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Wouters, J.

Publications and source records attributed to Wouters, J..

At least 19 recordsLinked to original sources
bioRxiv2026

Routine FFPE sections support clinically compatible single-nucleus transcriptomics across six human cancer types

Tumor cellular composition--including malignant cell states, immune populations, and stromal populations--is increasingly recognized as a determinant of therapeutic response and resistance to anti-cancer agents, yet comprehensive cellular profiling remains largely confined to research settings. Here, we present a clinically compatible sample-to-report workflow for tumor composition profiling from routine formalin-fixed paraffin-embedded (FFPE) clinical specimens. By combining low-input single-nucleus RNA sequencing with foundation model- based automated cell annotation, this workflow enables prospective sample-by-sample analysis without dedicated research material or cohort-based processing. Across 116 clinical specimens representing six cancer types, we generated reproducible measurements of cellular composition and cell-type-specific gene expression, demonstrated high technical reproducibility, and showed concordance with pathological assessment of immune infiltration. The workflow was similarly applicable to archival FFPE material and ultra-low-input biopsy specimens. Together, these findings establish a practical framework for routine single-cell profiling from standard pathology specimens and open the perspective of prospective evaluation of cellular composition as a clinical biomarker in precision oncology.

cancer biology↗
bioRxiv2026

Neural Tracking of Speech Envelope as an Index of Spatial Release from Masking

Understanding speech in noisy environments relies strongly on binaural cues such as interaural time and level differences (ITDs and ILDs), which support spatial hearing and the segregation of competing sound sources. When these cues are degraded, listeners experience substantial difficulty in complex acoustic environments. Behavioural measures of binaural benefit, such as binaural masking level differences, binaural intelligibility level differences, and spatial release from masking (SRM), are well established in normal-hearing (NH) listeners, but they require an active behavioural response. Neural speech tracking using electroencephalography (EEG) has emerged as a promising approach for quantifying neural processing of continuous speech, yet its sensitivity to spatial hearing cues remains insufficiently characterised. We investigated the neural correlates of spatial release from masking in NH listeners using EEG-based neural speech tracking. Nineteen participants listened to continuous Dutch speech stories presented with masking noise under two spatial configurations, collocated (S0N0) and spatially separated (S0N90), across multiple signal-to-noise ratios (SNRs). Neural tracking of the speech envelope was quantified using both envelope reconstruction and temporal response function (TRF) analyses. Spatial separation enhanced neural tracking of the target speech envelope, particularly at challenging SNRs where behavioural SRM was also observed. TRF analysis further revealed increased amplitudes and decreased latencies of late cortical components consistent with spatial unmasking effects. These findings demonstrate that neural speech tracking captures cortical signatures of spatial unmasking and closely reflects behavioural improvements in speech understanding. Establishing these relationships in NH listeners supports the development of objective neural measures for evaluating binaural benefit in difficult-to-test populations.

neuroscience↗
bioRxiv2025

Neural Phoneme Processing in Children with and without Dyslexia

This study investigates the neural dynamics of phoneme processing in 7-year-old children with and without dyslexia (25;9 [male]), using EEG recordings collected during continuous speech listening. By applying temporal generalization to phonetic descriptor decoding, we can disentangle whether potential phoneme processing deficits are due to the maintenance of phonemes in verbal short-term memory and/or inferred differences in phonetic processing speed, both of which are thought to be impaired in dyslexia. We investigated whether phonetic processing depends on the phonemes position or its lexical competition. Our results reveal two key findings that may help explain the challenges faced by children with dyslexia. First, these children exhibit reduced decoding accuracy for word-onset phonemes, suggesting disruptions in either predictive, word-level anticipatory mechanisms or in the intrinsic rhythmic processing aligned with word boundaries. Second, they exhibit increased decoding accuracy for non-onset phonemes with low lexical competition approximately 400 ms after phoneme onset. This pattern suggests that children with dyslexia retain linguistically less relevant sounds longer in verbal short-term memory and process them more slowly compared to their typical reading peers. Together, these findings suggest that dyslexia is characterized by altered phonetic encoding strategies, specifically inefficient prioritization of relevant phonological information. This work provides new insight into the neural mechanisms underlying phonological deficits and contributes to a deeper understanding of the cognitive basis of dyslexia. Significance statementDyslexia is associated with difficulties in phonological processing. Investigating EEG during continuous speech listening, we show that children with dyslexia exhibit weaker encoding of word-onset phonemes and prolonged processing of less informative phonemes. These altered encoding strategies suggest inefficient prioritization of linguistic information, offering new insight into the neural basis of dyslexia.

