassin comprises three distinct domains: a His and Asp-rich (HD) domain, a Pro-rich (P) domain, and a His and Thr-rich (HT) domain. Here, we investigated the roles of these three domains in silica adsorption by using glutathione S-transferase (GST) fused with glassin or with each domain. GST fused with the HD domain exhibited tight adsorption, equivalent to that of GST fused with the full-length glassin sequence at values above 7.0. The apparent K

Background/aim We evaluated the efficacy of "the tumor immune microenvironment (TIME) classification" for predicting clinical response to immune checkpoint inhibitors (ICIs) in patients with non-small cell lung cancer (NSCLC). In addition, we aimed to evaluate the "modified TIME classification", which adds the vascular endothelial growth factor (VEGF) status to TIME. Materials and methods Programmed cell death receptor ligand-1 (PD-L1), CD8 T cell tumor-infiltrating lymphocytes (CD8+TILs) count and VEGF expression analyses were performed using immuno - histochemistry in 44 patients who had undergone ICI monotherapy. Results Regarding TIME classification, type-I (PD-L1 high and CD8+TILs high) had a significantly higher response than the other types. Using the modified TIME classification, type-IA (PD-L1 high, CD8+TILs high, and VEGF low) had a significantly higher response than the other types. Conclusion The modified TIME classification, which adds tumor VEGF expression to "the TIME classification", could be useful in predicting clinical response to ICI monotherapy.

Vitamin BTomato plants were grown for 70 days in hydroponic culture pots and treated with 5 μm of cyanocobalamin on days 1-24 after the fruiting, and then harvested for tomato fruits. The ripened tomato fruits contained 4.0 × 10The present study highlights the use of a cyanocobalamin-supplementation system for the production of B

Background Immune checkpoint inhibitors targeting the programmed cell death‐1 (PD‐1)/PD‐1 ligand 1 (PD‐L1) axis have shown promising results in patients with nonsmall cell lung cancer (NSCLC). One major PD‐L1 inducer is IFNγ, which is secreted by T cells and NK cells. Importantly, IFNγ‐induced PD‐L1 is one of the major mechanisms by which cancer cells escape host immunity. Methods Here, we found that the NSCLC cell line, LC‐2/ad, has a unique character; the PD‐L1 expression in these cells is up‐regulated by both IFNγ and epidermal growth factor (EGF). Results Comparative analysis of the cell signaling pathway showed that IFNγ activates STAT1 signaling, while EGF activates AKT, MAPK, and ribosomal protein S6 kinase in LC‐2/ad cells. IFNγ‐induced PD‐L1, but not EGF‐induced PD‐L1, was clearly blocked by the JAK‐STAT inhibitor tofacitinib. Interestingly, IFNγ decreased the expression of NK cell‐activating ligands while increasing the expression of MHC class I molecules, resulting in a phenotype that can easily escape from NK cells, theoretically. Finally, we showed that IFNγ stimuli attenuated NK cell‐mediated cytotoxicity in LC‐2/ad cells, which was, however, blocked by tofacitinib. Conclusions Taken together, our study shows that tofacitinib blocks the IFNγ‐induced transformation from an NK cell‐sensitive phenotype to an NK cell‐resistant one in IFNγ‐reacted LC‐2/ad cells, thereby implicating that tofacitinib may be a promising agent to overcome IFNγ‐induced tumor immune escape, although it may be adapted to the limited number of NSCLC patients., The JAK‐STAT inhibitor tofacitinib blocks the IFNγ‐induced transformation from an NK cell‐sensitive phenotype to an NK cell‐resistant one in IFNγ‐reacted LC‐2/ad cells, thereby implicating that tofacitinib may be a promising agent to overcome IFNγ‐induced tumor immune escape, although it could be adapted to the limited number of NSCLC patients.

Introduction: Programmed cell death-1 (PD-1) inhibitors effectively treat NSCLC and prolong survival. Robust biomarkers for predicting clinical benefits of good response and long survival with anti–PD-1 therapy have yet to be identified; therefore, predictive biomarkers are needed to select patients with benefits. Methods: We conducted a prospective study to explore whether serum antibody against NY-ESO-1 and/or XAGE1 cancer-testis antigens predicted primarily good clinical response and secondarily long survival with anti–PD-1 therapy for NSCLC. The serum antibody was detected by enzyme-linked immunosorbent assay, and tumor immune microenvironment and mutation burden were analyzed by immunohistochemistry and next-generation sequencing. Results: In the discovery cohort (n ¼ 13), six antibodypositive NSCLC cases responded to anti–PD-1 therapy (two complete and four partial responses), whereas seven antibody-negative NSCLC cases did not. Antibody positivity was associated with good response and survival, regardless of tumor programmed death ligand 1 (PD-L1) expression, mutation burden, and CD8þ T-cell infiltration. In the validation cohort (n ¼ 75), 17 antibody-positive NSCLC cases responded well to anti–PD-1 therapy as compared with 58 negative NSCLC cases (objective response rate 65% versus 19%, p ¼ 0.0006) and showed significantly prolonged progression-free survival and overall survival. Antibody titers highly correlated with tumor reduction rates. In the multivariate analysis, response biomarkers were tumor programmed death ligand 1 expression and antibody positivity, and only antibody positivity was a significantly better predictive biomarker of progression-free survival (hazard ratio ¼ 0.4, p ¼ 0.01) and overall survival (hazard ratio ¼ 0.2, p ¼ 0.004). Conclusions: Our results suggest that NY-ESO-1 and/or XAGE1 serum antibodies are useful biomarkers for predicting clinical benefits in anti–PD-1 therapy for NSCLC and probably for other cancers., This work was supported by grants from JSPS KAKENHI (15K09235, 16K18463, 16K21533, 18K15226, and 18K07306) to Drs. Oka, Ohue, and Kurose; by grants from the Takeda Science Foundation to Drs. Ohue and Kurose; by a grant from the Medical Research Encouragement Prize of The Japan Medical Association to Dr. Ohue; by a grant from the Okayama Health Foundation to Dr. Ohue; and by grants from Kawasaki Medical School to Drs. Oka, Ohue, and Kurose. Dr. Oka received collaborative research funds from the University of Tokyo and Thyas Co., Ltd. The Department of Immuno-Oncology is endowed by Pole Star Co., Ltd.

