Biology subjects

Spaink, H. P.

Publications and source records attributed to Spaink, H. P..

17 recordsLinked to original sources

bioRxiv2026

Accelerating drug development in infectious diseases using zebrafish disease models supported by pharmacokinetic pharmacodynamic modeling as new approach methodology (NAM)

The development of novel therapeutics for infectious diseases remains a global health priority. To accelerate the treatment development, innovative strategies through new approach methodology (NAM) are needed to bridge speed of in vitro with predictive power of in vivo studies, while reducing mammalian experiments. The zebrafish (Danio rerio), particularly the embryo/larva, has been established as a valuable non-mammalian in vivo model in biomedical research. We developed a standardized and streamlined workflow for the zebrafish as NAM, which consisted of 3 steps: drug selection and efficacy evaluation, internal exposure assessment, and PKPD modelling. Compounds with higher tolerated doses than minimum inhibitory concentration were selected. Drug efficacy was quantified through longitudinal individual fluorescence microscopy at baseline and 24 and 48h on treatment. Drug exposure was quantified in larval homogenates and exposure medium from 0-48h on treatment. The PKPD relationship was quantified by non-linear mixed effects modelling. For case study bedaquiline, PKPD was quantified using a one-compartment model with age-depending elimination, and an Emax concentration-response relationship on the delayed logistic bacterial growth function, with an EC50 of 26.6 {micro}g/mL and an Emax of 1.07-1.37. In the case of clarithromycin, in contrast, negligible internal exposure after waterborne treatment were observed, illustrating the risk of false negatives without internal exposure assessments. Bactericidal efficacy was confirmed by intravenous drug injections, showing a clear dose dependent antimycobacterial effect. The standardized zebrafish NAM workflow presented here facilitates the translation of drug efficacy to higher vertebrates, reducing rodent studies to confirmatory or replacing them completely, thus accelerating drug development.

pharmacology and toxicology↗

bioRxiv2025

Unprecedented Cytotoxicity of Transition Metal Metallacarboranes on Triple Negative Breast Cancer Cells and a Vertebrate Cancer Model

Metallacarboranes have long been the subject of attention in the context of medicinal chemistry because of their promising characteristics and unconventional interactions with biological entities. The metal centre has been shown to have a significant influence on the internalisation and cytotoxicity of compounds in human cell lines. Additionally, specific nanomolar concentrations of metallacarboranes have demonstrated toxic effects on MDA-MB-231 cells under in vitro and in vivo conditions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/635527v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@116eb95org.highwire.dtl.DTLVardef@1c4a181org.highwire.dtl.DTLVardef@474c3forg.highwire.dtl.DTLVardef@dec88c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

The human pathogen Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum trigger the same core set of late innate immune response genes in zebrafish larvae

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Granuloma-like structures carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post infection. The continued presence of viable M. tuberculosis in the zebrafish larvae was further confirmed using the molecular bacterial load (MBL) assay. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the response of zebrafish larvae infected with the surrogate species M. marinum. We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis.

immunology↗

bioRxiv2023

Comparison of robotic automated and manual injection methods in zebrafish embryos for high throughput RNA silencing using CRISPR-CasRx

Recently, the CRISPR-RfxCas13d (CasRx) system was proven to induce efficient mRNA knockdown in animal embryos. Here we compared the efficiency of CasRx-based RNA depletion with that of Cas9-mediated DNA targeting under the same conditions, using automated robotic and manual injection methods. As a proof-of-principle target, we used the no tail (tbxta) gene in zebrafish embryos, for which knockdown and knockout embryonic phenotypes were easy to be scored. Both Cas9 and CasRx systems induced loss of function phenotypes of tbxta gene. Higher percentage of severe phenotype was observed using Cas9 protein compared to the mRNA while the efficiency was similar in terms of Cas13d protein and mRNA. In addition, both the robotic and manual injection approaches yielded similar percentages of phenotypes and mortality rates. Therefore, our study not only showcases the potential of RNA-targeting CRISPR effectors for precise and potent gene knockdown, but also emphasizes automated microinjection in zebrafish embryos as an excellent alternative to manual methods for achieving gene knockdown at a high throughput level.

bioengineering↗

bioRxiv2022

Taxonomic and metabolic diversity of Actinobacteria isolated from faeces of a 28,000-year-old mammoth

