Publications by authors named "Reinhard Wimmer"

91 Publications

Cyclic, Hydrophobic Hexapeptide Fusahexin Is the Product of a Nonribosomal Peptide Synthetase in .

J Nat Prod 2021 08 22;84(8):2070-2080. Epub 2021 Jul 22.

Department of Chemistry and Bioscience, Aalborg University, Frederik Bajers Vej 7H, DK-9220 Aalborg, Denmark.

The plant pathogenic fungus is known to produce a wide array of secondary metabolites during plant infection. This includes several nonribosomal peptides. Recently, the fusaoctaxin (/9) and gramilin () gene clusters were shown to be induced by host interactions. To widen our understanding of this important pathogen, we investigated the involvement of the gene cluster during infection and oxidative and osmotic stress. Overexpression of led to the discovery of a new cyclic hexapeptide, fusahexin (), with the amino acid sequence cyclo-(d-Ala-l-Leu-d--Thr-l-Pro-d-Leu-l-Leu). The structural analyses revealed an unusual ether bond between a proline C to C of the preceding threonine resulting in an oxazine ring system. The comparative genomic analyses showed that the small gene cluster only encodes an ABC transporter in addition to the five-module nonribosomal peptide synthetase (NRPS). Based on the structure of fusahexin and the domain architecture of , we propose a biosynthetic model in which the terminal module is used to incorporate two leucine units. So far, iterative use of NRPS modules has primarily been described for siderophore synthetases, which makes a rare example of a fungal nonsiderophore NRPS with distinct iterative module usage.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00947DOI Listing
August 2021

Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper ().

J Nat Prod 2021 06 2;84(6):1787-1798. Epub 2021 Jun 2.

Center for Tropical Medicine, NMT, Faculty of Medicine, University of Brasilia, Brasília 70910-900, Brazil.

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead (). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of () . Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.
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http://dx.doi.org/10.1021/acs.jnatprod.1c00153DOI Listing
June 2021

Production and Selectivity of Key Fusarubins from due to Media Composition.

Toxins (Basel) 2021 05 25;13(6). Epub 2021 May 25.

Department of Chemistry and Bioscience, Aalborg University, 6700 Esbjerg, Denmark.

Natural products display a large structural variation and different uses within a broad spectrum of industries. In this study, we investigate the influence of carbohydrates and nitrogen sources on the production and selectivity of production of four different polyketides produced by , fusarubin, javanicin, bostrycoidin and anhydrofusarubin. We introduce four different carbohydrates and two types of nitrogen sources. Hereafter, a full factorial design was applied using combinations of three levels of sucrose and three levels of the two types of nitrogen. Each combination displayed different selectivity and production yields for all the compounds of interest. Response surface design was utilized to investigate possible maximum yields for the surrounding combinations of media. It was also shown that the maximum yields were not always the ones illustrating high selectivity, which is an important factor for making purification steps easier. We visualized the production over time for one of the media types, illustrating high yields and selectivity.
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http://dx.doi.org/10.3390/toxins13060376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230112PMC
May 2021

Metabolic changes during carbon monoxide poisoning: An experimental study.

J Cell Mol Med 2021 06 5;25(11):5191-5201. Epub 2021 May 5.

Department of Anaesthesiology and Intensive Care, Pulmonary Research Center, Aalborg University Hospital, Aalborg, Denmark.

Carbon monoxide (CO) is the leading cause of death by poisoning worldwide. The aim was to explore the effects of mild and severe poisoning on blood gas parameters and metabolites. Eleven pigs were exposed to CO intoxication and had blood collected before and during poisoning. Mild CO poisoning (carboxyhaemoglobin, COHb 35.2 ± 7.9%) was achieved at 32 ± 13 minutes, and severe poisoning (69.3 ± 10.2% COHb) at 64 ± 23 minutes from baseline (2.9 ± 0.5% COHb). Blood gas parameters and metabolites were measured on a blood gas analyser and nuclear magnetic resonance spectrometer, respectively. Unsupervised principal component, analysis of variance and Pearson's correlation tests were applied. A P-value ≤ .05 was considered statistically significant. Mild poisoning resulted in a 28.4% drop in oxyhaemoglobin (OHb) and 12-fold increase in COHb, while severe poisoning in a 65% drop in OHb and 24-fold increase in COHb. Among others, metabolites implicated in regulation of metabolic acidosis (lactate, P < .0001), energy balance (pyruvate, P < .0001; 3-hydroxybutyrc acid, P = .01), respiration (citrate, P = .007; succinate, P = .0003; fumarate, P < .0001), lipid metabolism (glycerol, P = .002; choline, P = .0002) and antioxidant-oxidant balance (glutathione, P = .03; hypoxanthine, P < .0001) were altered, especially during severe poisoning. Our study adds new insights into the deranged metabolism of CO poisoning and leads the way for further investigation.
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http://dx.doi.org/10.1111/jcmm.16522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178256PMC
June 2021

