| 1. |
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| 2. |
- Bi, Huijuan, et al.
(author)
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A frame-shift mutation in COMTD1 is associated with impaired pheomelanin pigmentation in chicken
- 2023
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In: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 19:4
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Journal article (peer-reviewed)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. |
- Carpintero, Guillermo, et al.
(author)
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Interconnection challenges on integrated terahertz photonic systems
- 2021
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In: OPTICAL INTERCONNECTS XXI. - : SPIE-Intl Soc Optical Eng.
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Conference paper (peer-reviewed)abstract
- We present the current challenges for high frequency interconnects, especially for calibrated measures of the frequency response of components operating above 100 GHz. This is the challenge addressed by the TERAmeasure Future and Emerging Technologies project, aiming to combine photonics and electronics to develop new paradigm in the millimetre and Terahertz frequency ranges, overcoming the current obstacles to better measurements, eliminating the frequency banded nature of rectangular waveguides and providing metrology-grade results across the full frequency range.
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| 4. |
- Cheng, Liqin, et al.
(author)
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The protective role of commensal gut microbes and their metabolites against bacterial pathogens
- 2024
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In: Gut microbes. - : Taylor & Francis. - 1949-0976 .- 1949-0984. ; 16:1
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Journal article (peer-reviewed)abstract
- Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.
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| 5. |
- Conway, Louis P., et al.
(author)
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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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In: Chemical Communications. - : ROYAL SOC CHEMISTRY. - 1359-7345 .- 1364-548X. ; 55:62, s. 9080-9083
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Journal article (peer-reviewed)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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| 6. |
- Conway, Louis P., et al.
(author)
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Unexpected Acetylation of Endogenous Aliphatic Amines by Arylamine N-Acetyltransferase NAT2
- 2020
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In: Angewandte Chemie International Edition. - : John Wiley & Sons. - 1433-7851 .- 1521-3773. ; 59:34, s. 14342-14346
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Journal article (peer-reviewed)abstract
- N-Acetyltransferases play critical roles in the deactivation and clearance of xenobiotics, including clinical drugs. NAT2 has been classified as an arylamine N-acetyltransferase that mainly converts aromatic amines, hydroxylamines, and hydrazines. Herein, we demonstrate that the human arylamine N-acetyltransferase NAT2 also acetylates aliphatic endogenous amines. Metabolomic analysis and chemical synthesis revealed increased intracellular concentrations of mono- and diacetylated spermidine in human cell lines expressing the rapid compared to the slow acetylator NAT2 phenotype. The regioselective N8 -acetylation of monoacetylated spermidine by NAT2 answers the long-standing question of the source of diacetylspermidine. We also identified selective acetylation of structurally diverse alkylamine-containing drugs by NAT2, which may contribute to variations in patient responses. The results demonstrate a previously unknown functionality and potential regulatory role for NAT2, and we suggest that this enzyme should be considered for re-classification.
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| 7. |
- Correia, Mario S. P., et al.
(author)
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Comparative dietary sulfated metabolome analysis reveals unknown metabolic interactions of the gut microbiome and the human host
- 2020
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In: Free Radical Biology & Medicine. - : ELSEVIER SCIENCE INC. - 0891-5849 .- 1873-4596. ; 160, s. 745-754
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Journal article (peer-reviewed)abstract
- The gut microbiome converts dietary compounds that are absorbed in the gastrointestinal tract and further metabolized by the human host. Sulfated metabolites are a major compound class derived from this co-metabolism and have been linked to disease development. In the present multidisciplinary study, we have investigated human urine samples from a dietary intervention study with 22 individuals collected before and after consumption of a polyphenol rich breakfast. These samples were analyzed utilizing our method combining enzymatic metabolite hydrolysis using an arylsulfatase and mass spectrometric metabolomics. Key to this study is the validation of 235 structurally diverse sulfated metabolites. We have identified 48 significantly upregulated metabolites upon dietary intervention including 11 previously unknown sulfated metabolites for this diet. We observed a large variation in subjects based on their potential to sulfate metabolites, which may be the foundation for classification of subjects as high and low sulfate metabolizers in future large cohort studies. The reported sulfatase-based method is a robust tool for the discovery of unknown microbiota-derived metabolites in human samples.
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| 8. |
- Correia, Mário S. P., et al.
(author)
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Comprehensive kinetic and substrate specificity analysis of an arylsulfatase from Helix pomatia using mass spectrometry
- 2019
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In: Bioorganic & Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 27:6, s. 955-962
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Journal article (peer-reviewed)abstract
- Sulfatases hydrolyze sulfated metabolites to their corresponding alcohols and are present in all domains of life. These enzymes have found major application in metabolic investigation of drugs, doping control analysis and recently in metabolomics. Interest in sulfatases has increased due to a link between metabolic processes involving sulfated metabolites and pathophysiological conditions in humans. Herein, we present the first comprehensive substrate specificity and kinetic analysis of the most commonly used aryl sulfatases extracted from the snail Helix pomatia. In the past, this enzyme has been used in the form of a crude mixture of enzymes, however, recently we have purified this sulfatase for a new application in metabolomics-driven discovery of sulfated metabolites. To evaluate the substrate specificity of this promiscuous sulfatase, we have synthesized a series of new sulfated metabolites of diverse structure and employed a mass spectrometric assay for kinetic substrate hydrolysis evaluation. Our analysis of the purified enzyme revealed that the sulfatase has a strong preference for metabolites with a bi- or tricyclic aromatic scaffold and to a lesser extent for monocyclic aromatic phenols. This metabolite library and mass spectrometric method can be applied for the characterization of other sulfatases from humans and gut microbiota to investigate their involvement in disease development.
