| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
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| 3. |
- Hu, Jiwen, et al.
(författare)
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Rapid detection of mercury (II) ions and water content by a new rhodamine B-based fluorescent chemosensor
- 2020
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Ingår i: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy. - : PERGAMON-ELSEVIER SCIENCE LTD. - 1386-1425 .- 1873-3557. ; 241
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Tidskriftsartikel (refereegranskat)abstract
- A rhodamine B-based sensor (RS) was designed and synthesized by a combination of the spirolacton rhodamine B (fluorophore) and multidentate chelates (ionophore) with high affinity towards Hg2+. In the presence of Hg2+, the resulting red-orange fluorescence (under UV light) and naked eye red color of IDS are supposed to be used for quantitative and qualitative measurement of Hg2+. Further fluorescent titration and analysis demonstrate that RS can selectively detect Hg2+ within 1 s with a low limit of detection (LOD) of 16 nM in acetonitrile media, meanwhile, the association constant (K-a) was calculated to be 0.32 x 10(5) M-1. More importantly, the resultant complex (RSHg) of RS and Hg2+ has also been successfully applied to detect limited water content in acetonitrile solution. (C) 2020 Published by Elsevier B.V.
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| 4. |
- Chang, Jui-Che, et al.
(författare)
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Orthorhombic Ta3-xN5-yOy thin films grown by unbalanced magnetron sputtering : The role of oxygen on structure, composition, and optical properties
- 2021
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Ingår i: Surface & Coatings Technology. - : Elsevier. - 0257-8972 .- 1879-3347. ; 406
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Tidskriftsartikel (refereegranskat)abstract
- Direct growth of orthorhombic Ta3N5-type Ta-O-N compound thin films, specifically Ta3-xN5-yOy, on Si and sapphire substrates with various atomic fractions is realized by unbalanced magnetron sputtering. Low-degree fiber-textural Ta3-xN5-yOy films were grown through reactive sputtering of Ta in a gas mixture of N-2, Ar, and O-2 with keeping a partial pressure ratio of 3:2:0.1 in a total working pressure range of 5-30 mTorr. With increasing total pressure from 5 to 30 mTorr, the atomic fraction of O in the as-grown Ta3-xN5-yOy films was found to increase from 0.02 to 0.15 while that of N and Ta decrease from 0.66 to 0.54 and 0.33 to 0.31, respectively, leading to a decrease in b lattice constant up to around 1.3%. Metallic TaNx phases were formed without oxygen. For a working pressure of 40 mTorr, an amorphous, O-rich Ta-N-O compound film with a high O fraction of similar to 0.48, was formed, mixed with non-stoichiometric TaON and Ta2O5. By analyzing the plasma discharge, the increasing O incorporation is associated with oxide formation on top of the Ta target due to a higher reactivity of Ta with O than with N. The increase of O incorporation in the films also leads to a optical bandgap widening from similar to 2.22 to similar to 2.96 eV, which is in agreement with the compositional and structural changes from a crystalline Ta3-xN5-yOy to an amorphous O-rich Ta-O-N compound.
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| 5. |
- Frisk, Junmei Hu, et al.
(författare)
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Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly : Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity
- 2021
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Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 6:49, s. 33943-33952
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Tidskriftsartikel (refereegranskat)abstract
- Deoxythymidylate kinase (TMPK) is a key enzyme in the synthesis of deoxythymidine triphosphate (dTTP). Four TMPK variants (P81L, A99T, D128N, and a frameshift) have been identified in human patients who suffered from severe neurodegenerative diseases. However, the impact of these mutations on TMPK function has not been clarified. Here we show that in fibroblasts derived from a patient, the P81L and D128N mutations led to a complete loss of TMPK activity in mitochondria and extremely low and unstable TMPK activity in cytosol. Despite the lack of TMPK activity, the patient-derived fibroblasts apparently grew normal. To investigate the impact of the mutations on the enzyme function, the mutant TMPKs were expressed, purified, and characterized. The wild-type TMPK mainly exists as a dimer with high substrate binding affinity, that is, low Km value and high catalytic efficiency, that is, k(cat)/K-M. In contrast, all mutants were present as monomers with dramatically reduced substrate binding affinity and catalytic efficiencies. Based on the human TMPK structure, none of the mutated amino acids interacted directly with the substrates. By structural analysis, we could explain why the respective amino acid substitutions could drastically alter the enzyme structure and catalytic function. In conclusion, TMPK mutations identified in patients represent loss of function mutations but surprisingly the proliferation rate of the patient-derived fibroblasts was normal, suggesting the existence of an alternative and hitherto unknown compensatory TMPK-like enzyme for dTTP synthesis. Further studies of the TMPK enzymes will help to elucidate the role of TMPK in neuropathology.