Metabolic fingerprint of progression of chronic hepatitis B: changes in the metabolome and novel diagnostic possibilities.

Metabolomics 2021 01 25;17(2):16. Epub 2021 Jan 25.

Department of Molecular Diagnostics, Aalborg University Hospital, Reberbansgade 15, 9000, Aalborg, Denmark.

Introduction: Chronic hepatitis B (CHB) affects 257 million individuals worldwide with an annual estimated mortality rate of 880,000 individuals. Accurate diagnosis of the stage of disease is difficult, and there is considerable uncertainty concerning the optimal point in time, when treatment should be started.

Objectives: By analyzing and comparing the metabolomes of patients at different stages of CHB and comparing them to healthy individuals, we want to determine the metabolic signature of disease progression and develop a more accurate metabolome-based method for diagnosis of disease progression ultimately giving a better basis for treatment decisions.

Methods: In this study, we used the combination of transient elastography and serum metabolomics of 307 serum samples from a group of 90 patients with CHB before and under treatment (with a follow-up time up to 10 years) at different progression stages over the clinical phases and 43 healthy controls..

Results: Our data show that the metabolomics approach can successfully discover CHB changing from the immune tolerance to the immune clearance phase and show distinctive metabolomes from different medical treatment stages. Perturbations in ammonia detoxification, glutamine and glutamate metabolism, methionine metabolism, dysregulation of branched-chain amino acids, and the tricarboxylic acid (TCA) cycle are the main factors involved in the progression of the disease. Fluctuations increasing in aspartate, glutamate, glutamine, methionine and 13 other metabolites are fingerprints of progression.

Conclusions: The metabolomics approach may expand the diagnostic armamentarium for patients with CHB. This method can provide a more detailed decision basis for starting medical treatment.
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http://dx.doi.org/10.1007/s11306-020-01767-yDOI Listing
January 2021

Tissue, urine and serum NMR metabolomics dataset from a 5/6 nephrectomy rat model of chronic kidney disease.

Data Brief 2020 Dec 23;33:106567. Epub 2020 Nov 23.

Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark.

Serum, urine and tissue from a rat model of chronic kidney disease (CKD) were analysed using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics methods, and compared with samples from sham operated rats. Both urine and serum were sampled at multiple timepoints, and the results have been reported elsewhere (https://doi.org/10.1007/s11306-019-1569-3[1]). The data could be useful to researchers working with human CKD or rat models of the disease. In addition, several different types of NMR spectra were recorded, including 1D NOESY, CPMG, and 2D J-resolved spectra, and the data could be useful for method comparison and algorithm development, both in terms of NMR spectroscopy and multivariate analysis.
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http://dx.doi.org/10.1016/j.dib.2020.106567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708935PMC
December 2020

Heterologous Expression of the Core Genes in the Complex Fusarubin Gene Cluster of .

Int J Mol Sci 2020 Oct 14;21(20). Epub 2020 Oct 14.

Aalborg University Esbjerg, Department of Chemistry and Bioscience, Niels Bohrs Vej 8, 6700 Esbjerg, Denmark.

Through stepwise recreation of the biosynthetic gene cluster containing from , it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in . This was achieved through sequential transformation associated recombination (TAR) cloning of , , , and into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in . The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1-4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L.
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http://dx.doi.org/10.3390/ijms21207601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589453PMC
October 2020

Mechanistic basis of substrate-O coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study.