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| 9. |
- Correia, Mario S. P., et al.
(author)
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Coupled Enzymatic Treatment and Mass Spectrometric Analysis for Identification of Glucuronidated Metabolites in Human Samples
- 2019
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In: ChemBioChem. - : WILEY-V C H VERLAG GMBH. - 1439-4227 .- 1439-7633. ; 20:13, s. 1678-1683
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Journal article (peer-reviewed)abstract
- Glucuronidation is the most common phase II modification and plays an important role in human clearance metabolism. Glucuronidated metabolites have also been linked to disease development and microbiota-host co-metabolism. Although many of these compounds have been identified, the total number of unknown glucuronides and their impact on the human host's physiology can only be estimated. Herein, we describe the combination of an untargeted metabolomics analysis and enzymatic metabolic conversion for the selective detection of glucuronide conjugates by using UPLC-MS/MS in human urine samples. Our study demonstrates that this powerful strategy can be used for the selective identification of glucuronidated molecules and to discover unknown natural metabolites. In total, we identified 191 metabolites in a single sample including microbiota-derived compounds as well as previously unidentified molecules.
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| 10. |
- Correia, Mario S. P.
(author)
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Development of chemical biology tools for metabolomics analysis : Phase II modifications as a link between host and microbiota co-metabolism
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Biomarker discovery is an extremely important field for early disease detection. New selective molecular markers are in high demand, as an earlier disease detection can lead to improved patient care. Furthermore, with the increased knowledge of the importance of the microbiota for human physiology and disease, markers for the interaction between the microbiome and the human host are required. A biomarker can be any biomolecule including metabolites that is altered between a “healthy” state and a disease state. Furthermore, the metabolic interplay between the consortium of trillion of microorganisms and the human is still largely unexplored. The investigation of the co-metabolism represents a tremendous opportunity for the discovery of new metabolites.Metabolomics is the most recent of the major ‘omics sciences and provides a readout of the phenotype at the time of sample collection. The techniques of choice for metabolomics analysis are mass spectrometry (MS) and NMR spectroscopy. With the most recent developments in both hardware and software, data collected from liquid chromatography-MS can analyze thousands of metabolites at the same time. Despite the technical advances, metabolomics is still at a developmental stage in comparison to genomics or proteomics. Chemical biology tools are required for the selective analysis of functional groups to improve metabolite detection.We have developed new chemical biological tools for the combination with metabolomics analysis. Using an arylsulfatase from the snail Helix pomatia, we have selectively identified sulfated metabolites in urine and fecal samples. We identified more than 200 sulfates and many these were previously undetected. Several of the identified molecules were products of the co-metabolism of the human host and its microbiome, which have partly been linked to disease development.We also utilized a purified glucuronidase for identification of glucuronides in urine samples, respectively. We were able to identify close to 200 glucuronides. Additionally, we have developed a new strategy for metabolite validation, based on the enzymatic cleavage of glucuronides.We have also prepared a comprehensive sulfated metabolite library for efficient structure validation. The understanding of the human and the microbiota co-metabolism was also increased through applying a modified method on a dietary intervention study. This diet was based on (poly)phenolic compounds that are commonly digested by the bacteria and then further metabolized by the human host to their sulfated conjugates. These findings demonstrate the importance of these newly developed tools for metabolomics and the study of these metabolic interactions between the human and the microbiota.
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| 11. |
- Correia, Mario S. P., et al.
(author)
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Investigation of the individual human sulfatome in plasma and urine samples reveals an age-dependency
- 2021
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In: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 11:55, s. 34788-34794
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Journal article (peer-reviewed)abstract
- Metabolic microbiome interaction with the human host has been linked to human physiology and disease development. The elucidation of this interspecies metabolite exchange will lead to identification of beneficial metabolites and disease modulators. Their discovery and quantitative analysis requires the development of specific tools and analysis of specific compound classes. Sulfated metabolites are considered a readout for the co-metabolism of the microbiome and their host. This compound class is part of the human phase II clearance process of xenobiotics and is the main focus in drug or doping metabolism and also includes dietary components and microbiome-derived compounds. Here, we report the targeted analysis of sulfated metabolites in plasma and urine samples in the same individuals to identify the core sulfatome and similarities between these two sample types. This analysis of 27 individuals led to the identification of the core sulfatome of 41 metabolites in plasma and urine samples as well as an age effect for 15 metabolites in both sample types.
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| 12. |
- Correia, Mario S. P., et al.
(author)
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Rapid Preparation of a Large Sulfated Metabolite Library for Structure Validation in Human Samples
- 2020
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In: Metabolites. - : MDPI. - 2218-1989. ; 10:10
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Journal article (peer-reviewed)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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| 13. |
- Garg, Neeraj, et al.
(author)
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Chemoselective Probe Containing a Unique Bioorthogonal Cleavage Site for Investigation of Gut Microbiota Metabolism
- 2018
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In: Angewandte Chemie International Edition. - : WILEY-V C H VERLAG GMBH. - 1433-7851 .- 1521-3773. ; 57:42, s. 13805-13809
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Journal article (peer-reviewed)abstract
- While metabolites derived from gut microbiota metabolism have been linked to disease development in the human host, the chemical tools required for their detailed analysis and the discovery of biomarkers are limited. A unique and multifunctional chemical probe for mass spectrometric analysis, which contains p-nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site has been designed and synthesized. Coupled to magnetic beads, this chemical probe allows for straightforward extraction of metabolites from human samples and release under mild conditions. This isolation from the sample matrix results in significantly reduced ion suppression, an increased mass spectrometric sensitivity, and facilitates the detection of metabolites in femtomole quantities. The chemoselective probe was applied to the analysis of human fecal samples, resulting in the discovery of four metabolites previously unreported in this sample type and confirmation of the presence of medically relevant gut microbiota-derived metabolites.