Cellulosimicrobium sp. NTK2 (NTK2 strain) was isolated as a chitinolytic bacterium from mature compost derived from chitinous waste. The growth of the NTK2 strain was enhanced by supplementation of the culture medium with 2% crystalline chitin. Approximately 70% of the supplemented crystalline chitin was degraded during cultivation. Whole genome analysis of the NTK2 strain identified eight chitinases and two chitin-binding proteins. The NTK2 strain secreted two bacterial extracellular solute-binding proteins, three family 18 glycosyl hydrolases and one lytic polysaccharide monooxygenase specifically in the presence of crystalline chitin. A chitinolytic enzyme with a molecular mass of 29 kDa on SDS-PAGE under native conditions was also secreted. This chitinolytic enzyme exhibited the largest band upon zymography but could not be identified. In an attempt to identify all the chitinases secreted by the NTK2 strain, we expressed recombinant versions of the proteins exhibiting chitinolytic activity in Escherichia coli. Our results suggest that the 29 kDa protein belonging to family 19 glycosyl hydrolase was expressed specifically in the presence of 2% crystalline chitin.

d-Stereospecific amidohydrolase from Streptomyces sp. 82F2 (DAH) recognizes d-amino acyl ester derivatives as substrates and catalyzes hydrolysis and aminolysis to yield d-amino acids and d-amino acyl peptides or amide derivatives, respectively. Crystallographic analysis has revealed that DAH possesses a large cavity with a small pocket at the bottom. Because the pocket is close to the catalytic center and is thought to interact with substrates, we examined the function of the eight residues that form the pocket in terms of substrate recognition and aminolysis via mutational analysis. Formation of the acyl-enzyme intermediate and catalysis of aminolysis by DAH were changed by substitutions of selected residues with Ala. In particular, I338A DAH exhibited a significant increase in the condensation product of Ac-d-Phe methyl ester and 1,8-diaminooctane (Ac-d-Phe-1,8-diaminooctane) compared with the wild-type DAH. A similar effect was observed by the mutation of Ile338 to Gly and Ser. The pocket shapes and local flexibility of the mutants I338G, I338A, and I338S are thought to resemble each other. Thus, changes in the shape and local flexibility of the pocket of DAH by mutation presumably alter substrate recognition for aminolysis.

Recent research has shown that several mechanisms induce tumor progression and a novel cause is the escape of tumor cells from immune surveillance (1). Natural killer (NK) cells play an important role in immunosurveillance against cancer cells (2), and the immunosurveillance by NK cells involves NK cell-mediated tumor cytotoxicity, which depends on the balance between NK cell-activating and -inhibiting ligands expressed in tumor cells (3).

In this study, we investigate the nature of values change of business professionals in four powerbroker countries (China, Japan, Russia, United States) over a two-decade period (1992–2011; Young, Ahlstrom, Bruton, & Rubanik, 2011). We revisit the crossvergence argument for values evolution as initially proposed by Ralston, Gustafson, Cheung, and Terpstra (1993) and Ralston, Holt, Terpstra, and Yu (1997) in their cross-sectional studies. However, in this study we use a multi-period perspective that allows us to investigate Ralston’s (2008) further sophistication of crossvergence theory, which includes static-, conforming-, and deviating-crossvergence as the various forms that crossvergence might take over time. Our findings indicate that values orientations across the Chinese, Japanese and American business professionals have been fairly consistent (static) or have become more similar (conforming). Conversely, Russian managerial values have often become significantly more different (deviating) from those in the other three countries. We conclude with a discussion of the implications that this study has for the crossvergence theory of values evolution and the future research opportunities that it identifies.

