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Background For patients with locally advanced head and neck squamous cell carcinoma (LAHNSCC), surgery (S) followed by radiotherapy (RT) is a standard of care. Randomized controlled trials have shown that postoperative chemoradiation (CRT) increased the locoregional control (LRC) and overall survival (OS) in patient with R1-resection margin and/or extranodal extension (ENE). ENE has been introduced in the 8th TNM staging classification since its presence has been shown to have an independent adverse prognostic impact. The data supporting this finding were however mainly collected in the pre-CRT era. Objectives The objective of this study was to challenge the adverse prognostic factor of ENE in the era of CRT. Methods A retrospective cohort study was performed to evaluate patients diagnosed with LAHNSCC and undergoing a treatment by S and postoperative RT or CRT in Centre Leon Berard, Lyon, France between 2003 and 2018. Patients with oral cavity, oropharyngeal, laryngeal and hypopharyngeal SCC were included. Results 439 patients were included in the study. For patients with non-oropharyngeal p16-positive tumors without ENE, five-year OS, local control, and regional control (RC) reached 63.7%, 86.1%, and 94.9%, respectively; corresponding figures for patients with ENE reached, 42.6%, 77.5%, and 81.1%, respectively (p-value of 0.0006, 0.167, and 0.0005). In multivariable analysis, for non-oropharyngeal p16-positive tumors, ENE remained a poor prognostic factor for OS (RR = 1.74, 95%, CI = 1.16–2.61, p = 0.0069) and RC (RR 3.60, 95% CI =: 1.64–7.87, p = 0.0013). Conclusion In the era or postoperative chemoradiation, pathological ENE remains an adverse prognostic factor for OS and RC.

For the first time, blood samples were collected in all athletes participating in a major sporting event of the International Association of Athletics Federations (IAAF) (Athletics World Championships 2011, Daegu, Korea). All variables obtained from blood analyses were incorporated into the individual blood profiles of each athlete for the so-called "Athlete Biological Passport" (ABP). This unprecedented data collection highlighted differences for a few blood biomarkers commonly measured and reported for the ABP on some group of athletes. Subsequently, blood tests analyses for all athletes were repeated during the following World Championships (2013, Moscow, Russia). Both sets of blood tests were then used to set up the distribution of blood values for track and field athletes considering potential confounding factors such as gender, age, discipline, origin of the athlete (continental classification) and time of blood collection. Implementation of well-defined distribution of blood values will allow to improve the estimation of blood doping prevalence among a specific population of athletes in track and field.

RNA polymerase III (Pol III) synthesizes short noncoding RNAs, many of which are essential for translation. Accordingly, Pol III activity is tightly regulated with cell growth and proliferation by factors such as MYC, RB1, TRP53, and MAF1. MAF1 is a repressor of Pol III transcription whose activity is controlled by phosphorylation; in particular, it is inactivated through phosphorylation by the TORC1 kinase complex, a sensor of nutrient availability. Pol III regulation is thus sensitive to environmental cues, yet a diurnal profile of Pol III transcription activity is so far lacking. Here, we first use gene expression arrays to measure mRNA accumulation during the diurnal cycle in the livers of (1) wild-type mice, (2) arrhythmic Arntl knockout mice, (3) mice fed at regular intervals during both night and day, and (4) mice lacking the Maf1 gene, and so provide a comprehensive view of the changes in cyclic mRNA accumulation occurring in these different systems. We then show that Pol III occupancy of its target genes rises before the onset of the night, stays high during the night, when mice normally ingest food and when translation is known to be increased, and decreases in daytime. Whereas higher Pol III occupancy during the night reflects a MAF1-dependent response to feeding, the rise of Pol III occupancy before the onset of the night reflects a circadian clock-dependent response. Thus, Pol III transcription during the diurnal cycle is regulated both in response to nutrients and by the circadian clock, which allows anticipatory Pol III transcription.

