Research Interests:
Research Interests:
Research Interests:
Background and Aims To 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... more
Background and Aims To 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. Methods Paired 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. Results The 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 ami...
Research Interests:
Research Interests:
Research Interests:
<p>(<i>A</i>) shows that the cell death provoked by SecTRAPs is significantly decreased upon preincubation of either A549 or HeLa cells for 30 min with 100 µM of the general caspase inhibitor zVAD before the SecTRAP... more
<p>(<i>A</i>) shows that the cell death provoked by SecTRAPs is significantly decreased upon preincubation of either A549 or HeLa cells for 30 min with 100 µM of the general caspase inhibitor zVAD before the SecTRAP treatment (**, p<0.01; ***, p<0.001). In (<i>B</i>) HeLa or A549 cells were incubated 30 min with 25 µM of inhibitors for caspase-2 (zVDVAD-fmk), caspase-3 (zDEVD-fmk) or caspase-8 (zIETD-fmk) before SecTRAP treatment. In all of these cases a significantly lower cell death was observed, as indicated in the figure, suggesting that the three caspases may be involved in propagating the cell death triggered by SecTRAPs, as further discussed in the text (*, p<0.05; **, p<0.01; ***, p<0.001).</p
Research Interests:
<p>(<i>A</i>) Morphological features and staining of HeLa and A549 cells after incubation for 4 h with <i>BioPORTER</i> (BP) alone, 100 ng full-length TrxR1/<i>BioPORTER-</i>complex, 100 ng... more
<p>(<i>A</i>) Morphological features and staining of HeLa and A549 cells after incubation for 4 h with <i>BioPORTER</i> (BP) alone, 100 ng full-length TrxR1/<i>BioPORTER-</i>complex, 100 ng SecTRAP/<i>BioPORTER-</i>complex or 1 µM staurosporine (STS), as indicated. Hoechst 33342 was used to visualize the shape or condensation of the nuclei and PI was used as a probe for lack of membrane integrity. Assessment of cell death was performed as described in the text. The percentage of cells denoted as dead, counting a total of 700–1000 cells in this particular experiment, is also given in italics in the lower part of the figure. (<i>B</i>) Exposure of phospatidylserine was evaluated after 3 h treatment of HeLa cells with either only <i>BioPORTER</i>, SecTRAP/<i>BioPORTER-</i>complex or 1 µM staurosporine, staining cells with Annexin-V and PI as described in the text. Magnification was x 40 in all panels.</p
Research Interests:
a,<p>The specific activity was determined with the standard DTNB assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001846#pone.0001846-Arnr5" target="_blank">[50]</a> using... more
a,<p>The specific activity was determined with the standard DTNB assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001846#pone.0001846-Arnr5" target="_blank">[50]</a> using 10 nM enzyme preparation.</p>b,<p>Sec content was estimated from a combined assessment of specific activity, <sup>75</sup>Se incorporation, production and purification method and comparisons to earlier determinations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001846#pone.0001846-Arnr4" target="_blank">[43]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001846#pone.0001846-Cheng1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001846#pone.0001846-Rengby2" target="_blank">[49]</a>.</p>c,<p>Recombinant rat TrxR1 deriva...
Research Interests:
Research Interests:
This is a digital copy of the poster I, Stefanie Prast-Nielsen, presented at IHMC 2018 with the title: "The ketogenic diet influences taxonomic and functional composition of the gut microbiota in children with epilepsy".
Background: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its... more
Background: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. Principal Findings: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADP...
Research Interests:
Mammalian thioredoxin reductase (TrxR; EC 1.8.1.9) is a homodimeric NADPH-dependent selenium-containing flavoenzyme with disulfide oxidoreductase activity. The mammalian thioredoxin (Trx) system exerts an impressive spectrum of functions... more
Mammalian thioredoxin reductase (TrxR; EC 1.8.1.9) is a homodimeric NADPH-dependent selenium-containing flavoenzyme with disulfide oxidoreductase activity. The mammalian thioredoxin (Trx) system exerts an impressive spectrum of functions either directly through reactions catalyzed by TrxR or by its prime substrate Trx. It is involved in redox regulation, antioxidant defense, cell growth, replication and regulation of transcription factors. TrxR was found to be overexpressed in a variety of tumor cells and may be involved in several, if not all, steps of carcinogenesis. Therefore, TrxR has been proposed as a potential target for anticancer therapy. In fact, inhibition of TrxR has been shown by a number of chemotherapeutic drugs as part of their mechanism of action. The rather unique biochemistry of TrxR indeed supports efficient targeting by electrophiles. TrxR has an N-terminal active site with two cysteines (Cys) and a Cterminal active site with a Cys and a selenocysteine (Sec). Bo...