neuroscience↗
bioRxiv2023

Neural envelope tracking predicts speech intelligibility and hearing aid benefit in children with hearing loss

Early assessment of hearing aid benefit is crucial, as the extent to which hearing aids provide audible speech information predicts speech and language outcomes. A growing body of research has proposed neural envelope tracking as an objective measure of speech, particularly for individuals unable to provide reliable behavioral feedback. However, its potential for evaluating speech intelligibility and hearing aid benefit in hearing-impaired children remains unexplored. This study examined neural envelope tracking in hearing-impaired children through two separate experiments. EEG data was recorded while children listened to age-appropriate stories (experiment 1) or an animated movie (experiment 2) under aided and unaided conditions (using personal hearing aids) at multiple stimulus intensities. Results in the delta band demonstrated that neural tracking increased with increasing stimulus intensity, but only in the unaided condition. In the aided condition, neural tracking remained stable across a wide range of intensities, as long as speech intelligibility was maintained. This suggests that acoustic degradation of the speech signal does not necessarily impact neural tracking. Additionally, the use of personal hearing aids significantly enhanced neural envelope tracking, particularly in challenging speech conditions (which would be inaudible when unaided). Furthermore, neural envelope tracking strongly correlated with behaviorally measured speech intelligibility. Altogether, these findings indicate that neural envelope tracking could be a valuable tool for predicting speech intelligibility benefits derived from personal hearing aids in hearing-impaired children. Incorporating narrated stories or engaging movies expands the accessibility of these methods even in clinical settings, offering new avenues for using objective speech measures to guide pediatric audiology decision-making.

neuroscience↗
bioRxiv2023

Cardiopulmonary structural, functional and immune-alterations in a Down syndrome mouse model and upon modulation of EGCG

In individuals with Down syndrome (DS), cardiovascular and pulmonary diseases are the most common health problem and result in increased mortality and morbidity. Although these clinical comorbidities are well described, no preclinical models for DS are fully characterized for cardiopulmonary alterations, preventing research to understanding the development and pharmacological modulation of lungs, heart and immune system. Our objective is to characterize the cardiopulmonary and immunological phenotype in Ts65Dn mice and investigate the modulatory effects green tea extract enriched in epigallocatechin 3 gallate (GTE-EGCG). GTE-EGCG administration started at embryonic day 9 and was discontinued at postnatal day (PD) 180. Newborns were longitudinally monitored until PD210 using micro-computed tomography. At endpoint, we characterized the structural, functional and immunological alterations and persistent effects of GTE-EGCG administration. This study revealed normal lung development in the Ts65Dn mice and highlighted RV hypertrophy and immunological alterations. GTE-EGCG administration resulted in genotype-specific and genotype-independent alterations resulting in lung immaturation and airway hyperreactivity. Our results highlight the cardiovascular and immunological phenotype of Ts65Dn mice and potential use for safety studies of therapeutic agents in a DS-specific context. Summary statementThis study longitudinally follows respiratory and cardiac alterations in the Ts65Dn mouse model and describes the impact of prenatal EGCG modulation on the euploid and trisomic phenotype

systems biology↗
bioRxiv2022

SCENIC+: single-cell multiomic inference of enhancers and gene regulatory networks

