Coffee polyphenols change the expression of STAT5b and ATF-2 modifying cyclin D1 levels in cancer cells

Background: Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective: To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole genome microarrays. Results: ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were dowregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions: Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2.

Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

Comparative genomics of Hedgehog loci in chordates and the origin of Shh regulatory novelties

The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications.

Co-evolutionary dynamics between public good producers and cheats in the bacterium Pseudomonas aeruginosa.

The production of beneficial public goods is common in the microbial world, and so is cheating - the exploitation of public goods by nonproducing mutants. Here, we examine co-evolutionary dynamics between cooperators and cheats and ask whether cooperators can evolve strategies to reduce the burden of exploitation, and whether cheats in turn can improve their exploitation abilities. We evolved cooperators of the bacterium Pseudomonas aeruginosa, producing the shareable iron-scavenging siderophore pyoverdine, together with cheats, defective in pyoverdine production but proficient in uptake. We found that cooperators managed to co-exist with cheats in 56% of all replicates over approximately 150 generations of experimental evolution. Growth and competition assays revealed that co-existence was fostered by a combination of general adaptions to the media and specific adaptions to the co-evolving opponent. Phenotypic screening and whole-genome resequencing of evolved clones confirmed this pattern, and suggest that cooperators became less exploitable by cheats because they significantly reduced their pyoverdine investment. Cheats, meanwhile, improved exploitation efficiency through mutations blocking the costly pyoverdine-signalling pathway. Moreover, cooperators and cheats evolved reduced motility, a pattern that likely represents adaptation to laboratory conditions, but at the same time also affects social interactions by reducing strain mixing and pyoverdine sharing. Overall, we observed parallel evolution, where co-existence of cooperators and cheats was enabled by a combination of adaptations to the abiotic and social environment and their interactions.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

The High Prevalence and Diversity of Chlamydiales DNA within Ixodes ricinus Ticks Suggest a Role for Ticks as Reservoirs and Vectors of Chlamydia-Related Bacteria.

The Chlamydiales order is composed of nine families of strictly intracellular bacteria. Among them, Chlamydia trachomatis, C. pneumoniae, and C. psittaci are established human pathogens, whereas Waddlia chondrophila and Parachlamydia acanthamoebae have emerged as new pathogens in humans. However, despite their medical importance, their biodiversity and ecology remain to be studied. Even if arthropods and, particularly, ticks are well known to be vectors of numerous infectious agents such as viruses and bacteria, virtually nothing is known about ticks and chlamydia. This study investigated the prevalence of Chlamydiae in ticks. Specifically, 62,889 Ixodes ricinus ticks, consolidated into 8,534 pools, were sampled in 172 collection sites throughout Switzerland and were investigated using pan-Chlamydiales quantitative PCR (qPCR) for the presence of Chlamydiales DNA. Among the pools, 543 (6.4%) gave positive results and the estimated prevalence in individual ticks was 0.89%. Among those pools with positive results, we obtained 16S rRNA sequences for 359 samples, allowing classification of Chlamydiales DNA at the family level. A high level of biodiversity was observed, since six of the nine families belonging to the Chlamydiales order were detected. Those most common were Parachlamydiaceae (33.1%) and Rhabdochlamydiaceae (29.2%). "Unclassified Chlamydiales" (31.8%) were also often detected. Thanks to the huge amount of Chlamydiales DNA recovered from ticks, this report opens up new perspectives on further work focusing on whole-genome sequencing to increase our knowledge about Chlamydiales biodiversity. This report of an epidemiological study also demonstrates the presence of Chlamydia-related bacteria within Ixodes ricinus ticks and suggests a role for ticks in the transmission of and as a reservoir for these emerging pathogenic Chlamydia-related bacteria.

Finding and characterizing Partially Methylated Domains in human haematopoietic cells

DNA cytosine methylation has been demonstrated to be a central epigenetic modification that has essential roles in a myriad of cellular processes. Some examples of these include gene regulation, DNA-protein interactions, cellular differentiation, X-inactivation, maintenance of genome integrity by suppressing transposable elements and viruses, embryogenesis, genomic imprinting and tumourigenesis. This list is increasingly growing thanks to recent advances in genome-wide technologies, like Whole Genome Bisulfite Sequencing (WGBS-Seq). The development of this technology in research has allowed the identification of new features of the DNA methylation landscape that was not possible using previous technologies, like Partially Methylated Domains (PMDs). PMDs have been found in several cell lines, as well as in both healthy and cancer primary samples. They have been described as regions with high variability in methylation levels across individual CpG sites and intermediate methylation levels on average with respect to the genome. Here, we performed an extensive search of PMDs in a big dataset of different haematopoietic primary cells from both myeloid and lymphoid lineages. We found and characterized significant PMDs in plasma B cells, confirming that PMDs are a phenomenon that is restricted to certain differentiated cells. Additionally, we found loci aberrantly hypomethylated in a myeloma sample which overlapped with plasma B cell PMDs. Genome-wide comparison of the myeloma and plasma B cell sample revealed that this is probably also the case for other loci.

Robust DNA Methylation in the Clonal Raider Ant Brain.

Social insects are promising model systems for epigenetics due to their immense morphological and behavioral plasticity. Reports that DNA methylation differs between the queen and worker castes in social insects [1-4] have implied a role for DNA methylation in regulating division of labor. To better understand the function of DNA methylation in social insects, we performed whole-genome bisulfite sequencing on brains of the clonal raider ant Cerapachys biroi, whose colonies alternate between reproductive (queen-like) and brood care (worker-like) phases [5]. Many cytosines were methylated in all replicates (on average 29.5% of the methylated cytosines in a given replicate), indicating that a large proportion of the C. biroi brain methylome is robust. Robust DNA methylation occurred preferentially in exonic CpGs of highly and stably expressed genes involved in core functions. Our analyses did not detect any differences in DNA methylation between the queen-like and worker-like phases, suggesting that DNA methylation is not associated with changes in reproduction and behavior in C. biroi. Finally, many cytosines were methylated in one sample only, due to either biological or experimental variation. By applying the statistical methods used in previous studies [1-4, 6] to our data, we show that such sample-specific DNA methylation may underlie the previous findings of queen- and worker-specific methylation. We argue that there is currently no evidence that genome-wide variation in DNA methylation is associated with the queen and worker castes in social insects, and we call for a more careful interpretation of the available data.

No Accumulation of Transposable Elements in Asexual Arthropods.

Transposable elements (TEs) and other repetitive DNA can accumulate in the absence of recombination, a process contributing to the degeneration of Y-chromosomes and other nonrecombining genome portions. A similar accumulation of repetitive DNA is expected for asexually reproducing species, given their entire genome is effectively nonrecombining. We tested this expectation by comparing the whole-genome TE loads of five asexual arthropod lineages and their sexual relatives, including asexual and sexual lineages of crustaceans (Daphnia water fleas), insects (Leptopilina wasps), and mites (Oribatida). Surprisingly, there was no evidence for increased TE load in genomes of asexual as compared to sexual lineages, neither for all classes of repetitive elements combined nor for specific TE families. Our study therefore suggests that nonrecombining genomes do not accumulate TEs like nonrecombining genomic regions of sexual lineages. Even if a slight but undetected increase of TEs were caused by asexual reproduction, it appears to be negligible compared to variance between species caused by processes unrelated to reproductive mode. It remains to be determined if molecular mechanisms underlying genome regulation in asexuals hamper TE activity. Alternatively, the differences in TE dynamics between nonrecombining genomes in asexual lineages versus nonrecombining genome portions in sexual species might stem from selection for benign TEs in asexual lineages because of the lack of genetic conflict between TEs and their hosts and/or because asexual lineages may only arise from sexual ancestors with particularly low TE loads.

The Stealthy Superbug: the Role of Asymptomatic Enteric Carriage in Maintaining a Long-Term Hospital Outbreak of ST228 Methicillin-Resistant Staphylococcus aureus.

UNLABELLED: Whole-genome sequencing (WGS) of 228 isolates was used to elucidate the origin and dynamics of a long-term outbreak of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 228 (ST228) SCCmec I that involved 1,600 patients in a tertiary care hospital between 2008 and 2012. Combining of the sequence data with detailed metadata on patient admission and movement confirmed that the outbreak was due to the transmission of a single clonal variant of ST228, rather than repeated introductions of this clone into the hospital. We note that this clone is significantly more frequently recovered from groin and rectal swabs than other clones (P < 0.0001) and is also significantly more transmissible between roommates (P < 0.01). Unrecognized MRSA carriers, together with movements of patients within the hospital, also seem to have played a major role. These atypical colonization and transmission dynamics can help explain how the outbreak was maintained over the long term. This "stealthy" asymptomatic colonization of the gut, combined with heightened transmissibility (potentially reflecting a role for environmental reservoirs), means the dynamics of this outbreak share some properties with enteric pathogens such as vancomycin-resistant enterococci or Clostridium difficile. IMPORTANCE: Using whole-genome sequencing, we showed that a large and prolonged outbreak of methicillin-resistant Staphylococcus aureus was due to the clonal spread of a specific strain with genetic elements adapted to the hospital environment. Unrecognized MRSA carriers, the movement of patients within the hospital, and the low detection with clinical specimens were also factors that played a role in this occurrence. The atypical colonization of the gut means the dynamics of this outbreak may share some properties with enteric pathogens.

