Biology subjects

de Boer, M.

Publications and source records attributed to de Boer, M..

13 recordsLinked to original sources

bioRxiv2025

Comparative analysis of metabarcoding and metagenomics for fish biodiversity estimates using standard and novel high-flow filtration methods

Molecular techniques involving environmental DNA (eDNA) are increasingly used for aquatic species detection. Metabarcoding, a widely adapted technique, suffers from primer bias: uneven amplification of species due to primer mismatches. The primer bias can be eliminated by omitting PCR, thereby sequencing all eDNA in a sample. This method, known as metagenomics, offers potential benefits for relative abundance estimates and epigenetic modifications, but is seldom applied to eukaryotic communities and eDNA. This study uses an expanded two-by-two design to compare fish species detection between multi-marker metabarcoding and metagenomics using two filter types (conventional versus high-flow). Environmental DNA was collected in a controlled setup and two field settings, which contained several fish species including European sturgeon (Acipenser sturio). Moreover, we explore methylation patterns obtained from nanopore native sequencing. All species present in the controlled environment were detected using both metabarcoding and metagenomics. In field settings, metagenomics detected more species than metabarcoding. High-flow filters recovered more species across all sequencing datasets, except in metabarcoding of field settings. Relative read counts between metabarcoding and metagenomics illustrate primer bias is present in the used primer sets. Most fish metagenomic sequences were identified as A. sturio across all eDNA samples. We observed three base modifications on the 18S region of A. sturio, where three sites showed different methylation patterns between eDNA samples. Our results demonstrate that metabarcoding and metagenomics function complementary in species detection and metagenomics provides additional insights into base modifications. Moreover, high-flow filters offer strong potential for improved species detection in various environments.

molecular biology↗

bioRxiv2025

Bispecific CD20xCD40 Antibodies Achieve Multi-Lineage Modulation of Humoral and Cellular Immunity

Monoclonal antibodies targeting CD20 and CD40 modulate humoral and cellular immunity and have shown therapeutic benefit in B cell malignancies and autoimmune diseases. Here, we describe the generation and preclinical evaluation of two novel bispecific antibodies (DFI201 and DFI205) that combine CD20-mediated B cell depletion inhibition of CD40-mediated influencing populations of B cells, myeloid cells, and T cells. Both bispecifics retained potent B cell cytotoxicity and uniquely extended depletion to non-classical monocytes. A study in cynomolgus macaques showed that administration of bispecific at 5 and 50 mg/kg, or combination therapy with ofatumumab and the anti-CD40 mAb DFI105, achieved rapid and sustained depletion of CD19+CD20+ B cells in blood and lymphoid tissues, with superior memory B cell clearance compared to monospecific controls Both bispecifics transiently reduced non-classical monocytes and potently depleted dendritic cell subsets (DC1 and DC2). The bispecifics expand regulatory T cells and induce T cell exhaustion without activating T Cells. No cytokine release syndrome or adverse safety signals were observed. Functional immunization with keyhole limpet hemocyanin demonstrated that CD40 engagement was essential to suppress KLH-specific IgG formation. These results establish CD20xCD40 bispecific antibodies as a safe and effective approach to achieve deep, multi-lineage immunomodulation with potential for autoimmune indications.

immunology↗

bioRxiv2025

NEXT-FRET: A solution-based smFRET platform to resolve folding intermediates under native conditions

Folding intermediates are promising therapeutic targets in protein misfolding, bacterial virulence, and drug discovery. Yet, directly observing these transient, non-equilibrium states under physiological conditions remains challenging. Here, we introduce NEXT-FRET (Non-Equilibrium miXTure modeling of smFRET), a solution-based single-molecule FRET (smFRET) platform integrating accessible instrumentation and a time-variant Gaussian Mixture Model (tvGMM) to identify folding intermediates without microfluidics, surface tethering, or denaturants. Using maltose-binding protein (MBP) and its biologically relevant precursor (pre-MBP) for benchmarking and validation, we uncover previously elusive intermediates, including a long-hypothesized closed conformation undetected by existing single-molecule and ensemble methods. Furthermore, we demonstrate that chaperones modulate folding landscapes by stabilizing distinct intermediates, some effectively arresting folding progression; a mechanism increasingly recognized as a therapeutic strategy for targeted protein inactivation. Broadly applicable beyond folding, NEXT-FRET provides a generalizable, high-resolution analytical method for resolving transient biomolecular intermediates, offering a screening-compatible platform in biotechnology and elucidating proteostasis collapse in conformational diseases.

biophysics↗

bioRxiv2024

Safeguarding Iguana diversity: Enabling rapid and low-effort tracking of non-native iguanas through terrestrial eDNA innovations