Proc Natl Acad Sci U S A 2020 08 28;117(32):19178-19189. Epub 2020 Jul 28.

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology and Food Science, Norwegian University of Science and Technology, N-7491 Trondheim, Norway;

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of -LPMO10A from , along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and N-HYSCORE spectroscopy study on the uniformly isotope-labeled Cu(II)-bound N-LPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x-y) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O activation in chitin-active AA10 LPMOs.
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http://dx.doi.org/10.1073/pnas.2004277117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431007PMC
August 2020

Undefeated-Changing the phenamacril scaffold is not enough to beat resistant Fusarium.

PLoS One 2020 29;15(6):e0235568. Epub 2020 Jun 29.

Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark.

Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops. Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium. However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril. In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains. Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235568PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323951PMC
September 2020

A longitudinal serum NMR-based metabolomics dataset of ischemia-reperfusion injury in adult cardiac surgery.

Sci Data 2020 06 24;7(1):198. Epub 2020 Jun 24.

Department of Anaesthesia and Intensive Care, Aalborg University Hospital, 9000, Aalborg, Denmark.

Cardiovascular disease is the leading cause of death worldwide and cardiac surgery is a key treatment. This study explores metabolite changes as a consequence of ischemia-reperfusion due to cardiac surgery with the use of cardiopulmonary bypass (CPB). To describe the ischemia-reperfusion injury, metabolite changes were monitored in fifty patients before and after CPB at multiple time points. We describe a longitudinal metabolite dataset containing nearly 600 serum nuclear magnetic resonance (NMR) spectra obtained from samples collected simultaneously from the pulmonary artery (deoxygenated blood) and left atrium (oxygenated blood) before ischemia (pre-CPB), immediately after reperfusion (end-CPB), and the following 2, 4, 8, and 20 hours postoperatively. In addition, a longitudinal dataset including 57 quantified metabolites is also provided. These datasets will help researchers studying ischemia-reperfusion injury, as well as the time-dependent alterations related to the surgical trauma and the subsequent processes required in regaining metabolite balance. The datasets could also be used for the development of processing algorithms for NMR-based metabolomics studies and methods for the analysis of longitudinal multivariate data.
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http://dx.doi.org/10.1038/s41597-020-0545-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314852PMC
June 2020

The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor.

J Biol Chem 2020 05 21;295(22):7620-7634. Epub 2020 Apr 21.

Aalborg University, Department of Chemistry and Bioscience, 9220 Aalborg, Denmark

Mutations in the genes encoding the highly conserved Ca-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia-causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca coordination and has only a minor impact on binding affinity toward Ca and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca channel, Ca1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84-2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.
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http://dx.doi.org/10.1074/jbc.RA120.013430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261784PMC
May 2020

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca binding and cause misfolding.

J Physiol 2020 03 18;598(6):1169-1186. Epub 2020 Feb 18.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, V6T 1Z3 Vancouver, BC, Canada.

Key Points: Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage-gated calcium channel Ca 1.2. Arrhythmia mutations in CaM can lead to loss of Ca binding, uncoupling of Ca binding cooperativity, misfolding of the EF-hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease.

Abstract: Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage-gated Ca channel Ca 1.2 and the ryanodine receptor (RyR2), in a mutation-specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C-terminal lobe mutants and an N-terminal lobe mutant in complex with the Ca 1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF-hands within the C-lobe and loss of Ca binding in EF-hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca -saturating conditions when unbound to the IQ domain. Ca binding to a pair of EF-hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca ions bound to the C-lobe, possibly disrupting the cooperativity. An N-lobe variant (N54I), which causes severe stress-induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of Ca 1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease.
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http://dx.doi.org/10.1113/JP279307DOI Listing
March 2020

A new vector system for targeted integration and overexpression of genes in the crop pathogen .

Fungal Biol Biotechnol 2019 11;6:25. Epub 2019 Dec 11.

1Department of Chemistry and Bioscience, Aalborg University, Niels Bohrs Vej 8, 6700 Esbjerg, Denmark.

