| 1. |
- Jansson, Erik, 1996, et al.
(författare)
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CONVERGENCE OF THE VERTICAL GRADIENT FLOW FOR THE GAUSSIAN MONGE PROBLEM
- 2024
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Ingår i: Journal of Computational Dynamics. - 2158-2505 .- 2158-2491. ; 11:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- We investigate a matrix dynamical system related to optimal mass transport in the linear category, namely, the problem of finding an optimal invertible matrix by which two covariance matrices are congruent. We first review the differential geometric structure of the problem in terms of a principal fiber bundle. The dynamical system is a gradient flow restricted to the fibers of the bundle. We prove global existence of solutions to the flow, with convergence to the polar decomposition of the matrix given as initial data. The convergence is illustrated in a numerical example.
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| 2. |
- Jin, Zhe, et al.
(författare)
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GABA-mediated inhibition of human CD4+ T cell functions is enhanced by insulin but impaired by high glucose levels
- 2024
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Ingår i: EBioMedicine. - : Elsevier. - 2352-3964. ; 105
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: γ-aminobutyric acid (GABA), known as the main inhibitory neurotransmitter in the brain, exerts immunomodulatory functions by interaction with immune cells, including T cells. Metabolic programs of T cells are closely linked to their effector functions including proliferation, differentiation, and cytokine production. The physiological molecules glucose and insulin may provide environmental cues and guidance, but whether they coordinate to regulate GABA-mediated T cell immunomodulation is still being examined.METHODS: CD4+ T cells that were isolated from blood samples from healthy individuals and from patients with type 1 diabetes (T1D) were activated in vitro. We carried out metabolic assays, multiple proximity extension assay (PEA), ELISA, qPCR, immunoblotting, immunofluorescence staining, flow cytometry analysis, MS-based proteomics, as well as electrophysiology and live-cell Ca2+ imaging.FINDINGS: We demonstrate that GABA-mediated reduction of metabolic activity and the release of inflammatory proteins, including IFNγ and IL-10, were abolished in human CD4+ T cells from healthy individuals and patients with T1D when the glucose concentration was elevated above levels typically observed in healthy people. Insulin increased GABAA receptor-subunit ρ2 expression, enhanced the GABAA receptors-mediated currents and Ca2+ influx. GABA decreased, whereas insulin sustained, hexokinase activity and glycolysis in a glucose concentration-dependent manner.INTERPRETATION: These findings support that metabolic factors, such as glucose and insulin, influence the GABA-mediated immunomodulation of human primary T cells effector functions.FUNDING: The Swedish Children's Diabetes Foundation, The Swedish Diabetes Foundation, The Swedish Research Council 2018-02952, EXODIAB, The Ernfors Foundation, The Thurings Foundation and the Science for Life Laboratory.
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| 3. |
- Nezhyva, Mariya, et al.
(författare)
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Inquiry-based Learning of Proteomics and Metabolomics
- 2024
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Ingår i: Journal of Chemical Education. - : American Chemical Society (ACS). - 0021-9584 .- 1938-1328. ; 101:2, s. 521-529
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Tidskriftsartikel (refereegranskat)abstract
- Given the rapid development of data-driven experimental procedures within the life sciences, it is important to equip students with proper skills and knowledge on how to obtain and interpret complex data. While laboratory exercises have for a long time been well established as a teaching method for life sciences, we consider there is room for improvement in how laboratory exercises are conducted. Hence, we designed a laboratory exercise course in which students at the graduate level in the European education system (M.Sc.) are challenged to pose their own biological question and write their own laboratory protocol for a proteomic study to investigate their hypothesis. Here, students are supported in their task with lectures and seminars that take them through the required details on experimental sample preparation, analysis with LC-MS, and proteomic data evaluation of biological function and relevance. According to student interviews, the inquiry-based learning concept we used here provided a deeper understanding of the laboratory protocols they wrote, according to which they eventually performed their own experiments.