In elite sport, the Athlete Biological Passport (ABP) was invented to tackle cheaters by monitoring closely changes in biological parameters, flagging atypical variations. The haematological module of the ABP was indeed adopted in 2011 by the International Association of Athletics Federations (IAAF). This study estimates the prevalence of blood doping based on haematological parameters in a large cohort of track & field athletes measured at two international major events (2011 & 2013 IAAF World Championships) with a hypothesized decrease in prevalence due to the ABP introduction.A total of 3683 blood samples were collected and analysed from all participating athletes originating from 209 countries. The estimate of doping prevalence was obtained by using a Bayesian network with seven variables, as well as “doping” as a variable mimicking doping with low-doses of recombinant human erythropoietin (rhEPO), to generate reference cumulative distribution functions (CDFs) for the Abnormal Blood Profile Score (ABPS) from the ABP.Our results from robust haematological parameters indicate an estimation of an overall blood doping prevalence of 18% in average in endurance athletes (95% Confidence Interval (C.I.) 14-22%). A higher prevalence was observed in female athletes (22%, C.I. 16-28%) than in male athletes (15%, C.I. 9-20%). In conclusion, this study presents the first comparison of blood doping prevalence in elite athletes based on biological measurements from major international events that may help scientists and experts to use the ABP in a more efficient and deterrent way.What are the new findings ?This study presents the first comparison of blood doping prevalence in elite track & field athletes based on biological measurements from major international eventsOur results from robust haematological parameters indicate an estimation of an overall blood doping prevalence of 18% in average in endurance athletes.The confidence intervals for blood doping prevalence range from 9-28% with wide discrepancies between certain countries.How might it impact on clinical practice in the near futureThe further development of the Athlete Biological Passport with a careful monitoring of biological parameters still represents the most consistent approach to thwart athletes using undetectable prohibited substances or methods.This study describes a method to define blood doping prevalence with the analysis of robust haematological parametersEstimates of doping prevalence in subpopulations of athletes may represent a valuable tool for the antidoping authorities to perform a risk assessment in their sport.

Because plants do not possess a defined germline, deleterious somatic mutations can be passed to gametes, and a large number of cell divisions separating zygote from gamete formation may lead to many mutations in long-lived plants. We sequenced the genome of two terminal branches of a 234-year-old oak tree and found several fixed somatic single-nucleotide variants whose sequential appearance in the tree could be traced along nested sectors of younger branches. Our data suggest that stem cells of shoot meristems in trees are robustly protected from the accumulation of mutations.

In human transcriptional regulation, DNA-sequence-specific factors can associate with intermediaries that orchestrate interactions with a diverse set of chromatin-modifying enzymes. One such intermediary is HCFC1 (also known as HCF-1). HCFC1, first identified in herpes simplex virus transcription, has a poorly defined role in cellular transcriptional regulation. We show here that, in HeLa cells, HCFC1 is observed bound to 5400 generally active CpG-island promoters. Examination of the DNA sequences underlying the HCFC1-binding sites revealed three sequence motifs associated with the binding of (1) ZNF143 and THAP11 (also known as Ronin), (2) GABP, and (3) YY1 sequence-specific transcription factors. Subsequent analysis revealed colocalization of HCFC1 with these four transcription factors at ∼90% of the 5400 HCFC1-bound promoters. These studies suggest that a relatively small number of transcription factors play a major role in HeLa-cell transcriptional regulation in association with HCFC1.

A large-scale comparative gene expression study reveals the different ways in which the chromosome-wide gene dosage reductions resulting from sex chromosome differentiation events were compensated during mammalian and avian evolution., As a result of sex chromosome differentiation from ancestral autosomes, male mammalian cells only contain one X chromosome. It has long been hypothesized that X-linked gene expression levels have become doubled in males to restore the original transcriptional output, and that the resulting X overexpression in females then drove the evolution of X inactivation (XCI). However, this model has never been directly tested and patterns and mechanisms of dosage compensation across different mammals and birds generally remain little understood. Here we trace the evolution of dosage compensation using extensive transcriptome data from males and females representing all major mammalian lineages and birds. Our analyses suggest that the X has become globally upregulated in marsupials, whereas we do not detect a global upregulation of this chromosome in placental mammals. However, we find that a subset of autosomal genes interacting with X-linked genes have become downregulated in placentals upon the emergence of sex chromosomes. Thus, different driving forces may underlie the evolution of XCI and the highly efficient equilibration of X expression levels between the sexes observed for both of these lineages. In the egg-laying monotremes and birds, which have partially homologous sex chromosome systems, partial upregulation of the X (Z in birds) evolved but is largely restricted to the heterogametic sex, which provides an explanation for the partially sex-biased X (Z) expression and lack of global inactivation mechanisms in these lineages. Our findings suggest that dosage reductions imposed by sex chromosome differentiation events in amniotes were resolved in strikingly different ways., Author Summary Mammalian sex chromosomes (the X and Y) evolved from an ordinary pair of ancestral somatic chromosomes (the proto-sex chromosomes). The process that led to emergence of distinct sex chromosomes involved the degeneration of the Y chromosome, leaving males with only one copy of most proto-sex chromosomal genes on their single X chromosome. It has remained unclear whether mechanisms evolved that compensate for this dosage reduction. Here we trace the evolution of sex chromosomal expression levels in all major mammalian lineages and in birds. We find that the X has become globally upregulated in response to the dosage reduction in marsupials, whereas in placental mammals, genes resident on autosomal (non-sex) chromosomes that interact with X-linked genes have instead become downregulated. These mechanisms restore ancestral gene expression balances and also presumably drove the evolution of secondary compensation mechanisms (i.e., female X-inactivation) in these mammalian lineages. In egg-laying mammals and birds, sex chromosomes have become partially upregulated specifically in the heterogametic sex, i.e., in male monotremes (which are XY) and female birds (which are WZ). This probably explains why the evolution of inactivation mechanisms in the homogametic sexes in these lineages (XX and ZZ, respectively) was not necessary. Our findings suggest that gene dosage alterations associated with the emergence of sex chromosome systems can be compensated in various different ways.