現職中高数学教員のための数学科教育法の研修について考察する。特に、数学教育におけるアクティブラーニングの指導法ならびにそのための教材の研修に焦点をあてる。研修の実践として、神奈川県の私学数学科教員の研修団体「神奈川県私立中学高等学校協会 研究部 数学科専門委員会」所属の中学高校数学教員を対象とする研修を行った。研修の実施の経験を踏まえて、現職教員教育に対する方法と内容のあり方を検討・考察した。

The immune microenvironment of primary tumors has been reported to be one of the factors influencing the prognosis of patients with cancer. The tumor-infiltrating regulatory T cell (Treg) count has previously been revealed to be positively correlated with intratumoral cyclooxygenase-2 (Cox-2) expression, and was also associated with poor survival among patients with non-small cell lung cancer (NSCLC). In addition, the urinary levels of a prostaglandin E2 (PGE2) metabolite (PGE-M) were used as a biomarker in clinical trials of the Cox-2 inhibitor celecoxib. In the current prospective study, the association of urinary PGE2 and PGE-M levels with intratumoral Cox-2 expression and Treg count was examined in patients with NSCLC. A total of 21 patients with NSCLC who underwent complete resection of the tumor at Kawasaki Medical School Hospital (Kurashiki, Japan) were enrolled. Urine specimens were obtained prior to surgery in order to examine urinary PGE2 and PGE-M levels. A significant positive association was observed between urinary PGE2 levels and the intratumoral Treg count (P=0.023), but not the intratumoral Cox-2 expression levels. No significant associations were identified between urinary PGE2 levels and any of the other clinicopathological characteristics examined, including age, sex, smoking history, histology, tumor size, nodal status and disease stage. However, no significant association was observed between urinary PGE-M levels and the intratumoral Treg count (P=0.069) or Cox-2 expression. In conclusion, urinary PGE2 levels were positively correlated with intratumoral Treg counts in patients with NSCLC in the current study. This indicates that urinary PGE2 may be an improved biomarker, compared with PGE-M, for the prediction of intratumoral Treg numbers.

Glassin, a protein occluded in biosilica of the hexactinellid sponge Euplectela, promotes silica formation from silicic acid at room temperature and neutral pH and is characterized by its primary structure which consists of a tandem repeat carrying three distinct domains, histidine and aspartic acid-rich (HD) domain, proline-rich (P) domain, and histidine and threonine-rich (HT) domain. The present study aims to clarify the domain responsible for the promotion of silica formation and to demonstrate usefulness of glassin and its domain as a tag for purification and immobilization of recombinant proteins. When each domain was mixed with silicic acid at neutral pH, silica was formed with HD domain as well as glassin, or a single repeat, but not with P or HT domain. Neither of amino or carboxy-terminal half of HD domain induced silica formation. The amount of silica formed with HD domain was significantly lower than that of glassin or a single repeat. HD domain fused with HT domain raised the amount of silica formed, while a HD domain fused with P domain, a mixture of HD and P domains, or a mixture of HD and HT domains has little effect on the promotion of silica formation. Collectively, a minimum sequence for promotion of silica formation is HD domain, whose activity can be enhanced by HT domain through a topological effect. In addition, practicality of glassin and HD domain was demonstrated by fusion of these sequences to green fluorescent protein which was successfully purified with Ni affinity chromatography and immobilized on silica.

UL16-binding protein (ULBP) 1-6 and MHC class I chain-related molecule A and B (MICA/B) are NK group 2, member D (NKG2D) ligands, which are specifically expressed in infected or transformed cells and are recognized by NK cells via NKG2D-NKG2D ligand interactions. We previously reported that MICA/B overexpression predicted improved clinical outcomes in patients with resected non-small cell lung cancer (NSCLC). However, the clinicopathological features and prognostic significance of ULBPs in NSCLC remain unclear. Here,ULBP1-6 expression was evaluated based on immunohistochemistry of 91 NSCLC samples from patients following radical surgery. ULBPs were expressed by the majority of NSCLC. Either ULBP1 or ULBP2/5/6 overexpression was associated with squamous-cell carcinoma histology, whereas ULBP4 overexpression was associated with younger age and adenocarcinoma histology. Although overexpression of ULBP1-6 did not impact clinical outcomes in NSCLC patients, integrative profiling with cluster analysis classified patients into 3 subgroups based on the expression pattern of NKG2D ligands. The subgroup characterized by ULBP1 or ULBP2/5/6 high expressing but ULBP4 low expressing tumors showed poor overall survival. Taken together with previous results, NSCLC histological subtype strongly correlates with NKG2D ligands expression pattern. NKG2D ligands expression levels assessed by multiple immune parameters could predict clinical outcomes of patients with NSCLC.

Platinum‑based chemotherapy improves the clinical outcome of patients with non‑small cell lung cancer (NSCLC), although tumors often become refractory after treatment. Immunohistochemical staining was performed to investigate the expression levels of natural killer group 2 member D (NKG2D) ligands, programmed cell death‑1 ligand 1 (PD‑L1), and human leucocyte antigen (HLA)‑class I in tissue samples collected from 10 NSCLC patients who received platinum‑based chemotherapy followed by surgery. Additionally, the effects of repeated exposure to cisplatin on the expression of NKG2D ligands, PD‑L1 and HLA‑class I in NSCLC cell lines were assessed by flow cytometry. We found upregulation of PD‑L1 or downregulation of NKG2D ligands in 5 of the 10 NSCLC cases, leading to the attenuation of NK cell‑mediated tumor cell death. Moreover, upregulation of PD‑L1 or downregulation of HLA‑class I were observed in 6 cases, supporting tumor escape from T cell immunity. An in vitro assay showed that repeated exposure to cisplatin enhanced the expression of PD‑L1 and NKG2D ligands in NSCLC cell lines. Notably, interferon gamma (IFNγ) stimuli enhanced PD‑L1 expression while attenuated that of NKG2D ligands in NSCLC cell lines, which mimicked the results of the clinical study. Both IFNγ‑induced upregulation of PD‑L1 and downregulation of NKG2D ligands were blocked by the JAK‑STAT inhibitor tofacitinib. These findings suggested that the expression levels of NKG2D ligands, PD‑L1 and HLA‑class I in residual tumors after chemotherapy were affected by host immunity, resulting in an immunoescape phenotype. Blocking IFNγ‑induced tumor immunoescape by a JAK‑STAT inhibitor might be a promising treatment strategy for NSCLC.

