Endogenous Elements Research Articles

Non-canonical nematode endogenous retroviruses resulting from RNA virus glycoprotein gene capture by a metavirus.

Reverse-transcribing retroviruses exist as horizontally transmitted infectious agents or vertically transmitted endogenous retroviruses (ERVs) resident in eukaryotic genomes, and they are phylogenetically related to the long terminal repeat (LTR) class of retrotransposons. ERVs and retrotransposons are often distinguished only by the presence or absence of a gene encoding the envelope glycoprotein (env). Endogenous elements of the virus family Metaviridae include the insect-restricted Errantivirus genus of ERVs, for which some members possess env, and the pan-eukaryotic Metavirus genus that lacks an envelope glycoprotein gene. Here we report a novel Nematoda endogenous retrovirus (NERV) clade with core retroviral genes arranged uniquely as a continuous gag-env-pro-pol ORF. Reverse transcriptase sequences were phylogenetically related to metaviruses, but envelope glycoprotein sequences resembled those of the Nyamiviridae and Chrysoviridae RNA virus families, suggesting env gene capture during host cell infection by an RNA virus. NERVs were monophyletic, restricted to the nematode subclass Chromadoria, and included additional ORFs for a small hypothetical protein or a large Upf1-like RNA-dependent AAA-ATPase/helicase indicative of viral transduction of a host gene. Provirus LTR identity, low copy number, ORF integrity and segregation of three loci in Meloidogyne incognita, taken together with detection of NERV transcriptional activity, support potential infectivity of NERVs, along with their recent emergence and integration. Altogether, NERVs constitute a new and distinct Metaviridae lineage demonstrating retroviral evolution through sequential heterologous gene capture events.

Homing in on Endogenous Badnaviral Elements: Development of Multiplex PCR-DGGE for Detection and Rapid Identification of Badnavirus Sequences in Yam Germplasm.

Viruses of the genus Badnavirus (family Caulimoviridae) are double-stranded DNA-reverse transcribing (dsDNA-RT) plant viruses and have emerged as serious pathogens of tropical and temperate crops globally. Endogenous badnaviral sequences are found integrated in the genomes of several economically important plant species. Infection due to activation of replication-competent integrated copies of the genera Badnavirus, Petuvirus and Cavemovirus has been described. Such endogenous badnaviral elements pose challenges to the development of nucleic acid-based diagnostic methods for episomal virus infections and decisions on health certification for international movement of germplasm and seed. One major food security crop affected is yam (Dioscorea spp.). A diverse range of Dioscorea bacilliform viruses (DBVs), and endogenous DBV (eDBV) sequences have been found to be widespread in yams cultivated in West Africa and other parts of the world. This study outlines the development of multiplex PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE) to assist in the detection and analysis of eDBVs, through the example of analysing yam germplasm from Nigeria and Ghana. Primers targeting the three most prevalent DBV monophyletic species groups in West Africa were designed to improve DGGE resolution of complex eDBV sequence fingerprints. Multiplex PCR-DGGE with the addition of a tailor-made DGGE sequence marker enables rapid comparison of endogenous badnaviral sequence diversity across germplasm, as illustrated in this study for eDBV diversity in yam.

A simple preparation protocol for shipping and storage of tissue sections for laser ablation-inductively coupled plasma-mass spectrometry imaging.