Research Interests:
The scientific community currently defines the human microbiome as all the bacteria, viruses, fungi, archaea, and eukaryotes that occupy the human body. When considering the variable locations, composition, diversity, and abundance of our... more
The scientific community currently defines the human microbiome as all the bacteria, viruses, fungi, archaea, and eukaryotes that occupy the human body. When considering the variable locations, composition, diversity, and abundance of our microbial symbionts, the sheer volume of microorganisms reaches hundreds of trillions. With the onset of next generation sequencing (NGS), also known as high-throughput sequencing (HTS) technologies, the barriers to studying the human microbiome lowered significantly, making in-depth microbiome research accessible. Certain locations on the human body, such as the gastrointestinal, oral, nasal, and skin microbiomes have been heavily studied through community-focused projects like the Human Microbiome Project (HMP). In particular, the gastrointestinal microbiome (GM) has received significant attention due to links to neurological, immunological, and metabolic diseases, as well as cancer. Though HTS technologies allow deeper exploration of the GM, dat...
Research Interests:
Mammalian thioredoxin reductase (TrxR; EC 1.8.1.9) is a homodimeric NADPH-dependent selenium-containing flavoenzyme with disulfide oxidoreductase activity. The mammalian thioredoxin (Trx) system exerts an impressive spectrum of functions... more
Mammalian thioredoxin reductase (TrxR; EC 1.8.1.9) is a homodimeric NADPH-dependent selenium-containing flavoenzyme with disulfide oxidoreductase activity. The mammalian thioredoxin (Trx) system exerts an impressive spectrum of functions either directly through reactions catalyzed by TrxR or by its prime substrate Trx. It is involved in redox regulation, antioxidant defense, cell growth, replication and regulation of transcription factors. TrxR was found to be overexpressed in a variety of tumor cells and may be involved in several, if not all, steps of carcinogenesis. Therefore, TrxR has been proposed as a potential target for anticancer therapy. In fact, inhibition of TrxR has been shown by a number of chemotherapeutic drugs as part of their mechanism of action. The rather unique biochemistry of TrxR indeed supports efficient targeting by electrophiles. TrxR has an N-terminal active site with two cysteines (Cys) and a Cterminal active site with a Cys and a selenocysteine (Sec). Bo...
Research Interests: Immunology and Allergy
Research Interests:
Background Anti-tuberculosis therapy requires at least six-month treatment with continuous administration of combined antibiotics, including isoniazid, rifampicin, pyrazinamide, and ethambutol. The long-term exposure to antibiotics could... more
Background Anti-tuberculosis therapy requires at least six-month treatment with continuous administration of combined antibiotics, including isoniazid, rifampicin, pyrazinamide, and ethambutol. The long-term exposure to antibiotics could cause consequent changes in gut microbiota, which may alter the gastrointestinal function and drug absorption in patients, thereby affect the outcome of treatment. The study aims to characterize the longitudinal changes of gut microbiota among tuberculosis (TB) patients under standardized first-line treatment and provide an understanding of the association between alterations in gut microbiota composition and unfavorable clinical outcomes. Methods The study is a multicenter, observational prospective cohort study. Three study sites are purposively selected in the western (Sichuan Province) and eastern (Jiangsu Province and Shanghai) parts of China. Three-hundred patients with bacteriologically confirmed pulmonary TB are enrolled. All eligible patien...
Research Interests:
Research Interests:
Research Interests:
Research Interests: Organic Chemistry, Medicinal Chemistry, Drug Discovery, Pharmacokinetics, Enzyme Inhibitors, and 15 moreIn Vitro, Humans, Nitric oxide, Schistosomiasis, Animals, Solubility, Rats, Medicinal, Multienzyme complexes, Protein Conformation, ADME, Structure activity Relationship, Biological Availability, Chemical Synthesis, and Pharmacology and pharmaceutical sciences
The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we... more
The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K(i)=2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k(inact)) of (0.73 ± 0.07) × 10⁻³ μM⁻¹ min⁻¹. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.
