miRNA repertoire and host immune factor regulation upon avian coronavirus infection in eggs.

Avian infectious bronchitis virus (IBV) is a coronavirus with great economic impact on the poultry industry, causing an acute and highly contagious disease in chickens that primarily affects the respiratory and reproductive systems. The cellular regulation of IBV pathogenesis and the host immune responses involved remain to be fully elucidated. MicroRNAs (miRNAs) have emerged as a class of crucial regulators of numerous cellular processes, including responses to viral infections. Here, we employed a high-throughput sequencing approach to analyze the miRNA composition of the spleen and the lungs of chicken embryos upon IBV infection. Compared to healthy chicken embryos, 13 and six miRNAs were upregulated in the spleen and the lungs, respectively, all predicted to influence viral transcription, cytokine production, and lymphocyte functioning. Subsequent downregulation of NFATC3, NFAT5, SPPL3, and TGFB2 genes in particular was observed only in the spleen, demonstrating the biological functionality of the miRNAs in this lymphoid organ. This is the first study that describes the modulation of miRNAs and the related host immune factors by IBV in chicken embryos. Our data provide novel insight into complex virus-host interactions and specifically highlight components that could affect the host's immune response to IBV infection.

Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development.

There is mounting evidence that the ribosome is not a static translation machinery, but a cell-specific, adaptive system. Ribosomal variations have mostly been studied at the protein level, even though the essential transcriptional functions are primarily performed by rRNAs. At the RNA level, oocyte-specific 5S rRNAs are long known for Xenopus. Recently, we described for zebrafish a similar system in which the sole maternal-type 5S rRNA present in eggs is replaced completely during embryonic development by a somatic-type. Here, we report the discovery of an analogous system for the 45S rDNA elements: 5.8S, 18S, and 28S. The maternal-type 5.8S, 18S, and 28S rRNA sequences differ substantially from those of the somatic-type, plus the maternal-type rRNAs are also replaced by the somatic-type rRNAs during embryogenesis. We discuss the structural and functional implications of the observed sequence differences with respect to the translational functions of the 5.8S, 18S, and 28S rRNA elements. Finally, in silico evidence suggests that expansion segments (ES) in 18S rRNA, previously implicated in ribosome-mRNA interaction, may have a preference for interacting with specific mRNA genes. Taken together, our findings indicate that two distinct types of ribosomes exist in zebrafish during development, each likely conducting the translation machinery in a unique way.

Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci.

Identifying small RNAs derived from maternal- and somatic-type rRNAs in zebrafish development.

rRNAs are non-coding RNAs present in all prokaryotes and eukaryotes. In eukaryotes there are four rRNAs: 18S, 5.8S, 28S, originating from a common precursor (45S), and 5S. We have recently discovered the existence of two distinct developmental types of rRNA: a maternal-type, present in eggs and a somatic-type, expressed in adult tissues. Lately, next-generation sequencing has allowed the discovery of new small-RNAs deriving from longer non-coding RNAs, including small-RNAs from rRNAs (srRNAs). Here, we systemically investigated srRNAs of maternal- or somatic-type 18S, 5.8S, 28S, with small-RNAseq from many zebrafish developmental stages. We identified new srRNAs for each rRNA. For 5.8S, we found srRNA consisting of the 5' or 3' halves, with only the latter having different sequence for the maternal- and somatic-types. For 18S, we discovered 21 nt srRNA from the 5' end of the 18S rRNA with a striking resemblance to microRNAs; as it is likely processed from a stem-loop precursor and present in human and mouse Argonaute-complexed small-RNA. For 28S, an abundant 80 nt srRNA from the 3' end of the 28S rRNA was found. The expression levels during embryogenesis of these srRNA indicate they are not generated from rRNA degradation and might have a role in the zebrafish development.

Transcriptome dynamics in early zebrafish embryogenesis determined by high-resolution time course analysis of 180 successive, individual zebrafish embryos.

