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Article ; Online: Optimizing in vitro expression balance of central dogma-related genes using parallel reaction monitoring.

Nishizawa, Chisato / Aburaya, Shunsuke / Kosaka, Yuishin / Sugase, Kenji / Aoki, Wataru

Journal of bioscience and bioengineering

2024

Abstract: The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms in vitro. In a living cell, ... ...

Abstract	The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms in vitro. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma in vitro have been conducted. However, in vitro reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the in vitro expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired in vitro expression profiles of these genes. Additionally, we demonstrated that the in vitro expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the in vitro expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.
Language	English
Publishing date	2024-05-17
Publishing country	Japan
Document type	Journal Article
ZDB-ID	1465387-4
ISSN	1347-4421 ; 1389-1723
ISSN (online)	1347-4421
ISSN	1389-1723
DOI	10.1016/j.jbiosc.2024.04.006
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Article ; Online: Optimizing conditions to construct artificial cells using commercial in vitro transcription-translation system (PUREfrex2.0)

Sato, Toshiko / Matsuda, Shuhei / Aoki, Wataru

Journal of Bioscience and Bioengineering.

2023

Abstract: Artificial cells containing in vitro transcription and translation (IVTT) systems inside liposomes are important for the reconstruction and analysis of various biological systems. To improve the accessibility of artificial cell research, it is important ... ...

Abstract	Artificial cells containing in vitro transcription and translation (IVTT) systems inside liposomes are important for the reconstruction and analysis of various biological systems. To improve the accessibility of artificial cell research, it is important that artificial cells can be constructed using only commercially available components. Here, we optimized the construction of artificial cells containing PUREfrex2.0, a commercially available IVTT with high transcriptional and translational activity. Specifically, the composition of the inner and outer s olutions of the liposomes and the concentrations of lipids, glucose/sucrose, potassium glutamate, and magnesium acetate were systematically optimized, and finally we found a protocol for the stable construction of artificial cells containing PUREfrex2.0. These findings are expected to be important in expanding the artificial cell research community.
Keywords	glucose ; glutamic acid ; magnesium acetate ; potassium ; sucrose ; transcription (genetics) ; Artificial cells ; liposomes ; in vitro transcription-translation ; PUREfrex2.0 ; constructive biology
Language	English
Publishing place	Elsevier B.V.
Document type	Article ; Online
Note	Pre-press version
ZDB-ID	1465387-4
ISSN	1347-4421 ; 1389-1723
ISSN (online)	1347-4421
ISSN	1389-1723
DOI	10.1016/j.jbiosc.2023.07.004
Database	NAL-Catalogue (AGRICOLA)

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Article: Ammonia Production Using Bacteria and Yeast toward a Sustainable Society.

Watanabe, Yukio / Aoki, Wataru / Ueda, Mitsuyoshi

Bioengineering (Basel, Switzerland)

2023 Volume 10, Issue 1

Abstract: Ammonia is an important chemical that is widely used in fertilizer applications as well as in the steel, chemical, textile, and pharmaceutical industries, which has attracted attention as a potential fuel. Thus, approaches to achieve sustainable ammonia ... ...

Abstract	Ammonia is an important chemical that is widely used in fertilizer applications as well as in the steel, chemical, textile, and pharmaceutical industries, which has attracted attention as a potential fuel. Thus, approaches to achieve sustainable ammonia production have attracted considerable attention. In particular, biological approaches are important for achieving a sustainable society because they can produce ammonia under mild conditions with minimal environmental impact compared with chemical methods. For example, nitrogen fixation by nitrogenase in heterogeneous hosts and ammonia production from food waste using microorganisms have been developed. In addition, crop production using nitrogen-fixing bacteria has been considered as a potential approach to achieving a sustainable ammonia economy. This review describes previous research on biological ammonia production and provides insights into achieving a sustainable society.
Language	English
Publishing date	2023-01-07
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2746191-9
ISSN	2306-5354
ISSN	2306-5354
DOI	10.3390/bioengineering10010082
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Article ; Online: Optimizing conditions to construct artificial cells using commercial in vitro transcription-translation system (PUREfrex2.0).

Sato, Toshiko / Matsuda, Shuhei / Aoki, Wataru

Journal of bioscience and bioengineering

2023 Volume 136, Issue 4, Page(s) 334–339

Abstract	Artificial cells containing in vitro transcription and translation (IVTT) systems inside liposomes are important for the reconstruction and analysis of various biological systems. To improve the accessibility of artificial cell research, it is important that artificial cells can be constructed using only commercially available components. Here, we optimized the construction of artificial cells containing PUREfrex2.0, a commercially available IVTT with high transcriptional and translational activity. Specifically, the composition of the inner and outer s olutions of the liposomes and the concentrations of lipids, glucose/sucrose, potassium glutamate, and magnesium acetate were systematically optimized, and finally we found a protocol for the stable construction of artificial cells containing PUREfre×2.0. These findings are expected to be important in expanding the artificial cell research community.
MeSH term(s)	Liposomes ; Artificial Cells
Chemical Substances	Liposomes
Language	English
Publishing date	2023-07-28
Publishing country	Japan
Document type	Journal Article
ZDB-ID	1465387-4
ISSN	1347-4421 ; 1389-1723
ISSN (online)	1347-4421
ISSN	1389-1723
DOI	10.1016/j.jbiosc.2023.07.004
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Visualizing the spatial distribution of ustalic acid in the fruiting body of Tricholoma kakishimeji.

Ito, Tetsuro / Taira, Syu / Aoki, Wataru / Nagai, Hiroyuki / Fukaya, Masashi / Ryu, Kaori / Yamada, Akiyoshi

Journal of natural medicines

2024

Abstract: Imaging mass spectrometry (IMS) was conducted for the first time using ustalic acid (UA) and the fruiting body of Tricholoma kakishimeji to localize mushroom toxins. The mushroom materials were systematically collected in Japan, and analysis of the cross ...

Abstract	Imaging mass spectrometry (IMS) was conducted for the first time using ustalic acid (UA) and the fruiting body of Tricholoma kakishimeji to localize mushroom toxins. The mushroom materials were systematically collected in Japan, and analysis of the cross sections of the materials at a resolution of 120 μm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-IMS) revealed the localization of UA and its biogenically related metabolites. MALDI-IMS confirmed that UA was predominantly located on the entire surface of the fruiting body and accumulated in higher amounts in younger fruiting bodies than in mature ones. UA is the first toxic secondary metabolite in the genus Tricholoma locally identified using IMS in mushrooms.
Language	English
Publishing date	2024-05-18
Publishing country	Japan
Document type	Journal Article
ZDB-ID	2227540-X
ISSN	1861-0293 ; 1340-3443
ISSN (online)	1861-0293
ISSN	1340-3443
DOI	10.1007/s11418-024-01823-0
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Zs.A 2692: Show issues

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bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
Jg. 1995 - 2021: Lesesall (2.OG)
ab Jg. 2022: Lesesaal (EG)

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Article ; Online: Application of peptide barcoding to obtain high-affinity anti-PD-1 nanobodies.

Miyazaki, Takumi / Aoki, Wataru / Koike, Naoki / Sato, Toshiko / Ueda, Mitsuyoshi

Journal of bioscience and bioengineering

2023 Volume 136, Issue 3, Page(s) 173–181

Abstract: Cancer treatment has been revolutionized by immune checkpoint inhibitors, which regulate immune cell function by blocking the interactions between immune checkpoint molecules and their ligands. The interaction between programmed cell death-1 (PD-1) and ... ...

Abstract	Cancer treatment has been revolutionized by immune checkpoint inhibitors, which regulate immune cell function by blocking the interactions between immune checkpoint molecules and their ligands. The interaction between programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a target for immune checkpoint inhibitors. Nanobodies, which are recombinant variable domains of heavy-chain-only antibodies, can replace existing immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 conventional antibodies. However, the screening process for high-affinity nanobodies is laborious and time-consuming. Here, we identified high-affinity anti-PD-1 nanobodies using peptide barcoding, which enabled reliable and efficient screening by distinguishing each nanobody with a peptide barcode that was genetically appended to each nanobody. We prepared a peptide-barcoded nanobody (PBNb) library with thousands of variants. Three high-affinity PBNbs were identified from the PBNb library by quantifying the peptide barcodes derived from high-affinity PBNbs. Furthermore, these three PBNbs neutralized the interaction between PD-1 and PD-L1. Our results demonstrate the utility of peptide barcoding and the resulting nanobodies can be used as experimental tools and antitumor agents.
MeSH term(s)	Single-Domain Antibodies/chemistry ; Immune Checkpoint Inhibitors ; Peptides/chemistry ; Peptide Library ; Antineoplastic Agents
Chemical Substances	Single-Domain Antibodies ; Immune Checkpoint Inhibitors ; Peptides ; Peptide Library ; Antineoplastic Agents
Language	English
Publishing date	2023-07-22
Publishing country	Japan
Document type	Journal Article
ZDB-ID	1465387-4
ISSN	1347-4421 ; 1389-1723
ISSN (online)	1347-4421
ISSN	1389-1723
DOI	10.1016/j.jbiosc.2023.07.002
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Article ; Online: Production of Single-Domain Antibodies in Pichia pastoris.

Matsuzaki, Yusei / Kajiwara, Kaho / Aoki, Wataru / Ueda, Mitsuyoshi

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2446, Page(s) 181–203

Abstract: Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a ... ...

Abstract	Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogenic contamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.
MeSH term(s)	Escherichia coli/metabolism ; Pichia/genetics ; Pichia/metabolism ; Recombinant Proteins/chemistry ; Saccharomycetales/metabolism ; Single-Domain Antibodies/metabolism
Chemical Substances	Recombinant Proteins ; Single-Domain Antibodies
Language	English
Publishing date	2022-02-14
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2075-5_9
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Article ; Online: Enhancement of PET degradation by PET depolymerase with the microbe addition.

