Oral immune priming with Bacillus thuringiensis induces a shift in the gene expression of Tribolium castaneum larvae.

BACKGROUND: The phenomenon of immune priming, i.e. enhanced protection following a secondary exposure to a pathogen, has now been demonstrated in a wide range of invertebrate species. Despite accumulating phenotypic evidence, knowledge of its mechanistic underpinnings is currently very limited. Here we used the system of the red flour beetle, Tribolium castaneum and the insect pathogen Bacillus thuringiensis (Bt) to further our molecular understanding of the oral immune priming phenomenon. We addressed how ingestion of bacterial cues (derived from spore supernatants) of an orally pathogenic and non-pathogenic Bt strain affects gene expression upon later challenge exposure, using a whole-transcriptome sequencing approach. RESULTS: Whereas gene expression of individuals primed with the orally non-pathogenic strain showed minor changes to controls, we found that priming with the pathogenic strain induced regulation of a large set of distinct genes, many of which are known immune candidates. Intriguingly, the immune repertoire activated upon priming and subsequent challenge qualitatively differed from the one mounted upon infection with Bt without previous priming. Moreover, a large subset of priming-specific genes showed an inverse regulation compared to their regulation upon challenge only. CONCLUSIONS: Our data demonstrate that gene expression upon infection is strongly affected by previous immune priming. We hypothesise that this shift in gene expression indicates activation of a more targeted and efficient response towards a previously encountered pathogen, in anticipation of potential secondary encounter.

Genome-wide analysis of LXRα activation reveals new transcriptional networks in human atherosclerotic foam cells.

Increased physiological levels of oxysterols are major risk factors for developing atherosclerosis and cardiovascular disease. Lipid-loaded macrophages, termed foam cells, are important during the early development of atherosclerotic plaques. To pursue the hypothesis that ligand-based modulation of the nuclear receptor LXRα is crucial for cell homeostasis during atherosclerotic processes, we analysed genome-wide the action of LXRα in foam cells and macrophages. By integrating chromatin immunoprecipitation-sequencing (ChIP-seq) and gene expression profile analyses, we generated a highly stringent set of 186 LXRα target genes. Treatment with the nanomolar-binding ligand T0901317 and subsequent auto-regulatory LXRα activation resulted in sequence-dependent sharpening of the genome-binding patterns of LXRα. LXRα-binding loci that correlated with differential gene expression revealed 32 novel target genes with potential beneficial effects, which in part explained the implications of disease-associated genetic variation data. These observations identified highly integrated LXRα ligand-dependent transcriptional networks, including the APOE/C1/C4/C2-gene cluster, which contribute to the reversal of cholesterol efflux and the dampening of inflammation processes in foam cells to prevent atherogenesis.

Infection routes matter in population-specific responses of the red flour beetle to the entomopathogen Bacillus thuringiensis.

BACKGROUND: Pathogens can infect their hosts through different routes. For studying the consequences for host resistance, we here used the entomopathogen Bacillus thuringiensis and the red flour beetle Tribolium castaneum for oral and systemic (i. e. pricking the cuticle) experimental infection. In order to characterize the molecular mechanisms underpinning the two different infection routes, the transcriptomes of beetles of two different T. castaneum populations--one recently collected population (Cro1) and a commonly used laboratory strain (SB)--were analyzed using a next generation RNA sequencing approach. RESULTS: The genetically more diverse population Cro1 showed a significantly larger number of differentially expressed genes. While both populations exhibited similar reactions to pricking, their expression patterns in response to oral infection differed remarkably. In particular, the Cro1 population showed a strong response of cuticular proteins and developmental genes, which might indicate an adaptive developmental flexibility that was lost in the SB population presumably as a result of inbreeding. The immune response of SB was primarily based on antimicrobial peptides, while Cro1 relied on responses mediated by phenoloxidase and reactive oxygen species, which may explain the higher resistance of this strain against oral infection. CONCLUSIONS: Our data demonstrate that immunological and physiological processes underpinning the two different routes of infection are clearly distinct, and that host populations particularly differ in responses to oral infection. Furthermore, gene expression upon pricking infection entailed a strong signal of wounding, highlighting the importance of pricking controls in future infection studies.

Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development.

Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES) cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas "gene regulatory hotspots" which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints.

Brief Report: Predicting Sex Differences and Diagnosis from Early Parent Concerns.

Autism spectrum disorder (ASD) research is largely based on males, and females with ASD are at risk for under-identification. Research recommends listening to parent concerns since these are often predictive of a child's eventual diagnosis. This study examined how patterns of parent concerns predicted sex differences and eventual child diagnosis (ASD or developmental delay [DD]). We performed a secondary analysis with n = 273 children ages 36-72 months. Results suggested males with ASD had a higher likelihood of repetitive behavior and speech and language concerns compared to females with ASD. Females with DD were significantly more likely to have problem-solving concerns; whereas, males with DD were significantly less likely to have social communication concerns compared to females with ASD.

Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila.

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease.This paper has an associated First Person interview with the co-first authors of the article.

Accuracy of MRI volume measurements of breast lesions: comparison between automated, semiautomated and manual assessment.