A rapid and cost-efficient tissue preparation protocol for laser ablation-inductively coupled plasma-mass spectrometry imaging (LA–ICP–MSI) has been developed within this study as an alternative to the current gold standard using fresh-frozen samples or other preparation techniques such as formalin fixation (FFix) and formalin-fixed paraffin-embedding (FFPE). Samples were vacuum dried at room temperature (RT) and stored in sealed vacuum containers for storage and shipping between collaborating parties. We compared our new protocol to established methods using prostate tissue sections investigating typical endogenous elements such as zinc, iron, and phosphorous with LA–ICP–MSI. The new protocol yielded comparable imaging results as fresh-frozen sections. FFPE sections were also tested due to the wide use and availability of FFPE tissue. However, the FFPE protocol and the FFix alone led to massive washout of the target elements on the sections tested in this work. Therefore, our new protocol presents an easy and rapid alternative for tissue preservation with comparable results to fresh-frozen sections for LA–ICP–MSI. It overcomes washout risks of commonly used tissue fixation techniques and does not require expensive and potentially unstable and time-critical shipping of frozen material on dry ice. Additionally, this protocol is likely applicable for several bioimaging approaches, as the dry condition may act comparable to other dehydrating fixatives, such as acetone or methanol, preventing degradation while avoiding washout effects.

Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella.

Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans.

Comparative Expression Analysis of Human Endogenous Retrovirus Elements in Blood Samples of Children with Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a highly diverse group of developmental disorders with a multicausal etiology and a strong genetic basis. About 8% of the human genome consists of human endogenous retrovirus elements (HERVs). HERV transcription appears to be altered not only in specific brain regions but also in the peripheral blood of patients with psychiatric conditions such as schizophrenia, multiple sclerosis (MS), ASD, and different types of cancer. This study aims to investigate the blood-based expression of five distinct human endogenous retrovirus elements in a patient cohort of 31 children with Autism Spectrum Disorder (ASD) and a control group of 27 children with typical development. The comparative HERV expression analysis in 31 children with a diagnosis of ASD and 27 age- and sex-matched healthy controls was performed via a whole transcriptome sequencing approach combined with qRT-PCR analysis. Two HERV-encoded genes: HERV-P env, and HERV-R env, were found to be significantly downregulated in the group of children with ASD. Another HERV gene, HERV-W pol appeared to be significantly upregulated. Receiver operating characteristic (ROC) analysis indicated that the observed changes in transcript levels of HERV-P env and HERV-R env can discriminate between ASD patients and healthy controls with considerable sensitivity and specificity. Our results alongside a large number of expression studies conducted by other researchers suggest that human endogenous retrovirus elements may participate in important physiological processes and serve as key factors and markers for many conditions including ASD.

Bracoviruses, ichnoviruses, and virus-like particles from parasitoid wasps retain many features of their virus ancestors.

Animal genomes commonly contain genes or sequences that have been acquired from different types of viruses. The vast majority of these endogenous virus elements (EVEs) are inactive or consist of only a small number of components that show no evidence of cooption for new functions or interaction. Unlike most EVEs, bracoviruses (BVs), ichnoviruses (IVs) and virus-like particles (VLPs) in parasitoid wasps have evolved through retention and interaction of many genes from virus ancestors. Here, we discuss current understanding of BV, IV and VLP evolution along with associated implications for what constitutes a virus. We suggest that BVs and IVs are domesticated endogenous viruses (DEVs) that differ in several important ways from other known EVEs.

A novel and accurate full-length HTT mouse model for Huntington's disease.

Here, we report the generation and characterization of a novel Huntington's disease (HD) mouse model BAC226Q by using a bacterial artificial chromosome (BAC) system, expressing full-length human HTT with ~226 CAG-CAA repeats and containing endogenous human HTT promoter and regulatory elements. BAC226Q recapitulated a full-spectrum of age-dependent and progressive HD-like phenotypes without unwanted and erroneous phenotypes. BAC226Q mice developed normally, and gradually exhibited HD-like psychiatric and cognitive phenotypes at 2 months. From 3 to 4 months, BAC226Q mice showed robust progressive motor deficits. At 11 months, BAC226Q mice showed significant reduced life span, gradual weight loss and exhibited neuropathology including significant brain atrophy specific to striatum and cortex, striatal neuronal death, widespread huntingtin inclusions, and reactive pathology. Therefore, the novel BAC226Q mouse accurately recapitulating robust, age-dependent, progressive HD-like phenotypes will be a valuable tool for studying disease mechanisms, identifying biomarkers, and testing gene-targeting therapeutic approaches for HD.