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| 4. |
- Nezhyva, Mariya
(författare)
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Integrative Approaches in Shotgun Proteomics : From sample preparation to multifaceted data analysis
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bottom-up proteomics mass-spectrometry gives an opportunity to shed light on var-ious biological aspects/characteristics such as protein composition, its modifications,interactions, and dynamics. Its applications span a wide range of biological sciencesaiming to eventually answer fundamental disease related questions or evaluate drugperformance and predict adverse effects.Bottom-up proteomics experiments are worth the time and effort because it is a comprehensive and multi-level approach to address research questions from a single dataset. Hence, we can gain a thorough understanding of the processes in the studied system and their interconnected changes, thereby confirming results from different perspectives. While the majority of research is focused on the improvement of analytical techniques, it remains challenging to derive meaningful biological insights from the data.This Ph.D. thesis contributes to various proteomics challenges, including the development of a high-throughput micro-scale proteomic workflow and comprehensive multifaceted analysis. In particular, the data on the thermal proteome profiling of melanocyte-stimulating hormone interactions with melanocortin receptors was analyzed using a newly developed workflow. This workflow uniquely combines thermal stabilization data, inferred transcription factor activity, and pathway analysis. Additionally, this thesis integrates omics approaches such as metabolomics and proteomics. Another dimension to the dataset was added by combining metabolic mass spectrometry imaging with region-specific proteomics for the characterization of cocaine’s neurotoxicity.
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| 5. |
- Aerts, Jordan
(författare)
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Capillary electrophoresis mass spectrometry applied to structural proteomics and small molecule analysis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Capillary electrophoresis with mass spectrometric (CE–MS) detection offers a separation method without equal in terms of flexibility, utility, and cost efficiency. Here we demonstrate precisely this through the application of several laboratory-built CE–MS instruments for the separation of brain metabolites in non-primates, enantioselective separations of synthetic anesthetic metabolites in fractionated pony urine, application in structural proteomics workflows, and identification of exogenous alkaloid biotransformationproducts in human cerebrospinal fluid (CSF).We outline a method for quickly and affordably etching austenitic steel tubing, which is widely used in electrospray sources for CE–MS. The stainless steel tapered tip emitters provide robust electrospray with low sheath liquid flow rates and can be easily fabricated in-house, offering flexibility and cost-efficiency when commercial options areunavailable. We contribute a CE–MS method for enantiomer separation, specifically targeting 6-hydroxynorketamine (HNK). By introducing chiral selectors into the separation capillary, the method enables efficient enantiomer separation and offers a newtool to assist with research on HNK as a cure for depression.We explore the feasibility of cold CE–MS in hydrogen deuterium exchange workflows. The utilization of a lab-designed Peltier-cooled CE device achieves deuterium back exchange rates on par with commercial liquid chromatography-based platforms, offering new possibilities for studying protein structures and interactions.We also demonstrate the wide ranging versatility of CE–MS with contributions to the identification of specific tobacco related metabolites in CSF samples during the development of a high throughput mass spectrometry diagnostic tool for Parkinson’sDisease.This thesis showcases the versatility and value of CE–MS in various applications, a true blessing for analytical chemistry.
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| 6. |
- Aerts, Jordan, et al.
(författare)
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Electrochemically Etched Tapered-Tip Stainless-Steel Electrospray-Ionization Emitters for Capillary Electrophoresis-Mass Spectrometry
- 2023
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 22:4, s. 1377-1380
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Tidskriftsartikel (refereegranskat)abstract
- We have used household consumables to facilitate electrochemical etching of stainless-steel hypodermic tubing to produce tapered-tip emitters suitable for electrospray ionization for use in mass spectrometry. The process involves the use of 1% oxalic acid and a 5 W USB power adapter, commonly known as a phone charger. Further, our method avoids the otherwise commonly used strong acids that entail chemical hazards: concentrated HNO3 for etching stainless steel, or concentrated HF for etching fused silica. Hence, we here provide a convenient and self-inhibiting procedure with minimal chemical hazards to manufacture tapered-tip stainless-steel emitters. We show its performance in metabolomic analysis with CE-MS of a tissue homogenate where the metabolites acetylcarnitine, arginine, carnitine, creatine, homocarnosine, and valerylcarnitine were identified, all with basepeak separated electropherograms, within <6 min of separation. The mass spectrometry data are freely available through the MetaboLight public data repository via access number MTBLS7230.