Background: Besides their ability to produce several interesting bioactive secondary metabolites, members of the species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation.

Results: To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in through -mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)Cys transcriptional factor controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin.

Conclusion: By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (: ) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the species complex, and is obtainable from Addgene catalog #133094.

Graphic Abstract:
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http://dx.doi.org/10.1186/s40694-019-0089-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905090PMC
December 2019

Citrate NMR peak irreproducibility in blood samples after reacquisition of spectra.

Metabolomics 2019 12 19;16(1). Epub 2019 Dec 19.

Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220, Aalborg, Denmark.

Background: In our metabolomics studies we have noticed that repeated NMR acquisition on the same sample can result in altered metabolite signal intensities.

Aims: To investigate the reproducibility of repeated NMR acquisition on selected metabolites in serum and plasma from two large human metabolomics studies.

Methods: Two peak regions for each metabolite were integrated and changes occurring after reacquisition were correlated.

Results: Integral changes were generally small, but serum citrate signals decreased significantly in some samples.

Conclusions: Several metabolite integrals were not reproducible in some of the repeated spectra. Following established protocols, randomising analysis order and biomarker validation are important.
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http://dx.doi.org/10.1007/s11306-019-1629-8DOI Listing
December 2019

Characterization of Eight Novel Spiroleptosphols from .

Molecules 2019 Sep 26;24(19). Epub 2019 Sep 26.

Department of Chemistry and Bioscience-Section for Biotechnology, Aalborg University, Frederik Bajers Vej 7H, 9220 Aalborg Ø, Denmark.

Chemical analyses of grown on banana medium resulted in eight novel spiroleptosphols, T1, T2 and U-Z (-). The structures were elucidated by a combination of high-resolution mass spectrometric data and 1- and 2-D NMR experiments. The relative stereochemistry was assigned by H coupling and NOESY/ROESY experiments. Absolute stereochemistry established for by vibrational circular dichroism was found analogous to that of the putative polyketide spiroleptosphol from .
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http://dx.doi.org/10.3390/molecules24193498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804164PMC
September 2019

Tissue, urine and blood metabolite signatures of chronic kidney disease in the 5/6 nephrectomy rat model.

Metabolomics 2019 08 17;15(8):112. Epub 2019 Aug 17.

Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220, Aalborg, Denmark.

Introduction: Progressive chronic kidney disease (CKD) is an important cause of morbidity and mortality. It has a long asymptomatic phase, where routine blood tests cannot identify early functional losses, and therefore identifying common mechanisms across the many etiologies is an important goal.

Objectives: Our aim was to characterize serum, urine and tissue (kidney, lung, heart, spleen and liver) metabolomics changes in a rat model of CKD.

Methods: A total of 17 male Wistar rats underwent 5/6 nephrectomy, whilst 13 rats underwent sham operation. Urine samples were collected weekly, for 6 weeks; blood was collected at weeks 0, 3 and 6; and tissue samples were collected at week 6. Samples were analyzed on a nuclear magnetic resonance spectroscopy platform with multivariate and univariate data analysis.

Results: Changes in several metabolites were statistically significant. Allantoin was affected in all compartments. Renal asparagine, creatine, hippurate and trimethylamine were significantly different; in other tissues creatine, dimethylamine, dimethylglycine, trigonelline and trimethylamine were significant. Benzoate, citrate, dimethylglycine, fumarate, guanidinoacetate, malate, myo-inositol and oxoglutarate were altered in urine or serum.

Conclusion: Although the metabolic picture is complex, we suggest oxidative stress, the gut-kidney axis, acid-base balance, and energy metabolism as promising areas for future investigation.
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http://dx.doi.org/10.1007/s11306-019-1569-3DOI Listing
August 2019

Aerobic dissipation of the novel cyanoacrylate fungicide phenamacril in soil and sludge incubations.

Chemosphere 2019 Oct 5;233:873-878. Epub 2019 Jun 5.