Nanopotassium, Nanosilicon, and Biochar Applications Improve Potato Salt Tolerance by Modulating Photosynthesis, Water Status, and Biochemical Constituents

Salinity is one of the main environmental stresses, and it affects potato growth and productivity in arid and semiarid regions by disturbing physiological process, such as the photosynthesis rate, the absorption of essential nutrients and water, plant hormonal functions, and vital metabolic pathways. Few studies are available on the application of combined nanomaterials to mitigate salinity stress on potato plants (Solanum tuberosum L. cv. Diamont). In order to assess the effects of the sole or combined application of silicon (Si) and potassium (K) nanoparticles and biochar (Bc) on the agro-physiological properties and biochemical constituents of potato plants grown in saline soil, two open-field experiments were executed on a randomized complete block design (RCBD), with five replicates. The results show that the biochar application and nanoelements (n-K and n-Si) significantly improved the plant heights, the fresh and dry plant biomasses, the numbers of stems/plant, the leaf relative water content, the leaf chlorophyll content, the photosynthetic rate (Pn), the leaf stomatal conductance (Gc), and the tuber yields, compared to the untreated potato plants (CT). Moreover, the nanoelements and biochar improved the content of the endogenous elements of the plant tissues (N, P, K, Mg, Fe, Mn, and B), the leaf proline, and the leaf gibberellic acid (GA3), in addition to reducing the leaf abscisic acid content (ABA), the activity of catalase (CAT), and the peroxidase (POD) and polyphenol oxidase (PPO) in the leaves of salt-stressed potato plants. The combined treatment achieved maximum plant growth parameters, physiological parameters, and nutrient concentrations, and minimum transpiration rates (Tr), leaf abscisic acid content (ABA), and activities of the leaf antioxidant enzymes (CAT, POD, and PPO). Furthermore, the combined treatment also showed the highest tuber yield and tuber quality, including the contents of carbohydrates, proteins, and the endogenous nutrients of the tuber tissues (N, P, and K), and the lowest starch content. Moreover, Pearson’s correlation showed that the plant growth and the tuber yields of potato plants significantly and positively correlated with the photosynthesis rate, the internal CO2 concentration, the relative water content, the proline, the chlorophyll content, and the GA3, and that they were negatively correlated with the leaf Na content, PPO, CAT, ABA, MDA, and Tr. It might be concluded that nanoelement (n-K and n-Si) and biochar applications are a promising method to enhance the plant growth and crop productivity of potato plants grown under salinity conditions.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain–containing protein 3, and apoptosis-associated speck-like protein–containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA–driven diseases.

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.

Cisplatin Uptake in Macrophage Subtypes at the Single-Cell Level by LA-ICP-TOFMS Imaging.

A high-throughput laser ablation–inductively coupled plasma–time-of-flight mass spectrometry (LA-ICP-TOFMS) workflow was implemented for quantitative single-cell analysis following cytospin preparation of cells. For the first time, in vitro studies on cisplatin exposure addressed human monocytes and monocyte-derived macrophages (undifferentiated THP-1 monocytic cells, differentiated M0 macrophages, as well as further polarized M1 and M2 phenotypes) at the single-cell level. The models are of particular interest as macrophages comprise the biggest part of immune cells present in the tumor microenvironment and play an important role in modulating tumor growth and progression. The introduced bioimaging workflow proved to be universally applicable to adherent and suspension cell cultures and fit-for-purpose for the quantitative analysis of several hundreds of cells within minutes. Both, cross-validation of the method with single-cell analysis in suspension for THP-1 cells and with LA-ICP-TOFMS analysis of adherent M0 cells grown on chambered glass coverslips, revealed agreeing platinum concentrations at the single-cell level. A high incorporation of cisplatin was observed in M2 macrophages compared to the M0 and M1 macrophage subtypes and the monocyte model, THP-1. The combination with bright-field images and monitoring of highly abundant endogenous elements such as phosphorus and sodium at a high spatial resolution allowed assessing cell size and important morphological cell parameters and thus straightforward control over several cell conditions. This way, apoptotic cells and cell debris as well as doublets or cell clusters could be easily excluded prior to data evaluation without additional staining.