BACKGROUND: Recently, much progress has been made in the field of gene-expression in early embryogenesis. However, the dynamic behaviour of transcriptomes in individual embryos has hardly been studied yet and the time points at which pools of embryos are collected are usually still quite far apart. Here, we present a high-resolution gene-expression time series with 180 individual zebrafish embryos, obtained from nine different spawns, developmentally ordered and profiled from late blastula to mid-gastrula stage. On average one embryo per minute was analysed. The focus was on identification and description of the transcriptome dynamics of the expressed genes in this embryonic stage, rather than to biologically interpret profiles in cellular processes and pathways. RESULTS: In the late blastula to mid-gastrula stage, we found 6,734 genes being expressed with low variability and rather gradual changes. Ten types of dynamic behaviour were defined, such as genes with continuously increasing or decreasing expression, and all expressed genes were grouped into these types. Also, the exact expression starting and stopping points of several hundred genes during this developmental period could be pinpointed. Although the resolution of the experiment was so high, that we were able to clearly identify four known oscillating genes, no genes were observed with a peaking expression. Additionally, several genes showed expression at two or three distinct levels that strongly related to the spawn an embryo originated from. CONCLUSION: Our unique experimental set-up of whole-transcriptome analysis of 180 individual embryos, provided an unparalleled in-depth insight into the dynamics of early zebrafish embryogenesis. The existence of a tightly regulated embryonic transcriptome program, even between individuals from different spawns is shown. We have made the expression profile of all genes available for domain experts. The fact that we were able to separate the different spawns by their gene-expression variance over all expressed genes, underlines the importance of spawn specificity, as well as the unexpectedly tight gene-expression regulation in early zebrafish embryogenesis.

Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.

There is an increasing interest in complementing RNA-seq experiments with small-RNA (sRNA) expression data to obtain a comprehensive view of a transcriptome. Currently, two main experimental challenges concerning sRNA-seq exist: how to check the size distribution of isolated sRNAs, given the sensitive size-selection steps in the protocol; and how to normalize data between samples, given the low complexity of sRNA types. We here present two separate sets of synthetic RNA spike-ins for monitoring size-selection and for performing data normalization in sRNA-seq. The size-range quality control (SRQC) spike-in set, consisting of 11 oligoribonucleotides (10-70 nucleotides), was tested by intentionally altering the size-selection protocol and verified via several comparative experiments. We demonstrate that the SRQC set is useful to reproducibly track down biases in the size-selection in sRNA-seq. The external reference for data-normalization (ERDN) spike-in set, consisting of 19 oligoribonucleotides, was developed for sample-to-sample normalization in differential-expression analysis of sRNA-seq data. Testing and applying the ERDN set showed that it can reproducibly detect differential expression over a dynamic range of 2(18). Hence, biological variation in sRNA composition and content between samples is preserved while technical variation is effectively minimized. Together, both spike-in sets can significantly improve the technical reproducibility of sRNA-seq.

WRKY40 and WRKY6 act downstream of the green leaf volatile E-2-hexenal in Arabidopsis.

Plants are known to be responsive to volatiles, but knowledge about the molecular players involved in transducing their perception remains scarce. We study the response of Arabidopsis thaliana to E-2-hexenal, one of the green leaf volatiles (GLV) that is produced upon wounding, herbivory or infection with pathogens. We have taken a transcriptomics approach to identify genes that are induced by E-2-hexenal, but not by defence hormones or other GLVs. Furthermore, by studying the promoters of early E-2-hexenal-induced genes we determined that the only statistically enriched cis-element was the W-box motif. Since members of the plant-specific family of WRKY transcription factors act in trans on this cis-element, we focused on WRKY6, 40 and 53 that were most strongly induced by E-2-hexenal. Root elongation of Arabidopsis seedlings of the wrky40 wrky6 double mutant was much less inhibited than in wt plants, similar to the E-2-hexenal-responsive mutant her1, which is perturbed in γ-amino butyric acid (GABA) metabolism. The induction of several of the E-2-hexenal-specific genes was much higher in the wrky40, wrky6 or wrky40 wrky6 mutants, including GAD4, a glutamate decarboxylase that catalyzes the formation of GABA from glutamate. In conclusion, WRKY6 and 40 seem to act as important players transducing E-2-hexenal perception.

Absence/presence calling in microarray-based CGH experiments with non-model organisms.