Department of Environmental Science Aarhus University, Frederiksborgvej 399, 4000, Roskilde, Denmark. Electronic address:

The cyanoacrylate ethyl (2Z)-3-amino-2-cyano-3-phenylacrylate (phenamacril), has been introduced as an effective agent against several fungi species belonging to the Fusarium genus. However, in current literature, knowledge about the environmental behavior of this fungicide is limited and there are no data on the degradation in the environment. By performing tests on inherent degradability as well as degradation studies in soils this study provides the only published information regarding the environmental stability and degradation kinetics of this compound. Tests for inherent/ready biodegradation revealed the phenamacril is inherently degradable with zero order kinetics, even though the degradation is comparatively slow. Degradation of phenamacril in soil was found to occur following first order kinetics with a final plateau with a half live of 17.1 days (i.e. more rapidly than tebuconazole but less rapidly than octylisothiazolinone).
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http://dx.doi.org/10.1016/j.chemosphere.2019.06.015DOI Listing
October 2019

Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum.

Fungal Genet Biol 2019 11 4;132:103248. Epub 2019 Jul 4.

Department of Chemistry and Bioscience, Aalborg University, Niels Bohrs Vej 8, 6700 Esbjerg, Denmark. Electronic address:

Filamentous fungi such as species from the genus Fusarium are capable of producing a wide palette of interesting metabolites relevant to health, agriculture and biotechnology. Secondary metabolites are formed from large synthase/synthetase enzymes often encoded in gene clusters containing additional enzymes cooperating in the metabolite's biosynthesis. The true potential of fungal metabolomes remain untapped as the majority of secondary metabolite gene clusters are silent under standard laboratory growth conditions. One way to achieve expression of biosynthetic pathways is to clone the responsible genes and express them in a well-suited heterologous host, which poses a challenge since Fusarium polyketide synthase and non-ribosomal peptide synthetase gene clusters can be large (e.g. as large as 80 kb) and comprise several genes necessary for product formation. The major challenge associated with heterologous expression of fungal biosynthesis pathways is thus handling and cloning large DNA sequences. In this paper we present the successful workflow for cloning, reconstruction and heterologous production of two previously characterized Fusarium pseudograminearum natural product pathways in Fusarium graminearum. In vivo yeast recombination enabled rapid assembly of the W493 (NRPS32-PKS40) and the Fusarium Cytokinin gene clusters. F. graminearum transformants were obtained through protoplast-mediated and Agrobacterium tumefaciens-mediated transformation. Whole genome sequencing revealed isolation of transformants carrying intact copies the gene clusters was possible. Known Fusarium cytokinin metabolites; fusatin, 8-oxo-fusatin, 8-oxo-isopentenyladenine, fusatinic acid together with cis- and trans-zeatin were detected by liquid chromatography and mass spectrometry, which confirmed gene functionality in F. graminearum. In addition the non-ribosomal lipopeptide products W493 A and B was heterologously produced in similar amounts to that observed in the F. pseudograminearum doner. The Fusarium pan-genome comprises more than 60 uncharacterized putative secondary metabolite gene clusters. We nominate the well-characterized F. graminearum as a heterologous expression platform for Fusarium secondary metabolite gene clusters, and present our experience cloning and introducing gene clusters into this species. We expect the presented methods will inspire future endevours in heterologous production of Fusarium metabolites and potentially aid the production and characterization of novel natural products.
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http://dx.doi.org/10.1016/j.fgb.2019.103248DOI Listing
November 2019

Fusaoctaxin A, an Example of a Two-Step Mechanism for Non-Ribosomal Peptide Assembly and Maturation in Fungi.

Toxins (Basel) 2019 05 16;11(5). Epub 2019 May 16.

Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark.

Fungal non-ribosomal peptide synthetase ( clusters are spread across the chromosomes, where several modifying enzyme-encoding genes typically flank one . However, a recent study showed that the octapeptide fusaoctaxin A is tandemly synthesized by two NRPSs in . Here, we illuminate parts of the biosynthetic route of fusaoctaxin A, which is cleaved into the tripeptide fusatrixin A and the pentapeptide fusapentaxin A during transport by a cluster-specific ABC transporter with peptidase activity. Further, we deleted the histone H3K27 methyltransferase , which induced the production of fusaoctaxin A.
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http://dx.doi.org/10.3390/toxins11050277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563249PMC
May 2019

The Proteome of is adapted to Changing Conditions in Wastewater Treatment Plants.