Characterisation of novel endogenous geminiviral elements in macadamia

BackgroundThe presence of geminivirus sequences in a preliminary analysis of sRNA sequences from the leaves of macadamia trees with abnormal vertical growth (AVG) syndrome was investigated.ResultsA locus of endogenous geminiviral elements (EGE) in the macadamia genome was analysed, and the sequences revealed a high level of deletions and/or partial integrations, thus rendering the EGE transcriptionally inactive. The replication defective EGE in the macadamia genome indicates its inability to be the source of new viral infections and thus cause AVG or any other disease in macadamia. The EGE sequences were detected in two edible Macadamia species that constitute commercial cultivars and the wild germplasm of edible and inedible species of Macadamia. This strongly suggests that the integration preceded speciation of the genus Macadamia. A draft genome of a locus of EGE in Macadamia was developed. The findings of this study provide evidence to suggest the endogenization of the geminiviral sequences in the macadamia genome and the ancestral relationship of EGE with Macadamia in the Proteaceae family. Random mutations accumulating in the EGE inform that the sequence is evolving.ConclusionsThe EGE in Macadamia is inactive and thus not a direct cause of any diseases or syndromes including AVG in macadamia. The insertion of the EGE in the macadamia genome preceded speciation of the genus Macadamia.

Synaptotagmin-13 Is a Neuroendocrine Marker in Brain, Intestine and Pancreas.

Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.

Mutagenesis of human genomes by endogenous mobile elements on a population scale.

Several large-scale Illumina whole-genome sequencing (WGS) and whole-exome sequencing (WES) projects have emerged recently that have provided exceptional opportunities to discover mobile element insertions (MEIs) and study the impact of these MEIs on human genomes. However, these projects also have presented major challenges with respect to the scalability and computational costs associated with performing MEI discovery on tens or even hundreds of thousands of samples. To meet these challenges, we have developed a more efficient and scalable version of our mobile element locator tool (MELT) called CloudMELT. We then used MELT and CloudMELT to perform MEI discovery in 57,919 human genomes and exomes, leading to the discovery of 104,350 nonredundant MEIs. We leveraged this collection (1) to examine potentially active L1 source elements that drive the mobilization of new Alu, L1, and SVA MEIs in humans; (2) to examine the population distributions and subfamilies of these MEIs; and (3) to examine the mutagenesis of GENCODE genes, ENCODE-annotated features, and disease genes by these MEIs. Our study provides new insights on the L1 source elements that drive MEI mutagenesis and brings forth a better understanding of how this mutagenesis impacts human genomes.

Impact of Tet2 Deficiency, and of TET2 Mutations in Clonal Hematopoiesis, on Neutrophil/Granulocyte Immune Function

