| 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. |
- Bi, Huijuan, et al.
(författare)
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A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken
- 2023
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Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 19:4
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Tidskriftsartikel (refereegranskat)abstract
- The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.
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| 3. |
- Conway, Louis P., et al.
(författare)
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Chemoselective probe for detailed analysis of ketones and aldehydes produced by gut microbiota in human samples
- 2019
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Ingår i: Chemical Communications. - : ROYAL SOC CHEMISTRY. - 1359-7345 .- 1364-548X. ; 55:62, s. 9080-9083
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Tidskriftsartikel (refereegranskat)abstract
- New strategies are required for the discovery of unknown bioactive molecules produced by gut microbiota in the human host. Herein, we utilize a chemoselective probe immobilized to magnetic beads for analysis of carbonyls in human fecal samples. We identified 112 metabolites due to femtomole analysis and an increased mass spectrometric sensitivity by up to six orders of magnitude.
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| 4. |
- Correia, Mario S. P., et al.
(författare)
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Rapid Preparation of a Large Sulfated Metabolite Library for Structure Validation in Human Samples
- 2020
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Ingår i: Metabolites. - : MDPI. - 2218-1989 .- 2218-1989. ; 10:10
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Tidskriftsartikel (refereegranskat)abstract
- Metabolomics analysis of biological samples is widely applied in medical and natural sciences. Assigning the correct chemical structure in the metabolite identification process is required to draw the correct biological conclusions and still remains a major challenge in this research field. Several metabolite tandem mass spectrometry (MS/MS) fragmentation spectra libraries have been developed that are either based on computational methods or authentic libraries. These libraries are limited due to the high number of structurally diverse metabolites, low commercial availability of these compounds, and the increasing number of newly discovered metabolites. Phase II modification of xenobiotics is a compound class that is underrepresented in these databases despite their importance in diet, drug, or microbiome metabolism. The O-sulfated metabolites have been described as a signature for the co-metabolism of bacteria and their human host. Herein, we have developed a straightforward chemical synthesis method for rapid preparation of sulfated metabolite standards to obtain mass spectrometric fragmentation pattern and retention time information. We report the preparation of 38 O-sulfated alcohols and phenols for the determination of their MS/MS fragmentation pattern and chromatographic properties. Many of these metabolites are regioisomers that cannot be distinguished solely by their fragmentation pattern. We demonstrate that the versatility of this method is comparable to standard chemical synthesis. This comprehensive metabolite library can be applied for co-injection experiments to validate metabolites in different human sample types to explore microbiota-host co-metabolism, xenobiotic, and diet metabolism.
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| 5. |
- Dong, Yihui, et al.
(författare)
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Complementary Powerful Techniques for Investigating the Interactions of Proteins with Porous TiO2 and Its Hybrid Materials: A Tutorial Review
- 2022
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Ingår i: Membranes. - : MDPI. - 2077-0375. ; 12:4
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Forskningsöversikt (refereegranskat)abstract
- Understanding the adsorption and interaction between porous materials and protein is of great importance in biomedical and interface sciences. Among the studied porous materials, TiO2 and its hybrid materials, featuring distinct, well-defined pore sizes, structural stability and excellent biocompatibility, are widely used. In this review, the use of four powerful, synergetic and complementary techniques to study protein-TiO2-based porous materials interactions at different scales is summarized, including high-performance liquid chromatography (HPLC), atomic force microscopy (AFM), surface-enhanced Raman scattering (SERS), and Molecular Dynamics (MD) simulations. We expect that this review could be helpful in optimizing the commonly used techniques to characterize the interfacial behavior of protein on porous TiO2 materials in different applications.
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| 6. |
- Kaur, Amanpreet, et al.
(författare)
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Chemoselective bicyclobutane-based mass spectrometric detection of biological thiols uncovers human and bacterial metabolites
- 2023
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Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 14:20, s. 5291-5301
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Tidskriftsartikel (refereegranskat)abstract
- Sulfur is an essential element of life. Thiol-containing metabolites in all organisms are involved in the regulation of diverse biological processes. Especially, the microbiome produces bioactive metabolites or biological intermediates of this compound class. The analysis of thiol-containing metabolites is challenging due to the lack of specific tools, making these compounds difficult to investigate selectively. We have now developed a new methodology comprising bicyclobutane for chemoselective and irreversible capturing of this metabolite class. We utilized this new chemical biology tool immobilized onto magnetic beads for the investigation of human plasma, fecal samples, and bacterial cultures. Our mass spectrometric investigation detected a broad range of human, dietary and bacterial thiol-containing metabolites and we even captured the reactive sulfur species cysteine persulfide in both fecal and bacterial samples. The described comprehensive methodology represents a new mass spectrometric strategy for the discovery of bioactive thiol-containing metabolites in humans and the microbiome.
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| 7. |
- Lin, Weifeng, et al.
