Biology subjects

de Jong, L.

Publications and source records attributed to de Jong, L..

6 recordsLinked to original sources

bioRxiv2025

Parameter optimisation for mitigating somatosensory confounds during transcranial ultrasonic stimulation

Transcranial ultrasonic stimulation (TUS) redefines what is possible with non-invasive neuromodulation by oaering unparalleled spatial precision and flexible targeting capabilities. However, peripheral confounds pose a significant challenge to reliably implementing this technology. While auditory confounds during TUS have been studied extensively, the somatosensory confound has been overlooked thus far. It will become increasingly vital to quantify and manage this confound as the field shifts towards higher doses, more compact stimulation devices, and more frequent stimulation through the temple where co-stimulation is more pronounced. Here, we provide a systematic characterisation of somatosensory co-stimulation during TUS. We also identify the conditions under which this confound can be mitigated most eaectively by mapping the confound-parameter space. Specifically, we investigate dose-response eaects, pulse shaping characteristics, and transducer-specific parameters. We demonstrate that somatosensory confounds can be mitigated by avoiding near-field intensity peaks in the scalp, spreading energy across a greater area of the scalp, ramping the pulse envelope, and delivering equivalent doses via longer, lower-intensity pulses rather than shorter, higher-intensity pulses. Additionally, higher pulse repetition frequencies and fundamental frequencies reduce somatosensory eaects. Through our systematic mapping of the parameter space, we also find preliminary evidence that particle displacement (strain) may be a primary biophysical driving force behind peripheral somatosensory co-stimulation. This study provides actionable strategies to minimise somatosensory confounds, which will support the thorough experimental control required to unlock the full potential of TUS for scientific research and clinical interventions. Highlights- Tactile, thermal, and even painful somatosensory confounds may occur during TUS. - Confounds can be mitigated via pulse shaping & transducer-specific parameters. - Valid and replicable TUS research requires control for peripheral confounds. - Particle displacement may be a primary driving force for somatosensory confounds.

neuroscience↗

bioRxiv2022

Cross-link scrambling in peptide pairs

Identification of peptides and their linked amino acids from chemically cross-linked protein complexes with bifunctional N-hydroxysuccinimidyl (NHS) esters can reveal interacting proteins and their spatial arrangements. With NHS esters both amide- and ester cross-links can be formed. Retention time prediction for strong cation exchange chromatography (SCXC) of cross-linked peptides at pH 3 can distinguish between ester and amide cross-links based on their charge differences. By this approach we show that about 98 % of cross-links are formed by two amide bonds. However MS/MS analysis revealed the presence of an ester linkage in more than 5% of peptide pairs predicted by SCXC to be linked by amide bonds. This discrepancy can be explained by intra-peptide amide-ester rearrangement in the gas phase during MS/MS analysis. So, SCXC retention time prediction can be used to distinguish amide-amide, amide-ester and ester-ester linkages actually formed in the cross-linking reaction and to detect scrambling of cross-linked sites. This information is important for studies aimed to understand the spatial arrangement of protein complexes by cross-linking at the highest possible resolution.

biochemistry↗

bioRxiv2020

A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking

In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at a proteome wide-scale and at the peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker that enables isolation of target peptides and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 for analysis of a previously obtained LCMSMS dataset from exponentially growing Bacillus subtilis treated in culture with the cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 in very short time at an overall FDR of < 5%. To also obtain a FDR < 5% for inter-protein cross-linked peptide pairs additional thresholds values were applied for matched fragment intensity and for the numbers of unambiguous y and b ions to be assigned for both composite peptides. Threshold values were based on a set of decoy sequences from yeast and human sequence databases. Also the mass- and charge-dependent retention times of target peptides purified by diagonal strong cation exchange chromatography were used as a criterion to distinguish true from false positives. After this filtering, pLink 2 identified more than 80% of previously reported protein-protein interactions. In addition the use of pLink 2 revealed interesting new inter-protein cross-linked peptide pairs, among others showing interactions between the global transcriptional repressor AbrB and elongation factor Tu and between the essential protein YlaN of unknown function and the ferric uptake repressor Fur. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/097956v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@974dd1org.highwire.dtl.DTLVardef@1760d10org.highwire.dtl.DTLVardef@1539d5eorg.highwire.dtl.DTLVardef@808e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIImproved protocol for identification of PPIs at low FDR by in vivo cross-linking with BAMG C_LIO_LIThe use of all intra-protein cross-linked peptide pairs as true positives C_LIO_LIThe cytosolic aminopeptidase (AMPA_BACSU) interacts with the 50S ribosomal protein L17 C_LIO_LIThe transition state regulator AbrB interacts with elongation factor Tu C_LIO_LIThe essential protein YlaN of unknown function interacts with the iron uptake repressor Fur C_LI SignificanceImportant for reliable identification of PPIs by chemical cross-linking in vivo is a low FDR of non-redundant inter-protein peptide pairs. Here we describe how to recognize the presence of spurious interactions in a dataset of cross-linked peptide pairs enriched by 2D strong cation exchange chromatography and identified by LCMSMS by taking into account chromatographic behavior of cross-linked peptide pairs and protein abundance of corresponding peptides. Based on these criteria we assessed that the FDR of the fraction of non-redundant inter-protein cross-linked peptide pairs was approx. 20-25% by interrogating an entire species specific database at an overall FDR of 5% or 0.1% with a search engine that otherwise scores best in sensitivity among other search engines. We have defined a composite filter to decrease this high FDR of inter-protein cross-linked peptide pairs to only about 2%.

biochemistry↗

bioRxiv2019

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

Clostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold. In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors. ImportanceTranscription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor. Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain. Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.

microbiology↗

bioRxiv2017

Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events

Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. For the first time, we resolve the exon structure of whole BRCA1 transcripts using MinION nanopore sequencing of long-range PCR amplicons. Our study identified 32 BRCA1 isoforms, including 18 novel isoforms which comprised skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as {Delta}(9,10) and {Delta}21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Our results highlight complexity in BRCA1 transcript structure that has not previously been described. This finding has key implications for predicting translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants.

genomics↗

bioRxiv2016

In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at peptide level

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the {delta} and {beta}' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how {delta} might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

biochemistry↗