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| 14. |
- Garg, Neeraj, et al.
(author)
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Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control
- 2018
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In: Organic and biomolecular chemistry. - : ROYAL SOC CHEMISTRY. - 1477-0520 .- 1477-0539. ; 16:5, s. 698-702
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Journal article (peer-reviewed)abstract
- Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography- mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.
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| 15. |
- Globisch, Daniel, et al.
(author)
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Validation of onchocerciasis biomarker N-acetyltyramine-O-glucuronide (NATOG)
- 2017
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In: Bioorganic & Medicinal Chemistry Letters. - : PERGAMON-ELSEVIER SCIENCE LTD. - 0960-894X .- 1464-3405. ; 27:15, s. 3436-3440
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Journal article (peer-reviewed)abstract
- The Neglected Tropical Disease onchocerciasis is a parasitic disease. Despite many control programmes by the World Health Organization (WHO), large communities in West and Central Africa are still affected. Besides logistic challenges during biannual mass drug administration, the lack of a robust, point-of-care diagnostic is limiting successful eradication of onchocerciasis. Towards the implementation of a non-invasive and point-of-care diagnostic, we have recently reported the discovery of the biomarker N-acetyltyramine-O-glucuronide (NATOG) in human urine samples using a metabolomics-mining approach. NATOG's biomarker value was enhanced during an investigation in a rodent model. Herein, we further detail the specificity of NATOG in active onchocerciasis infections as well as the co-infecting parasites Loa loa and Mansonella perstans. Our results measured by liquid chromatography coupled with mass spectrometry (LC-MS) reveal elevated NATOG values in mono-and co-infection samples only in the presence of the nematode Onchocerca volvulus. Metabolic pathway investigation of L-tyrosine/tyramine in all investigated nematodes uncovered an important link between the endosymbiotic bacterium Wolbachia and O. volvulus for the biosynthesis of NATOG. Based on these extended studies, we suggest NATOG as a biomarker for tracking active onchocerciasis infections and provide a threshold concentration value of NATOG for future diagnostic tool development.
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| 16. |
- Globisch, Maria A., et al.
(author)
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Immunothrombosis and vascular heterogeneity in cerebral cavernous malformation
- 2022
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In: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 140:20, s. 2154-2169
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Journal article (peer-reviewed)abstract
- Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies, but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA sequencing to investigate the transcriptome of mouse brain endothelial cells with an inducible endothelial-specific Ccm3 knock-out (Ccm3-iECKO). We found that Ccm3-deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared with wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. We created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3 expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model, this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCMs. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.
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| 17. |
- Jain, Abhishek, et al.
(author)
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Comparison of two arylsulfatases for targeted mass spectrometric analysis of microbiota-derived metabolites
- 2021
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In: Journal of Pharmaceutical and Biomedical Analysis. - : Elsevier. - 0731-7085 .- 1873-264X. ; 195
-
Journal article (peer-reviewed)abstract
- Sulfation of metabolites is the second highest phase II modification in humans, which plays a critical role in the xenobiotics clearance process and gut microbiota-host co-metabolism. Besides the main function to remove xenobiotics from the body, sulfated metabolites have also been linked to inflammation, bacterial pathogenesis and metabolic disorders. A better understanding of how these metabolites impact the human body has turned into an important research area. Analytical methods for selective identification of this metabolite class are scarce. We have recently developed an assay utilizing the arylsulfatase from Helix pomatia due to a high substrate promiscuity combined with state-of-the-art metabolomics bioinformatic analysis for the selective identification of O-sulfated metabolites in human samples. This enzyme requires a multistep purification process as highest purity is needed for the developed mass spectrometric assay. In this study, we have utilized a new and recombinant overexpressed arylsulfatase (ASPC) for the selective identification of organic sulfate esters in human urine samples. We have compared the substrate conversion in urine samples and substrate specificity of this enzyme with purified arylsulfatase from Helix pomatia. Our analysis of urine samples revealed that both enzymes can be utilized for the selective analysis and discovery of sulfated metabolites with high promiscuity as demonstrated by equal hydrolysis of 108 substrates including sulfated conjugates of 27 metabolites of microbial origin. Importantly, we also identified 21 substrates in human urine samples that are exclusively hydrolyzed by ASPC and application of this enzyme increases the discovery of unknown sulfated metabolites with a higher scaffold diversity.
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| 18. |
- Joffré, Enrique, et al.