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| 6. |
- Frisk, Junmei Hu, et al.
(författare)
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Differential expression of enzymes in thymidylate biosynthesis in zebrafish at different developmental stages : implications for dtymk mutation-caused neurodegenerative disorders
- 2022
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Ingår i: BMC Neuroscience. - : Springer Nature. - 1471-2202. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundDeoxythymidine triphosphate (dTTP) is an essential building block of DNA, and defects in enzymes involved in dTTP synthesis cause neurodegenerative disorders. For instance, mutations in DTYMK, the gene coding for thymidylate kinase (TMPK), cause severe microcephaly in human. However, the mechanism behind this is not well-understood. Here we used the zebrafish model and studied (i) TMPK, an enzyme required for both the de novo and the salvage pathways of dTTP synthesis, and (ii) thymidine kinases (TK) of the salvage pathway in order to understand their role in neuropathology.ResultsOur findings reveal that maternal-stored dNTPs are only sufficient for 6 cell division cycles, and the levels of dNTPs are inversely correlated to cell cycle length during early embryogenesis. TMPK and TK activities are prominent in the cytosol of embryos, larvae and adult fish and brain contains the highest TMPK activity. During early development, TMPK activity increased gradually from 6 hpf and a profound increase was observed at 72 hpf, and TMPK activity reached its maximal level at 96 hpf, and remained at high level until 144 hpf. The expression of dtymk encoded Dtymk protein correlated to its mRNA expression and neuronal development but not to the TMPK activity detected. However, despite the high TMPK activity detected at later stages of development, the Dtymk protein was undetectable. Furthermore, the TMPK enzyme detected at later stages showed similar biochemical properties as the Dtymk enzyme but was not recognized by the Dtymk specific antibody.ConclusionsOur results suggest that active dNTP synthesis in early embryogenesis is vital and that Dtymk is essential for neurodevelopment, which is supported by a recent study of dtymk knockout zebrafish with neurological disorder and lethal outcomes. Furthermore, there is a novel TMPK-like enzyme expressed at later stages of development.
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| 7. |
- Frisk, Junmei Hu, et al.
(författare)
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Identification of a novel thymidylate kinase activity.
- 2020
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 39:10-12, s. 1359-1368
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Tidskriftsartikel (refereegranskat)abstract
- Thymidylate kinase (TMPK, EC2.7.4.9) is the enzyme that converts deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) in the synthesis of dTTP, an essential building block of DNA. To date, there is only one gene (TYMK) known to encode TMPK in mammalian cells. In this study, we investigated the distribution of TMPK activity and protein in subcellular fractions by using activity measurements and by using a specific antibody against TYMK-encoded TMPK (canonical TMPK). TMPK activity was detected in all subcellular fractions, of which the mitochondrial outer membrane contained the highest activity. High levels of canonical TMPK protein were detected in the cytosolic fraction, whereas low levels were found in the nuclear and mitochondrial matrix fractions. Strikingly, despite the detection of high TMPK activity in the mitochondrial outer membrane, canonical TMPK protein was not detected in this fraction. These results suggest that the TMPK activity detected in the outer membrane fraction may originate from a novel dTMP kinase, distinct from the canonical TYMK.
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| 8. |
- Frisk, Junmei Hu, et al.
