Search

Background: Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization., Methods: EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte ® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse ® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting., Results: EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells., Conclusion: Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype., (© 2024. The Author(s).)

Background: For over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO 2 -baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates ® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV., Methods: In the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR., Results: A total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus., Conclusion: The suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory., (© 2021. The Author(s).)

Background: We investigated parent-of-origin and allele-specific expression effects on obesity and hepatic gene expression in reciprocal crosses between the Berlin Fat Mouse Inbred line (BFMI) and C57Bl/6NCrl (B6N)., Results: We found that F1-males with a BFMI mother developed 1.8 times more fat mass on a high fat diet at 10 weeks than F1-males of a BFMI father. The phenotype was detectable from six weeks on and was preserved after cross-fostering. RNA-seq data of liver provided evidence for higher biosynthesis and elongation of fatty acids (p = 0.00635) in obese male offspring of a BFMI mother versus lean offspring of a BFMI father. Furthermore, fatty acid degradation (p = 0.00198) and the peroxisome pathway were impaired (p = 0.00094). The circadian rhythm was affected as well (p = 0.00087). Among the highest up-regulated protein coding genes in obese males were Acot4 (1.82 fold, p = 0.022), Cyp4a10 (1.35 fold, p = 0.026) and Cyp4a14 (1.32 fold, p = 0.012), which hydroxylize fatty acids and which are known to be increased in liver steatosis. Obese males showed lower expression of the genetically imprinted and paternally expressed 3 (Peg3) gene (0.31 fold, p = 0.046) and higher expression of the androgen receptor (Ar) gene (2.38 fold, p = 0.068). Allelic imbalance was found for expression of ATP-binding cassette transporter gene Abca8b. Several of the differentially expressed genes contain estrogen response elements., Conclusions: Parent-of-origin effects during gametogenesis and/or fetal development in an obese mother epigenetically modify the transcription of genes that lead to enhanced fatty acid synthesis and impair β-oxidation in the liver of male, but not female F1 offspring. Down-regulation of Peg3 could contribute to trigger this metabolic setting. At puberty, higher amounts of the androgen receptor and altered access to estrogen response elements in affected genes are likely responsible for male specific expression of genes that were epigenetically triggered. A suggestive lack of estrogen binding motifs was found for highly down-regulated genes in adult hepatocytes of obese F1 males (p = 0.074).

Background: Gene expression analysis by RNA sequencing is now widely used in a number of applications surveying the whole transcriptomes of cells and tissues. The recent introduction of ribosomal RNA depletion protocols, such as RiboZero, has extended the view of the polyadenylated transcriptome to the poly(A)- fraction of the RNA. However, substantial amounts of intronic transcriptional activity has been reported in RiboZero protocols, raising issues regarding their potential nuclear origin and the impact on the actual sequence depth in exonic regions., Results: Using HEK293 human cells as source material, we assessed here the impact of the two commonly used RNA extraction methods and of the library construction protocols (rRNA depletion versus mRNA) on 1) the relative abundance of intronic reads and 2) on the estimation of gene expression values. We benchmarked the rRNA depletion-based sequencing with a specific analysis of the cytoplasmic and nuclear transcriptome fractions, suggesting that the large majority of the intronic reads correspond to unprocessed nuclear transcripts rather than to independent transcriptional units. We show that Qiagen or TRIzol extraction methods retain differentially nuclear RNA species, and that consequently, rRNA depletion-based RNA sequencing protocols are particularly sensitive to the extraction methods., Conclusions: We could show that the combination of Trizol-based RNA extraction with rRNA depletion sequencing protocols led to the largest fraction of intronic reads, after the sequencing of the nuclear transcriptome. We discuss here the impact of the various strategies on gene expression and alternative splicing estimation measures. Further, we propose guidelines and a double selection strategy for minimizing the expression biases, without loss of information.