The aim of this study was to investigate the efficacy of a dedicated software tool for automated and semiautomated volume measurement in contrast-enhanced (CE) magnetic resonance mammography (MRM). Ninety-six breast lesions with histopathological workup (27 benign, 69 malignant) were re-evaluated by different volume measurement techniques. Volumes of all lesions were extracted automatically (AVM) and semiautomatically (SAVM) from CE 3D MRM and compared with manual 3D contour segmentation (manual volume measurement, MVM, reference measurement technique) and volume estimates based on maximum diameter measurement (MDM). Compared with MVM as reference method MDM, AVM and SAVM underestimated lesion volumes by 63.8%, 30.9% and 21.5%, respectively, with significantly different accuracy for benign (102.4%, 18.4% and 11.4%) and malignant (54.9%, 33.0% and 23.1%) lesions (p < 0.05). Inter- and intraobserver reproducibility was best for AVM (mean difference +/- 2SD, 1.0 +/- 9.7% and 1.8 +/- 12.1%) followed by SAVM (4.3 +/- 25.7% and 4.3 +/- 7.9%), MVM (2.3 +/- 38.2% and 8.6 +/- 31.8%) and MDM (33.9 +/- 128.4% and 9.3 +/- 55.9%). SAVM is more accurate for volume assessment of breast lesions than MDM and AVM. Volume measurement is less accurate for malignant than benign lesions.

Survey of the visual exploration and analysis of perfusion data.

Dynamic contrast-enhanced image data (perfusion data) are used to characterize regional tissue perfusion. Perfusion data consist of a sequence of images, acquired after a contrast agent bolus is applied. Perfusion data are used for diagnostic purposes in oncology, ischemic stroke assessment or myocardial ischemia. The diagnostic evaluation of perfusion data is challenging, since the data is complex and exhibits various artifacts, e.g., motion artifacts. We provide an overview on existing methods to analyze, and visualize CT and MR perfusion data. The integrated visualization of several 2D parameter maps, the 3D visualization of parameter volumes and exploration techniques are discussed. An essential aspect in the diagnosis of perfusion data is the correlation between perfusion data and derived time-intensity curves as well as with other image data, in particular with high resolution morphologic image data. We discuss visualization support with respect to the three major application areas: ischemic stroke diagnosis, breast tumor diagnosis and the diagnosis of coronary heart disease.

Respiratory Influenza A Virus Infection Triggers Local and Systemic Natural Killer Cell Activation via Toll-Like Receptor 7.

The innate immune system senses influenza A virus (IAV) through different pathogen-recognition receptors including Toll-like receptor 7 (TLR7). Downstream of viral recognition natural killer (NK) cells are activated as part of the anti-IAV immune response. Despite the known decisive role of TLR7 for NK cell activation by therapeutic immunostimulatory RNAs, the contribution of TLR7 to the NK cell response following IAV infection has not been addressed. We have analyzed lung cytokine responses as well as the activation, interferon (IFN)-γ production, and cytotoxicity of lung and splenic NK cells following sublethal respiratory IAV infection in wild-type and TLR7ko mice. Early airway IFN-γ levels as well as the induction of lung NK cell CD69 expression and IFN-γ production in response to IAV infection were significantly attenuated in TLR7-deficient hosts. Strikingly, respiratory IAV infection also primed splenic NK cells for IFN-γ production, degranulation, and target cell lysis, all of which were fully dependent on TLR7. At the same time, lung type I IFN levels were significantly reduced in TLR7ko mice early following IAV infection, displaying a potential upstream mechanism of the attenuated NK cell activation observed. Taken together, our data clearly demonstrate a specific role for TLR7 signaling in local and systemic NK cell activation following respiratory IAV infection despite the presence of redundant innate IAV-recognition pathways.

Computer assistance for MR based diagnosis of breast cancer: present and future challenges.

MR based methods have gained an important role for the clinical detection and diagnosis of breast cancer. Dynamic contrast-enhanced MRI of the breast has become a robust and successful method, especially for diagnosis of high-risk cases due to its higher sensitivity compared to X-ray mammography. The application of MR based imaging methods depends on various automated image processing routines. The combination of techniques for preprocessing, quantification and visualization of datasets is necessary to achieve fast and solid assessment of valuable parameters for diagnosis. In this paper, different aspects such as registration methods for the reduction of motion artifacts, segmentation issues, as well as morphologic and dynamic lesion analysis will be reviewed with a focus on breast MRI, MR spectroscopy and MR guided biopsies of the breast, their implications and technical challenges from a computer assistance point of view.

Current analysis of host-parasite interactions with a focus on next generation sequencing data.

Among the most common forms of interaction between species are those between hosts and their parasites and they have important implications for evolutionary theory. Understanding both the phenotypic and genotypic processes governing such interactions is a major endeavour in biology, but is a complex and challenging task. The development of next generation sequencing technologies has recently opened up this field from a molecular perspective, allowing us access to the genomic data underlying laboratory or wild phenotypes. The data obtained from such technologies has many advantages over previous methods, such as being more abundant, often more accurate, less labour intensive to generate and more cost effective to produce. We present a review of the impact of next generation sequencing data on the study of host-parasite evolution and current topics being explored with this data. We focus on two main data types, genomic and transcriptomic. We discuss popular computational approaches which can help us characterise the molecular forces driving host-parasite systems and highlight some studies which have utilised such approaches to gain information about particular immune processes. We furthermore highlight some promising perspectives from emerging and new technologies which will allow researchers to reach a deeper understanding of these interactions.

