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Pharmacogenomics (PGx) studies the impact of interindividual genomic variation on drug response, allowing the opportunity to tailor the dosing regimen for each patient. Current targeted PGx testing platforms are mainly based on microarray, polymerase chain reaction, or short-read sequencing. Despite demonstrating great value for the identification of single nucleotide variants (SNVs) and insertion/deletions (INDELs), these assays do not permit identification of large structural variants, nor do they allow unambiguous haplotype phasing for star-allele assignment. Here, we used Oxford Nanopore Technologies' adaptive sampling to enrich a panel of 1,036 genes with well-documented PGx relevance extracted from the Pharmacogenomics Knowledge Base (PharmGKB). By evaluating concordance with existing truth sets, we demonstrate accurate variant and star-allele calling for five Genome in a Bottle reference samples. We show that up to three samples can be multiplexed on one PromethION flow cell without a significant drop in variant calling performance, resulting in 99.35% and 99.84% recall and precision for the targeted variants, respectively. This work advances the use of nanopore sequencing in clinical PGx settings., Competing Interests: KD has received free congress access from Oxford Nanopore Technologies (ONT) to present his findings at a scientific meeting. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Deserranno, Tilleman, Rubben, Deforce and Van Nieuwerburgh.)

The digestive tract constitutes an important interface between an animal's internal and external environment. In insects, available gut transcriptome studies are mostly exploratory or look at changes upon infection or upon exposure to xenobiotics, mainly performed in species belonging to holometabolan orders, such as Diptera, Lepidoptera or Coleoptera. By contrast, studies focusing on gene expression changes after food uptake and during digestion are underrepresented. We have therefore compared the gene expression profiles in the midgut of the desert locust, Schistocerca gregaria , between three different time points after feeding, i.e., 24 h (no active digestion), 10 min (the initial stage of feeding), and 2 h (active food digestion). The observed gene expression profiles were consistent with the polyphagous herbivorous lifestyle of this hemimetabolan (orthopteran) species. Our study reveals the upregulation of 576 genes 2 h post-feeding. These are mostly predicted to be associated with digestive physiology, such as genes encoding putative digestive enzymes or nutrient transporters, as well as genes putatively involved in immunity or in xenobiotic metabolism. The 10 min time point represented an intermediate condition, suggesting that the S. gregaria midgut can react rapidly at the transcriptional level to the presence of food. Additionally, our study demonstrated the critical importance of two transcripts that exhibited a significant upregulation 2 h post-feeding: the vacuolar-type H(+)-ATPase and the sterol transporter Niemann-Pick 1b protein, which upon RNAi-induced knockdown resulted in a marked increase in mortality. Their vital role and accessibility via the midgut lumen may make the encoded proteins promising insecticidal target candidates, considering that the desert locust is infamous for its huge migrating swarms that can devastate the agricultural production in large areas of Northern Africa, the Middle East, and South Asia. In conclusion, the transcriptome datasets presented here will provide a useful and promising resource for studying the midgut physiology of S. gregaria , a socio-economically important pest species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Van Lommel, Holtof, Tilleman, Cools, Vansteenkiste, Polgun, Verdonck, Van Nieuwerburgh and Vanden Broeck.)

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The currently used pharmacogenetic genotyping assays offer limited haplotype information, which can potentially cause specific functional effects to be missed. This study tested if Targeted Locus Amplification (TLA), when using non-patient-specific primers combined with Illumina or Nanopore sequencing, can offer an advantage in terms of accurate phasing. The TLA method selectively amplifies and sequences entire genes based on crosslinking DNA in close physical proximity. This way, DNA fragments that were initially further apart in the genome are ligated into one molecule, making it possible to sequence distant variants within one short read. In this study, four pharmacogenes, CYP2D6, CYP2C19, CYP1A2 and BRCA1, were sequenced after enrichment using different primer pairs. Only 24% or 38% of the nucleotides mapped on target when using Illumina or Nanopore sequencing, respectively. With an average depth of more than 1000X for the regions of interest, none of the genes were entirely covered with either sequencing method. For three of the four genes, less than half of the variants were phased correctly compared to the reference. The Nanopore dataset with the optimized primer pair for CYP2D6 resulted in the correct haplotype, showing that this method can be used for reliable genotyping and phasing of pharmacogenes but does require patient-specific primer design and optimization to be effective., (© 2022. The Author(s).)