Structural variations in genomes are commonly studied by (micro)array-based comparative genomic hybridization. The data analysis methods to infer copy number variation in model organisms (human, mouse) are established. In principle, the procedures are based on signal ratios between test and reference samples and the order of the probe targets in the genome. These procedures are less applicable to experiments with non-model organisms, which frequently comprise non-sequenced genomes with an unknown order of probe targets. We therefore present an additional analysis approach, which does not depend on the structural information of a reference genome, and quantifies the presence or absence of a probe target in an unknown genome. The principle is that intensity values of target probes are compared with the intensities of negative-control probes and positive-control probes from a control hybridization, to determine if a probe target is absent or present. In a test, analyzing the genome content of a known bacterial strain: Staphylococcus aureus MRSA252, this approach proved to be successful, demonstrated by receiver operating characteristic area under the curve values larger than 0.9995. We show its usability in various applications, such as comparing genome content and validating next-generation sequencing reads from eukaryotic non-model organisms.

A novel toxicogenomics-based approach to categorize (non-)genotoxic carcinogens.

Alternative methods to detect non-genotoxic carcinogens are urgently needed, as this class of carcinogens goes undetected in the current testing strategy for carcinogenicity under REACH. A complicating factor is that non-genotoxic carcinogens act through several distinctive modes of action, which makes prediction of their carcinogenic property difficult. We have recently demonstrated that gene expression profiling in primary mouse hepatocytes is a useful approach to categorize non-genotoxic carcinogens according to their modes of action. In the current study, we improved the methods used for analysis and added mouse embryonic stem cells as a second in vitro test system, because of their features complementary to hepatocytes. Our approach involved an unsupervised analysis based on the 30 most significantly up- and down-regulated genes per chemical. Mouse embryonic stem cells and primary mouse hepatocytes were exposed to a selected set of chemicals and subsequently subjected to gene expression profiling. We focused on non-genotoxic carcinogens, but also included genotoxic carcinogens and non-carcinogens to test the robustness of this approach. Application of the optimized comparison approach resulted in improved categorization of non-genotoxic carcinogens. Mouse embryonic stem cells were a useful addition, especially for genotoxic substances, but also for detection of non-genotoxic carcinogens that went undetected by primary hepatocytes. The approach presented here is an important step forward to categorize chemicals, especially those that are carcinogenic.

A day and night difference in the response of the hepatic transcriptome to cyclophosphamide treatment.

Application of omics-based technologies is a widely used approach in research aiming to improve testing strategies for human health risk assessment. In most of these studies, however, temporal variations in gene expression caused by the circadian clock are a commonly neglected pitfall. In the present study, we investigated the impact of the circadian clock on the response of the hepatic transcriptome after exposure of mice to the chemotherapeutic agent cyclophosphamide (CP). Analysis of the data without considering clock progression revealed common responses in terms of regulated pathways between light and dark phase exposure, including DNA damage, oxidative stress, and a general immune response. The overall response, however, was stronger in mice exposed during the day. Use of time-matched controls, thereby eliminating non-CP-responsive circadian clock-controlled genes, showed that this difference in response was actually even more pronounced: CP-related responses were only identified in mice exposed during the day. Only minor differences were found in acute toxicity pathways, namely lymphocyte counts and kidney weights, indicating that gene expression is subject to time of day effects. This study is the first to highlight the impact of the circadian clock on the identification of toxic responses by omics approaches.

Staphylococcus aureus ST398 gene expression profiling during ex vivo colonization of porcine nasal epithelium.

BACKGROUND: Staphylococcus aureus is a common human and animal opportunistic pathogen. In humans nasal carriage of S. aureus is a risk factor for various infections. Methicillin-resistant S. aureus ST398 is highly prevalent in pigs in Europe and North America. The mechanism of successful pig colonization by MRSA ST398 is poorly understood. Previously, we developed a nasal colonization model of porcine nasal mucosa explants to identify molecular traits involved in nasal MRSA colonization of pigs. RESULTS: We report the analysis of changes in the transcription of MRSA ST398 strain S0462 during colonization on the explant epithelium. Major regulated genes were encoding metabolic processes and regulation of these genes may represent metabolic adaptation to nasal mucosa explants. Colonization was not accompanied by significant changes in transcripts of the main virulence associated genes or known human colonization factors. Here, we documented regulation of two genes which have potential influence on S. aureus colonization; cysteine extracellular proteinase (scpA) and von Willebrand factor-binding protein (vWbp, encoded on SaPIbov5). Colonization with isogenic-deletion strains (Δvwbp and ΔscpA) did not alter the ex vivo nasal S. aureus colonization compared to wild type. CONCLUSIONS: Our results suggest that nasal colonization with MRSA ST398 is a complex event that is accompanied with changes in bacterial gene expression regulation and metabolic adaptation.