Ancient microbial communities of permafrost soils and frozen animal remains represent an archive that has barely been explored. This yet unexplored microbial world is a vast resource that can provide us with new evolutionary insights, metabolic pathways and novel chemistry. Here, we reveal that Actinobacteria isolated from a faecal sample from the intestinal tract of a 28,000-year-old Siberian mammoth are phylogenetically and metabolically distinct from currently known modern siblings. Ancient Micromonospora, Oerskovia, Saccharopolyspora, Sanguibacter and Streptomyces species were successfully revived and their genome sequences resolved. Surprisingly, the genomes of the ancestors show a large phylogenetic distance to strains isolated today and harbour many novel biosynthetic gene clusters that may well represent uncharacterised biosynthetic potential. Metabolic profiles of the strains display production of known molecules like antimycin, conglobatin and macrotetrolides, but the majority of the mass features could not be dereplicated. Our work provides a snapshot into Actinobacteria of the past, yielding unexplored genomic information that is not yet present in current databases.

microbiology↗

bioRxiv2022

Cellular Factors Involved in Transcriptome Dynamics in Early Zebrafish Embryogenesis

At gastrulation in the zebrafish embryogenesis, the embryonic genome is switched on to produce transcripts that are used for the maintenance and development of the embryo. In a previous study from late blastula to mid gastrula on the transcriptomes of 179 individual embryos, we capture the transcriptome dynamics via ten gene-expression types. Here we study the factors that regulate these transcriptome dynamics by in extensive silico analyses and two small-RNA sequencing experiments. We analyzed mechanisms that would make it possible for the embryo to achieve the tight regulation of gene expression that was observed, not only during development, but also when individual embryos were compared. We found that many of the gene-expression regulatory factors that are available to the embryo are operational in the different gene-expression types and act concurrently with not one mechanism prevailing in this developmental phase. We also saw that at least one of the regulatory mechanisms, the expression of members of the miRNA-430 family again is very tightly regulated, both during development as well as when miRNA expression from individual embryos is compared.

developmental biology↗

bioRxiv2022

Loss of Shp1 impairs myeloid cell function and causes lethal inflammation in zebrafish larvae

PTPN6 encodes SHP1, a protein tyrosine phosphatase that has an essential role in immune cell function. SHP1 missense and splice site mutations are associated with neutrophilic dermatoses and emphysema in humans, which resembles the phenotype seen in mice that lack functional SHP1 partially. Complete lack of SHP1 function in mouse motheaten mutants leads to severe defects and lethality at 2-6 weeks after birth. Here, we investigated the function of Shp1 in developing zebrafish embryos. We generated a ptpn6 knockout zebrafish line lacking functional Shp1. Zebrafish develop ex utero and are transparent, which facilitates analysis of the function of Shp1 during early hematopoietic development. Shp1 knockout caused severe inflammation and lethality around 17 days post fertilization (dpf). During early development the myeloid lineage was affected, which resulted in a decrease in the number of neutrophils, and a concomitant increase in the number of macrophages. The number of emerging hematopoietic stem and progenitor cells (HSPCs) was decreased, but due to hyperproliferation, the number of HSPCs was higher in ptpn6 mutants than in siblings at 5 dpf. Finally, directional migration of neutrophils and macrophages was decreased in response to wounding and less macrophages were recruited to the wound site. Yet, regeneration of the caudal fin fold was normal. We conclude that loss of Shp1 impairs neutrophil and macrophage function and caused severe inflammation and lethality at the larval stage.

developmental biology↗

bioRxiv2020

Tuberculosis causes highly conserved metabolic changes in human patients, mycobacteria-infected mice and zebrafish larvae

Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.

molecular biology↗

bioRxiv2020

Immunoglobulin switch-like recombination regions implicated in the formation of extrachromosomal circular 45S rDNA involved in the maternal-specific translation system of zebrafish

Cellular translation is essential to all life on earth and in recent years we have reported on the discovery of a unique dual translation system in zebrafish. In this system, a maternal-type variant shows absolute expression in eggs and is progressively replaced during embryogenesis by a somatic-type variant. There are several translation system components, all with a non-coding RNA part, that show this dual characteristic: snRNA, snoRNA, rRNA, RNaseP, tRNA, and SRP-RNA. To produce sufficient ribosomes during oogenesis, zebrafish amplify their 45S locus (18S-5.8S-28S tandem repeat) by means of extrachromosomal circular DNA (eccDNA) organized in extrachromosomal rDNA circles (ERCs). Although this cellular process is discovered quite some time ago, still little is known about the mechanisms involved. Yet, because only the 45S maternal-type (45S-M) rRNA is expressed during oogenesis, the zebrafish genome provides a rare opportunity to compare an ERC 45S locus to a non-ERC 45S locus. In this study, we analyzed the genomic composition of the 45S-M and 45S-S (somatic-type) loci in combination with ultra-long read Nanopore sequencing of ERCs present in total DNA isolated from zebrafish eggs. We discovered 45S-M flanking sequences that were absent in the 45S-S locus and showed high homology to immunoglobulin (Ig) switch regions. Also, several other unique G-quadruplex DNA containing regions were found in the 45S-M locus. Some of those auxiliary regions showed different sizes in the sequenced ERCs, although within each ERC they appear to have identical sizes. These results point to a two-step system for ERC synthesis in zebrafish oogenesis: first the 45S-M repeat is excised from the chromosome into an ERC by recombination that uses the flanking Ig switch-like regions, after which the initial ECR is multiplied and extended into many ECRs with a varying number of 45S-M repeats.