Joint profiling of chromatin accessibility and gene expression of individual cells provides an opportunity to decipher enhancer-driven gene regulatory networks (eGRN). Here we present a new method for the inference of eGRNs, called SCENIC+. SCENIC+ predicts genomic enhancers along with candidate upstream transcription factors (TF) and links these enhancers to candidate target genes. Specific TFs for each cell type or cell state are predicted based on the concordance of TF binding site accessibility, TF expression, and target gene expression. To improve both recall and precision of TF identification, we curated and clustered more than 40,000 position weight matrices that we could associate with 1,553 human TFs. We validated and benchmarked each of the SCENIC+ components on diverse data sets from different species, including human peripheral blood mononuclear cell types, ENCODE cell lines, human melanoma cell states, and Drosophila retinal development. Next, we exploit SCENIC+ predictions to study conserved TFs, enhancers, and GRNs between human and mouse cell types in the cerebral cortex. Finally, we provide new capabilities that exploit the inferred eGRNs to study the dynamics of gene regulation along differentiation trajectories; to map regulatory activities onto tissues using spatial omics data; and to predict the effect of TF perturbations on cell state. SCENIC+ provides critical insight into gene regulation, starting from multiome atlases of scATAC-seq and scRNA-seq. The SCENIC+ suite is available as a set of Python modules at https://scenicplus.readthedocs.io.

bioinformatics↗
bioRxiv2021

Analysis of long and short enhancers in melanoma cell states

Understanding how enhancers drive cell type specificity and efficiently identifying them is essential for the development of innovative therapeutic strategies. In melanoma, the melanocytic (MEL) and the mesenchymal-like (MES) states present themselves with different responses to therapy, making the identification of specific enhancers highly relevant. Using massively parallel reporter assay (MPRA) in a panel of patient-derived melanoma lines (MM lines), we set to identify and decipher melanoma enhancers by first focusing on regions with state specific H3K27 acetylation close to differentially expressed genes. A more in-depth evaluation of those regions was then pursued by investigating the activity of ATAC-seq peaks found therein along with a full tiling of the acetylated regions with 190 bp sequences. Activity was observed in more than 60% of the selected regions and we were able to precisely locate the active regions within ATAC-seq peaks. Comparison of sequence content with activity, using the deep learning model DeepMEL2, revealed that AP-1 alone is responsible for the MES enhancer activity, while SOX and MITF both influence MEL enhancer activity with SOX being required to achieve high levels of activity. Overall, our MPRA assays shed light on the relationship between long and short sequences in terms of their sequence content, enhancer activity, and specificity as reporters across melanoma cell states.

genomics↗
bioRxiv2021

Antimicrobial activity of a repurposed harmine-derived compound on extensively drug-resistant Acinetobacter baumannii clinical isolates

SynopsisO_ST_ABSObjectivesC_ST_ABSThe spread of antibiotic resistant bacteria is an important threat for human healthcare. Acinetobacter baumannii bacteria impose one of the major issues, as multidrug- to pandrug-resistant strains have been found, rendering some infections untreatable. In addition, A. baumannii is a champion in surviving in harsh environments, being capable of resisting to disinfectants and to persist prolonged periods of desiccation. Due to the high degree of variability found in A. baumannii isolates, the search for new antibacterials is challenging. Here, we screened a compound library to identify compounds active against recent isolates of A. baumannii bacteria. MethodsA repurposing drug screen was undertaken to identify A. baumannii growth inhibitors. One hit was further characterized by determining its IC50 and testing its activity on 43 recent clinical A. baumannii isolates, amongst which 40 are extensively drug- and carbapenem-resistant strains. ResultsThe repurposing screen led to the identification of a harmine-derived compound, called HDC1, which proved to have bactericidal activity on the multidrug-resistant AB5075-VUB reference strain with an IC50 of 48.23 {micro}M. In addition, HDC1 impairs growth of all 43 recent clinical A. baumannii isolates. ConclusionsWe identified a compound with inhibitory activity on all tested, extensively drug-resistant clinical A. baumannii isolates.