Mergers and acquisitions (M&As) are increasingly important globally. In this environment, we observe that Japanese firms have started voluntarily announcing M&A budgets since the 2000s. Given that ...

本研究開発では、教科「情報」における実習の重要性を指摘し、大学の「情報科教育法」において3 つの実習の導入の重要性と必要性を指摘した。特に、教科「情報」では自立的な協調学習ができる生徒の育成は実習の効果の成否を決める重要な要素であり、そのため本授業「情報科教育法」では3 つの実習を導入した研究開発をしていること、その実際の研究開発内容として、2 つのロング・プロジェクトと代表的なショート実習の概略を示し、3 つの実習の中身を具体的に示した。

本論文では科学技術振興財団のグローバルサイエンスキャンパスの採択を受けて2015 年に東京理科大学で行われている卓越した意欲・能力を有する生徒の教育プログラムを分析し、それに基づき数学分野における才能教育の提案とそれを目的とする教材の提案をするものである。本論文ではアクティブラーニングを通じた才能教育に注目する。

As environmental change accelerates and future uncertainty increases, implementation of strategy inherently involves continuous adjustment and modification. To meet the need for further research on the critical role of communication, this article contributes to the literature by examining the relationship between communication and strategy implementation. I propose that senders’ bias, which refers to the overestimation of the quality of communication (i.e., degree of sharing) with organizational members by senders (i.e., top managers), is a fundamental implementation problem. Thus, top managers’ perceived degree of communication with organizational members is expected to have limited effects on the degree of value sharing and resulting effectiveness of strategy implementation up to a certain threshold point. Additionally, I argue that the relationship between top managers’ perceived degree of communication and strategy implementation are moderated by the type of communication (i.e., whether storytelling is used in the communication), communication medium (i.e., the use of e-mails), and top managers’ openness to the voices of organizational members. The idea of senders’ bias should provide insights into why many organizations struggle with strategy implementation.

Here, we report a patient with deciduoid type malignant pleural mesothelioma (MPM), which rapidly progressed. A 55-year-old man who might have been exposed to asbestos a few decades ago had severe back pain. The chest X-ray scanning and computed tomography (CT) revealed pleural thickness on his right thoracic space, without the presence of a lung mass. A pleural biopsy was performed and the patient was histologically diagnosed with deciduoid type MPM. Although he received two cycles of chemotherapy, his disease rapidly progressed and he died within two months of the diagnosis of deciduoid type MPM.

Katsuhiko Shimizu, Riki Okita, Shinsuke Saisho, Ai Maeda, Yuji Nojima, Masao NakataDepartment of General Thoracic Surgery, Kawasaki Medical School, Kurashiki, Okayama, JapanIntroduction: Numerous studies conducted until date have reported that the chemotherapy regimen could affect the programmed cell death ligand 1 (PD-L1) expression status in patients with non-small cell lung cancer (NSCLC).Materials and methods: A total of 36 NSCLC patients for whom both the surgically resected specimens of the primary tumors and re-biopsy specimens of the recurrent tumors were available were enrolled in this study. The PD-L1 expression status and tumor-infiltrating CD8-positive T lymphocytes (CD8+TILs) count were measured in paired samples by immunohistochemistry. The concordance rate in the tumor immune microenvironment (TIME) classification based on the PD-L1 expression status and CD8+TILs count was analyzed.Results: While the PD-L1 expression levels were similar between the surgical and re-biopsy specimens in 77.8% of cases, in 16.7% of cases, the expression levels were higher in the re-biopsy specimens. When the analysis was confined to patients who had received platinum-based chemotherapy, the percentage increased to 42.9%. The TIME classification changed in the re-biopsy specimens as compared to the surgical specimens in one-third of the patients, especially in those who had received chemotherapy previously. The TIME classification in the re-biopsy specimens more closely resembled that in the metastatic lymph nodes as compared to that in the primary tumor.Conclusion: In patients with recurrent NSCLC, especially those who have received chemotherapy previously, a recent re-biopsy sample is required to determine whether PD-1/PD-L1 inhibitors should be used for treatment or not.Keywords: non-small cell lung cancer, NSCLC, programmed cell death ligand 1 (PD-L1), tumor-infiltrating T lymphocytes, tumor immunemicroenvironment (TIME), re-biopsy

D-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of D/L configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound D-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with D-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of D/L-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of D/L-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.

Silicatein, a silica-synthesizing, catalytic triad hydrolase, was discovered in the silica spicules comprising the skeletons of certain marine sponges. Sequence similarity is closest to that of the mammalian cathepsin L, a catalytic triad hydrolase and protease. Genetic substitutions of residues in the catalytic triad, the predictive activities of polymeric and small-molecule analogs of the enzyme, and the wide range of structures accepted as substrates all support a reaction mechanism closely analogous to that established for the classical catalytic triad hydrolases. In this mechanism, hydrogen bonding of residues in the catalytic site is required to enhance nucleophilic attack and consequent hydrolysis of silicon alkoxide (and a wide range of other precursors), enabling subsequent polycondensation. Experimental and computational analyses revealed a novel pathway of self-assembly, in which the silicatein subunits first form a fractally patterned intermediate before entropic rearrangement to the hexagonally close-packed, macroscopic filament that serves both as the catalyst of silica synthesis in the sponge, and as a template guiding the deposition and emergent structure of the macroscopic silica filaments that form the sponge skeleton. Silicatein also proves capable of catalyzing the synthesis of organic silicones, metal oxides, metal phosphates, polylactides, and polymeric materials composed of organic metal compounds from their corresponding precursors, suggesting an evolutionary relaxation of structural substrate specificity that may have been necessary to accommodate the organic adducts of silicic acid suggested to comprise the natural precursor of the biogenic silica. Methods for purification, characterization, assay, and multiple uses of the enzyme are described.

In the rapidly changing and globalizing environment, mergers and acquisitions (M&As) have become increasingly important. In this study, we paid specific attention to the voluntary announcements of M&A budgets by Japanese firms. We discussed the antecedents and consequences of such announcements by incorporating the context of Japan, which has experienced an enduring economic downturn since 1990 and is in the process of adopting a Western style of governance. Drawing on signaling theory and impression management theory, this exploratory study intended to contribute to the literature by incorporating the influence of the social context and by arguing for the possibility that announcements of M&A budgets may be used not only for strategic purposes but also for impression management and to reduce information asymmetry.

Family S9 prolyl oligopeptidases (POPs) are of interest as pharmacological targets. We recently found that an S9 POP from Pleurotus eryngii showed altered substrate specificity following H2O2 treatment. Oxidation of Met203 on the non-catalytic β-propeller domain resulted in decreased activity toward non-aromatic aminoacyl-para-nitroanilides (pNAs) while maintaining its activity toward aromatic aminoacyl-pNAs. Given that the other Met residues should also be oxidized by H2O2 treatment, we constructed mutants in which all the Met residues were substituted with other amino acids. Analysis of the mutants showed that Met570 in the catalytic domain is another potent residue for the altered substrate specificity following oxidation. Met203 and Met570 lie on the surfaces of two different domains and form part of a funnel from the surface to the active center. Our findings indicate that the funnel forms the substrate pathway and plays a role in substrate recognition. Possible substrate entry route of prolyl oligopeptidase.

Silk produced by the silkworm Bombyx mori is an attractive material because of its luster, smooth and soft texture, conspicuous mechanical strength, good biocompatibility, slow biodegradation, and carbon neutral synthesis. Silkworms have been domesticated and bred for production of better quality and quantity of silk, resulting in the development of sericulture and the textile industry. Silk is generally white, so dyeing is required to obtain colored fiber. However, the dyeing process involves harsh conditions and generates a large volume of waste water, which have environmentally and economically negative impacts. Although some strains produce cocoons that contain pigments derived from the mulberry leaves that they eat, the pigments are distributed in the sericin layer and are lost during gumming. In trials for production of colored silk by feeding silkworms on diets containing dyes, only limited species of dye molecules were incorporated into the silk threads. A method for the generation of transgenic silkworm was established in conjunction with the discovery of green fluorescent protein (GFP), and silkworms carrying the GFP gene spun silk threads that formed cocoons that glowed bright green and still retained the original properties of silk. A wide range of color variation of silk threads has been obtained by replacing the GFP gene with the genes of other fluorescent proteins chosen from the fluorescent protein palette. The genetically modified silk with photonic properties can be processed to form various products including linear threads, 2D fabrics, and 3D materials. The transgenic colored silk could be economically advantageous due to addition of a new value to silk and reduction of cost for water waste, and environmentally preferable for saving water. Here, I review the literature regarding the production methods of fluorescent silk from transgenic silkworms and present examples of genetically modified color silk.