Proteomes 2019 Apr 25;7(2). Epub 2019 Apr 25.

Center for Microbial Communities, Department of Chemistry and Bioscience, Fredrik Bajers Vej 7H, Aalborg University, DK-9220 Aalborg, Denmark.

The activated sludge in wastewater treatment plants (WWTP) designed for enhanced biological phosphorus removal (EBPR) experiences periodically changing nutrient and oxygen availability. is the most abundant genus in Danish WWTP and represents up to 20-30% of the activated sludge community based on 16S rRNA amplicon sequencing and quantitative fluorescence in situ hybridization analyses, although the genus is in low abundance in the influent wastewater. Here we investigated how can successfully out-compete most other microorganisms in such highly dynamic ecosystems. To achieve this, we analyzed the physiological adaptations of the WWTP isolate str. LP2 during an aerobic to anoxic shift by label-free quantitative proteomics and NMR-metabolomics. was used as reference organism as it shares several metabolic capabilities and is regularly introduced to wastewater treatment plants without succeeding there. When compared to only minor changes in the proteome of were observed after the switch to anoxic conditions. This indicates that metabolic pathways for anaerobic energy harvest were already expressed during the aerobic growth. This allows continuous growth of immediately after the switch to anoxic conditions. Metabolomics furthermore revealed that the substrates provided were exploited far more efficiently by than by . These results suggest that prospers in the dynamic WWTP environment due to adaptation to the changing environmental conditions.
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http://dx.doi.org/10.3390/proteomes7020016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630437PMC
April 2019

F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by F NMR.

J Biomol NMR 2019 Apr 18;73(3-4):167-182. Epub 2019 Mar 18.

Section for Biotechnology, Department of Chemistry and Bioscience, Aalborg University, Frederik Bajers Vej 7H, 9220, Aalborg, Denmark.

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.
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http://dx.doi.org/10.1007/s10858-019-00239-3DOI Listing
April 2019

Self-assembled nanoparticles based on cyclodextrin-modified pullulan: Synthesis, and structural characterization using SAXS.

Carbohydr Polym 2019 Jun 30;213:403-410. Epub 2019 Jan 30.

Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220, Aalborg Ø, Denmark. Electronic address:

Synthesis of novel host-guest functionalized polymers is presented along with structural characterization using small-angle X-ray scattering (SAXS) of the resulting nanoparticles. Mono-6-deoxy-mono-6-azidoβCD (NβCD) was grafted onto alkyne-functionalized pullulan via the "click" reaction copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and an adamantane-modified dextran was prepared via the same strategy. Characterization of the polymers was carried out using nuclear magnetic resonance (NMR) spectroscopy, gel filtration chromatography (GFC), isothermal titration calorimetry (ITC) and SAXS. Nanoparticles were created via host-guest interactions between the well-defined βCD-pullulans and adamantane-modified dextran. Characterization was carried out using dynamic light scattering (DLS) and SAXS, which revealed spherical particles in the sub-100 nm range. The studies shed light on the importance of molecular structure and host-guest ratio on crucial properties such as particle size, size distribution, porosity and stability towards aggregation.
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http://dx.doi.org/10.1016/j.carbpol.2019.01.106DOI Listing
June 2019

There it is! Fusarium pseudograminearum did not lose the fusaristatin gene cluster after all.

Fungal Biol 2019 01 28;123(1):10-17. Epub 2018 Oct 28.

Department of Chemistry and Bioscience, Aalborg University, Esbjerg, Denmark. Electronic address:

Fusarium pseudograminearum is a significant pathogen of cereals in arid regions worldwide and has the ability to produce numerous bioactive secondary metabolites. The genome sequences of seven F. pseudograminearum strains have been published and in one of these strains, C5834, we identified an intact gene cluster responsible for biosynthesis of the cyclic lipopeptide fusaristatin A. The high level of sequence identity of the fusaristatin cluster remnant in strains that do not produce fusaristatin suggests that the absence of the cluster evolved once, and subsequently the resulting locus with the cluster fragments became widely dispersed among strains of F. pseudograminearum in Australia. We examined a selection of 99 Australian F. pseudograminearum isolates to determine how widespread the ability to produce fusaristatin A is in F. pseudograminearum. We identified 15 fusaristatin producing strains, all originating from Western Australia. Phylogenetic analyses could not support a division of F. pseudograminearum into fusaristatin producing and nonproducing populations, which could indicate the loss has occurred relatively recent.
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http://dx.doi.org/10.1016/j.funbio.2018.10.004DOI Listing
January 2019

Lung Protection Strategies during Cardiopulmonary Bypass Affect the Composition of Blood Electrolytes and Metabolites-A Randomized Controlled Trial.