Saito, Hiroki / Ueda, Mitsuyoshi / Aoki, Wataru

Bioscience, biotechnology, and biochemistry

2022 Volume 86, Issue 10, Page(s) 1482–1484

Abstract: The degradation of polyethylene terephthalate (PET) by modified PET depolymerase has recently attracted much attention. We found that mixing a PET depolymerase with non-genetically modified Thermus sp. can enhance its PET-degrading activity by 7.7-fold. ... ...

Abstract	The degradation of polyethylene terephthalate (PET) by modified PET depolymerase has recently attracted much attention. We found that mixing a PET depolymerase with non-genetically modified Thermus sp. can enhance its PET-degrading activity by 7.7-fold. This approach is attractive for constructing a sustainable PET recycling system.
MeSH term(s)	Enzymes/metabolism ; Polyethylene Terephthalates/metabolism ; Thermus
Chemical Substances	Enzymes ; Polyethylene Terephthalates
Language	English
Publishing date	2022-09-26
Publishing country	England
Document type	Journal Article
ZDB-ID	1106450-x
ISSN	1347-6947 ; 0916-8451
ISSN (online)	1347-6947
ISSN	0916-8451
DOI	10.1093/bbb/zbac129
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Zs.A 4647: Show issues

Location:
Je nach Verfügbarkeit (siehe Angabe bei Bestand)
bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
Jg. 1995 - 2021: Lesesall (2.OG)
ab Jg. 2022: Lesesaal (EG)

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Article ; Online: Application of peptide barcoding to obtain high-affinity anti-PD-1 nanobodies

Miyazaki, Takumi / Aoki, Wataru / Koike, Naoki / Sato, Toshiko / Ueda, Mitsuyoshi

Journal of Bioscience and Bioengineering.

2023

Abstract	Cancer treatment has been revolutionized by immune checkpoint inhibitors, which regulate immune cell function by blocking the interactions between immune checkpoint molecules and their ligands. The interaction between programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a target for immune checkpoint inhibitors. Nanobodies, which are recombinant variable domains of heavy-chain-only antibodies, can replace existing immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 conventional antibodies. However, the screening process for high-affinity nanobodies is laborious and time-consuming. Here, we identified high-affinity anti-PD-1 nanobodies using peptide barcoding, which enabled reliable and efficient screening by distinguishing each nanobody with a peptide barcode that was genetically appended to each nanobody. We prepared a peptide-barcoded nanobody (PBNb) library with thousands of variants. Three high-affinity PBNbs were identified from the PBNb library by quantifying the peptide barcodes derived from high-affinity PBNbs. Furthermore, these three PBNbs neutralized the interaction between PD-1 and PD-L1. Our results demonstrate the utility of peptide barcoding and the resulting nanobodies can be used as experimental tools and antitumor agents.
Keywords	barcoding ; cancer therapy ; ligands ; peptides ; Nanobody ; Screening ; Peptide barcode ; Immune checkpoint inhibitor ; Programmed cell death-1 ; Size-exclusion chromatography ; Mass spectrometry
Language	English
Publishing place	Elsevier B.V.
Document type	Article ; Online
Note	Pre-press version
ZDB-ID	1465387-4
ISSN	1347-4421 ; 1389-1723
ISSN (online)	1347-4421
ISSN	1389-1723
DOI	10.1016/j.jbiosc.2023.07.002
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Evaluation of a library of loxP variants with a wide range of recombination efficiencies by Cre.

Yamauchi, Yuji / Matsukura, Hidenori / Motone, Keisuke / Ueda, Mitsuyoshi / Aoki, Wataru

PloS one

2022 Volume 17, Issue 10, Page(s) e0276657

Abstract: Sparse labeling of individual cells is an important approach in neuroscience and many other fields of research. Various methods have been developed to sparsely label only a small population of cells; however, there is no simple and reproducible strategy ... ...

Abstract	Sparse labeling of individual cells is an important approach in neuroscience and many other fields of research. Various methods have been developed to sparsely label only a small population of cells; however, there is no simple and reproducible strategy for managing the probability of sparse labeling at desired levels. Here, we aimed to develop a novel methodology based on the Cre-lox system to regulate sparseness at desired levels, and we purely analyzed cleavage efficiencies of loxP mutants by Cre. We hypothesized that mutations in the loxP sequence reduce the recognition efficiency by Cre, which enables the regulation of the sparseness level of gene expression. In this research, we mutagenized the loxP sequence and analyzed a library of loxP variants. We evaluated more than 1000 mutant loxP sequences, including mutants with reduced excision efficiencies by Cre ranging from 0.51% to 59%. This result suggests that these mutant loxP sequences can be useful in regulating the sparseness of genetic labeling at desired levels.
MeSH term(s)	Recombination, Genetic ; Integrases/genetics ; Integrases/metabolism ; Gene Library ; Mutation
Chemical Substances	Cre recombinase (EC 2.7.7.-) ; Integrases (EC 2.7.7.-)
Language	English
Publishing date	2022-10-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0276657
Database	MEDical Literature Analysis and Retrieval System OnLINE

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