In vivo murine hepatic microRNA and mRNA expression signatures predicting the (non-)genotoxic carcinogenic potential of chemicals.

There is a high need to improve the assessment of, especially non-genotoxic, carcinogenic features of chemicals. We therefore explored a toxicogenomics-based approach using genome-wide microRNA and mRNA expression profiles upon short-term exposure in mice. For this, wild-type mice were exposed for seven days to three different classes of chemicals, i.e., four genotoxic carcinogens (GTXC), seven non-genotoxic carcinogens (NGTXC), and five toxic non-carcinogens. Hepatic expression patterns of mRNA and microRNA transcripts were determined after exposure and used to assess the discriminative power of the in vivo transcriptome for GTXC and NGTXC. A final classifier set, discriminative for GTXC and NGTXC, was generated from the transcriptomic data using a tiered approach. This appeared to be a valid approach, since the predictive power of the final classifier set in three different classifier algorithms was very high for the original training set of chemicals. Subsequent validation in an additional set of chemicals revealed that the predictive power for GTXC remained high, in contrast to NGTXC, which appeared to be more troublesome. Our study demonstrated that the in vivo microRNA-ome has less discriminative power to correctly identify (non-)genotoxic carcinogen classes. The results generally indicate that single mRNA transcripts do have the potential to be applied in risk assessment, but that additional (genomic) strategies are necessary to correctly predict the non-genotoxic carcinogenic potential of a chemical.

Genome Sequence of a New Carnation Small Viroid-Like RNA, CarSV-1.

Here, we report the genome sequence of a new circular viroid-like RNA (CarSV-1) derived from Dianthus caryophyllus (carnation) leaves. The CarSV-1 genome has notable sequence similarity (62%) to the well-studied CarSV viroid-like RNA and comprises the complete hammerhead consensus sequences involved in self-cleavage. CarSV-1 co-occurs with carnation viruses, such as CarMV.

Genome sequences of 10 new carnation mottle virus variants.

Here, we report the genome sequences of 10 Carnation mottle virus variants. Six variants originated from a single proprietary carnation cultivar, and four were derived from four different proprietary cultivars. All variants showed nucleotide differences, but the last four did not show any variation at the amino acid level.

Interorgan coordination of the murine adaptive response to fasting.

Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0-72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted "carbohydrate-lipid-protein" succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.

Transcriptome analysis of a long-lived natural Drosophila variant: a prominent role of stress- and reproduction-genes in lifespan extension.

BACKGROUND: While studying long-lived mutants has advanced our understanding of the processes involved in ageing, the mechanisms underlying natural variation in lifespan and ageing rate remain largely unknown. Here, we characterise genome-wide expression patterns of a long-lived, natural variant of Drosophila melanogaster resulting from selection for starvation resistance (SR) and compare it with normal-lived control flies (C). We do this at two time points representing middle age (90% survival) and old age (10% survival) respectively, in three adult diets (malnutrition, optimal food, and overfeeding). RESULTS: We found profound differences between Drosophila lines in their age-related expression. Most of the age-associated changes in normal-lived flies were abrogated in long-lived Drosophila. The stress-related genes, including those involved in proteolysis and cytochrome P450, were generally higher expressed in SR flies and showed a smaller increase in expression with age compared to C flies. The genes involved in reproduction showed a lower expression in middle-aged SR than in C flies and, unlike C flies, a lack of their downregulation with age. Further, we found that malnutrition strongly affected age-associated transcript patterns overriding the differences between the lines. However, under less stressful dietary conditions, line and diet affected age-dependent expression similarly. Finally, we present lists of candidate markers of ageing and lifespan extension. CONCLUSIONS: Our study unveils transcriptional changes associated with lifespan extension in SR Drosophila. The results suggest that natural genetic variation for SR and lifespan can operate through similar transcriptional mechanisms as those of dietary restriction and life-extending mutations.