Although excessive exposure to UV is widely recognized as a major factor leading to skin perturbations and cancer, the complex mechanisms underlying inflammatory skin disorders resulting from UV exposure remain incompletely characterized. The nuclear hormone receptor PPARβ/δ is known to control mouse cutaneous repair and UV‐induced skin cancer development. Here, we describe a novel PPARβ/δ‐dependent molecular cascade involving TGFβ1 and miR‐21‐3p, which is activated in the epidermis in response to UV exposure. We establish that the passenger miRNA miR‐21‐3p, that we identify as a novel UV‐induced miRNA in the epidermis, plays a pro‐inflammatory function in keratinocytes and that its high level of expression in human skin is associated with psoriasis and squamous cell carcinomas. Finally, we provide evidence that inhibition of miR‐21‐3p reduces UV‐induced cutaneous inflammation in ex vivo human skin biopsies, thereby underlining the clinical relevance of miRNA‐based topical therapies for cutaneous disorders.

International audience; Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

A preliminary understanding into the phenotypic effect of DNA segment copy number variation (CNV) is emerging. These rearrangements were demonstrated to influence, in a somewhat dose-dependent manner, the expression of genes that map within them. They were also shown to modify the expression of genes located on their flanks and sometimes those at a great distance from their boundary. Here we demonstrate, by monitoring these effects at multiple life stages, that these controls over expression are effective throughout mouse development. Similarly, we observe that the more specific spatial expression patterns of CNV genes are maintained through life. However, we find that some brain-expressed genes mapping within CNVs appear to be under compensatory loops only at specific time points, indicating that the effect of CNVs on these genes is modulated during development. Notably, we also observe that CNV genes are significantly enriched within transcripts that show variable time courses of expression between strains. Thus, modifying the copy number of a gene may potentially alter not only its expression level, but also the timing of its expression.

In yeast TOR and PKA pathways both control cell growth but how TORC1 and PKA signaling are linked is unknown. Here we show that TORC1 inhibition prevents the phosphorylation of some but not all PKA targets. We further demonstrate that TORC1 controls PKA by inhibiting the phosphorylation of the PKA regulatory subunit BCY1 by the MAP kinase MPK1., Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogenesis, translation, nutrient metabolism, and autophagy. In yeast, the protein kinases Target of Rapamycin (TOR) and protein kinase A (PKA) regulate these processes and are thereby the main activators of cell growth in response to nutrients. How TOR, PKA, and their corresponding signaling pathways are coordinated to control the same cellular processes is not understood. Quantitative analysis of the rapamycin-sensitive phosphoproteome combined with targeted analysis of PKA substrates suggests that TOR complex 1 (TORC1) activates PKA but only toward a subset of substrates. Furthermore, we show that TORC1 signaling impinges on BCY1, the negative regulatory subunit of PKA. Inhibition of TORC1 with rapamycin leads to BCY1 phosphorylation on several sites including T129. Phosphorylation of BCY1 T129 results in BCY1 activation and inhibition of PKA. TORC1 inhibits BCY1 T129 phosphorylation by phosphorylating and activating the S6K homolog SCH9 that in turn inhibits the MAP kinase MPK1. MPK1 phosphorylates BCY1 T129 directly. Thus, TORC1 activates PKA toward some substrates by preventing MPK1-mediated activation of BCY1.