Accurate estimations of evolutionary times in the context of strong CpG hypermutability.

We consider the substitution model T92+CpG of DNA sequence evolution which takes into account the hypermutability of CpG dinucleotides, an effect that can be especially observed in vertebrate genomes. We provide an exact method to simulate the evolution of finite DNA sequences under this model and numerical procedures to infer evolutionary times in two cases: between an ancestral and a present sequence and between two homologous sequences. We show on simulated data that our new numerical method yields very accurate estimations of divergence times. In a context of strong CpG hypermutability, it clearly outperforms the classical estimation procedure that is solely based on the model T92 without CpG influence. Supplementary Material is available at www.liebertonline.com/cmb .

An automaton approach for waiting times in DNA evolution.

In a recent article, Behrens and Vingron (J. Comput. Biol. 17/12, 2010) compute waiting times for k-mers to appear during DNA evolution under the assumption that the considered k-mers do not occur in the initial DNA sequence, an issue arising when studying the evolution of regulatory DNA sequences with regard to transcription factor (TF) binding site emergence. The mathematical analysis underlying their computation assumes that occurrences of words under interest do not overlap. We relax here this assumption by use of an automata approach. In an alphabet of size 4 like the DNA alphabet, most words have no or a low autocorrelation; therefore, globally, our results confirm those of Behrens and Vingron. The outcome is quite different when considering highly autocorrelated k-mers; in this case, the autocorrelation pushes down the probability of occurrence of these k-mers at generation 1 and, consequently, increases the waiting time for apparition of these k-mers up to 40%. An analysis of existing TF binding sites unveils a significant proportion of k-mers exhibiting autocorrelation. Thus, our computations based on automata greatly improve the accuracy of predicting waiting times for the emergence of TF binding sites to appear during DNA evolution. We do the computation in the Bernoulli or M0 model; computations in the M1 model, a Markov model of order 1, are more costly in terms of time and memory but should produce similar results. While Behrens and Vingron considered specifically promoters of length 1000, we extend the results to promoters of any size; we exhibit the property that the probability that a k-mer occurs at generation time 1 while being absent at time 0 behaves linearly with respect to the length of the promoter, which induces a hyperbolic behaviour of the waiting time of any k-mer with respect to the length of the promoter. The C code is available at www.lipn.univ-paris13.fr/∼nicodeme/ .

CpG deamination creates transcription factor-binding sites with high efficiency.

The formation of new transcription factor-binding sites (TFBSs) has a major impact on the evolution of gene regulatory networks. Clearly, single nucleotide mutations arising within genomic DNA can lead to the creation of TFBSs. Are molecular processes inducing single nucleotide mutations contributing equally to the creation of TFBSs? In the human genome, a spontaneous deamination of methylated cytosine in the context of CpG dinucleotides results in the creation of thymine (C → T), and this mutation has the highest rate among all base substitutions. CpG deamination has been ascribed a role in silencing of transposons and induction of variation in regional methylation. We have previously shown that CpG deamination created thousands of p53-binding sites within genomic sequences of Alu transposons. Interestingly, we have defined a ∼30 bp region in Alu sequence, which, depending on a pattern of CpG deamination, can be converted to functional p53-, PAX-6-, and Myc-binding sites. Here, we have studied single nucleotide mutational events leading to creation of TFBSs in promoters of human genes and in genomic regions bound by such key transcription factors as Oct4, NANOG, and c-Myc. We document that CpG deamination events can create TFBSs with much higher efficiency than other types of mutational events. Our findings add a new role to CpG methylation: We propose that deamination of methylated CpGs constitutes one of the evolutionary forces acting on mutational trajectories of TFBSs formation contributing to variability in gene regulation.

Studying the evolution of promoter sequences: a waiting time problem.

To gain a better understanding of the evolutionary dynamics of regulatory DNA sequences, we address the following questions: (1) How long does it take until a given transcription factor (TF) binding site emerges at random in a promoter sequence? and (2) How does the composition of a TF binding site affect this waiting time? Using two different probabilistic models (an i.i.d. model and a neighbor dependent model), we can compute the expected waiting time for every k-mer, k ranging from 5 to 10, until it appears in a promoter of a species. Our findings indicate that new TF binding sites can be created on a short evolutionary time scale, i.e. in a time span below the speciation time of human and chimp. Furthermore, one can conclude that the composition of a TF binding site plays a crucial role concerning the waiting time until it appears and that the CpG methylation-deamination substitution process probably accelerates the creation of new TF binding sites. A screening of existing TF binding sites moreover reveals that k-mers predicted to have short waiting times occur more frequently than others. Supplementary Material is available at www.libertonline.com/cmb .