Gene expression patterns and life cycle responses of toxicant-exposed chironomids.

Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle end points such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive end points has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle end points indirect. Therefore we aimed to directly compare the effects on growth, survival, and gene expression of the nonbiting midge Chironomus riparius. To this purpose, we simultaneously analyzed life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle end points of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular end life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle end points, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment.

Dissecting modes of action of non-genotoxic carcinogens in primary mouse hepatocytes.

Under REACH, the European Community Regulation on chemicals, the testing strategy for carcinogenicity is based on in vitro and in vivo genotoxicity assays. Given that non-genotoxic carcinogens are negative for genotoxicity and chronic bioassays are no longer regularly performed, this class of carcinogens will go undetected. Therefore, test systems detecting non-genotoxic carcinogens, or even better their modes of action, are required. Here, we investigated whether gene expression profiling in primary hepatocytes can be used to distinguish different modes of action of non-genotoxic carcinogens. For this, primary mouse hepatocytes were exposed to 16 non-genotoxic carcinogens with diverse modes of action. Upon profiling, pathway analysis was performed to obtain insight into the biological relevance of the observed changes in gene expression. Subsequently, both a supervised and an unsupervised comparison approach were applied to recognize the modes of action at the transcriptomic level. These analyses resulted in the detection of three of eight compound classes, that is, peroxisome proliferators, metalloids and skin tumor promotors. In conclusion, gene expression profiles in primary hepatocytes, at least in rodent hepatocytes, appear to be useful to detect some, certainly not all, modes of action of non-genotoxic carcinogens.

Operon structure of Staphylococcus aureus.

In bacteria, gene regulation is one of the fundamental characteristics of survival, colonization and pathogenesis. Operons play a key role in regulating expression of diverse genes involved in metabolism and virulence. However, operon structures in pathogenic bacteria have been determined only by in silico approaches that are dependent on factors such as intergenic distances and terminator/promoter sequences. Knowledge of operon structures is crucial to fully understand the pathophysiology of infections. Presently, transcriptome data obtained from growth curves in a defined medium were used to predict operons in Staphylococcus aureus. This unbiased approach and the use of five highly reproducible biological replicates resulted in 93.5% significantly regulated genes. These data, combined with Pearson's correlation coefficients of the transcriptional profiles, enabled us to accurately compile 93% of the genome in operon structures. A total of 1640 genes of different functional classes were identified in operons. Interestingly, we found several operons containing virulence genes and showed synergistic effects for two complement convertase inhibitors transcribed in one operon. This is the first experimental approach to fully identify operon structures in S. aureus. It forms the basis for further in vitro regulation studies that will profoundly advance the understanding of bacterial pathophysiology in vivo.

Finding maximal transcriptome differences between reprotoxic and non-reprotoxic phthalate responses in rat testis.

The chemical legislation of the EU, Registration, Evaluation, and Authorization of Chemicals (REACH), stipulates that about 30 000 chemical substances are to be assessed on their possible risks. Toxicological evaluation of these compounds will at least partly be based on animal testing. In particular, the assessment of reproductive toxicity is a very complicated, time-consuming and animal-demanding process. Introducing microarray-based technologies can potentially refine in vivo toxicity testing. If compounds of a distinct chemical class induce reproducible gene-expression responses with a recognizable overlap, these gene-expression signatures may indicate intrinsic features of certain compounds, including specific toxicity. In the present study, we have set out the first steps towards this approach for the reproductive toxicity of phthalates. Male rats were treated with a single dose of either reprotoxic or non-reprotoxic phthalates, and were analyzed 24 h afterwards. Subsequently, histopathological and gene-expression profiling analyses were performed. Despite ambiguous histopathological observations, we were able to identify genes with differential expression profiles between the reprotoxic phthalates and the non-reprotoxic counterparts. This shows that differences in gene-expression profiles, indicative of the type of exposure, may be detected earlier, or at lower doses, than classical pathological endpoints. These findings are promising for 'early warning' biomarker analyses and for using toxicogenomics in a category approach. Ultimately, this could lead to a more cost-effective approach for prioritizing the toxicity testing of large numbers of chemicals in a short period of time in hazard assessment of chemicals, which is one of the objectives of the REACH chemical legislation.