(author)
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Analysis of Growth Phases of Enterotoxigenic Escherichia coli Reveals a Distinct Transition Phase before Entry into Early Stationary Phase with Shifts in Tryptophan, Fucose, and Putrescine Metabolism and Degradation of Neurotransmitter Precursors
- 2022
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In: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 10:4
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Journal article (peer-reviewed)abstract
- Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and adults in endemic areas. Gene regulation of ETEC during growth in vitro and in vivo needs to be further evaluated, and here we describe the full transcriptome and metabolome of ETEC during growth from mid-logarithmic growth to early stationary phase in rich medium (LB medium). We identified specific genes and pathways subjected to rapid transient alterations in gene expression and metabolite production during the transition from logarithmic to stationary growth. The transient phase was found to be different from the subsequent induction of early stationary phase-induced genes. The transient phase was characterized by the repression of genes and metabolites involved in organic substance transport. Genes involved in fucose and putrescine metabolism were upregulated, and genes involved in iron transport were repressed. Expression of toxins and colonization factors were not changed, suggesting retained virulence from mid-logarithmic to the start of the stationary phase. Metabolomic analyses showed that the transient phase was characterized by a drop of intracellular amino acids, e.g., l-tyrosine, l-tryptophan, l-phenylalanine, l-leucine, and l-glutamic acid, followed by increased levels at induction of stationary phase. A pathway enrichment analysis of the entire combined transcriptome and metabolome revealed that significant pathways during progression from logarithmic to early stationary phase are involved in the degradation of neurotransmitters aminobutyrate (GABA) and precursors of 5-hydroxytryptamine (serotonin). This work provides a comprehensive framework for further studies on transcriptional and metabolic regulation in pathogenic E. coli.IMPORTANCE We show that E. coli, exemplified by the pathogenic subspecies enterotoxigenic E. coli (ETEC), undergoes a stepwise transcriptional and metabolic transition into the stationary phase. At a specific entry point, E. coli induces activation and repression of specific pathways. This leads to a rapid decrease of intracellular levels of certain amino acids. The resulting metabolic activity leads to an intense but short peak of indole production, suggesting that this is the previously described “indole peak,” rapid decrease of intermediate molecules of bacterial neurotransmitters, increased putrescine and fucose uptake, increased glutathione levels, and decreased iron uptake. This specific transient shift in gene expression and metabolome is short-lived and disappears when bacteria enter the early stationary phase. We suggest that these changes mainly prepare bacteria for ceased growth, but based on the pathways involved, we could suggest that this transient phase substantially influences survival and virulence.
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| 19. |
- Kaur, Amanpreet, et al.
(author)
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Chemoselective bicyclobutane-based mass spectrometric detection of biological thiols uncovers human and bacterial metabolites
- 2023
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In: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 14:20, s. 5291-5301
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Journal article (peer-reviewed)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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| 20. |
- Kundu, Snehangshu, et al.
(author)
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Common and mutation specific phenotypes of KRAS and BRAF mutations in colorectal cancer cells revealed by integrative -omics analysis
- 2021
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In: Journal of Experimental & Clinical Cancer Research. - : BioMed Central (BMC). - 1756-9966. ; 40
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Journal article (peer-reviewed)abstract
- Background: Genes in the Ras pathway have somatic mutations in at least 60 % of colorectal cancers. Despite activating the same pathway, the BRAF V600E mutation and the prevalent mutations in codon 12 and 13 of KRAS have all been linked to different clinical outcomes, but the molecular mechanisms behind these differences largely remain to be clarified.Methods: To characterize the similarities and differences between common activating KRAS mutations and between KRAS and BRAF mutations, we used genome editing to engineer KRAS G12C/D/V and G13D mutations in colorectal cancer cells that had their mutant BRAF V600E allele removed and subjected them to transcriptome sequencing, global proteomics and metabolomics analyses.Results: By intersecting differentially expressed genes, proteins and metabolites, we uncovered (i) two-fold more regulated genes and proteins when comparing KRAS to BRAF mutant cells to those lacking Ras pathway mutation, (ii) five differentially expressed proteins in KRAS mutants compared to cells lacking Ras pathway mutation (IFI16, S100A10, CD44, GLRX and AHNAK2) and 6 (CRABP2, FLNA, NXN, LCP1, S100A10 and S100A2) compared to BRAF mutant cells, (iii) 19 proteins expressed differentially in a KRAS mutation specific manner versus BRAF V600E cells, (iv) regulation of the Integrin Linked Kinase pathway by KRAS but not BRAF mutation, (v) regulation of amino acid metabolism, particularly of the tyrosine, histidine, arginine and proline pathways, the urea cycle and purine metabolism by Ras pathway mutations, (vi) increased free carnitine in KRAS and BRAF mutant RKO cells.Conclusions: This comprehensive integrative -omics analysis confirms known and adds novel genes, proteins and metabolic pathways regulated by mutant KRAS and BRAF signaling in colorectal cancer. The results from the new model systems presented here can inform future development of diagnostic and therapeutic approaches targeting tumors with KRAS and BRAF mutations.
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| 21. |
- Lillja, Johan
(author)
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Novel strategies to increase throughput and differentiate lipid isomers in mass spectrometry imaging : Development of computational tools and complex mass spectrometric methods for nanospray desorption electrospray ionization
- 2023
-
Doctoral thesis (other academic/artistic)abstract
- In this thesis, method development for improved analyte identification and throughput in mass spectrometry imaging (MSI) is discussed. In MSI, the spatial distribution of analytes from a sample is determined and visualized, information about the detected molecules interaction within the sample can thereby the deduced. Most MSI methods utilize high resolution accurate mass (HRAM) to assign an identity to a feature by its mass-to-charge (m/z) value. However, HRAM cannot distinguish isomeric species. I have therefore developed novel tools for annotation and separation of lipid isomer for MSI with nanospray desorption electrospray ionization (nano-DESI). Specifically, I show that tandem mass spectrometry (MSn) of silver ion species of lipids can be used for the separation of both fatty acid and phospholipid isomers. Additionally, I developed a method for parallelized MSn experiments, by performing multiple ion trap MSn in parallel to a fourier transform mass spectrometry (FTMS) transient. The ion trap MSn, albeit with lower resolution, has orthogonal specificity to FTMS and therefore generates a data set where the analytes identity can be deduced. Because the ITMS is executed in parallel to the typically used FTMS scan the imaging parameters are kept constant, thus generating a richer data set without increasing spatial resolution or experimental runtime.Lastly, data sets generated with nano-DESI MSI are complex and require specialized software tools for processing. I also discuss an open-source tool for data processing with high flexibility and fast processing speeds. With the newly developed tool we were able to process and interrogate data sets, thereby making better use of the acquired data.
