Background: Composition of high-density lipoproteins (HDL) is emerging as an important determinant in the development of microvascular complications in type 2 diabetes mellitus (T2DM). Dutch South... Show moreBackground: Composition of high-density lipoproteins (HDL) is emerging as an important determinant in the development of microvascular complications in type 2 diabetes mellitus (T2DM). Dutch South Asian (DSA) individuals with T2DM display an increased risk of microvascular complications compared with Dutch white Caucasian (DwC) individuals with T2DM. In this study, we aimed to investigate whether changes in HDL composition associate with increased microvascular risk in this ethnic group and lead to new lipoprotein biomarkers. Materials and Methods: Using H-1 nuclear magnetic resonance spectroscopy and Bruker IVDr Lipoprotein Subclass Analysis (B.I.LISA) software, plasma lipoprotein changes were determined in 51 healthy individuals (30 DwC, 21 DSA) and 92 individuals with T2DM (45 DwC, 47 DSA) in a cross-sectional, case-control study. Differential HDL subfractions were investigated using multinomial logistic regression analyses, adjusting for possible confounders including BMI and diabetes duration. Results: We identified HDL compositional differences between healthy and diabetic individuals in both ethnic groups. Specifically, levels of apolipoprotein A2 and HDL-4 subfractions were lower in DSA compared with DwC with T2DM. Apolipoprotein A2 and HDL-4 subfractions also negatively correlated with waist circumference, waistto-hip ratio, haemoglobin A1c, glucose levels and disease duration in DSA with T2DM, and associated with increased incidence of microvascular complications .Conclusion: While HDL composition differed between controls and T2DM in both ethnic groups, the lower levels of lipid content in the smallest HDL subclass (HDL-4) in DSA with T2DM appeared to be more clinically relevant, with higher odds of having diabetes-related pan-microvascular complications such as retinopathy and neuropathy. These typical differences in HDL could be used as ethnicity-specific T2DM biomarkers. Show less
Alarcon-Barrera, J.C.; Kostidis, S.; Ondo-Mendez, A.; Giera, M. 2022
The pharmaceutical industry adapted proteomics and other 'omics technologies for drug research early following their initial introduction. Although metabolomics lacked behind in this development,... Show moreThe pharmaceutical industry adapted proteomics and other 'omics technologies for drug research early following their initial introduction. Although metabolomics lacked behind in this development, it has now become an accepted and widely applied approach in early drug development. Over the past few decades, metabolomics has evolved from a pure exploratory tool to a more mature and quantitative biochemical technology. Several metabolomics-based platforms are now applied during the early phases of drug discovery. Metabolomics analysis assists in the definition of the physiological response and target engagement (TE) markers as well as elucidation of the mode of action (MoA) of drug candidates under investigation. In this review, we highlight recent examples and novel developments of metabolomics analyses applied during early drug development. Show less
Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis... Show moreLipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated Drhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O- deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition. Show less
Mucins are large glycoproteins characterized by the abundant O-linked oligosaccharides (O-glycans) clustered on a protein backbone. Most of the circulating mucins are rapidly cleared by glycan... Show moreMucins are large glycoproteins characterized by the abundant O-linked oligosaccharides (O-glycans) clustered on a protein backbone. Most of the circulating mucins are rapidly cleared by glycan-recognizing hepatic clearance receptors in the liver. Those mucins that remain in the bloodstream are most commonly used as markers in clinical diagnostics. One of such circulating mucins is MUC16; a peptide epitope of which is known as CA125 antigen - a marker for ovarian cancer. Here, using a targeted 1H-NMR profiling of plasma we are exploring a link between the measured CA125 values and the systemic metabolism of the patients within a group with confirmed high-grade ovarian cancer. The study allowed identifying statistically significant associations between the measured values of CA125 epitope and the plasma concentrations of glucose, glutamine, alanine, betaine and serine. The significance of the identified associations for the listed compounds is below 0.01. This, in turn, enables us to hypothesize about a possibility of including the metabolic measures into a composite score of the ovarian cancer based on the CA125 epitope of MUC16. Show less
Context: There is a need for novel biomarkers and better understanding of the pathophysiology of diabetic kidney disease.Objective: To investigate associations between plasma metabolites and kidney... Show moreContext: There is a need for novel biomarkers and better understanding of the pathophysiology of diabetic kidney disease.Objective: To investigate associations between plasma metabolites and kidney function in people with type 2 diabetes (T2D).Design: 3089 samples from individuals with T2D, collected between 1999 and 2015, from 5 independent Dutch cohort studies were included. Up to 7 years follow-up was available in 1100 individuals from 2 of the cohorts.Main outcome measures: Plasma metabolites (n = 149) were measured by nuclear magnetic resonance spectroscopy. Associations between metabolites and estimated glomerular filtration rate (eGFR), urinary albumin-to-creatinine ratio (UACR), and eGFR slopes were investigated in each study followed by random effect meta-analysis. Adjustments included traditional cardiovascular risk factors and correction for multiple testing.Results: In total, 125 metabolites were significantly