microbiology↗
bioRxiv2021

HyDrop: droplet-based scATAC-seq and scRNA-seq using dissolvable hydrogel beads

Single-cell RNA-seq and single-cell ATAC-seq technologies are being used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost, and allow for more specialized multi-ome assays, custom droplet microfluidics may provide complementary solutions to commercial setups. We developed HyDrop, a flexible and generic droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost non-commercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 8,502 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9,508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNAs high capture rate to analyse a small population of FAC-sorted neurons from the Drosophila brain, confirming the protocols applicability to low-input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi-) omics protocols.

genomics↗
bioRxiv2021

Ahead of Maturation: Enhanced Speech Envelope Training Boosts Rise Time Discrimination in Pre-Readers at Cognitive Risk for Dyslexia

Dyslexia has frequently been related to atypical auditory temporal processing and speech perception. Results of studies emphasizing speech onset cues and reinforcing the temporal structure of the speech envelope, i.e. envelope enhancement, demonstrated reduced speech perception deficits in individuals with dyslexia. The use of this strategy as an auditory intervention might thus reduce some of the deficits related to dyslexia. Importantly, interventions are most effective when they are provided during kindergarten and first grade. Hence, we provided a tablet-based 12-week preventive auditory and phonics-based intervention to pre-readers at cognitive risk for dyslexia and investigated the effect on auditory temporal processing with a rise time discrimination task. Ninety-one pre-readers at cognitive risk for dyslexia (aged 5-6) were assigned to two groups receiving a phonics-based intervention and playing a story listening game either with (n = 31) or without (n = 31) envelope enhancement or a third group playing control games and listening to non-enhanced stories (n = 29). Rise time discrimination was measured directly before, directly after and one year after the intervention. While the groups listening to non-enhanced stories mainly improved after the intervention during first grade, the group listening to enhanced stories improved during the intervention in kindergarten and subsequently remained stable during first grade. Hence, an envelope enhancement intervention improves auditory processing skills important for the development of phonological skills. This occurred before the onset of reading instruction, preceding the maturational improvement of these skills, hence giving at risk children a head start when learning to read. Research highlightsO_LIThe first investigation of speech envelope enhancement as a potential preventive intervention strategy in pre-readers at cognitive risk for dyslexia C_LIO_LISpeech envelope enhancement increases the rise time sensitivity of children at cognitive risk for dyslexia C_LIO_LIRise time discrimination can be enhanced before formal reading instruction, a crucial period in development C_LI

neuroscience↗
bioRxiv2021

Cortical compensation for hearing loss, but not age, in neural tracking of the fundamental frequency of the voice

Auditory processing is affected by advancing age and hearing loss, but the underlying mechanisms are still unclear. We investigated the effects of age and hearing loss on temporal processing of naturalistic stimuli in the auditory system. We analysed neural phase-locking to the fundamental frequency of the voice (f0) in 54 normal-hearing and 14 hearing-impaired adults between 17 and 82 years old. We found that both subcortical and cortical neural sources contributed to the responses. Results indicated that advancing age was related to smaller responses with less cortical response contributions, consistent with an age-related decrease in neural phase-locking ability. Conversely, hearing impaired subjects displayed larger responses compared to age-matched normal hearing controls. This was due to additional cortical response contributions which were stronger for participants with more severe hearing loss. This is consistent with the recruitment of additional cortical sources for auditory processing in persons with hearing impairment.

neuroscience↗
bioRxiv2020

Enhanced neural tracking of the fundamental frequency of the voice

Objective F0 tracking is a novel method that investigates the neural processing of the fundamental frequency of the voice (f0) in continuous speech. Through linear modelling, a feature that reflects the stimulus f0 is predicted from the EEG data. Then, the neural response strength is evaluated through the correlation between the predicted and actual f0 feature. The aim of this study was to improve upon this f0 tracking method by optimizing the f0 feature. ApproachSpecifically, we aimed to design a feature that approximates the expected EEG responses to the f0. We hypothesized that this would improve neural tracking results, because the more similar the feature and the neural response are, the easier it will be to reconstruct the one from the other. Two techniques were explored: a phenomenological model to simulate neural processing in the auditory periphery and a low-pass filter to approximate the effect of more central processing on the f0 response. Since these optimizations target different aspects of the auditory system, they were also applied in a cumulative fashion. ResultsResults obtained from EEG evoked by a Flemish story in 34 subjects indicated that both the use of the auditory model and the addition of the low-pass filter significantly improved the correlations between the actual and reconstructed feature. The combination of both strategies almost doubled the mean correlation over subjects, from 0.78 to 0.13. Moreover, canonical correlation analysis with the modelled feature revealed two distinct processes contributing to the f0 response: one driven by the compound activity of auditory nerve fibers with center frequency up to 8 kHz and one driven predominantly by the auditory nerve fibers with center frequency below 1 kHz. SignificanceThe optimized f0 features developed in this study enhance the analysis of f0-tracking responses and facilitate future research and applications.