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| 22. |
- Lin, Weifeng, et al.
(author)
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Chemoselective and Highly Sensitive Quantification of Gut Microbiome and Human Metabolites
- 2021
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In: Angewandte Chemie International Edition. - : John Wiley & Sons. - 1433-7851 .- 1521-3773. ; 60:43, s. 23232-23240
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Journal article (other academic/artistic)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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| 23. |
- Lin, Weifeng
(author)
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Development of advanced chemical biology tools for microbiome metabolism : Chemoselective probes for enhanced metabolomics analysis
- 2023
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Doctoral thesis (other academic/artistic)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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| 24. |
- Lin, Weifeng, et al.
(author)
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Identification of nutritional biomarkers through highly sensitive and chemoselective metabolomics
- 2023
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In: Food Chemistry. - : Elsevier. - 0308-8146 .- 1873-7072. ; 425
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Journal article (peer-reviewed)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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| 25. |
- Lin, Weifeng, et al.
(author)
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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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In: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773.
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Journal article (peer-reviewed)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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| 26. |
- Lin, Weifeng, et al.
(author)
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Sensitive mass spectrometric analysis of carbonyl metabolites in human urine and fecal samples using chemoselective modification
- 2020
-
In: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 145:11, s. 3822-3831
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Journal article (peer-reviewed)abstract
- Metabolites with ketone or aldehyde functionalities comprise a large proportion of the human metabolome, most notably in the form of sugars. However, these reactive molecules are also generated through oxidative stress or gut microbiota metabolism and have been linked to disease development. The discovery and structural validation of this class of metabolites over the large concentration range found in human samples is crucial to identify their links to pathogenesis. Herein, we have utilized an advanced chemoselective probe methodology alongside bioinformatic analysis to identify carbonyl-metabolites in urine and fecal samples. In total, 99 metabolites were identified in urine samples and the chemical structure for 40 metabolites were unambiguously validated using a co-injection procedure. We also describe the preparation of a metabolite-conjugate library of 94 compounds utilized to efficiently validate these ketones and aldehydes. This method was used to validate 33 metabolites in a pooled fecal sample extract to demonstrate the potential for rapid and efficient metabolite detection over a wide metabolite concentration range. This analysis revealed the presence of six metabolites that have not previously been detected in either sample type. The constructed library can be utilized for straightforward, large-scale, and expeditious analysis of carbonyls in any sample type.
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| 27. |
- Lin, Weifeng, et al.
(author)
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Sensitive quantification of short-chain fatty acids combined with global metabolomics in microbiome cultures
- 2023
-
In: Chemical Communications. - : Royal Society of Chemistry. - 1359-7345 .- 1364-548X. ; 59:39, s. 5843-5846
-
Journal article (peer-reviewed)abstract
- The microbiome has been identified to have a key role for the physiology of their human host. One of the major impacts is the clearance of bacterial pathogens. We have now developed a chemoselective probe methodology for the absolute quantification of short-chain fatty acids at low nM concentrations, with high reproducibility and spiked isotope labelled internal standards. Immobilization to magnetic beads allows for separation from the matrix and the tagged metabolites upon bioorthogonal cleavage can be analyzed via UHPLC-MS. The major advantage of our sensitive method is the simple combination with global metabolomics analysis as only a small sample volume is required. We have applied this chemical metabolomics strategy for targeted SCFA analysis combined with global metabolomics on gut microbiome co-cultures with Salmonella and investigated the effect of antibiotic treatment.
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| 28. |
- Lin, Weifeng, et al.
(author)
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Squaric acid as a new chemoselective moiety for mass spectrometry-based metabolomics analysis of amines
- 2021
-
In: RSC CHEMICAL BIOLOGY. - : Royal Society of Chemistry. - 2633-0679. ; 2:5, s. 1479-1483
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Journal article (peer-reviewed)abstract
- The investigation of microbiome-derived metabolites is important to understand metabolic interactions with their human host. New methodologies for mass spectrometric discovery of undetected metabolites with unknown bioactivity are required. Herein, we introduce squaric acid as a new chemoselective moiety for amine metabolite analysis in human fecal samples.
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| 29. |
- Marklund, Emil, et al.
(author)
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DNA surface exploration and operator bypassing during target search
- 2020
-
In: Nature. - : NATURE RESEARCH. - 0028-0836 .- 1476-4687. ; 583:7818, s. 858-
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Journal article (peer-reviewed)abstract
- Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA(1-5). Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 mu s. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude. Single-molecule fluorescence resonance energy transfer and real-time confocal laser tracking with fluorescence correlation spectroscopy together characterize how individual lac repressor molecules bypass operator sites while exploring the DNA surface at microsecond timescales.
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| 30. |
- Marklund, Emil, et al.
(author)
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Mechanism of DNA surface exploration and operator bypassing during target search
-
Other publication (other academic/artistic)abstract
- Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 μs. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.