CYP2D6 is a very important pharmacogene as it is responsible for the metabolization or bioactivation of 20 to 30% of the clinically used drugs. However, despite its relatively small length of only 4.4 kb, it is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants., Competing Interests: The authors have declared that no competing interests exist.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Acetaminophen (APAP) intoxication is the foremost cause of drug-induced liver failure in developed countries. The only pharmacologic treatment option, N-acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti-MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP-intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti-MT antibodies. Anti-MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS-stimulated bone marrow-derived macrophages. Importantly, NAC and anti-MT antibodies were equally effective whereas administration of anti-MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP-induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono- or add-on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization., (©2021 Society for Leukocyte Biology.)

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as β-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation., (© 2021. The Author(s).)

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation., (©2021 The Authors. Stem Cells published by Wiley Periodicals LLC on behalf of AlphaMed Press 2021.)

Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.

This study reports the comparative analyses of four Flavobacterium columnare isolates that have different virulence and antimicrobial resistance patterns. The main research goal was to reveal new insights into possible virulence genes by comparing the genomes of bacterial isolates that could induce tissue damage and mortality versus the genome of a non-virulent isolate. The results indicated that only the genomes of the virulent isolates possessed unique genes encoding amongst others a methyl-accepting chemotaxis protein possibly involved in the initial colonization of tissue, and several VgrG proteins engaged in interbacterial competition. Furthermore, comparisons of genes unique for the genomes of the highly virulent (HV) carp and trout isolates versus the, respectively, low and non-virulent carp and trout isolates were performed. An important part of the identified unique virulence genes of the HV-trout isolate was located in one particular gene region identified as a genomic island. This region contained araC and nodT genes, both linked to pathogenic and multidrug-resistance, and a luxR-gene, functional in bacterial cell-to-cell communication. Furthermore, the genome of the HV-trout isolate possessed unique sugar-transferases possibly important in bacterial adhesion. The second research goal was to obtain insights into the genetic basis of acquired antimicrobial resistance. Several point-mutations were discovered in gyrase-genes of an isolate showing phenotypic resistance towards first and second-generation quinolones, which were absent in isolates susceptible to quinolones. Tetracycline-resistance gene tetA was found in an isolate displaying acquired phenotypic resistance towards oxytetracycline. Although not localized on a prophage, several flanking genes were indicative of the gene's mobile character.

In both mouse and human, Notch1 activation is the main initial driver to induce T-cell development in hematopoietic progenitor cells. The initiation of this developmental process coincides with Notch1-dependent repression of differentiation towards other hematopoietic lineages. Although well described in mice, the role of the individual Notch1 target genes during these hematopoietic developmental choices is still unclear in human, particularly for HES4 since no orthologous gene is present in the mouse. Here, we investigated the functional capacity of the Notch1 target genes HES1 and HES4 to modulate human Notch1-dependent hematopoietic lineage decisions and their requirement during early T-cell development. We show that both genes are upregulated in a Notch-dependent manner during early T-cell development and that HES1 acts as a repressor of differentiation by maintaining a quiescent stem cell signature in CD34+ hematopoietic progenitor cells. While HES4 can also inhibit natural killer and myeloid cell development like HES1, it acts differently on the T- versus B-cell lineage choice. Surprisingly, HES4 is not capable of repressing B-cell development, the most sensitive hematopoietic lineage with respect to Notch-mediated repression. In contrast to HES1, HES4 promotes initiation of early T-cell development, but ectopic expression of HES4, or HES1 and HES4 combined, is not sufficient to induce T-lineage differentiation. Importantly, knockdown of HES1 or HES4 significantly reduces human T-cell development. Overall, we show that the Notch1 target genes HES1 and HES4 have non-redundant roles during early human T-cell development which may relate to differences in mediating Notch-dependent human hematopoietic lineage decisions.

Aim: This study provides clinicians and researchers with an informed choice between current commercially available targeted sequencing panels and exome sequencing panels in the context of pan-cancer pharmacogenetics. Materials & methods: Nine contemporary commercially available targeted pan-cancer panels and the xGen Exome Research Panel v2 were investigated to determine to what extent they cover the pharmacogenetic variant-drug interactions in five available cancer knowledgebases, and the driver mutations and fusion genes in the Cancer Genome Atlas. Results: xGen Exome Research Panel v2 and TrueSight Oncology 500 target 71.0 and 68.9% of the pharmacogenetic interactions in the available knowledgebases; and 93.7 and 86.0% of the driver mutations in the Cancer Genome Atlas, respectively. All other studied panels target lower percentages. Conclusion: Exome sequencing outperforms pan-cancer targeted sequencing panels in terms of covered cancer pharmacogenetic variant-drug interactions and pharmacogenetic cancer variants.