developmental biology↗

bioRxiv2020

Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

Currently the majority of non-culturable microbes in sea water are yet to be discovered, Nanopore offers a solution to overcome the challenging tasks to identify the genomes and complex composition of oceanic microbiomes. In this study we evaluate the utility of Oxford Nanopore Technologies (ONT) sequencing to characterize microbial diversity in seawater from multiple locations. We compared the microbial species diversity of retrieved environmental samples from two different locations and time points. With only three ONT flow cells we were able to identify thousands of organisms, including bacteriophages, from which a large part at species level. It was possible to assemble genomes from environmental samples with Flye. In several cases this resulted in >1 Mbp contigs and in the particular case of a Thioglobus singularis species it even produced a near complete genome. k-mer analysis reveals that a large part of the data represents species of which close relatives have not yet been deposited to the database. These results show that our approach is suitable for scalable genomic investigations such as monitoring oceanic biodiversity and provides a new platform for education in biodiversity

bioinformatics↗

bioRxiv2019

New observations on non-coding RNAs involved in the dual translation system in zebrafish development.

Cellular translation relies heavily on the involvements of several types of non-coding RNAs. In previous studies we have identified a dual translation system in zebrafish development, involving maternal-type and somatic-type rRNAs, snoRNAs, and snRNAs. In this study we focused on several remaining non-coding RNAs involved in the translation system; tRNAs, RNase P, and SRP RNA. Even though our studies have been limited in extent, for all three types of non-coding RNA we were able to identify a maternal-specific type, with substantial sequence differences as compared to the somatic-type variant. Hence, these RNA types complement the previously discovered RNA types in the unique dual translation system in zebrafish development.

developmental biology↗

bioRxiv2019

Maternal- and Somatic-type snoRNA Expression and Processing in Zebrafish Development

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that play an important role in the complex maturation process of ribosomal RNAs (rRNAs). SnoRNAs are categorized in classes, with each class member having several variants present in a genome. Similar to our finding of specific rRNA expression types in zebrafish embryogenesis, we discovered preferential maternal- and somatic-expression for snoRNAs. Most snoRNAs and their variants have higher expression levels in somatic tissues than in eggs, yet we identified three snoRNAs; U3, U8 and snoZ30 of which specific variants show maternal- or somatic-type expression. For U3 and U8 we also found small-derived snoRNAs that lack their 5 rRNA recognition part and are essentially Domain II hairpin structures (U-DII). These U-DII snoRNAs from variants showed similar preferential expression, in which maternal-type variants are prominently expressed in eggs and subsequently replaced by a somatic-type variants during embryogenesis. This differential expression is related to the organization in tandem repeats (maternal type) or solitary (somatic-type) genes of the involved U snoRNA loci. The collective data showed convincingly that the preferential expression of snoRNAs is achieved by transcription regulation, as well as through RNA processing. Finally, we observed small-RNAs derived from internal transcribed spacers (ITSs) of a U3 snoRNA loci that via complementarity binding, may be involved in the biosynthesis of U3-DII snoRNAs. Altogether, the here described maternal- and somatic-type snoRNAs are the latest addition to the developing story about the dual ribosome system in zebrafish development.

developmental biology↗

bioRxiv2019

An alternative spliceosome defined by distinct snRNAs in early zebrafish embryogenesis