Copy number variation (CNV) is a key source of genetic diversity, but a comprehensive understanding of its phenotypic effect is only beginning to emerge. We have generated a CNV map in wild mice and classical inbred strains. Genome-wide expression data from six major organs show not only that expression of genes within CNVs tend to correlate with copy number changes, but also that CNVs influence the expression of genes in their vicinity, an effect that extends up to half a megabase. Genes within CNVs show lower expression and more specific spatial expression patterns than genes mapping elsewhere. Our analyses reveal differential constraint on copy number changes of genes expressed in different tissues. Dosage alterations of brain-expressed genes are less frequent than those of other genes and are buffered by tighter transcriptional regulation. Our study provides initial evidence that CNVs shape tissue transcriptomes on a global scale.

Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it.

The taxonomic composition of egg-associated microbial communities can play a crucial role in the development of fish embryos. In response, hosts increasingly influence the composition of their associated microbial communities during embryogenesis, as concluded from recent field studies and laboratory experiments. However, little is known about the taxonomic composition and the diversity of egg-associated microbial communities within ecosystems; e.g., river networks. We sampled late embryonic stages of naturally spawned brown trout at nine locations within two different river networks and applied 16S rRNA pyrosequencing to describe their bacterial communities. We found no evidence for a significant isolation-by-distance effect on the composition of bacterial communities and no association between neutral genetic divergence of fish host (based on 11 microsatellites) and phylogenetic distances of the composition of their associated bacterial communities. We characterized core bacterial communities on brown trout eggs and compared them to corresponding water samples with regard to bacterial composition and its presumptive function. Bacterial diversity was positively correlated with water temperature at the spawning locations. We discuss this finding in the context of the increased water temperatures that have been recorded during the last 25 years in the study area.

MS/MS and associated database search algorithms are essential proteomic tools for identifying peptides. Due to their widespread use, it is now time to perform a systematic analysis of the various algorithms currently in use. Using blood specimens used in the HUPO Plasma Proteome Project, we have evaluated five search algorithms with respect to their sensitivity and specificity, and have also accurately benchmarked them based on specified false-positive (FP) rates. Spectrum Mill and SEQUEST performed well in terms of sensitivity, but were inferior to MASCOT, X!Tandem, and Sonar in terms of specificity. Overall, MASCOT, a probabilistic search algorithm, correctly identified most peptides based on a specified FP rate. The rescoring algorithm, PeptideProphet, enhanced the overall performance of the SEQUEST algorithm, as well as provided predictable FP error rates. Ideally, score thresholds should be calculated for each peptide spectrum or minimally, derived from a reversed-sequence search as demonstrated in this study based on a validated data set. The availability of open-source search algorithms, such as X!Tandem, makes it feasible to further improve the validation process (manual or automatic) on the basis of "consensus scoring", i.e., the use of multiple (at least two) search algorithms to reduce the number of FPs. complement.

Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from approximately 10(6) for cells to approximately 10(10) for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by approximately 0.02-0.10 pH unit depending on the gradient created, followed by rapid (approximately 6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720.

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution. published

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.

Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus–1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

The characterization of mice with different number of copies of the same genomic segment shows that structural changes influence the phenotypic outcome independently of gene dosage., A large fraction of genome variation between individuals is comprised of submicroscopic copy number variation of genomic DNA segments. We assessed the relative contribution of structural changes and gene dosage alterations on phenotypic outcomes with mouse models of Smith-Magenis and Potocki-Lupski syndromes. We phenotyped mice with 1n (Deletion/+), 2n (+/+), 3n (Duplication/+), and balanced 2n compound heterozygous (Deletion/Duplication) copies of the same region. Parallel to the observations made in humans, such variation in gene copy number was sufficient to generate phenotypic consequences: in a number of cases diametrically opposing phenotypes were associated with gain versus loss of gene content. Surprisingly, some neurobehavioral traits were not rescued by restoration of the normal gene copy number. Transcriptome profiling showed that a highly significant propensity of transcriptional changes map to the engineered interval in the five assessed tissues. A statistically significant overrepresentation of the genes mapping to the entire length of the engineered chromosome was also found in the top-ranked differentially expressed genes in the mice containing rearranged chromosomes, regardless of the nature of the rearrangement, an observation robust across different cell lineages of the central nervous system. Our data indicate that a structural change at a given position of the human genome may affect not only locus and adjacent gene expression but also “genome regulation.” Furthermore, structural change can cause the same perturbation in particular pathways regardless of gene dosage. Thus, the presence of a genomic structural change, as well as gene dosage imbalance, contributes to the ultimate phenotype., Author Summary Mammalian genomes contain many forms of genetic variation. For example, some genome segments were shown to vary in their number of copies between individuals of the same species, i.e. there is a range of number of copies in the normal population instead of the usual two copies (one per chromosome). These genetic differences play an important role in determining the phenotype (the observable characteristics) of each individual. We do not know, however, if such influences are brought about solely through changes in the number of copies of the genomic segments (and of the genes that map within) or if the structural modification of the genome per se also plays a role in the outcome. We use mouse models with different number of copies of the same genomic region to show that rearrangements of the genetic materials can affect the phenotype independently of the dosage of the rearranged region.

Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that ∼50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that ∼40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

The biochemical mechanisms controlling the diverse functional outcomes of human central memory (CM) and effector memory (EM) T-cell responses triggered through the T-cell receptor (TCR) remain poorly understood. We implemented reverse phase protein arrays to profile TCR signaling components in human CD8 and CD4 memory T-cell subsets isolated ex vivo. As compared with CD4 CM cells, EM cells express statistically significant increased amounts of SLP-76 and reduced levels of c-Cbl, Syk, Fyn, and LAT. Moreover, in EM cells reduced expression of negative regulator c-Cbl correlates with expression of c-Cbl kinases (Syk and Fyn), PI3K, and LAT. Importantly, consistent with reduced expression of c-Cbl, EM cells display a lower functional threshold than CM cells. Increasing c-Cbl content of EM cells to the same level as that of CM cells using cytosolic transduction, we impaired their proliferation and cytokine production. This regulatory mechanism depends primarily on c-Cbl E3 ubiquitin ligase activity as evidenced by the weaker impact of enzymatically deficient c-Cbl C381A mutant on EM cell functions. Our study reports c-Cbl as a critical regulator of the functional responses of memory T cell subsets and identifies for the first time in humans a mechanism controlling the functional heterogeneity of memory CD4 cells.

INTRODUCTION: Breast cancer subtyping and prognosis have been studied extensively by gene expression profiling, resulting in disparate signatures with little overlap in their constituent genes. Although a previous study demonstrated a prognostic concordance among gene expression signatures, it was limited to only one dataset and did not fully elucidate how the different genes were related to one another nor did it examine the contribution of well-known biological processes of breast cancer tumorigenesis to their prognostic performance. METHOD: To address the above issues and to further validate these initial findings, we performed the largest meta-analysis of publicly available breast cancer gene expression and clinical data, which are comprised of 2,833 breast tumors. Gene coexpression modules of three key biological processes in breast cancer (namely, proliferation, estrogen receptor [ER], and HER2 signaling) were used to dissect the role of constituent genes of nine prognostic signatures. RESULTS: Using a meta-analytical approach, we consolidated the signatures associated with ER signaling, ERBB2 amplification, and proliferation. Previously published expression-based nomenclature of breast cancer 'intrinsic' subtypes can be mapped to the three modules, namely, the ER-/HER2- (basal-like), the HER2+ (HER2-like), and the low- and high-proliferation ER+/HER2- subtypes (luminal A and B). We showed that all nine prognostic signatures exhibited a similar prognostic performance in the entire dataset. Their prognostic abilities are due mostly to the detection of proliferation activity. Although ER- status (basal-like) and ERBB2+ expression status correspond to bad outcome, they seem to act through elevated expression of proliferation genes and thus contain only indirect information about prognosis. Clinical variables measuring the extent of tumor progression, such as tumor size and nodal status, still add independent prognostic information to proliferation genes. CONCLUSION: This meta-analysis unifies various results of previous gene expression studies in breast cancer. It reveals connections between traditional prognostic factors, expression-based subtyping, and prognostic signatures, highlighting the important role of proliferation in breast cancer prognosis., Journal Article, Meta-Analysis, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Mass spectrometry–based methods for the identification of proteins are fundamental platform technologies for proteomics. One comprehensive approach is to subject trypsinized peptides to tandem mass spectrometry (MS/MS) to obtain detailed structural information. Different strategies are available for interpreting MS/MS data and hence deducing the amino acid sequence of the peptides. The most common method is to use a search algorithm to identify peptides by correlating experimental and theoretical MS/MS data (the latter being generated from possible peptides in the protein sequence database). Identified peptides are collated and protein entries from the sequence database inferred. This unit focuses on the most widely used tandem MS peptide identification search algorithms (commercial and open source), their availability, ease of use, strengths, speed and scoring, as well as their relative sensitivity and specificity. Curr. Protoc. Protein Sci. 49:25.2.1-25.2.19. © 2007 by John Wiley & Sons, Inc. Keywords: SEQUEST; Mascot; X!Tandem; OMSSA; PLGS; Sorcerer; ProteinPilot; Phenyx; SpectrumMill

Background Remodeling of quiescent vessels with increases in permeability, vasodilatation, and edema are hallmarks of inflammatory disorders. Factors involved in this type of remodeling represent potential therapeutic targets. Objectives We investigated whether the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) β/δ, a regulator of metabolism, fibrosis, and skin homeostasis, is involved in regulation of this type of remodeling. Methods Wild-type and various Pparb/d mutant mice were used to monitor dermal acute vascular hyperpermeability (AVH) and passive systemic anaphylaxis–induced hypothermia and edema. PPARβ/δ-dependent kinase activation and remodeling of endothelial cell-cell junctions were addressed by using human endothelial cells. Results AVH and dilatation of dermal microvessels stimulated by vascular endothelial growth factor A, histamine, and thrombin are severely compromised in PPARβ/δ-deficient mice. Selective deletion of the Pparb/d -encoding gene in endothelial cells in vivo similarly limits dermal AVH and vasodilatation, providing evidence that endothelial PPARβ/δ is the major player in regulating acute dermal microvessel remodeling. Furthermore, endothelial PPARβ/δ regulatory functions are not restricted to the skin vasculature because its deletion in the endothelium, but not in smooth muscle cells, also leads to reduced systemic anaphylaxis, the most severe form of allergic reaction, in which an acute vascular response plays a key role. PPARβ/δ-dependent AVH activation likely involves the activation of mitogen-activated protein kinase and Akt pathways and leads to downstream destabilization of endothelial cell-cell junctions. Conclusion These results unveil not only a novel function of PPARβ/δ as a direct regulator of acute vessel permeability and dilatation but also provide evidence that antagonizing PPARβ/δ represents an important strategy to consider for moderating diseases with altered endothelial integrity, such as acute inflammatory and allergic disorders.

Improved search algorithms and scoring functions are required before the identification of peptide tandem MS data can be considered to be fully reliable and automatable. The development of models that can accurately predict product ion spectra from a peptide sequence would certainly help achieve this goal, but this firstly requires a better understanding of the process of fragmentation of peptides in the gas-phase. We summarize recent developments in this area and show that the prediction of product ion spectra is feasible and should improve the identification of peptide tandem MS data, especially for peptides that currently give low or insignificant scores with current search algorithms.

A database of 5500 unique peptide tandem mass spectra acquired in an ion trap mass spectrometer was assembled for peptides derived from proteins digested with trypsin. Peptides were identified initially from their tandem mass spectra by the SEQUEST algorithm and subsequently validated manually. Two different statistical methods were used to identify sequence-dependent fragmentation patterns that could be used to improve fragmentation models incorporated into current peptide sequencing and database search algorithms. The currently accepted "mobile proton" model was expanded to derive a new classification scheme for peptide mass spectra, the "relative proton mobility" scale, which considers peptide ion charge state and amino acid composition to categorize peptide mass spectra into peptide ions containing "nonmobile", "partially mobile", or "mobile" protons. Quantitation of amide bond fragmentation, both N- and C-terminal to any given amino acid, as well as the positional effect of an amino acid in a peptide and peptide length on such fragmentation, has been determined. Peptide bond cleavage propensities, both positive (i.e., enhanced) and negative (i.e., suppressed), were determined and ranked in order of their cleavage preferences as primary, secondary, or tertiary cleavage effects. For example, primary positive cleavage effects were observed for Xaa-Pro and Asp-Xaa bond cleavage for mobile and nonmobile peptide ion categories, respectively. We also report specific pairwise interactions (e.g., Asn-Gly) that result in enhanced amide bond cleavages analogous to those observed in solution-phase chemistry. Peptides classified as nonmobile gave low or insignificant scores, below reported MS/MS score thresholds (cutoff filters), indicating that incorporation of the relative proton mobility scale classification would lead to improvements in current MS/MS scoring functions.

Summary: Hidden Markov models (HMMs) are probabilistic models that are well adapted to many tasks in bioinformatics, for example, for predicting the occurrence of specific motifs in biological sequences. MAMOT is a command-line program for Unix-like operating systems, including MacOS X, that we developed to allow scientists to apply HMMs more easily in their research. One can define the architecture and initial parameters of the model in a text file and then use MAMOT for parameter optimization on example data, decoding (like predicting motif occurrence in sequences) and the production of stochastic sequences generated according to the probabilistic model. Two examples for which models are provided are coiled-coil domains in protein sequences and protein binding sites in DNA. A wealth of useful features include the use of pseudocounts, state tying and fixing of selected parameters in learning, and the inclusion of prior probabilities in decoding. Availability: MAMOT is implemented in C++, and is distributed under the GNU General Public Licence (GPL). The software, documentation, and example model files can be found at http://bcf.isb-sib.ch/mamot Contact: Mauro.Delorenzi@isb-sib.ch

A paradigm shift is underway in the field of quantitative liquid chromatography-mass spectrometry (LC-MS) analysis thanks to the arrival of recent high-resolution mass spectrometers (HRMS). The capability of HRMS to perform sensitive and reliable quantifications of a large variety of analytes in HR-full scan mode is showing that it is now realistic to perform quantitative and qualitative analysis with the same instrument. Moreover, HR-full scan acquisition offers a global view of sample extracts and allows retrospective investigations as virtually all ionized compounds are detected with a high sensitivity. In time, the versatility of HRMS together with the increasing need for relative quantification of hundreds of endogenous metabolites should promote a shift from triple-quadrupole MS to HRMS. However, a current "pitfall" in quantitative LC-HRMS analysis is the lack of HRMS-specific guidance for validated quantitative analyses. Indeed, false positive and false negative HRMS detections are rare, albeit possible, if inadequate parameters are used. Here, we investigated two key parameters for the validation of LC-HRMS quantitative analyses: the mass accuracy (MA) and the mass-extraction-window (MEW) that is used to construct the extracted-ion-chromatograms. We propose MA-parameters, graphs, and equations to calculate rational MEW width for the validation of quantitative LC-HRMS methods. MA measurements were performed on four different LC-HRMS platforms. Experimentally determined MEW values ranged between 5.6 and 16.5 ppm and depended on the HRMS platform, its working environment, the calibration procedure, and the analyte considered. The proposed procedure provides a fit-for-purpose MEW determination and prevents false detections.

Type 2 diabetes mellitus (T2DM) is a major disease affecting nearly 280 million people worldwide. Whilst the pathophysiological mechanisms leading to disease are poorly understood, dysfunction of the insulin-producing pancreatic beta-cells is key event for disease development. Monitoring the gene expression profiles of pancreatic beta-cells under several genetic or chemical perturbations has shed light on genes and pathways involved in T2DM. The EuroDia database has been established to build a unique collection of gene expression measurements performed on beta-cells of three organisms, namely human, mouse and rat. The Gene Expression Data Analysis Interface (GEDAI) has been developed to support this database. The quality of each dataset is assessed by a series of quality control procedures to detect putative hybridization outliers. The system integrates a web interface to several standard analysis functions from R/Bioconductor to identify differentially expressed genes and pathways. It also allows the combination of multiple experiments performed on different array platforms of the same technology. The design of this system enables each user to rapidly design a custom analysis pipeline and thus produce their own list of genes and pathways. Raw and normalized data can be downloaded for each experiment. The flexible engine of this database (GEDAI) is currently used to handle gene expression data from several laboratory-run projects dealing with different organisms and platforms. Database URL: http://eurodia.vital-it.ch., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

BackgroundTheRUNX1transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia.ResultsHere we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in eitherRUNX1or its cofactor,CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygousRUNX1point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes.ConclusionThis work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.