Short Tandem Repeat (STR-) and Single Nucleotide Polymorphism (SNP-) genotyping have been extensively studied within forensic kinship analysis. Nevertheless, no results have been reported on kinship analysis after whole genome amplification (WGA) of single cells. This WGA step is a necessary procedure in several applications, such as cell-based non-invasive prenatal testing (cbNIPT) and pre-implantation genetic diagnosis (PGD). In cbNIPT, all putative fetal cells must be discriminated from maternal cells after enrichment from whole blood. This study investigates the efficacy and evidential value of STR- and SNP-genotyping methods for the discrimination of 24 single cells after WGA, within three families. Formaldehyde-fixed and unfixed cells are assessed in offspring-parent duos and offspring-mother-father trios. Results demonstrate that both genotyping methods can be used in all tested conditions and scenarios with 100% sensitivity and 100% specificity, with a similar evidential value for fixed and unfixed cells. Moreover, sequence-based SNP-genotyping results in a higher evidential value than length-based STR-genotyping after WGA, which is not observed using high-quality offspring bulk DNA samples. Finally, it is also demonstrated that the availability of the DNA genotypes of both parents strongly increases the evidential value of the results.

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation., (© 2020 by The American Society of Hematology.)

Background and Aims: Obesity, diabetes and associated non-alcoholic steatohepatitis (NASH) are rising risk factors for hepatocellular carcinoma (HCC). Macrophages are important immune cells involved in inflammation and tumour development. Macrophage inositol-requiring enzyme 1 alpha (IRE1α), an ER-stress protein, has been shown to be involved in macrophage cytokine production, and myeloid-specific IRE1α knock-out (myeloid IRE1α-KO) mice showed reduced weight gain during high-fat diet feeding. However, the effect of myeloid IRE1α on NASH and subsequent HCC development has not been examined. Here, we characterized the transcriptional profile of the hepatic macrophage population in a diabetes-NASH-HCC mouse model, and investigated the effect of myeloid-specific IRE1α deletion on the phenotype of hepatic macrophage subsets and experimental NASH-HCC development., Methods: Mice with non-functional myeloid IRE1α were created by crossing Ire1a floxed mice with Lysm-Cre mice. Two-day old myeloid IRE1α-KO and wild type (WT) mice were subcutaneously injected with streptozotocin (STZ), and male mice were fed a high-fat, -sucrose, -cholesterol diet (Western diet, WD) from the age of 4 weeks until 21 weeks. Control myeloid IRE1α-KO and WT mice received a PBS injection and were fed a matched control diet. These mice were evaluated for obesity, diabetes, NASH and HCC. The hepatic macrophage population was evaluated by flow cytometry and RNA sequencing on FACS-isolated macrophage subsets., Results: STZ-injection and WD feeding resulted in an impaired glucose tolerance, advanced NASH with fibrosis, and HCC development. Myeloid IRE1α-KO STZ mice showed lower fasting glucose levels at the start of WD feeding, and an improved glucose tolerance and attenuated HCC development after 17 weeks of WD feeding despite a similar degree of liver steatosis and inflammation compared to WT mice. Transcriptomic analysis of WT liver Kupffer cells, macrophages and monocytes revealed phenotypical changes in those cell subsets during NASH-HCC development. Isolated liver Kupffer cells and macrophages from mice with a myeloid IRE1α deletion showed downregulated pathways involved in immune system activation and metabolic pathways (only in Kupffer cells), whereas pathways involved in cell division and metabolism were upregulated in monocytes. These transcriptional differences were attenuated during NASH-HCC development., Conclusion: Our results show that myeloid-specific IRE1α deletion results in an altered transcriptional profile of hepatic macrophages and dampens diabetes-induced NASH-HCC development, possibly by attenuated diabetes induction., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo-maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro-RNA-Sequencing was performed on all collected samples, mRNA-Sequencing on the same tissue samples and mass spectrometry was conducted previously on the same fluid samples. Differential expression of miRNA, mRNA and proteins showed high conformity with literature and confirmed involvement in pregnancy establishment, embryo quality, steroid synthesis and prostaglandin regulation, but the link between differential miRNAs and their targets was limited and did not indicate the identity of an unequivocal signal for MRP in the horse. Differential expression at the embryo-maternal interface was prominent, highlighting a potential role of miRNAs in embryo-maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies.