| 1. |
- Han, Won, et al.
(författare)
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Low-cost, open-source contact angle analyzer using a mobile phone, commercial tripods and 3D printed parts
- 2022
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Ingår i: HardwareX. - : Elsevier BV. - 2468-0672. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Measurement of contact angle is important in many areas of science and engineering research. Contact angle analyzers are however not easily accessible due to their expensive cost, which hinders their use in research and also in education. In this study we propose a low-cost contact angle analyzer that can be assembled with 3D printed parts. Mobile phone is used for imaging, and the image is analyzed using an open-source ImageJ plugin. Commercial camera tripods are used as platform that provides movement in many degrees of freedom, which are important in leveling of the substrate and proper imaging of droplets. We utilize the tripods to build imaging modules, sample plate module and volume metering module, each of which perform distinct tasks. Especially, we characterize the usefulness of the volume metering module, which helps users dispense same volume of liquid to reduce human error during measurement. The cost of an analyzer is $255.10, which is an order of magnitude lower compared to commercial products. With the advancement in open source software and upgrades in the hardware modules, we expect that the proposed contact angle analyzer to have a positive impact in resource limited research labs and educational environments.
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| 2. |
- Thang, Le Tran Huy, et al.
(författare)
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Disposable, pressure-driven, and self-contained cartridge with pre-stored reagents for automated nucleic acid extraction
- 2023
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Ingår i: Sensors and Actuators, B: Chemical. - : Elsevier BV. - 0925-4005. ; 375
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Tidskriftsartikel (refereegranskat)abstract
- Nucleic acid extraction is vital in many applications such as molecular diagnostics, genetic engineering, and deoxyribonucleic acid (DNA) sequencing. Although recent advances in nucleic acid extraction have been made to improve the process using microfluidics, it is still necessary to make it easy for end-users by having pre-loaded reagents, eliminating pipetting between steps, and automating the process for point-of-care (POC) testing. Herein, we present a pressure-driven and self-contained cartridge with pre-stored reagents for automating nucleic acid purification. To reduce operational complexity, reagents were transferred through a microfluidic chip using pressurized air stored inside the cartridge instead of an external pump or valving system. After performing cell lysis, the cartridge was inserted into the device, and the nucleic acid was purified automatically within 3 min Escherichia coli (E. coli) O157:H7 DNA extracted by our device showed similar concentration, purity, and real-time polymerase chain reaction (qPCR) results as the conventional column-based nucleic acid extraction method. Our device achieved a detection limit of 103 CFU for E. coli DNA, which is the same as that obtained using the conventional solid-phase extraction method. This study introduces a novel approach for automating the sample preparation process, which can help in facilitating POC molecular diagnostics.
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| 3. |
- Karlsson, Emma, 1983, et al.
(författare)
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In silico and in vitro studies of the reduction of unsaturated α,β bonds of trans-2-hexenedioic acid and 6-amino-trans-2-hexenoic acid – Important steps towards biobased production of adipic acid
- 2018
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 13:2
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Tidskriftsartikel (refereegranskat)abstract
- The biobased production of adipic acid, a precursor in the production of nylon, is of great interest in order to replace the current petrochemical production route. Glucose-rich lignocel-lulosic raw materials have high potential to replace the petrochemical raw material. A number of metabolic pathways have been proposed for the microbial conversion of glucose to adipic acid, but achieved yields and titers remain to be improved before industrial applications are feasible. One proposed pathway starts with lysine, an essential metabolite industrially produced from glucose by microorganisms. However, the drawback of this pathway is that several reactions are involved where there is no known efficient enzyme. By changing the order of the enzymatic reactions, we were able to identify an alternative pathway with one unknown enzyme less compared to the original pathway. One of the reactions lacking known enzymes is the reduction of the unsaturated α,β bond of 6-amino-trans-2-hexenoic acid and trans-2hexenedioic acid. To identify the necessary enzymes, we selected N-ethylmaleimide reductase from Escherichia coli and Old Yellow Enzyme 1 from Saccharomyces pastorianus. Despite successful in silico docking studies, where both target substrates could fit in the enzyme pockets, and hydrogen bonds with catalytic residues of both enzymes were predicted, no in vitro activity was observed. We hypothesize that the lack of activity is due to a difference in electron withdrawing potential between the naturally reduced aldehyde and the carboxylate groups of our target substrates. Suggestions for protein engineering to induce the reactions are discussed, as well as the advantages and disadvantages of the two metabolic pathways from lysine. We have highlighted bottlenecks associated with the lysine pathways, and proposed ways of addressing them.