neuroscience↗
bioRxiv2020

Neural tracking of the fundamental frequency of the voice: male voices preferred

Traditional electrophysiological methods to study temporal auditory processing of the fundamental frequency of the voice (f0) often use unnaturally repetitive stimuli. In this study, we investigated f0 processing of meaningful continuous speech. EEG responses evoked by stories in quiet were analysed with a novel method based on linear modelling that characterizes the neural tracking of the f0. We studied both the strength and the spatio-temporal properties of the f0-tracking response. Moreover, different samples of continuous speech (six stories by four speakers: two male and two female) were used to investigate the effect of voice characteristics on the f0 response. The results indicated that response strength is inversely related to f0 frequency and rate of f0 change throughout the story. As a result, the male-narrated stories in this study (low and steady f0) evoked stronger f0-tracking compared to female-narrated stories (high and variable f0), for which many responses were not significant. The spatio-temporal analysis revealed that f0-tracking response generators were not fixed in the brainstem but were voice-dependent as well. Voices with high and variable f0 evoked subcortically-dominated responses with a latency between 7 and 12 ms. Voices with low and steady f0 evoked responses that are both subcortically (latency of 13-15 ms) and cortically (latency of 23-26 ms) generated, with the right primary auditory cortex as a likely cortical source. Finally, additional experiments revealed that response strength greatly improves for voices with strong higher harmonics, which is particularly useful to boost the small responses evoked by voices with high f0.

neuroscience↗
bioRxiv2020

Antiviral treatment of SARS-CoV-2-infected hamsters reveals a weak effect of favipiravir and a complete lack of effect for hydroxychloroquine

SARS-CoV-2 rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus was able to infect millions of people. To date, close to half a million patients succumbed to the viral disease, COVID-19. The high need for treatment options, together with the lack of small animal models of infection has led to clinical trials with repurposed drugs before any preclinical in vivo evidence attesting their efficacy was available. We used Syrian hamsters to establish a model to evaluate antiviral activity of small molecules in both an infection and a transmission setting. Upon intranasal infection, the animals developed high titers of SARS-CoV-2 in the lungs and pathology similar to that observed in mild COVID-19 patients. Treatment of SARS-CoV-2-infected hamsters with favipiravir or hydroxychloroquine (with and without azithromycin) resulted in respectively a mild or no reduction in viral RNA and infectious virus. Micro-CT scan analysis of the lungs showed no improvement compared to non-treated animals, which was confirmed by histopathology. In addition, both compounds did not prevent virus transmission through direct contact and thus failed as prophylactic treatments. By modelling the PK profile of hydroxychloroquine based on the trough plasma concentrations, we show that the total lung exposure to the drug was not the limiting factor. In conclusion, we here characterized a hamster infection and transmission model to be a robust model for studying in vivo efficacy of antiviral compounds. The information acquired using hydroxychloroquine and favipiravir in this model is of critical value to those designing (current and) future clinical trials. At this point, the data here presented on hydroxychloroquine either alone or combined with azithromycin (together with previously reported in vivo data in macaques and ferrets) provide no scientific basis for further use of the drug in humans.