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| 7. |
- Aerts, Jordan, et al.
(författare)
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Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry
- 2023
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Ingår i: Analytical Chemistry. - : Springer Nature. - 0003-2700 .- 1520-6882. ; 95:2, s. 1149-1158
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Tidskriftsartikel (refereegranskat)abstract
- Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX–MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.
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| 8. |
- Sandbaumhüter, Friederike A., et al.
(författare)
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Enantioselective CE–MS analysis of ketamine metabolites in urine
- 2023
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Ingår i: Electrophoresis. - : Wiley-VCH Verlagsgesellschaft. - 0173-0835 .- 1522-2683. ; 44:1-2, s. 125-134
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Tidskriftsartikel (refereegranskat)abstract
- The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was used to develop a method for the enantioselective analysis of HNK stereoisomers with a special focus on the 6HNK stereoisomers. The partial filling approach was applied in order to avoid contamination of the MS with the chiral selector. Concentration of the chiral selector and the length of the separation zone were optimized. With 5% highly sulfated γ-CD in 20 mM ammonium formate with 10% formic acid and a 75% filling the four 6HNK stereoisomers could be separated with a resolution between 0.79 and 3.17. The method was applied to analyze fractionated equine urine collected after a ketamine infusion and to screen the fractions as well as unfractionated urine for the parent drug ketamine and other metabolites, including norketamine and dehydronorketamine.
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| 9. |
- Sandbaumhuter, Friederike A., et al.
(författare)
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Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling
- 2023
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:41, s. 15400-15408
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Tidskriftsartikel (refereegranskat)abstract
- Thermal proteome profiling with label-free quantitation using ion-mobility-enhanced LC-MS offers versatile data sets, providing information on protein differential expression, thermal stability, and the activities of transcription factors. We developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments that incorporates the aspects of gene set enrichment analysis, differential protein expression analysis, and inference of transcription factor activities from LC-MS data. We applied it to study the signaling processes downstream of melanocortin 3 receptor (MC3R) activation by endogenous agonists derived from the proopiomelanocortin prohormone: ACTH, alpha-MSH, and gamma-MSH. The obtained information was used to map signaling pathways downstream of MC3R and to deduce transcription factors responsible for cellular response to ligand treatment. Using our workflow, we identified differentially expressed proteins and investigated their thermal stability. We found in total 298 proteins with altered thermal stability, resulting from MC3R activation. Out of these, several proteins were transcription factors, indicating them as being downstream target regulators that take part in the MC3R signaling cascade. We found transcription factors CCAR2, DDX21, HMGB2, SRSF7, and TET2 to have altered thermal stability. These apparent target transcription factors within the MC3R signaling cascade play important roles in immune responses. Additionally, we inferred the activities of the transcription factors identified in our data set. This was done with Bayesian statistics using the differential expression data we obtained with label-free quantitative LC-MS. The inferred transcription factor activities were validated in our bioinformatic pipeline by the phosphorylated peptide abundances that we observed, highlighting the importance of post-translational modifications in transcription factor regulation. Our multidimensional data analysis workflow allows for a comprehensive characterization of the signaling processes downstream of MC3R activation. It provides insights into protein differential expression, thermal stability, and activities of key transcription factors. All proteomic data generated in this study are publicly available at DOI: 10.6019/PXD039945.
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| 10. |
- Stenum, Thomas Søndergaard, et al.
(författare)
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RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:9, s. 4572-4587
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Tidskriftsartikel (refereegranskat)abstract
- RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.
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