Genomic evidence for adaptive inversion clines in Drosophila melanogaster.

Clines in chromosomal inversion polymorphisms-presumably driven by climatic gradients-are common but there is surprisingly little evidence for selection acting on them. Here we address this long-standing issue in Drosophila melanogaster by using diagnostic single nucleotide polymorphism (SNP) markers to estimate inversion frequencies from 28 whole-genome Pool-seq samples collected from 10 populations along the North American east coast. Inversions In(3L)P, In(3R)Mo, and In(3R)Payne showed clear latitudinal clines, and for In(2L)t, In(2R)NS, and In(3R)Payne the steepness of the clinal slopes changed between summer and fall. Consistent with an effect of seasonality on inversion frequencies, we detected small but stable seasonal fluctuations of In(2R)NS and In(3R)Payne in a temperate Pennsylvanian population over 4 years. In support of spatially varying selection, we observed that the cline in In(3R)Payne has remained stable for >40 years and that the frequencies of In(2L)t and In(3R)Payne are strongly correlated with climatic factors that vary latitudinally, independent of population structure. To test whether these patterns are adaptive, we compared the amount of genetic differentiation of inversions versus neutral SNPs and found that the clines in In(2L)t and In(3R)Payne are maintained nonneutrally and independent of admixture. We also identified numerous clinal inversion-associated SNPs, many of which exhibit parallel differentiation along the Australian cline and reside in genes known to affect fitness-related traits. Together, our results provide strong evidence that inversion clines are maintained by spatially-and perhaps also temporally-varying selection. We interpret our data in light of current hypotheses about how inversions are established and maintained.

Variability of the 3' terminal of the polymerase gene of Grapevine leafroll-associated virus 3 isolates from Vale do São Francisco, Brazil

Many viral diseases, including leafroll, which is of great economic importance, affect grapevines (Vitis spp.). A complex of eight viruses [Grapevine leafroll-associated virus (GLRaV) -1 to 8] is associated with this disease. The objective of this study was to compare the variability of the 3' terminal region of the polymerase gene of three isolates of GLRaV-3 (Grapevine leafroll-associated virus-3), from Submédio do Vale do Rio São Francisco (Petrolina-PE) with that of other isolates available at the GenBank, including an isolate from North America and another from Southern Brazil. The viral RNA was extracted from three infected ELISA reactive plants and a fragment of 340 bp was amplified, by RT-PCR, using primers that recognize that portion of the polymerase gene found between nucleotides 8267 and 8606. The three isolates from Vale do Rio São Francisco named Pet-1, Pet-2 and Pet-3, showed similarities ranging from 98% and 94%, respectively to the isolates from North America (AF037268) and Southern Brazilian (AF438411). Considering the whole genome, the main variation found was one amino acid change at position 2766 (F2766Y). These preliminary data indicate the existence of a natural variation among GLRaV-3 isolates from grapevines. This could be due to the vegetative propagation and long cycle of the plant, associated with the error-prone nature of RNA-dependent RNA polymerase.

Molecular diagnostics of rare inherited SYNDROMES with a view on diagnostic test development