J Clin Med 2018 Nov 21;7(11). Epub 2018 Nov 21.

Department of Cardiothoracic Anesthesiology, Rigshospitalet, Copenhagen University Hospital, 2100 Copenhagen, Denmark.

Cardiac surgery with cardiopulmonary bypass (CPB) causes an acute lung ischemia-reperfusion injury, which can develop to pulmonary dysfunction postoperatively. This sub-study of the Pulmonary Protection Trial aimed to elucidate changes in arterial blood gas analyses, inflammatory protein interleukin-6, and metabolites of 90 chronic obstructive pulmonary disease patients following two lung protective regimens of pulmonary artery perfusion with either hypothermic histidine-tryptophan-ketoglutarate (HTK) solution or normothermic oxygenated blood during CPB, compared to the standard CPB with no pulmonary perfusion. Blood was collected at six time points before, during, and up to 20 h post-CPB. Blood gas analysis, enzyme-linked immunosorbent assay, and nuclear magnetic resonance spectroscopy were used, and multivariate and univariate statistical analyses were performed. All patients had decreased gas exchange, augmented inflammation, and metabolite alteration during and after CPB. While no difference was observed between patients receiving oxygenated blood and standard CPB, patients receiving HTK solution had an excess of metabolites involved in energy production and detoxification of reactive oxygen species. Also, patients receiving HTK suffered a transient isotonic hyponatremia that resolved within 20 h post-CPB. Additional studies are needed to further elucidate how to diminish lung ischemia-reperfusion injury during CPB, and thereby, reduce the risk of developing severe postoperative pulmonary dysfunction.
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http://dx.doi.org/10.3390/jcm7110462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262287PMC
November 2018

Enhancing the Production of the Fungal Pigment Aurofusarin in .

Toxins (Basel) 2018 Nov 21;10(11). Epub 2018 Nov 21.

Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark.

There is an increasing demand for products from natural sources, which includes a growing market for naturally-produced colorants. Filamentous fungi produce a vast number of chemically diverse pigments and are therefore explored as an easily accessible source. In this study we examine the positive regulatory effect of the transcription factor AurR1 on the aurofusarin gene cluster in . Proteomic analyses showed that overexpression of AurR1 resulted in a significant increase of five of the eleven proteins belonging to the aurofusarin biosynthetic pathway. Further, the production of aurofusarin was increased more than threefold in the overexpression mutant compared to the wild type, reaching levels of 270 mg/L. In addition to biosynthesis of aurofusarin, several yet undescribed putative naphthoquinone/anthraquinone analogue compounds were observed in the overexpression mutant. Our results suggest that it is possible to enhance the aurofusarin production through genetic engineering.
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http://dx.doi.org/10.3390/toxins10110485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266765PMC
November 2018

Arrhythmia mutations in calmodulin cause conformational changes that affect interactions with the cardiac voltage-gated calcium channel.

Proc Natl Acad Sci U S A 2018 11 22;115(45):E10556-E10565. Epub 2018 Oct 22.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in the genes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (Ca1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the Ca1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca-free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca-dependent inactivation of the voltage-gated calcium channel.
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http://dx.doi.org/10.1073/pnas.1808733115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233071PMC
November 2018

Lung Protection Strategies during Cardiopulmonary Bypass Affect the Composition of Bronchoalveolar Fluid and Lung Tissue in Cardiac Surgery Patients.

Metabolites 2018 Sep 21;8(4). Epub 2018 Sep 21.

Department of Anesthesia and Intensive Care Medicine, Aalborg University Hospital, 9000 Aalborg, Denmark.