BACKGROUNDNeutrophils, the most abundant leukocytes and granulocytes, are important regulators of cardiovascular, inflammatory and infectious diseases, yet their role in the pathophysiology of clonal hematopoiesis of indeterminate potential (CHIP) has not been adequately addressed. The effects of inactivating CHIP-driver mutations in the epigenetic regulator TET2 in neutrophils especially, are broadly unknown. HYPOTHESISTet2 inactivation in murine neutrophils, and TET2 mutations in CHIP in humans (CHIP TET2), perturb granulocyte immune effector functions. METHODSNeutrophils were obtained (EasySep™, StemCell) from the bone marrow of 2- to 4-months-old, sex-matched, control Tet2 f/f;Vav1-icre - (Tet2 f/f) and hematopoietic knockout Tet2 f/f;Vav1-icre + (Tet2 -/-) mice. Neutrophils were cultured (RPMI+10% mouse serum/FBS) and: i) stained with Mitotracker Deep Red/Nuc Blue, co-cultured and imaged (Leica SP8-X) for 30min with GFP-labeled Staphylococcus aureus (10:1 ratio) and analyzed in FIJI; ii) cultured for 3h with vehicle or 10μg/mL of S. aureus lipotechoic acid (LTA). RNA-Seq was generated (Illumina QuantSeq 3' mRNA, single-end 75bp read lengths, 5 million reads/sample), trimmed, aligned to GRCm39 using STAR.CHIP participant DNA and RNA were sequenced previously from whole blood (Cook et al., Bld Adv 2019; Cook et al., ASH 2018, with a 48-gene panel on Ion Proton, and ribo-depleted bulk RNA on Illumina, respectively). New CHIP TET2 vs. no CHIP, and murine RNA-Seq analyses were carried out in DESeq2. Human serum granule protein levels were quantified by ELISA (VersaMax).Mann-Whitney U tests were carried out in Prism. P<0.05 was considered statistically significant, and Benjamini-Hochberg multiple testing correction was applied as needed. RESULTSTet2 -/- mice had 1.34-fold more bone marrow CD11b +Ly6G + neutrophils than control Tet2 f/f mice (p=0.03), consistent with myeloid expansion. Compared to Tet2 f/f, Tet2 -/- neutrophils phagocytosed fewer S. aureus (Fig1A) and moved more slowly (Fig1B). Preliminary data suggest that Tet2 -/- neutrophil extracellular trap (NET) formation in response to S. aureus was also impaired, showing fewer and less extensive NETs (Fig1C).LTA-stimulated gene expression profiles were similar between Tet2 -/- and Tet2 f/f, suggesting pre-existing differences at baseline. Unexpectedly, the most significant GO term enrichment related to upregulated viral response pathways, including interferon-stimulated genes, (e.g. Ifitm1). The cause is unknown, but this is reminiscent of the constitutive interferon response seen in myelodysplastic syndrome (MDS) patients and TET2-mutant hematopoietic stem cells, where epigenetic dysregulation of endogenous retrotransposable elements leads to a viral mimicry response. Tet2 -/- neutrophils also overexpressed Asprv1, a regulator of inflammation ostensibly acquired from a retrotransposon. Interestingly, Ccdc80, which has been linked to Tet2 and Jak2 functions, was most significantly downregulated in Tet2 -/-, along with the Pnpla1 lipid phosphatase. Finally, Tesc, a promoter of granulocytic differentiation, was upregulated in Tet2 -/-, and there were perturbations of genes encoding neutrophil granule contents.Similarly, human RNA-Seq revealed that several leukocyte (de)granulation-related genes (e.g. lactoferrin LTF, myeloperoxidase MPO) were upregulated in CHIP TET2 subjects to those without CHIP, and these corresponded with higher LTF and MPO serum titers in an expanded cohort (Fig1D,E). Finally, there were striking decreases of gene expression associated with cytotoxic (T/NK) human lymphocytes (i.e. GZMM, TRGV8, etc.). Neutrophil, lymphocyte and monocyte counts were not significantly different between the groups. CONCLUSIONSTet2-deficient murine neutrophils have compromised immune function, possibly due to differences in pre-stimulus state. TET2-mutation carrying neutrophils in CHIP may exhibit similar abnormalities, as has been previously noted in neutrophils isolated from MDS patients. Indeed, CHIP is now known to associate with increased risk of bacterial and viral infections, and infection risk has also previously been noted for MDS. People with CHIP have elevated peripheral blood serum MPO and LTF levels, suggesting a difference in leukocyte granule biology, likely related to neutrophils. These data aid in understanding how CHIP alters immunity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The impact of laser-induced thermocoagulation effects on the optical spectra of brain tissues

In this work, the results of spectral and optical studies of the grey matter tissues of the brain by a laser hyperthermia action are presented. The coefficients of optical absorption and transport scattering in the spectral range of 250-1500 nm are determined, and the features of the spatial distribution of the laser radiation intensity in the studied objects are identified. It is shown that the coagulation effects lead to compaction and an increase in the concentration of endogenous chromophores and structural elements, which is reflected in the optical properties of biological objects.

Virophages and retrotransposons colonize the genomes of a heterotrophic flagellate.

Virophages can parasitize giant DNA viruses and may provide adaptive anti-giant virus defense in unicellular eukaryotes. Under laboratory conditions, the virophage mavirus integrates into the nuclear genome of the marine flagellate Cafeteria burkhardae and reactivates upon superinfection with the giant virus CroV. In natural systems, however, the prevalence and diversity of host-virophage associations has not been systematically explored. Here, we report dozens of integrated virophages in four globally sampled C. burkhardae strains that constitute up to 2% of their host genomes. These endogenous mavirus-like elements (EMALEs) separated into eight types based on GC-content, nucleotide similarity, and coding potential and carried diverse promoter motifs implicating interactions with different giant viruses. Between host strains, some EMALE insertion loci were conserved indicating ancient integration events, whereas the majority of insertion sites were unique to a given host strain suggesting that EMALEs are active and mobile. Furthermore, we uncovered a unique association between EMALEs and a group of tyrosine recombinase retrotransposons, revealing yet another layer of parasitism in this nested microbial system. Our findings show that virophages are widespread and dynamic in wild Cafeteria populations, supporting their potential role in antiviral defense in protists.

Rapid evolutionary turnover of mobile genetic elements drives bacterial resistance to phages.

Although it is generally accepted that phages drive bacterial evolution, how these dynamics play out in the wild remains poorly understood. We found that susceptibility to viral killing in marine Vibrio is mediated by large and highly diverse mobile genetic elements. These phage defense elements display exceedingly fast evolutionary turnover, resulting in differential phage susceptibility among clonal bacterial strains while phage receptors remain invariant. Protection is cumulative, and a single bacterial genome can harbor 6 to 12 defense elements, accounting for more than 90% of the flexible genome among close relatives. The rapid turnover of these elements decouples phage resistance from other genomic features. Thus, resistance to phages in the wild follows evolutionary trajectories alternative to those predicted from laboratory-based evolutionary experiments.

Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes.

The "toxicology in the twenty-first century" paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration-response curves. We named such a multi-level inhibitory mechanism that might mask effects as "maisonette squelching." A transient reporter gene assay in zebrafish cell lines utilizing endogenous regulatory gene elements shows increased in vitro toxicity testing performance. Synthetic and constitutive promotors interfere with signal transduction ("squelching") and might increase cellular stress (cytotoxicity). The squelching phenomenon might occur on multiple levels (toxicity pathway crosstalk and normalization vector), leading to a complete silencing of the reporter signal.

APOBEC3C, a nucleolar protein induced by genotoxins, is excluded from DNA damage sites.

The human genome contains 11 APOBEC (apolipoprotein B mRNA editing catalytic polypeptide‐like) cytidine deaminases classified into four families. These proteins function mainly in innate antiviral immunity and can also restrict endogenous retrotransposable element multiplication. The present study focuses on APOBEC3C (A3C), a member of the APOBEC3 subfamily. Some APOBEC3 proteins use their enzymatic activity on genomic DNA, inducing mutations and DNA damage, while other members facilitate DNA repair. Our results show that A3C is highly expressed in cells treated with DNA‐damaging agents. Its expression is regulated by p53. Depletion of A3C slightly decreases proliferation and does not affect DNA repair via homologous recombination or nonhomologous end joining. The A3C interactomes obtained from control cells and cells exposed to the genotoxin etoposide indicated that A3C is a nucleolar protein. This was confirmed by the detection of either endogenous or ectopic A3C in nucleoli. Interestingly, we show that A3C is excluded from areas of DNA breaks in live cells. Our data also indicate that the C‐terminal part of A3C is responsible for its nucleolar localization and exclusion from DNA damage sites.