associated (P-FDR = 1.5x10(-32) - 0.046; beta = -11.98-2.17) with eGFR. Inverse associations with eGFR were demonstrated for branched-chain and aromatic amino acids (AAAs), glycoprotein acetyls, triglycerides (TGs), lipids in very low-density lipoproteins (VLDL) subclasses, and fatty acids (P-FDR < 0.03). We observed positive associations with cholesterol and phospholipids in high-density lipoproteins (HDL) and apolipoprotein A1 (P-FDR < 0.05). Albeit some metabolites were associated with UACR levels (P < 0.05), significance was lost after correction for multiple testing. Tyrosine and HDL-related metabolites were positively associated with eGFR slopes before adjustment for multiple testing (P-Tyr = 0.003; P-HDLrelated < 0.05), but not after.Conclusions: This study identified metabolites associated with impaired kidney function in T2D, implying involvement of lipid and amino acid metabolism in the pathogenesis. Whether these processes precede or are consequences of renal impairment needs further investigation. Show less
Narcissus is an economically important crop with a large number of species, hybrids and/or varieties in cultivation. It is one of the most popular ornamental plants. The Netherlands is among the... Show moreNarcissus is an economically important crop with a large number of species, hybrids and/or varieties in cultivation. It is one of the most popular ornamental plants. The Netherlands is among the top producers of Narcissus in the world. The ornamental value of Narcissus is quite well known but they can also be a valuable resource of natural compounds. Among these compounds, galanthamine has been successfully commercialized as medicine for the treatment of early to mild Alzheimer’s Disease (AD). The characteristic symptoms of this disease are loss of memory and impairment of multiple cognitive and emotional functions. The objective was to find out how galanthamine levels could be enhanced to convert this floricultural crop into a medicinal crop. There are different factors that influence the production of galanthamine and other metabolites in the bulbs. Effect of various factors such as preanalytical methods, bulb location and age along with effect of biotic and abiotic factors on the growth and yield were studied. From the results, it is evident that factors have detrimental effect on the total bulb yield and galanthamine levels. Show less
Hameed, D.S.; Tilburg, G.B.A. van; Merkx, R.; Flierman, D.; Wienk, H.; Oualid, F. el; ... ; Ovaa, H. 2020
Ubiquitination is a process in which a protein is modified by the covalent attachment of the C-terminal carboxylic acid of ubiquitin (Ub) to the epsilon-amine of lysine or N-terminal methionine... Show moreUbiquitination is a process in which a protein is modified by the covalent attachment of the C-terminal carboxylic acid of ubiquitin (Ub) to the epsilon-amine of lysine or N-terminal methionine residue of a substrate protein or another Ub molecule. Each of the seven internal lysine residues and the N-terminal methionine residue of Ub can be linked to the C-terminus of another Ub moiety to form 8 distinct Ub linkages and the resulting differences in linkage types elicit different Ub signaling pathways. Cellular responses are triggered when proteins containing ubiquitin-binding domains (UBDs) recognize and bind to specific polyUb linkage types. To get more insight into the differences between polyUb chains, all of the seven lysine-linked di-ubiquitin molecules (diUbs) were prepared and used as a model to study their structural conformations in solution using NMR spectroscopy. We report the synthesis of diUb molecules, fully N-15-labeled on the distal (N-terminal) Ub moiety and revealed their structural orientation with respect to the proximal Ub. As expected, the diUb molecules exist in different conformations in solution, with multiple conformations known to exist for K6-, K48-, and K63-linked diUb molecules. These multiple conformations allow structural flexibility in binding with UBDs thereby inducing unique responses. One of the well-known but poorly understood UBD-Ub interaction is the recognition of K6 polyubiquitin by the ubiquitin-associated (UBA) domain of UBXN1 in the BRCA-mediated DNA repair pathway. Using our synthetic N-15-labeled diUbs, we establish here how a C-terminally extended UBA domain of UBXN1 confers specificity to K6 diUb while the non-extended version of the domain does not show any linkage preference. We show that the two distinct conformations of K6 diUb that exist in solution converge into a single conformation upon binding to this extended form of the UBA domain of the UBXN1 protein. It is likely that more of such extended UBA domains exist in nature and can contribute to linkage-specificity in Ub signaling. The isotopically labeled diUb compounds described here and the use of NMR to study their interactions with relevant partner molecules will help accelerate our understanding of Ub signaling pathways. Show less
BlaC is the β-lactamase of Mycobacterium tuberculosis. We show that it can recover from inhibition by clavulanic acid and that phosphate helps it do so. We also show that in solution, BlaC is a... Show moreBlaC is the β-lactamase of Mycobacterium tuberculosis. We show that it can recover from inhibition by clavulanic acid and that phosphate helps it do so. We also show that in solution, BlaC is a rigid protein on the pico-nanosecond timescale but shows dynamics around the active site on the catalytic timescale. These dynamics become more pronounced upon inhibitor binding. Lastly, we show that two mutations that both provide BlaC with inhibitor resistance have very different effects on the dynamic behaviour. Show less
The studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a... Show moreThe studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a degree of ambiguity, this approach allows to iteratively refine and validate the model and interpret its meaning for the molecular basis of protein function. Often all three points at the same time. This dynamic nature makes the use of structural models in the design of therapeutic compounds especially useful since the inhibition of a certain protein function might not require a structure to be accurate down to the last atom but rather highlight key interactions or structural features that can be addressed in context of small molecule or peptide inhibitors. Presented are the use of strucutral biochemistry techniques to investigate the mechanism of how the ubiquitine ligase PSIP1 obtains its target specificity. Furthermore, another epigenetic effector protein PSIP1 is investigated with the aim to develop a workflow for the design of potential peptide-based inhibitors. Show less
In the early nineties, Sidles (1991) came with a solution to combine the force microscopy techniques sensitive to atoms with that of magnetic resonance techniques: Magnetic Resonance Force... Show moreIn the early nineties, Sidles (1991) came with a solution to combine the force microscopy techniques sensitive to atoms with that of magnetic resonance techniques: Magnetic Resonance Force Microscopy (MRFM) was born. The technique was promising, big steps were taken, and the holy grail of atomic resolution imaging of biological tissues seemed within an arm’s reach. Unfortunately, the last steps are the most difficult. The technique is experimentally challenging and so far, the images of biological structures are no better than those obtained by other conventional techniques. In order to be an attractive technique, MRFM needs to be scientifically relevant while the technique is further improved towards the holy grail of imaging biological structures on the nanometer scale. In this thesis, we show how MRFM can usefully contribute to the field of condensed-matter. Show less
The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation... Show more The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation sequencing revealed that the biosynthetic potential of actinomycetes had been grossly underestimated. In this thesis, different antibiotics-eliciting strategies, including microbial cocultivation, streptomycin-resistant mutation, overexpression of pathway-specific activator, variation of culture conditions, were utilized to enforce fluctuations in the production of bioactive compounds in actinomycetes, after which, NMR-based metabolic profiling was used to facilitate uncovering those elicited molecules. This pipeline allowed the discovery of new antibiotics involving various chemical skeletons, such as 7-prenylisatin, methoxylated isocoumarins, endophenazines, and C-glycosylpyranonaphthoquinones. On the other hand, genome-mining methodology enabled the discovery of a group of endophenasides and leucanicidin in Kitasatospora sp. MBT66, whereby the rhamnosylation of both scaffold are executed by a same promiscuous glycosyltransferase. Last but not least, a novel antibiotic termed lugdunomycin with unprecedented chemical scaffold, as well as a number of new angucycline-type antibiotics, were characterized from Streptomyces sp. QL37. The biosynthetic pathway of lugdunomycin was deciphered by genetic knockout and OSMAC (One Strain MAny Compound) strategy. In summary, this thesis explores an interface of genomics and metabolomics to accelerate new antibiotics discovery. Show less
Kohler, I.; Verhoeven, A.; Derks, R.J.E.; Giera, M. 2016
Metabolomics-based strategies have become an integral part of modern clinical research, allowing for a better understanding of pathophysiological conditions and disease mechanisms, as well as... Show moreMetabolomics-based strategies have become an integral part of modern clinical research, allowing for a better understanding of pathophysiological conditions and disease mechanisms, as well as providing innovative tools for more adequate diagnostic and prognosis approaches. Metabolomics is considered an essential tool in precision medicine, which aims for personalized prevention and tailor-made treatments. Nevertheless, multiple pitfalls may be encountered in clinical metabolomics during the entire workflow, hampering the quality of the data and, thus, the biological interpretation. This review describes the challenges underlying metabolomics-based experiments, discussing step by step the potential pitfalls of the analytical process, including study design, sample collection, storage, as well as preparation, chromatographic and electrophoretic separation, detection and data analysis. Moreover, it offers practical solutions and strategies to tackle these challenges, ensuring the generation of high-quality data. Show less
Nuclear magnetic resonance force microscopy (MRFM) is a technique which combines magnetic resonance imaging (MRI) with scanning probe microscopy (SPM). The final goal is to develop this technique... Show moreNuclear magnetic resonance force microscopy (MRFM) is a technique which combines magnetic resonance imaging (MRI) with scanning probe microscopy (SPM). The final goal is to develop this technique to such a level that the atomic structure of a virus or protein can be revealed by this microscope. This thesis shows nuclear magnetic resonance force measurements on copper in which the interaction of the magnetic moments of the nuclei of copper with a magnetic cantilever has delivered a detectable signal at a temperature of 50 millikelvin. Furthermore, we show measurements, which support a new theory where at low magnetic field and low temperature, non contact friction between the magnetic cantilever and paramagnetic electron spins is described. These measurements were enabled by technical improvements such as vibration reduction in a cryogen free dilution refrigerator. As a benchmark for the low vibration, we show atomic resolution scanning tunneling microscopy at 15 millikelvin temperature on graphite. We also show a method to create small magnets for MRFM from a thin magnet film. With these small magnets the field gradient and therefore the sensitivity may be significantly enhanced. Show less