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| 31. |
- Mavroudakis, Leonidas, 1994-
(author)
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Analytical developments and applications of ambient mass spectrometry imaging : Visualizing challenging analytes and understanding chemical mechanisms of ischemic stroke
- 2023
-
Doctoral thesis (other academic/artistic)abstract
- Bioanalytical methods that provide spatial information on molecular distributions are important for deep understanding of chemical mechanisms in health and disease. For instance, the localized action of drugs and metabolites can provide valuable information on metabolism in specific cellular regions, which would not be possible with bulk analysis. Mass spectrometry imaging (MSI) is a powerful tool for realizing molecular distributions directly from the surface of biological samples e.g., tissue sections. Analytical and technological developments over the past years have constituted MSI as a valuable asset in the bioanalysis toolbox. Several MSI techniques exist for transferring the analytes from the tissue surface to the mass spectrometer. In this thesis, pneumatically assisted (PA) nanospray desorption electrospray ionization (nano-DESI) was used for ambient MSI of thin tissue sections. In PA nano-DESI MSI, molecules from the surface of the tissue section are desorbed into a liquid stream of solvent that is formed between two thin fused silica capillaries and electrosprayed directly into the mass spectrometer. The extraction solvent allows for addition of internal standards for matrix effects compensation and relative quantitation, and dopants for improved ionization efficiency of analytes as well as host-guest chemistry.In this work, PA nano-DESI MSI was evaluated for its ability to provide insights into chemical mechanisms in health and disease. The effect of preconditioning agents on the membrane lipid breakdown after stroke as well as the effect of ischemia on acylcarnitine metabolism, which is involved in oxidation of fatty acids, was studied. These results provided valuable information into the chemical mechanisms that are induced during ischemia. Additionally, analytical insights are provided on the extraction abilities of various solvents used in PA nano-DESI. Further, analytical methodologies have been developed for analyzing challenging analytes, particularly light alkali metal ions (Na+ and K+), which cannot be directly detected by modern high resolution mass spectrometers, and prostaglandins, which exhibit many isomers that are not distinguishable through mass spectrometry alone. Finally, PA nano-DESI MSI was combined in one analytical platform with surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS) to increase the coverage of information obtained from a single tissue section, i.e. elucidating isomeric species through electrophoretic separation.Overall, this thesis adds unique insights into understanding ischemic stroke and presents novel analytical methodologies for analyzing prostaglandins, and Na+ and K+ ions. All developed methods are fully compatible with liquid extraction based MSI techniques which expands the range of applications.
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| 32. |
- Nainyte, Milda, et al.
(author)
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Amino Acid Modified RNA Bases as Building Blocks of an Early Earth RNA-Peptide World
- 2020
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In: Chemistry - A European Journal. - : WILEY-V C H VERLAG GMBH. - 0947-6539 .- 1521-3765. ; 26:65, s. 14856-14860
-
Journal article (peer-reviewed)abstract
- Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA-peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA-peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem-loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world.
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| 33. |
- Nyström, Niklas, et al.
(author)
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Mucosal and Plasma Metabolomes in New-onset Paediatric Inflammatory Bowel Disease : Correlations with Disease Characteristics and Plasma Inflammation Protein Markers
- 2023
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In: Journal of Crohn's & Colitis. - : Oxford University Press. - 1873-9946 .- 1876-4479. ; 17:3, s. 418-432
-
Journal article (peer-reviewed)abstract
- Background and AimsTo advance the understanding of inflammatory bowel disease [IBD] pathophysiology, we compared the mucosal and plasma metabolomes between new-onset paediatric IBD patients and symptomatic non-IBD controls, and correlated plasma inflammation markers and disease characteristics with the altered metabolites.MethodsPaired colonic and ileal biopsies and plasma from 67 treatment-naïve children with incident Crohn’s disease [CD; n = 47], ulcerative colitis [UC; n = 9], and non-IBD controls [n = 11] were analysed using ultra-performance liquid chromatography-mass spectrometry [UPLC-MS/MS]. Inflammatory plasma proteins [n = 92] were assessed.ResultsThe metabolomes in inflamed mucosal biopsies differed between IBD patients and controls. In CD, mucosal levels of several lysophospholipids [lysophosphatidylcholines, lysophosphatidyletanolamines, lysophosphatidylinositols, and lysophosphatidylserines] were decreased, correlating with various plasma metabolites including amino acid analogues and N-acetylated compounds. In both CD and UC, mucosal sphingolipids, including ceramide [d18:2/24:1, d18:1/24:2], lactosyl-N-palmitoyl-sphingosine [d18:1/16:0], behenoyl sphingomyelin [d18:1/22:0], lignoceroyl sphingomyelin [d18:1/24:0], and/or sphingomyelin [d18:1/24:1, d18:2/24:0] were increased, correlating with sphingolipids, bile acids, and/or N-acetylated metabolites in plasma. Among proteins associated with CD, interleukin-24 correlated with plasma metabolites, including lactosyl-N-palmitoyl sphingosine [d18:1/16:0] and phosphatidyletanolamine [18:1/18:1], haemoglobin, and faecal calprotectin. In UC, interleukin-24, interleukin-17A, and C-C motif chemokine 11 correlated with several plasma metabolites, including N-acetyltryptophan, tryptophan, glycerate, and threonate, and with the Paediatric Ulcerative Colitis Activity Index, C-reactive protein, and faecal calprotectin.ConclusionsMucosal perturbations of lysophospholipids and sphingolipids characterised the metabolome in new-onset paediatric IBD and correlated with plasma metabolites. By integrating plasma metabolomics data with inflammatory proteins and clinical data, we identified clinical and inflammatory markers associated with metabolomic signatures for IBD.