Splicing removes intronic RNA sequences from pre-mRNA molecules and enables, by alternative splicing, the generation of multiple unique RNA molecules from a single gene. As such, splicing is an essential part of the whole translation system of a cell. The spliceosome is a ribonucleoprotein complex in which five small nuclear RNAs (snRNAs) are involved; U1, U2, U4, U5, and U6. For each of these snRNAs there are variant gene copies present in a genome. Furthermore, in many eukaryotic species there is an alternative, minor spliceosome that can splice a small number of specific introns. As we previously discovered an embryogenesis-specific ribosomal system in zebrafish early embryogenesis based on variant rRNA and snoRNA expression, we hypothesized that there may also be an embryogenesis-specific spliceosome. An inventory of zebrafish snRNA genes revealed clustered and dispersed loci for all but U2 major snRNAs. For each minor spliceosome snRNA, just one gene locus was found. Since complete snRNA molecules are hard to sequence, we employed a combined PCR-sequencing approach to measure the individual snRNA-variant presence. Analysis of egg and male-adult samples revealed embryogenesis-specific and somatic-specific variants for each major snRNA. These variants have substantial sequence differences, yet none in their mRNA binding sites. Given that many of the sequence differences are found in loop structures indicate possible alternative protein binding. Altogether, with this study we established that the spliceosome is also an element of the embryogenesis-specific translation system in zebrafish.

developmental biology↗

bioRxiv2019

Patterning of the vertebrate head in time and space by BMP signalling

How head patterning is regulated in vertebrates is yet to be understood. In this study, we show that frog embryos injected with Noggin at different blastula and gastrula stages had their head development sequentially arrested at different positions. When timed BMP inhibition was applied to BMP-overexpressing embryos, the expression of five genes: xcg-1 (a marker of the cement gland, which is the front-most structure in the frog embryo), six3 (a forebrain marker), otx2 (a forebrain and mid-brain marker), gbx2 (an anterior hindbrain marker) and hoxd1 (a posterior hindbrain marker) were sequentially fixed. These results suggest that timed interactions between BMP and anti-BMP are involved in patterning the vertebrate head progressively in time and space. Since the above genes are not expressed sequentially, there may be a BMP dependent gene sequence during head patterning that can be arrested by BMP inhibition and regulate the specification of positional values in the head.

developmental biology↗

bioRxiv2019

RNAseq profiling of leukocyte populations in zebrafish larvae reveals a cxcl11 chemokine gene as a marker of macrophage polarization during mycobacterial infection.

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polarization during inflammation has been visualized by real time imaging in larvae of the zebrafish. This model organism has also become widely used to study macrophage responses to microbial pathogens. To support the increasing use of zebrafish in macrophage biology, we set out to determine the complete transcriptome of zebrafish larval macrophages. We studied the specificity of the macrophage signature compared with other larval immune cells and the macrophage-specific expression changes upon infection. We made use of the well-established mpeg1, mpx, and lck fluorescent reporter lines to sort and sequence the transcriptome of larval macrophages, neutrophils, and lymphoid progenitor cells, respectively. Our results provide a complete dataset of genes expressed in these different immune cell types and highlight their similarities and differences. Major differences between the macrophage and neutrophil signatures were found within the families of proteinases. Furthermore, expression of genes involved in antigen presentation and processing was specifically detected in macrophages, while lymphoid progenitors showed expression of genes involved in macrophage activation. Comparison with datasets of in vitro polarized human macrophages revealed that zebrafish macrophages express a strongly homologous gene set, comprising both M1 and M2 markers. Furthermore, transcriptome analysis of low numbers of macrophages infected by the intracellular pathogen Mycobacterium marinum revealed that infected macrophages change their transcriptomic response by downregulation of M2-associated genes and overexpression of specific M1-associated genes. Among the infection-induced genes, a homolog of the human CXCL11 chemokine gene, cxcl11aa, stood out as the most strongly overexpressed M1 marker. Upregulation of cxcl11aa in Mycobacterium-infected macrophages was found to require the function of Myd88, a critical adaptor molecule in the Toll-like and interleukin 1 receptor pathways that are central to pathogen recognition and activation of the innate immune response. Altogether, our data provide a valuable data mining resource to support infection and inflammation research in the zebrafish model.

immunology↗

bioRxiv2018

Deep learning image recognition enables efficient genome editing in zebrafish by automated injections

One of the most popular techniques in zebrafish research is microinjection, as it is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes or tracers at larval stages.\n\nHere we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3.\n\nIn a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 {micro}m to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.

molecular biology↗

bioRxiv2017

Rapid de novo assembly of the European eel genome from nanopore sequencing reads

We have sequenced the genome of the endangered European eel using the MinION by Oxford Nanopore, and assembled these data using a novel algorithm specifically designed for large eukaryotic genomes. For this 860 Mbp genome, the entire computational process takes two days on a single CPU. The resulting genome assembly significantly improves on a previous draft based on short reads only, both in terms of contiguity (N50 1.2 Mbp) and structural quality. This combination of affordable nanopore sequencing and light-weight assembly promises to make high-quality genomic resources accessible for many non-model plants and animals.

genomics↗