Research Interests: Kinetics, Fluorescence, Enzyme Inhibitors, Free Radical, Humans, and 15 moreEscherichia coli, High throughput screening, Enzyme, Second Order, Time Dependent, Cell Proliferation, Recombinant Proteins, Molecular Targeted Therapy, Hemin, Biochemistry and cell biology, Anticancer Drug, Thioredoxin reductase, Free Radical Biology, Rate Constant, and lung neoplasms
The selenoprotein thioredoxin reductase 1 (TrxR1) is currently recognized as a plausible anticancer drug target. Here we analyzed the effects of TrxR1 targeting in the human A549 lung carcinoma cell line, having a very high basal TrxR1... more
The selenoprotein thioredoxin reductase 1 (TrxR1) is currently recognized as a plausible anticancer drug target. Here we analyzed the effects of TrxR1 targeting in the human A549 lung carcinoma cell line, having a very high basal TrxR1 expression. We determined the total cellular TrxR activity to be 271.4 +/- 39.5 nmol min(-1) per milligram of total protein, which by far exceeded the total thioredoxin activity (39.2 +/- 3.5 nmol min(-1) per milligram of total protein). Knocking down TrxR1 by approx 90% using siRNA gave only a slight effect on cell growth, irrespective of concurrent glutathione depletion (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; or = 98% decrease), and no increase in cell death or distorted cell cycle phase distributions. This apparent lack of phenotype could probably be explained by Trx functions being maintained by the remaining TrxR1 activity. TrxR1 knockdown nonetheless yielded drug-specific modulation of cytotoxic efficacy in response to various chemotherapeutic agents. No changes in response upon exposure to auranofin or juglone were seen after TrxR1 knockdown, whereas sensitivity to 1-chloro-2,4-dinitrobenzene or menadione became markedly increased. In contrast, a virtually complete resistance to cisplatin using concentrations up to 20 microM appeared upon TrxR1 knockdown. The results suggest that high overexpression of TrxR has an impact not necessarily linked to Trx function that nonetheless modulates drug-specific cytotoxic responses.
Research Interests: Cell Cycle, Apoptosis, Free Radical, Cell line, Humans, and 15 moreCell Death, Glutathione, Naphthoquinones, Cisplatin, Thioredoxin, Lung Carcinoma, Biochemistry and cell biology, Adenocarcinoma, Anticancer Drug, Thioredoxin reductase, Cell Growth, Free Radical Biology, Vitamin K, Total protein, and lung neoplasms
Palladium (Pd), platinum (Pt), and gold (Au) are noble metals, two of which have established medical use. Pt has anticancer efficacy, predominantly as cisplatin, whereas the gold compound auranofin is used against arthritis. Both... more
Palladium (Pd), platinum (Pt), and gold (Au) are noble metals, two of which have established medical use. Pt has anticancer efficacy, predominantly as cisplatin, whereas the gold compound auranofin is used against arthritis. Both compounds inhibit the selenoprotein thioredoxin reductase (TrxR), but Pd has not been studied in this regard. Using salts of Pd, Pt, and Au as well as cisplatin and auranofin we found that Pd and Au were strikingly more potent inhibitors of recombinant TrxR1 than Pt. The TrxR-related nonselenoprotein glutathione reductase in pure form (but less so in a cellular context), as well as cellular thioredoxin (Trx) activities, were inhibited by the gold salt KAuCl(4) but were little affected by auranofin or the other compounds. In an analysis of three cancer cell lines, the extent of inhibition of TrxR activity and decrease in cell viability depended upon the choice of both noble metal and ligand and also seemed independent of p53 status. During treatment of cells with cisplatin, covalent complexes of TrxR1 with either Trx1 or TRP14 (Trx-related protein of 14kDa) were formed, as verified by Western blot analyses and mass spectrometry. These results reveal that Au and Pd are strong inhibitors of TrxR, but Pt and cisplatin trigger highly specific cellular effects on the Trx system, including covalent cross-linking of TrxR1 with Trx1 and TRP14.
Research Interests:
Research Interests:
The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we... more
The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K(i)=2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k(inact)) of (0.73 ± 0.07) × 10⁻³ μM⁻¹ min⁻¹. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.