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| 34. |
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| 35. |
- Oldenburg, Joppe, et al.
(author)
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Propranolol Reduces the Development of Lesions and Rescues Barrier Function in Cerebral Cavernous Malformations : A Preclinical Study
- 2021
-
In: Stroke. - : Lippincott Williams & Wilkins. - 0039-2499 .- 1524-4628. ; 52:4, s. 1418-1427
-
Journal article (peer-reviewed)abstract
- Background and Purpose: Cerebral cavernous malformations (CCM) present as mulberry-like malformations of the microvasculature of the central nervous system. Current medical treatment of CCM lesions is limited to surgical removal of the vascular malformations. It is, therefore, important to identify therapeutic drug treatments for patients with CCM. Propranolol has shown great benefit in the treatment of infantile hemangioma. In addition, patients with CCM who receive propranolol have demonstrated a reduction of their lesions. Our investigation set out to provide preclinical data to support propranolol as a therapeutic treatment.Methods: An inducible endothelial-specific Ccm3 knockout murine model (CCM3(iECKO)) was used, with assessment of lesion quantity and size following oral treatment with propranolol. Scanning and transmission electron microscopy were used to characterize the CCM3(iECKO) lesions and the effects of propranolol on the disease. Immunofluorescent imaging was used to investigate pericyte coverage in the propranolol-treated CCM3(iECKO) mice.Results: With propranolol treatment, the lesion quantity, size, and volume decreased in both the brain and retina in the CCM3(iECKO) model. Novel characteristics of the CCM3(iECKO) lesions were discovered using electron microscopy, including plasmalemmal pits and thickening of the endothelial-pericyte basal membrane. These characteristics were absent with propranolol treatment. Pericyte coverage of the CCM3(iECKO) lesions increased after propranolol treatment, and vascular leakage was reduced.Conclusions: This study supports the concept that propranolol can be used to reduce and stabilize vascular lesions and can, therefore, be suggested as a pharmaceutical treatment for CCM.
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| 36. |
- Shirey, Ryan J., et al.
(author)
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Noninvasive Urine Biomarker Lateral Flow Immunoassay for Monitoring Active Onchocerciasis
- 2018
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In: ACS - Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 4:10, s. 1423-1431
-
Journal article (peer-reviewed)abstract
- The parasitic disease onchocerciasis is the second leading cause of preventable blindness, afflicting more than 18 million people worldwide. Despite an available treatment, ivermectin, and control efforts by the World Health Organization, onchocerciasis remains a burden in many regions. With an estimated 120 million people living in areas at risk of infection, efforts are now shifting from prevention to surveillance and elimination. The lack of a robust, point-of-care diagnostic for an active Onchocerca infection has been a limiting factor in these efforts. Previously, we reported the discovery of the biomarker N-acetyl-tyramine-O-glucuronide (NATOG) in human urine samples and its ability to track treatment progression between medicated patients relative to placebo; we also established its capability to monitor disease burden in a jird model. NATOG is a human-produced metabolite of tyramine, which itself is produced as a nematode neurotransmitter. The ability of NATOG to distinguish between active and past infection overcomes the limitations of antibody biomarkers and PCR methodologies. Lateral flow immunoassay (LFIA) diagnostics offer the versatility and simplicity to be employed in the field and are inexpensive enough to be utilized in large-scale screening efforts. Herein, we report the development and assessment of a NATOG-based urine LFIA for onchocerciasis, which accurately identified 85% of analyzed patient samples (N = 27).
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| 37. |
- Tsiara, Ioanna, et al.
(author)
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Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes
- 2023
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:33, s. 12565-12571
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Journal article (peer-reviewed)abstract
- The human body hasevolved to remove xenobiotics through a multistepclearance process. Non-endogenous metabolites are converted througha series of phase I and different phase II enzymes into compoundswith higher hydrophilicity. These compounds are important for diverseresearch fields such as toxicology, nutrition, biomarker discovery,doping control, and microbiome metabolism. One of the challenges inthese research fields has been the investigation of the two majorphase II modifications, sulfation and glucuronidation, and the correspondingunconjugated aglycon independently. We have now developed a new methodologyutilizing an immobilized arylsulfatase and an immobilized & beta;-glucuronidaseto magnetic beads for treatment of human urine samples. The enzymeactivities remained the same compared to the enzyme in solution. Theseparate mass spectrometric investigation of each metabolite classin a single sample was successfully applied to obtain the dietaryglucuronidation and sulfation profile of 116 compounds. Our new chemicalbiology strategy provides a new tool for the investigation of metabolitesin biological samples with the potential for broad-scale applicationin metabolomics, nutrition, and microbiome studies.
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| 38. |
- Vallianatou, Theodosia, et al.
(author)
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Differential regulation of oxidative stress, microbiota-derived, and energy metabolites in the mouse brain during sleep
- 2021
-
In: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. - 1559-7016.
-
Journal article (peer-reviewed)abstract
- Sleep has evolved as a universal core function to allow for restorative biological processes. Detailed knowledge of metabolic changes necessary for the sleep state in the brain is missing. Herein, we have performed an in-depth metabolic analysis of four mouse brain regions and uncovered region-specific circadian variations. Metabolites linked to oxidative stress were altered during sleep including acylcarnitines, hydroxylated fatty acids, phenolic compounds, and thiol-containing metabolites. These findings provide molecular evidence of a significant metabolic shift of the brain energy metabolism. Specific alterations were observed for brain metabolites that have previously not been associated with a circadian function including the microbiome-derived metabolite ergothioneine that suggests a regulatory function. The pseudopeptide β-citryl-glutamate has been linked to brain development and we have now discovered a previously unknown regioisomer. These metabolites altered by the circadian rhythm represent the foundation for hypothesis-driven studies of the underlying metabolic processes and their function.