Background Immune checkpoint inhibitors targeting PD-1/PD-L1 axis have shown promising results in patients with non-small-cell lung cancer (NSCLC); however, the objective response rate is less than 30%. One major PD-L1 inducer, interferon γ (IFNg), has anti-tumour properties and is secreted by T cells and NK cells. IFNg-induced PD-L1 is considered to be a major mechanism that facilitates cancer cell escape from host immunity. Therefore, a better understanding of how IFNg stimulus influences the regulation of PD-L1 expression in cancer cells is warranted. Methods NSCLC cell lines: A549, PC-9, and LC2/ad were used for in vitro assays. Comparative analysis between IFNg and EGF was conducted in LC2/ad cells because EGF stimuli also enhanced PD-L1 expression in this cell line. PD-L1 expression was evaluated using flow cytometry while cell signalling pathway analysis was assessed using phospho-receptor tyrosine kinase arrays. To assess the NK cell-mediated cytotoxicity, NK cells were collected from healthy donors. NK killing was evaluated by lactate dehydrogenase release assay. Apoptotic cells were assessed via flow cytometry using Annexin V. Results IFNg stimulus significantly upregulated PD-L1 expression in all cell lines. In LC2/ad cells, IFNg activated STAT1 signalling while EGF activated AKT, MAPK, and ribosomal protein S6 (RPS6). Although IFNg-induced PD-L1 was clearly blocked by the JAK-STAT inhibitor, tofacitinib, EGF-induced PD-L1 was not. Both IFNg- and EGF-induced PD-L1 were blocked by PI3K and MAPK inhibitors; however, they were not blocked by the RPS6 inhibitor. Interestingly, IFNg downregulated NK cell-activating ligand expression while upregulating MHC class I molecules, the phenotype of which can theoretically escape NK cells easily. Thus, we confirmed that IFNg stimulus attenuated NK cell-mediated cytotoxicity in LC2/ad cells; however, tofacitinib blocked IFNg-reduced NK killing. We also confirmed that tofacitinib, at concentrations less than 3 μM, did not cause NK cell apoptosis. Conclusions Tofacitinib blocks IFNg-induced immunoescape from NK cells in NSCLC cells, thereby suggesting that NK cell therapy combined with tofacitinib might be a promising strategy in overcoming tumour immune escape. Legal entity responsible for the study Riki Okita. Funding Japan Society for the Promotion of Science (JSPS) Kakenhi Grant (25462189 and 16K10696),. Disclosure M. Nakata: Research grant / Funding (institution), The sponsor had no control over the interpretation or presentation of this work.: Kyowa Kirin. All other authors have declared no conflicts of interest.

MHC class I chain-related molecule A and B (MICA/B) are NK group 2 member D (NKG2D) ligands, which are broadly expressed in transformed cells. Both DNA damage-induced ataxia-telangiectasia-mutated (ATM)- and ATM and Rad3-related protein kinases (ATM-ATR) signaling and oncogene-induced PI3K-AKT signaling regulate the expression of NKG2D ligands, which promote NK cell-mediated cytotoxicity via NKG2D-NKG2D ligand interactions. NKG2D ligand overexpression was recently reported to be correlated with good prognosis in several types of cancer. However, the prognostic significance of NKG2D ligands in non-small cell lung cancer (NSCLC) remains unclear. Here, MICA/B expression was evaluated based on immunohistochemistry of 91 NSCLC samples from patients following radical surgery. In addition, expression of MICA/B was assessed in NSCLC cell lines treated with cisplatin in order to evaluate the regulatory mechanisms of MICA/B expression. Overall, 28 out of 91 (30.8%) specimens showed high expression level of MICA/B, which was associated with low (18)F-fluorodeoxyglucose uptake and manifestation of adenocarcinoma. After a median follow-up of 48.2 months, high MICA/B expression was associated with good recurrence-free survival (p = 0.037). In vitro assays using cell lines revealed that MICA/B expression was upregulated by cisplatin via ATM-ATR signaling, resulting in enhanced NK cell-mediated cytotoxicity. Upregulated MICA/B expressions in patients with radically resected NSCLC are predictive of good disease prognosis. Cisplatin-induced MICA/B upregulation is possibly an indirect mechanism by which the innate immune system eliminates tumor cells. NKG2D-NKG2D ligand-targeting therapy is a promising avenue for future immune-chemotherapy development.

We found that opals covering a rice plant are composed of loosely packed silica nanoparticles ∼50 nm in diameter. The plant opal-mimetic structures of bunching nonspherical silica nanoparticles were produced through hydrolysis of tetraethoxysilane in an alcoholic solution containing a long-chain branched polyethyleneimine (BPEI). Bunching silica nanoparticles 10–50 nm in diameter were spontaneously grown in polymer-mediated polycondensation in the BPEI matrix that accumulated hydrolyzed silicate species.

EGFR/HER2 are frequently expressed in MPM tissues, however, no studies have shown the clinical benefit of using EGFR/HER2-targeting drugs in patients with malignant pleural mesothelioma (MPM). It was reported that the tyrosine kinase inhibitor (TKI) lapatinib enhanced trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) in HER2-positive breast cancer, suggesting that this combination is a promising strategy for MPM treatment. The aim of the present study was to explore the possibility of a TKI combined with trastuzumab to enhance ADCC in MPM cells. Five MPM cell lines were used to test the effects of TKIs targeting EGFR (gefitinib, afatinib and lapatinib) on cell proliferation and the expression of the HER family receptor. The combined effects of TKI with trastuzumab on ADCC were evaluated using the LDH release assay. Additionally, MPM cells were isolated from patients and evaluated for lapatinib-induced upregulation of HER family receptors and trastuzumab- or cetuximab‑mediated ADCC. In MPM cell lines, HER2 expression was upregulated by lapatinib, downregulated by afatinib and unaffected by gefitinib. As expected, more trastuzumab bound to MPM cells pretreated with lapatinib than untreated cells, resulting in the enhancement of trastuzumab-mediated ADCC in MPM cells. In patient-derived MPM cells, both HER2 and EGFR were upregulated by lapatinib, resulting in the enhancement of both trastuzumab- and cetuximab-mediated ADCC. Of the three TKIs, only lapatinib enhanced trastuzumab-mediated ADCC via the upregulation of HER2 expression in MPM cells, suggesting that sequential combination of lapatinib and trastuzumab may be a promising strategy for MPM treatment.