CHARGE syndrome, Sotos syndrome and 3p deletion syndrome are examples of rare inherited syndromes that have been recognized for decades but for which the molecular diagnostics only have been made possible by recent advances in genomic research. Despite these advances, development of diagnostic tests for rare syndromes has been hindered by diagnostic laboratories having limited funds for test development, and their prioritization of tests for which a (relatively) high demand can be expected. In this study, the molecular diagnostic tests for CHARGE syndrome and Sotos syndrome were developed, resulting in their successful translation into routine diagnostic testing in the laboratory of Medical Genetics (UTUlab). In the CHARGE syndrome group, mutation was identified in 40.5% of the patients and in the Sotos syndrome group, in 34%, reflecting the use of the tests in routine diagnostics in differential diagnostics. In CHARGE syndrome, the low prevalence of structural aberrations was also confirmed. In 3p deletion syndrome, it was shown that small terminal deletions are not causative for the syndrome, and that testing with arraybased analysis provides a reliable estimate of the deletion size but benign copy number variants complicate result interpretation. During the development of the tests, it was discovered that finding an optimal molecular diagnostic strategy for a given syndrome is always a compromise between the sensitivity, specificity and feasibility of applying a new method. In addition, the clinical utility of the test should be considered prior to test development: sometimes a test performing well in a laboratory has limited utility for the patient, whereas a test performing poorly in the laboratory may have a great impact on the patient and their family. At present, the development of next generation sequencing methods is changing the concept of molecular diagnostics of rare diseases from single tests towards whole-genome analysis.

Functional Genomic Approaches for Deciphering Plant-Virus Interactions

Plant-virus interactions are very complex in nature and lead to disease and symptom formation by causing various physiological, metabolic and developmental changes in the host plants. These interactions are mainly the outcomes of viral hijacking of host components to complete their infection cycles and of host defensive responses to restrict the viral infections. Viral genomes contain only a small number of genes often encoding for multifunctional proteins, and all are essential in establishing a viral infection. Thus, it is important to understand the specific roles of individual viral genes and their contribution to the viral life cycles. Among the most important viral proteins are the suppressors of RNA silencing (VSRs). These proteins function to suppress host defenses mediated by RNA silencing and can also serve in other functions, e.g. in viral movement, transactivation of host genes, virus replication and protein processing. Thus these proteins are likely to have a significant impact on host physiology and metabolism. In the present study, I have examined the plant-virus interactions and the effects of three different VSRs on host physiology and gene expression levels by microarray analysis of transgenic plants that express these VSR genes. I also studied the gene expression changes related to the expression of the whole genome of Tobacco mosaic virus (TMV) in transgenic tobacco plants. Expression of the VSR genes in the transgenic tobacco plants causes significant changes in the gene expression profiles. HC-Pro gene derived from the Potyvirus Y (PVY) causes alteration of 748 and 332 transcripts, AC2 gene derived from the African cassava mosaic virus (ACMV) causes alteration of 1118 and 251transcripts, and P25 gene derived from the Potyvirus X (PVX) causes alterations of 1355 and 64 transcripts in leaves and flowers, respectively. All three VSRs cause similar up-regulation in defense, hormonally regulated and different stress-related genes and down-regulation in the photosynthesis and starch metabolism related genes. They also induce alterations that are specific to each viral VSR. The phenotype and transcriptome alterations of the HC-Pro expressing transgenic plants are similar to those observed in some Potyvirus-infected plants. The plants show increased protein degradation, which may be due to the HC-Pro cysteine endopeptidase and thioredoxin activities. The AC2-expressing transgenic plants show a similar phenotype and gene expression pattern as HC-Pro-expressing plants, but also alter pathways related to jasmonic acid, ethylene and retrograde signaling. In the P25 expressing transgenic plants, high numbers of genes (total of 1355) were up-regulated in the leaves, compared to a very low number of down-regulated genes (total of 5). Despite of strong induction of the transcripts, only mild growth reduction and no other distinct phenotype was observed in these plants. As an example of whole virus interactions with its host, I also studied gene expression changes caused by Tobacco mosaic virus (TMV) in tobacco host in three different conditions, i.e. in transgenic plants that are first resistant to the virus, and then become susceptible to it and in wild type plants naturally infected with this virus. The microarray analysis revealed up and down-regulation of 1362 and 1422 transcripts in the TMV resistant young transgenic plants, and up and down-regulation of a total of 1150 and 1200 transcripts, respectively, in the older plants, after the resistance break. Natural TMV infections in wild type plants caused up-regulation of 550 transcripts and down-regulation of 480 transcripts. 124 up-regulated and 29 down-regulated transcripts were commonly altered between young and old TMV transgenic plants, and only 6 up-regulated and none of the down-regulated transcripts were commonly altered in all three plants. During the resistant stage, the strong down-regulation in translation-related transcripts (total of 750 genes) was observed. Additionally, transcripts related to the hormones, protein degradation and defense pathways, cell division and stress were distinctly altered. All these alterations may contribute to the TMV resistance in the young transgenic plants, and the resistance may also be related to RNA silencing, despite of the low viral abundance and lack of viral siRNAs or TMV methylation activity in the plants.

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.