Reptiles have among the highest extinction risk across terrestrial vertebrates, with habitat fragmentation, habitat destruction, and invasive alien species being the primary causes of reptile species loss on a global scale. Invasive hybridization (i.e. hybridization between native and invasive alien species) is increasing globally, causing the extinction of native genotypes, and this phenomenon is particularly pervasive in Caribbean iguanas. The Lesser Antillean Iguana (Iguana delicatissima), a keystone species of Caribbean coastal ecosystems, has become critically endangered mainly due to ongoing hybridization with the invasive Common Green Iguana (I. iguana). For impactful conservation intervention, the need for early detection of invasive animals and their progeny, or detection of surviving pure native animals, is urgent. We aimed to develop a novel environmental DNA (eDNA) toolkit using Kompetitive Allele Specific PCR (KASP) technology, a method of allele-specific amplification for cost-effective and efficient sampling of terrestrial substrates to aid in mapping the distribution of native I. delicatissima, invasive I. iguana, and signal potential invasive hybridization. We demonstrate proof-of-concept and successfully identified I. delicatissima, I. iguana, and their hybrids via blood samples using our primer sets, as well as successful detection of I. delicatissima in several ex-situ (Rotterdam Zoo) and in-situ (St. Eustatius) eDNA samples, collected with environmental swabs and tape-lifting. We found that sampling potential perching spots yielded the highest number of positive detections via environmental swabbing and tape-lifting. Our toolkit demonstrates the potential of terrestrial eDNA sampling for iguana conservation, enabling faster detection of potential invasive hybridization. Additionally, the method holds promise for other terrestrial cryptic species, contributing to broader collection of population-level information.

ecology↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2021

Sleep spindle dynamics suggest over-consolidation in post-traumatic stress disorder

Devastating and persisting traumatic memories are a central symptom of post-traumatic stress disorder (PTSD). Sleep problems are highly co-occurrent with PTSD and intertwined with its etiology. Notably, sleep hosts memory consolidation processes, supported by sleep spindles (11-16 Hz). Here we assess the hypothesis that intrusive memory symptoms in PTSD may arise from excessive memory consolidation, reflected in exaggerated spindling. We use a newly developed spindle detection method, entailing minimal assumptions regarding spindle phenotype, to assess spindle activity in PTSD patients and traumatized controls (n=2x14, matched on gender). Our results show increased spindle activity in PTSD, which positively correlates with daytime intrusive memory symptoms. Together, these findings provide a putative mechanism through which profound sleep disturbance in PTSD may contribute to memory problems. Due to its uniform and unbiased approach, the new, minimal assumption spindle detection method seems a promising tool to detect aberrant spindling in psychiatric disorders.

neuroscience↗

bioRxiv2021

Stability of ligand-induced protein conformation influences affinity in maltose-binding protein

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MPB) from Escherichia coli. We used singlemolecule Forster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.

biochemistry↗

bioRxiv2020

Triggering closure of a sialic acid TRAP transporter substrate binding protein through binding of natural or artificial substrates

The pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.

biophysics↗

bioRxiv2020

Evolution of structural dynamics in bilobed proteins

Novel biophysical tools allow the structural dynamics of proteins, and the regulation of such dynamics by binding partners, to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remains poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the type-II periplasmic binding protein domain family, have undergone divergent evolution leading to modification of their structural dynamics and function. We performed a structural analysis of ~600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Forster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enables distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extra-cytoplasmic transport-related proteins. Structural embellishments of the core created new interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately, altered substrate specificity. Our findings reveal an as yet unrecognized mechanism for the emergence of functional promiscuity during long periods of protein evolution and are applicable to a large number of domain architectures.

biophysics↗

bioRxiv2020

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Forster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the type-I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using the methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our study expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

biophysics↗

bioRxiv2020

The relation between intrinsic protein conformational changes and ligand binding

1Structural changes in proteins allow them to exist in several conformations. Non-covalent interactions with ligands drive the structural changes, thereby allowing the protein to perform its biological function. Recent findings suggest that many proteins are always in an equilibrium of different conformations and that each of these conformations can be formed by both the ligand-free and ligand-bound protein. By using classical statistical mechanics, we derived the equilibrium probabilities of forming a conformation with and without ligand. We found, under certain conditions, that increasing the probability of forming a conformation by the ligand-free protein also increases the probability of forming the same conformation when the protein has a ligand bound. Further, we found that changes in the conformational equilibrium of the ligand-free protein can increase or decrease the affinity for the ligand.

biophysics↗

bioRxiv2019

Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open-to a closed-conformation upon substrate binding providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

biophysics↗

bioRxiv2017

The Spectral Fingerprint of Sleep Problems in Post-Traumatic Stress Disorder

BACKGROUNDSleep problems are a core feature of post-traumatic stress disorder (PTSD). However, a robust objective measure for the sleep disturbance in patients has yet to be found.\n\nMETHODSThe current study assessed EEG power across a wide frequency range and multiple scalp locations, in matched trauma-exposed individuals with and without PTSD, during rapid eye movement (REM) and non-REM (NREM) sleep. In addition, a full polysomnographical evaluation was performed, including sleep staging and assessment of respiratory function, limb movements and heart rate. The occurrence of sleep disorders was also assessed.\n\nRESULTSIn PTSD patients, NREM sleep shows a substantial loss of slow oscillation power and increased higher frequency activity compared to controls. The change is most pronounced in right-frontal brain areas and correlates with insomnia. PTSD REM sleep shows a large power shift in the opposite direction, with increased slow oscillation power in occipital areas, which is strongly related to nightmare activity and to lesser extent with insomnia. These pronounced spectral changes occur in the context of severe subjective sleep problems, increased occurrence of various sleep disorders and modest changes in sleep macrostructure.\n\nCONCLUSIONSThis is the first study to show pronounced changes in EEG spectral topologies during both NREM and REM sleep in PTSD. Importantly, the observed power changes reflect the hallmarks of PTSD sleep problems: insomnia and nightmares and may thus be specific for PTSD. A spectral index derived from these data distinguishes patients from controls with high effect size, bearing promise as a candidate biomarker.

neuroscience↗