(författare)
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Structural and functional analysis of human thymidylate kinase isoforms
- 2022
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Taylor & Francis. - 1525-7770 .- 1532-2335. ; 41:3, s. 321-332
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Tidskriftsartikel (refereegranskat)abstract
- Thymidylate kinase (TMPK) phosphorylates deoxythymidine monophosphate (dTMP) and plays an important role in genome stability. Deficiency in TMPK activity due to genetic alterations of DTYMK, i.e., the gene coding for TMPK, causes severe microcephaly in humans. However, no defects were observed in other tissues, suggesting the existence of a compensatory enzyme for dTTP synthesis. In search for this compensatory enzyme we analyzed 6 isoforms of TMPK mRNA deposited in the GenBank. Of these, only isoform 1 has been characterized and represents the known human TMPK. Our results reveal that isoform 2, 3, 4 and 5 lack essential structural elements for substrate binding and, thus, they are considered as nonfunctional isoforms. Isoform 6, however, has intact catalytic centers, i.e., dTMP-binding, DRX motif, ATP-binding p-loop and lid region, which are the key structural elements of an active TMPK, suggesting that isoform 6 may function as TMPK. When isoform 6 was expressed and purified, it showed only minimal activity (<0.1%) as compared with isoform 1. A putative isoform 6 was detected in a cancer cell line, in addition to the dominant isoform 1. However, because of its low activity, isoform 6 is unlikely be able to compensate for the loss of TMPK activity caused by deletions and/or point mutations of the DTYMK gene. Thereby, future studies to identify and characterize the compensatory TMPK enzyme found in patients with DTYMK mutations may contribute to the understanding of dTTP synthesis and of the pathophysiological role of DTYMK mutations in neurodegenerative disorders.
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| 9. |
- Hu Frisk, Junmei, et al.
(författare)
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Heavy metal tolerance of Mesorhizobium delmotii thymidylate kinase
- 2022
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Taylor & Francis. - 1525-7770 .- 1532-2335. ; 41:12, s. 1305-1317
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Tidskriftsartikel (refereegranskat)abstract
- Metal ions play an important role in many metabolic processes in all living organisms. At low concentrations, heavy metals such as Fe2+, Cu2+ and Zn2+ are essential cofactors for many enzymes. However, at high concentrations they are toxic. Mesorhizobium species belong to the class α-proteobacteria and have high tolerance to soil acidity, salinity, temperature extremes, and metallicolous conditions. To identify factors responsible for this tolerance we have studied the effects of metal ions on Mesorhizobium delmotii thymidylate kinase (MdTMPK), an essential enzyme in the synthesis of dTTP, thus being vital for cell growth. We show that Mg2+ and Mn2+ are the divalent metal ions required for catalysis and that Mn2+ gives the highest catalytic efficiency. MdTMPK activity in the presence of Mg2+ was strongly inhibited by the co-presence of Zn2+, Ni2+ and Co2+. However, the addition of Cs+ caused >2-fold enhanced MdTMPK activity. For TMPK from Bacilus anthracis and humans, the effects of Mg2+ and Mn2+ were similar, whereas the effects of other divalent metal ions were different, and no stimulatory effect of Cs+ was observed. Together, our results demonstrate that MdTMPK and BaTMPK function well in the presence of high concentrations of heavy metal ions, introducing a potential contribution of these enzymes to the heavy metal tolerance of Mesorhizobium delmotii and Bacillus anthracis
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| 10. |
- Hu, Jiwen, et al.
(författare)
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Selective colorimetric detection of copper (II) by a protein-based nanoprobe
- 2021
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Ingår i: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy. - : PERGAMON-ELSEVIER SCIENCE LTD. - 1386-1425 .- 1873-3557. ; 252
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Tidskriftsartikel (refereegranskat)abstract
- In this work, we report a novel protein-based nanoprobe (PNP) that can be employed for quantitative analysis of Cu2+ in pure water medium and real samples. Structurally, the proposed nanoprobe comprises a biofriendly protein (hen egg-white lysozyme (HEWL)) and a Cu2+-specific chromogenic agent, where HEWL acts as a nanocarrier encapsulating a structurally tailored rhodamine B derivate. The resulting PNP exhibits a hydrodynamic diameter of similar to 106 nm and efficiently disperses in water, enabling the detection of Cu2+ in pure aqueous systems without the aid of any organic co-solvents. The high sensitivity and selectivity of PNP allow the colorimetric detection of Cu2+ in the presence of other metal interferents with a low detection limit of 160 nM. The satisfying recovery of trace level Cu2+ in environmental samples demonstrate the great potential of employing PNP for the determination of Cu2+ in actual applications. Most importantly, the simple co-grinding method employing proteins and chromogenic agents provides a novel strategy to generate sensing systems that are useful detection of pollutants in aqueous samples. (C) 2021 Elsevier B.V. All rights reserved.
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