The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure-function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named "glassin," the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6-8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.

Research in the field of mergers and acquisitions (M&A) has extensively examined potential sources of failure and success. However, despite increasing attention from scholars, only a handful of studies have actually investigated M&A processes. Adopting a process perspective is crucial to unraveling the underlying micro-behavioral foundations of M&A. This paper focuses on why M&As rarely reach their full potential. Specifically, we address these issues by providing an in-depth review of mechanisms contributing to M&A failure and developing a processual framework. We argue that behavioral factors at the individual and organizational levels impede rational and effective decision-making before, during and after an acquisition and suggest that communication climate plays a vital role in facilitating effective integration and may serve as a platform to improve M&A performance.

Silicatein is the first protein isolated from the silicon biominerals and characterized as constituent of the axial filament in the silica spicules of the demosponge Tethya aurantia, by significant sequence similarity with cathepsin L, an animal lysosomal protease, and as a catalyst of silica polycondensation at neutral pH and room temperature. This protein was then identified in a wide range of the class Demospongiae and in some species of the class Hexactinellida. Our attempt to isolate silicatein from the silica skeleton of Euplectella was unsuccessful, but instead we discovered glassin, a protein directing acceleration of silica polycondensation and sharing no significant relationship with any proteins including silicatein. The present study aims to verify the existence of silicatein by exploring the whole genome DNA sequence database of E. curvistellata with the sequence similarity search. Although we identified the sequences of glassin, cathepsin L and chitin synthetase, an enzyme synthesizing chitin, which has already been found in the silicon biominerals in E. aspergillum, silicatein failed to be identified. Our result indicates that silicatein is not essential for poriferan silicon biomineralization in the presence of glassin.

Silicatein, a silica-synthesizing, catalytic triad hydrolase, was discovered in the silica spicules comprising the skeletons of certain marine sponges. Sequence similarity is closest to that of the mammalian cathepsin L, a catalytic triad hydrolase and protease. Genetic substitutions of residues in the catalytic triad, the predictive activities of polymeric and small-molecule analogs of the enzyme, and the wide range of structures accepted as substrates all support a reaction mechanism closely analogous to that established for the classical catalytic triad hydrolases. In this mechanism, hydrogen bonding of residues in the catalytic site is required to enhance nucleophilic attack and consequent hydrolysis of silicon alkoxide (and a wide range of other precursors), enabling subsequent polycondensation. Experimental and computational analyses revealed a novel pathway of self-assembly, in which the silicatein subunits first form a fractally patterned intermediate before entropic rearrangement to the hexagonally close-packed, macroscopic filament that serves both as the catalyst of silica synthesis in the sponge, and as a template guiding the deposition and emergent structure of the macroscopic silica filaments that form the sponge skeleton. Silicatein also proves capable of catalyzing the synthesis of organic silicones, metal oxides, metal phosphates, polylactides, and polymeric materials composed of organic metal compounds from their corresponding precursors, suggesting an evolutionary relaxation of structural substrate specificity that may have been necessary to accommodate the organic adducts of silicic acid suggested to comprise the natural precursor of the biogenic silica. Methods for purification, characterization, assay, and multiple uses of the enzyme are described.

Organisms produce various organic/inorganic hybrid materials, which are called biominerals. They form through the self-organization of organic molecules and inorganic elements under ambient conditions. Biominerals often have highly organized and hierarchical structures from nanometer to macroscopic length scales, resulting in their remarkable physical and chemical properties that cannot be obtained by simple accumulation of their organic and inorganic constituents. These observations motivate us to create novel functional materials exhibiting properties superior to conventional materials--both synthetic and natural. Herein, we introduce recent progress in understanding biomineralization processes at the molecular level and the development of organic/inorganic hybrid materials by these processes. We specifically outline fundamental molecular studies on silica, iron oxide, and calcium carbonate biomineralization and describe material synthesis based on these mechanisms. These approaches allow us to design a variety of advanced hybrid materials with desired morphologies, sizes, compositions, and structures through environmentally friendly synthetic routes using functions of organic molecules.