(författare)
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Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites
- 2021
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Ingår i: Angewandte Chemie International Edition. - : John Wiley & Sons. - 1433-7851 .- 1521-3773. ; 60:43, s. 23232-23240
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.
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| 8. |
- Lin, Weifeng
(författare)
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Development of advanced chemical biology tools for microbiome metabolism : Chemoselective probes for enhanced metabolomics analysis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human microbiome has a profound impact on host physiology by generating highly reactive compounds that can contribute to the development of diseases. These microbial metabolites have a substantial potential that can serve as valuable indicators or biomarkers for different health conditions. Nevertheless, elucidating the microbiota composition and function remains challenging due to its remarkable diversity and complex. Furthermore, conducting a comprehensive analysis of the entire metabolome in a single analytical measurement is difficult. Researchers often employ derivatization techniques in analytical chemistry, which involve modifying the chemical structure of molecules to enhance their detectability, ionization properties and stability during analysis. However, derivatization carries the risk of introducing artifacts or chemical alterations that may compromise the accuracy of analytical results. Consequently, more advanced techniques are urgently required to improve the precision of derivatization-based metabolomics.In response to this challenge, we have developed chemoselective probes immobilized onto magnetic beads to capture metabolites within biological samples. This innovative method improves the mass spectrometric sensitivity by up to a factor of one million, due to the efficient removal of sample matrix background through magnetic separation and improved ionization properties of the metabolites via derivatization. Our approach, termed quant-SCHEMA, has demonstrated the qualitative detection of metabolites containing carbonyl and amine groups with exceptional sensitivity and reproducibility. Additionally, we have successfully applied this method with improved probe design to quantitatively analyse carbonyl-containing metabolites, leading to the discovery of four valuable nutritional biomarkers. Furthermore, we have developed a precise quantification method for short-chain fatty acids (SCFAs) based on this chemoselective probe. The successful implementation of our chemoselective probes highlights the importance of chemical biology tools in advancing metabolomics, which we have termed chemical metabolomics,This comprehensive mass spectrometric analysis expands the horizons of metabolomics-driven biomarker discovery. We envision that our innovative chemical biology tool will find widespread utility in metabolomics analysis, providing valuable insights into microbial interactions with the human host and the development of diseases.
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| 9. |
- Lin, Weifeng, et al.
(författare)
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Identification of nutritional biomarkers through highly sensitive and chemoselective metabolomics
- 2023
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Ingår i: Food Chemistry. - : Elsevier. - 0308-8146 .- 1873-7072. ; 425
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Tidskriftsartikel (refereegranskat)abstract
- The importance of a healthy diet for humans is known for decades. The elucidation of key molecules responsible for the beneficial and adverse dietary effects is slowly developing as the tools are missing. Carbonyl-containing metabolites are a common bioproducts through conversion of diet by the microbiome. In here, we have utilized our recently developed mass spectrometric methodology based on chemoselective conjugation of carbonyl-metabolites. The method has been applied for urine sample analysis from a dietary (poly)phenol intervention study (N = 78 individuals) for the first time. We have identified a series of carbonyl-metabolites of dietary origin and the chemical structure was validated for 30 metabolites. Our sensitive analysis led to the discovery of four unknown dietary markers with high sensitivity and selectivity (AUC > 0.91). Our chemical metabolomics method has been successfully applied for large-scale analysis and provides the basis for targeted metabolomics to identify unknown nutritional and disease-related biomarkers.
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| 10. |
- Lin, Weifeng, et al.
(författare)
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Rapid and Bifunctional Chemoselective Metabolome Analysis of Liver Disease Plasma Using the Reagent 4‐Nitrophenyl‐2H‐azirine
- 2024
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Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773.
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Tidskriftsartikel (refereegranskat)abstract
- Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts that has been associated with diverse metabolic carboxylic acids. Mass spectrometric techniques are the method of choice for their analysis. However, the broad investigation of this metabolite class remains challenging. Derivatization of carboxylic acids represents a strategy to overcome these limitations but available methods suffer from diverse analytical challenges. Herein, we have designed a novel strategy introducing 4-nitrophenyl-2H-azirine as a new chemoselective moiety for the first time for carboxylic acid metabolites. This moiety was selected as it rapidly forms a stable amide bond and also generates a new ketone, which can be analyzed by our recently developed quant-SCHEMA method specific for carbonyl metabolites. Optimization of this new method revealed a high reproducibility and robustness, which was utilized to validate 102 metabolic carboxylic acids using authentic synthetic standard conjugates in human plasma samples including nine metabolites that were newly detected. Using this sequential analysis of the carbonyl- and carboxylic acid-metabolomes revealed alterations of the ketogenesis pathway, which demonstrates the vast benefit of our unique methodology. We anticipate that the developed azirine moiety with rapid functional group transformation will find broad application in diverse chemical biology research fields.
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