Pulmonary dysfunction is among the most frequent complications to cardiac surgeries. Exposure of blood to the cardiopulmonary bypass (CPB) circuit with subsequent lung ischemia-reperfusion leads to the production of inflammatory mediators and increases in microvascular permeability. The study aimed to elucidate histological, cellular, and metabolite changes following two lung protective regimens during CPB with Histidine-Tryptophan-Ketoglutarate (HTK) enriched or warm oxygenated blood pulmonary perfusion compared to standard regimen with no pulmonary perfusion. A total of 90 patients undergoing CPB were randomized to receiving HTK, oxygenated blood or standard regimen. Of these, bronchoalveolar lavage fluid (BALF) and lung tissue biopsies were obtained before and after CPB from 47 and 25 patients, respectively. Histopathological scores, BALF cell counts and metabolite screening were assessed. Multivariate and univariate analyses were performed. Profound histological, cellular, and metabolic changes were identified in all patients after CPB. Histological and cellular changes were similar in the three groups; however, some metabolite profiles were different in the HTK patients. While all patients presented an increase in inflammatory cells, metabolic acidosis, protease activity and oxidative stress, HTK patients seemed to be protected against severe acidosis, excessive fatty acid oxidation, and inflammation during ischemia-reperfusion. Additional studies are needed to confirm these findings.
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http://dx.doi.org/10.3390/metabo8040054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316472PMC
September 2018

Who Needs Neighbors? Is a Stand-Alone Gene in Responsible for Production of Gibepyrones and Prolipyrone B.

Molecules 2018 Sep 2;23(9). Epub 2018 Sep 2.

Department of Chemistry and Bioscience, Aalborg University, 9100 Aalborg, Denmark.

Genome sequencing of the genus has revealed a great capacity for discovery of new natural products of potential economical and therapeutic importance. Several of these are unknown. In this study, we investigated the product of the gene in , which was recently linked to gibepyrones in . Genomic analyses showed that constitutes a stand-alone gene in and related species. Overexpression of resulted in production of gibepyrones A, B, D, G and prolipyrone B, which could not be detected in the wild type strain. Our results suggest that produces the entry compound gibepyrone A, which is subsequently oxidized by one or several non-clustering cytochrome P450 monooxygenases ending with prolipyrone B.
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http://dx.doi.org/10.3390/molecules23092232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225250PMC
September 2018

Genomic and Analyses Reveal the spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems.

Front Microbiol 2018 23;9:1004. Epub 2018 May 23.

Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on str. Lg2 and compared to the physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus , but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal - as they compete for substrates with the PAO and stored carbon is not made available to the " Accumulibacter" PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.
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http://dx.doi.org/10.3389/fmicb.2018.01004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974061PMC
May 2018

The cereal pathogen Fusarium pseudograminearum produces a new class of active cytokinins during infection.

Mol Plant Pathol 2018 05 24;19(5):1140-1154. Epub 2017 Oct 24.

Queensland Bioscience Precinct, Commonwealth Scientific and Industrial Research Organization (CSIRO) Agriculture and Food, Brisbane, 4067, Australia.

The fungal pathogen Fusarium pseudograminearum causes important diseases of wheat and barley. During a survey of secondary metabolites produced by this fungus, a novel class of cytokinins, herein termed Fusarium cytokinins, was discovered. Cytokinins are known for their growth-promoting and anti-senescence activities, and the production of a cytokinin mimic by what was once considered as a necrotrophic pathogen that promotes cell death and senescence challenges the simple view that this pathogen invades its hosts by employing a barrage of lytic enzymes and toxins. Through genome mining, a gene cluster in the F. pseudograminearum genome for the production of Fusarium cytokinins was identified and the biosynthetic pathway was established using gene knockouts. The Fusarium cytokinins could activate plant cytokinin signalling, demonstrating their genuine hormone mimicry. In planta analysis of the transcriptional response to one Fusarium cytokinin suggests extensive reprogramming of the host environment by these molecules, possibly through crosstalk with defence hormone signalling pathways.
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http://dx.doi.org/10.1111/mpp.12593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638160PMC
May 2018
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