Katsuhiko Shimizu,Riki Okita,Shinsuke Saisho,Ai Maeda,Yuji Nojima,Masao Nakata Department of General Thoracic Surgery, Kawasaki Medical School, Kurashiki, Okayama, Japan Abstract: Programmed cell death-1 ligand 1 (PD-L1), tumor-infiltrating CD8-positive T lymphocytes (CD8-positive TILs), and cyclooxygenase-2 (Cox-2) have been used as prognostic tools in patients with lung adenocarcinoma. We conducted a retrospective review of data from 170 patients who had undergone pulmonary resection as an initial treatment for clinical T1-2 N0 lung adenocarcinoma. We then investigated the expressions of three biomarkers using immunohistochemical analyses and compared the expression levels with the clinicopathological characteristics and outcomes of the patients. Next, we classified the tumors into four groups based on the PD-L1 and CD8-positive TILs statuses and evaluated the prognostic significance of Cox-2 expression according to the tumor immune microenvironment classification. Tumors with positive PD-L1 expression levels had a significantly larger number of CD8-positive TILs than tumors with negative PD-L1 expression levels, whereas tumors with high Cox-2 expressions had significantly fewer CD8-positive TILs than tumors with low Cox-2 expressions. A multivariate analysis showed that histological subtype, nodal metastasis, CD8-positive TILs count, and PD-L1 expression were independent predictors of patient outcome. Using a classification based on the PD-L1 and CD8-positive TILs statuses, the outcomes of patients with a negative PD-L1 expression and a high CD8-positive TIL count were significantly better than those with other classifications. In patients with negative PD-L1 and low CD8-positive TILs, the rate of EGFR mutation was significantly higher than that in other classifications, and Cox-2 expression was a powerful predictor of outcome. Clinical and pathological features in conjunction with the tumor immune microenvironment classification indicate that lung adenocarcinoma should be divided into different subgroups for prognosis and treatment. Classification according to the PD-L1 and CD8-positive TILs statuses might enable the effects of Cox-2 inhibitor to be predicted. Keywords: non-small-cell lung cancer, tumor microenvironment, programmed cell death-1 ligand 1 (PD-L1), tumor-infiltrating CD8-positive T lymphocytes, cyclooxygenase-2 (Cox-2)

Enzymes belonging to the S9 family of prolyl oligopeptidases are of interest because of their pharmacological importance and have a non-catalytic β-propeller domain. In this study, we found that the oxidation of Met203, which lies on surface of the β-propeller domain, leads to change in the substrate specificity of eryngase, an enzyme from Pleurotus eryngii and a member of the S9 family of prolyl oligopeptidases. The activity of eryngase for L-Phe-p-nitroanilide was maintained following hydrogen peroxide treatment but was dramatically reduced for other p-nitroanilide substrates. MALDI-TOF MS analysis using tryptic peptides of eryngase indicated that the change in substrate specificity was triggered by oxidizing Met203 to methionine sulfoxide. In addition, mutations of Met203 to smaller residues provided specificities similar to those observed following oxidation of the wild-type enzyme. Substitution of Met203 with Phe significantly decreased activity, indicating that Met203 may be involved in substrate gating.

Background: Programmed cell death-1 (PD-1) and PD-ligand1 (PD-L1) inhibitors effectively treat advanced non-small cell lung cancer (NSCLC) and prolong patient survival, resulting in standard therapies of NSCLC. Clinical benefits of good response and long survival with anti-PD-1/L1 therapy are limited in a small population of NSCLC patients, although combinations with chemotherapy have been extensively developed. Tumor PD-L1 expression and mutation burden are well known as response biomarkers for these therapies. On the other hand, biomarkers monitoring clinical responses during anti-PD-1/L1 therapy have yet to be identified. Very recently, we reported robust biomarkers of NY-ESO-1 and XAGE1 serum antibodies (Abs) predicting clinical benefits with anti-PD-1 monotherapy for NSCLC. NY-ESO-1 and XAGE1 are major cancer-testis antigens in NSCLC and play important roles in the immune surveillance. Then, we investigated whether these Abs became monitoring markers of clinical responses to anti-PD-1 monotherapy in NSCLC. Patients and Methods: Patient sera were serially obtained during anti-PD-1 therapy (nivolumab or pembrolizumab) of the first or later line as a standard care, and serum Abs against NY-ESO-1 and XAGE1 antigens and mutant p53 (as neoantigen) were detected by ELISA. Ab-positive was defined for serum with extrapolated titers exceeding or equal to 100 (≥100). Objective responses to anti-PD-1 therapy were assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Results: In patients responding to anti-PD-1 therapy, NY-ESO-1/XAGE1 serum Ab titers were increased with several spikes within two to three months after therapy and were gradually decreased with tumor shrinkage. The increase of Ab titers showed rapid or gradual type, and the former reflected rapid tumor shrinkage and the latter did slow one. The Ab titers in patients with complete response (CR) were gradually decreased to an undetectable level, and these patients experienced no recurrence. However, the decreased titers in patients with partial response (PR) remained plateau or under 100, and some of these patients experienced recurrence or regrowth. The Ab titers in patients with progressive disease (PD) were gradually increased and not decreased. On the other hand, p53 serum Ab titers were gradually increased after the peaks of NY-ESO-1/XAGE1 serum Ab titers, suggesting antigen-spreading of neoantigens associated with tumor destruction. Conclusions: Our results suggest that NY-ESO-1 and/or XAGE1 serum Abs are robust monitoring biomarkers of clinical responses to immune checkpoint inhibitors and are useful for decision making regarding the continuation or discontinuation of therapy in clinical settings. Citation Format: Mikio Oka, Koji Kurose, Yoshihiro Ohue, Takahiro Karasaki, Junichiro Futami, Takeshi Masuda, Masaaki Fukuda, Akitoshi Kinoshita, Katsuhiko Shimizu, Masao Nakata, Noboru Hattori, Hiroyuki Yamaguchi, Minoru Fukuda, Toru Oga, Kazuhiro Kakimi. Immunologic monitoring markers of clinical responses to anti-PD-1 therapy for non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B38.