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| 4. |
- Nakano, H., et al.
(författare)
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Haemogenic endocardium contributes to transient definitive haematopoiesis
- 2013
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Haematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial and haematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a haemogenic organ akin to the dorsal aorta. Here we examine the haemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Haemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the haemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells serve as a de novo source for transient definitive haematopoietic progenitors. © 2013 Macmillan Publishers Limited. All rights reserved.
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| 5. |
- Novy, Vera, 1986, et al.
(författare)
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Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases
- 2021
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 297:5
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Tidskriftsartikel (refereegranskat)abstract
- Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications, however, requires deeper knowledge of cutinases’ biodiversity and structure–function relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C2 to C16). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C12 and C16, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure–function analysis. All five enzymes showed a decrease in kcat values with increasing substrate chain length, whereas KM values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low KM values, resulting in high catalytic efficiencies toward pNP-C16. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.
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| 6. |
- Oster, Liya F., et al.
(författare)
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Rna Compaction in the Presence of Polyvalent Cations
- 2017
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495. ; 112:3, s. 367a-
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The effects of polyvalent cations on the effective size and charge of double-stranded DNA (dsDNA) have been well studied. In the presence of polyvalent cations, dsDNA in dilute solution undergoes a single-molecule, first-order phase transition, otherwise called condensation: more explicitly, upon onset of 90% neutralization of the phosphate backbone, the DNA undergoes discontinuous compaction into tightly wound toroids. However, the effects of these cations on long single-stranded RNAs (ssRNA) have not been well characterized. In this study we use centrifugation methods to examine the effective size of long ssRNAs in solutions of increasing concentration of the tetravalent cation spermine. In contrast to the case of dsDNA, we find only a continuous decrease in the size of ssRNA upon increase in spermine concentration. However, the decrease is significant enough to suggest that RNA molecules longer than viral genomes can be packaged in vitro into virus-like vectors for gene delivery.
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| 7. |
- Saez Jimenez, Veronica, 1985, et al.
(författare)
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Structure-function investigation of 3-methylaspartate ammonia lyase reveals substrate molecular determinants for the deamination reaction
- 2020
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 15:5
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Tidskriftsartikel (refereegranskat)abstract
- The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.
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| 8. |
- Shin, Jae Ho, 1987
(författare)
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A comprehensive metabolic map for production of bio-based chemicals
- 2019
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Ingår i: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 2:1, s. 18-33
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Forskningsöversikt (refereegranskat)abstract
- Production of industrial chemicals using renewable biomass feedstock is becoming increasingly important to address limited fossil resources, climate change and other environmental problems. To develop high-performance microbial cell factories, equivalent to chemical plants, microorganisms undergo systematic metabolic engineering to efficiently convert biomass-derived carbon sources into target chemicals. Over the past two decades, many engineered microorganisms capable of producing natural and non-natural chemicals have been developed. This Review details the current status of representative industrial chemicals that are produced through biological and/or chemical reactions. We present a comprehensive bio-based chemicals map that highlights the strategies and pathways of single or multiple biological reactions, chemical reactions and combinations thereof towards production of particular chemicals of interest. Future challenges are also discussed to enable production of even more diverse chemicals and more efficient production of chemicals from renewable feedstocks
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| 9. |
- Shin, Jae Ho, 1987
(författare)
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Bio‐based production of C2–C6 platform chemicals
- 2012
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 109:10, s. 2437-2459
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Tidskriftsartikel (refereegranskat)abstract
- Platform chemicals composed of 2-6 carbons derived from fossil resources are used as important precursors for making a variety of chemicals and materials, including solvents, fuels, polymers, pharmaceuticals, perfumes, and foods. Due to concerns regarding our environment and the limited nature of fossil resources, however, increasing interest has focused on the development of sustainable technologies for producing these platform chemicals from renewable resources. The techniques and strategies for developing microbial strains for chemicals production have advanced rapidly, and it is becoming feasible to develop microbes for producing additional types of chemicals, including non-natural molecules. In this study, we review the current status of the bio-based production of major C2-C6 platform chemicals, focusing on the microbial production of platform chemicals that have been used for the production of chemical intermediates, building block compounds, and polymers.
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| 10. |
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