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| 39. |
- Vallianatou, Theodosia, et al.
(author)
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Differential regulation of oxidative stress, microbiota-derived, and energy metabolites in the mouse brain during sleep
- 2021
-
In: Journal of Cerebral Blood Flow and Metabolism. - : Sage Publications. - 0271-678X .- 1559-7016. ; 41:12, s. 3324-3338
-
Journal article (other academic/artistic)abstract
- Sleep has evolved as a universal core function to allow for restorative biological processes. Detailed knowledge of metabolic changes necessary for the sleep state in the brain is missing. Herein, we have performed an in-depth metabolic analysis of four mouse brain regions and uncovered region-specific circadian variations. Metabolites linked to oxidative stress were altered during sleep including acylcarnitines, hydroxylated fatty acids, phenolic compounds, and thiol-containing metabolites. These findings provide molecular evidence of a significant metabolic shift of the brain energy metabolism. Specific alterations were observed for brain metabolites that have previously not been associated with a circadian function including the microbiome-derived metabolite ergothioneine that suggests a regulatory function. The pseudopeptide beta-citryl-glutamate has been linked to brain development and we have now discovered a previously unknown regioisomer. These metabolites altered by the circadian rhythm represent the foundation for hypothesis-driven studies of the underlying metabolic processes and their function.
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| 40. |
- Vallianatou, Theodosia, et al.
(author)
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Identification of New Ketamine Metabolites and Their Detailed Distribution in the Mammalian Brain
- 2024
-
In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 15:7, s. 1335-1341
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Journal article (peer-reviewed)abstract
- Ketamine is a common anesthetic used in human and veterinary medicine. This drug has recently received increased medical and scientific attention due to its indications for neurological diseases. Despite being applied for decades, ketamine's entire metabolism and pharmacological profile have not been elucidated yet. Therefore, insights into the metabolism and brain distribution are important toward identification of neurological effects. Herein, we have investigated ketamine and its metabolites in the pig brain, cerebrospinal fluid, and plasma using mass spectrometric and metabolomics analysis. We discovered previously unknown metabolites and validated their chemical structures. Our comprehensive analysis of the brain distribution of ketamine and 30 metabolites describes significant regional differences detected mainly for phase II metabolites. Elevated levels of these metabolites were identified in brain regions linked to clearance through the cerebrospinal fluid. This study provides the foundation for multidisciplinary studies of ketamine metabolism and the elucidation of neurological effects by ketamine.
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| 41. |
- Vallianatou, Theodosia, et al.
(author)
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Regional Brain Analysis of Modified Amino Acids and Dipeptides during the Sleep/Wake Cycle
- 2022
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In: Metabolites. - : MDPI. - 2218-1989. ; 12:1
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Journal article (peer-reviewed)abstract
- Sleep is a state in which important restorative and anabolic processes occur. Understanding changes of these metabolic processes during the circadian rhythm in the brain is crucial to elucidate neurophysiological mechanisms important for sleep function. Investigation of amino acid modifications and dipeptides has recently emerged as a valuable approach in the metabolic profiling of the central nervous system. Nonetheless, very little is known about the effects of sleep on the brain levels of amino acid analogues. In the present study, we examined brain regional sleep-induced alterations selective for modified amino acids and dipeptides using Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) based metabolomics. Our approach enabled the detection and identification of numerous amino acid-containing metabolites in the cortex, the hippocampus, the midbrain, and the cerebellum. In particular, analogues of the aromatic amino acids phenylalanine, tyrosine and tryptophan were significantly altered during sleep in the investigated brain regions. Cortical levels of medium and long chain N-acyl glycines were higher during sleep. Regional specific changes were also detected, especially related to tyrosine analogues in the hippocampus and the cerebellum. Our findings demonstrate a strong correlation between circadian rhythms and amino acid metabolism specific for different brain regions that provide previously unknown insights in brain metabolism.
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| 42. |
- Zhang, Xiaonan, et al.
(author)
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Enhanced cytotoxicity of a novel family of ATPase inhibitors in colorectal cancer cells with high NAT2 activity
- 2022
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In: Biochemical Pharmacology. - : Elsevier. - 0006-2952 .- 1356-1839 .- 1873-2968. ; 203
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Journal article (peer-reviewed)abstract
- Loss of heterozygosity (LOH) is a hallmark feature of cancer genomes that reduces allelic variation, thereby creating tumor specific vulnerabilities which could be exploited for therapeutic purposes. We previously reported that loss of drug metabolic arylamine N-acetyltransferase 2 (NAT2) activity following LOH at 8p22 could be targeted for collateral lethality anticancer therapy in colorectal cancer (CRC). Here, we report a novel compound CBK034026C that exhibits specific toxicity towards CRC cells with high NAT2 activity. Connectivity Map analysis revealed that CBK034026C elicited a response pattern related to ATPase inhibitors. Similar to ouabain, a potent inhibitor of the Na+/K+-ATPase, CBK034026C activated the Nf-kB pathway. Further metabolomic profiling revealed downregulation of pathways associated with antioxidant defense and mitochondrial metabolism in CRC cells with high NAT2 activity, thereby weakening the protective response to oxidative stress induced by CBK034026C. The identification of a small molecule targeting metabolic vulnerabilities caused by NAT2 activity provides novel avenues for development of anticancer agents.
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