microbiology↗
bioRxiv2019

Cross-species analysis of melanoma enhancer logic using deep learning

Genomic enhancers form the central nodes of gene regulatory networks by harbouring combinations of transcription factor binding sites. Deciphering the combinatorial code by which these binding sites are assembled within enhancers is indispensable to understand their regulatory involvement in establishing a cells phenotype, especially within biological systems with dysregulated gene regulatory networks, such as melanoma. In order to unravel the enhancer logic of the two most common melanoma cell states, namely the melanocytic and mesenchymal-like state, we combined comparative epigenomics with machine learning. By profiling chromatin accessibility using ATAC-seq on a cohort of 27 melanoma cell lines across six different species, we demonstrate the conservation of the two main melanoma states and their underlying master regulators. To perform an in-depth analysis of the enhancer architecture, we trained a deep neural network, called DeepMEL, to classify melanoma enhancers not only in the human genome, but also in other species. DeepMEL revealed the presence, organisation and positional specificity of important transcription factor binding sites. Together, this extensive analysis of the melanoma enhancer code allowed us to propose the concept of a core regulatory complex binding to melanocytic enhancers, consisting of SOX10, TFAP2A, MITF and RUNX, and to disentangle their individual roles in regulating enhancer accessibility and activity.

genomics↗
bioRxiv2019

From Modulated Noise to Natural Speech: the Effect of Stimulus Parameters on the Frequency Following Response

Frequency following responses (FFRs) can be evoked by a wide range of auditory stimuli, but for many stimulus parameters the effect on FFR strength is not fully understood. This complicates the comparison of earlier studies and the design of new studies. Furthermore, the most optimal stimulus parameters are unknown. To help resolve this issue, we investigated the effects of four important stimulus parameters and their interactions on the FFR. FFRs were measured in 16 normal hearing subjects evoked by stimuli with four levels of stimulus complexity (amplitude modulated noise, artificial vowels, natural vowels and nonsense words), three frequencies (around 105 Hz, 185 Hz and 245 Hz), three frequency contours (upward sweeping, downward sweeping and flat) and three vowels (Flemish /a:/, /u:/, and /i:/). We found that FFRs evoked by artificial vowels were on average 4 to 6 dB SNR larger than responses evoked by the other stimulus complexities, probably because of (unnaturally) strong higher harmonics. Moreover, response amplitude decreased with stimulus frequency but response SNR did not. Thirdly, frequency variation within the stimulus did not impact FFR strength, but only when rate of change remained low (e.g. not the case for sweeping natural vowels). Finally, the vowel /i:/ appeared to evoke larger response amplitudes compared to /a:/ and /u:/, but analysis power was too small to confirm this statistically. Differences in response strength between evoking vowels have been suggested to stem from destructive interference between response components. We show how a model of the auditory periphery can simulate these interference patterns and predict response strength. Altogether, the results of this study can guide stimulus choice for future FFR research and practical applications.

neuroscience↗
bioRxiv2019

Improving source modeling of auditory steady-state responses with frequency-specific brain maps

Auditory steady-state responses (ASSRs) are evoked brain responses to modulated or repetitive acoustic stimuli. Due to a wide range of clinical and research applications, there is a great (clinical) interest to investigate the underlying neural generators of ASSRs. The cortical sources of ASSRs mostly are located in the auditory cortex (AC), although some studies avoiding prior assumptions regarding the number and location of the sources have also reported activity of sources outside of the AC. However, little is known about the number and location of these sources. In this study, we present a novel extension to minimum-norm imaging (MNI) which facilitates ASSR source reconstruction and provides a comprehensive and consistent picture of sources in response to low- as well as high modulation frequencies, monaurally presented to the left and right ears. Results demonstrate that the proposed MNI approach is successful in reconstructing sources located both within (primary) and outside (non-primary) of the AC. The locations of the non-primary sources are consistent with the literature. Primary sources are detected in every experimental condition, thereby corroborating the robustness of the approach. Moreover, we show that the MNI approach is capable of reconstructing the subcortical activities of ASSRs. In summary, the results indicate that the MNI approach outperforms the previously used method of group-ICA, in terms of detection of sources in the AC, reconstructing the subcortical activities and reducing computational load.

neuroscience↗