Rho-dependent transcriptional switches regulate the bacterial response to cold shock.

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program. We identify Rut sites in 5'-untranslated regions of key CSR genes/operons (cspA, cspB, cspG, and nsrR-rnr-yjfHI) that trigger premature RDTT at 37°C but not at 15°C. High concentrations of RNA chaperone CspA or nucleotide changes in the cspA mRNA leader reduce RDTT efficiency, revealing how RNA restructuring directs Rho to activate CSR genes during the cold shock and to silence them during cold acclimation. These findings establish a paradigm for how RNA thermosensors can modulate gene expression.

A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches.

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA-protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme-Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)-that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (Rut) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating in vitro and in vivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.

Characterization of Influenza D Virus Reassortant Strain in Swine from Mixed Pig and Beef Farm, France.

Influenza D virus was isolated from pigs on a mixed pig and beef farm in France. Investigation suggested bull-to-pig transmission and spread among pigs. The swine influenza D virus recovered was a reassortant of D/660 and D/OK lineages. Reported mutations in the receptor binding site might be related to swine host adaptation.

An outbreak of botulism on a pig farm due to the newly described Clostridium botulinum type C.

This study reports a botulism outbreak on a pig farm. Clostridium botulinum type C was detected using PCR. The gene encoding the toxin corresponds to a novel type C neurotoxin recently described in a human botulism outbreak, raising the question of its prevalence in pigs and the related risks to humans.

NusG prevents transcriptional invasion of H-NS-silenced genes.

Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho's primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface.

A multivariate prediction model for Rho-dependent termination of transcription.

Bacterial transcription termination proceeds via two main mechanisms triggered either by simple, well-conserved (intrinsic) nucleic acid motifs or by the motor protein Rho. Although bacterial genomes can harbor hundreds of termination signals of either type, only intrinsic terminators are reliably predicted. Computational tools to detect the more complex and diversiform Rho-dependent terminators are lacking. To tackle this issue, we devised a prediction method based on Orthogonal Projections to Latent Structures Discriminant Analysis [OPLS-DA] of a large set of in vitro termination data. Using previously uncharacterized genomic sequences for biochemical evaluation and OPLS-DA, we identified new Rho-dependent signals and quantitative sequence descriptors with significant predictive value. Most relevant descriptors specify features of transcript C>G skewness, secondary structure, and richness in regularly-spaced 5'CC/UC dinucleotides that are consistent with known principles for Rho-RNA interaction. Descriptors collectively warrant OPLS-DA predictions of Rho-dependent termination with a ∼85% success rate. Scanning of the Escherichia coli genome with the OPLS-DA model identifies significantly more termination-competent regions than anticipated from transcriptomics and predicts that regions intrinsically refractory to Rho are primarily located in open reading frames. Altogether, this work delineates features important for Rho activity and describes the first method able to predict Rho-dependent terminators in bacterial genomes.

Bayesian estimation of the sensitivity and specificity of coprological and serological diagnostic tests for the detection of Ascaris suum infection on pig farms.

Coprological and serological diagnostic tests were compared to define the status of a pig farm with regard to Ascaris suum. On each of the 100 farms in France visited for the study, 10 blood samples were taken from pigs at the end of fattening (at least 22 weeks old) and 20 to 30 faecal samples were taken, depending on the category of animals present on the farm (10 sows, 10 piglets aged 10 to 12 weeks and 10 pigs at the end of fattening, aged at least 22 weeks). A SERASCA® ELISA test (Laboratory of Parasitology, Ghent University) was performed on each blood sample (cut-off 0.5) and a coprological analysis on each faecal sample. A Bayesian approach was used to estimate the sensitivity and specificity of the coprological and serological tests. A farm was considered positive if at least one A. suum egg was observed in the faecal samples. With regard to the serological test, various hypotheses were tested in order to define the number of seropositive animals required to consider a farm positive for A. suum. The coprological test has very good specificity in the search for A. suum, whether 20 or 30 samples are taken per farm. However, even with an increase in the number of samples, the sensitivity of this diagnostic approach is very low (less than 30%). On the other hand, the serological diagnostic method, which consists of taking blood samples from 10 animals at the end of fattening, has good sensitivity and seems better suited to defining the status of a farm with regard to A. suum, provided that a farm is considered seropositive only if two out of 10 samples are positive.

Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events.

Concomitant infections by different influenza A virus subtypes within pig farms increase the risk of new reassortant virus emergence. The aims of this study were to characterize the epidemiology of recurrent swine influenza virus infections and identify their main determinants. A follow-up study was carried out in 3 selected farms known to be affected by repeated influenza infections. Three batches of pigs were followed within each farm from birth to slaughter through a representative sample of 40 piglets per batch. Piglets were monitored individually on a monthly basis for serology and clinical parameters. When a flu outbreak occurred, daily virological and clinical investigations were carried out for two weeks. Influenza outbreaks, confirmed by influenza A virus detection, were reported at least once in each batch. These outbreaks occurred at a constant age within farms and were correlated with an increased frequency of sneezing and coughing fits. H1N1 and H1N2 viruses from European enzootic subtypes and reassortants between viruses from these lineages were consecutively and sometimes simultaneously identified depending on the batch, suggesting virus co-circulations at the farm, batch and sometimes individual levels. The estimated reproduction ratio R of influenza outbreaks ranged between 2.5 [1.9-2.9] and 6.9 [4.1-10.5] according to the age at infection-time and serological status of infected piglets. Duration of shedding was influenced by the age at infection time, the serological status of the dam and mingling practices. An impaired humoral response was identified in piglets infected at a time when they still presented maternally-derived antibodies.

Do Rubber Floor Mats Prevent Lameness in Gestating Sows Housed in Large Groups? A Field Experiment on Three Commercial Farms in France.

Lameness and foot disorders are major health and welfare issues in intensive swine production systems. They are exacerbated when sows are housed in large groups on slatted concrete floors during gestation. Our study aimed to assess the effect of rubber mats in the lying area of the gestation pen on lameness and leg health in gestating sows housed in large pens in commercial conditions. The study was conducted on three commercial farms over two successive gestations. A total of 582 Large White × Landrace sows, housed in 10 static groups, were enrolled: 5 groups in pens with rubber mats and 5 groups on slatted concrete floors. Lameness, bursitis, leg injuries, claw growth defects and claw lesions were measured at the beginning, middle and end of each gestation period. The rubber mats decreased the risk of suffering from bursitis, but had no effect on the risk of lameness, leg injuries, claw growth defects or claw lesions. Sows housed on rubber mats were heavily soiled compared with those on slatted concrete floors because the mats were not perforated for slurry evacuation. Locomotion disorders and foot lesions remained prevalent despite the rubber mats in the lying area of the gestation pens, but adding rubber mats in service rooms and farrowing crates may produce better results.

Natural viral co-infections in pig herds affect hepatitis E virus (HEV) infection dynamics and increase the risk of contaminated livers at slaughter.

Hepatitis E virus (HEV) is a zoonotic pathogen, in particular genotype 3 HEV is mainly transmitted to humans through the consumption of contaminated pork products. This study aimed at describing HEV infection patterns in pig farms and at assessing the impact of immunomodulating co-infections namely Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus Type 2 (PCV2), as well as other individual factors such as piglets' immunity and litters' characteristics on HEV dynamics. A longitudinal follow-up was conducted in three farrow-to-finish farms known to be HEV infected. Overall, 360 piglets were individually monitored from birth to slaughter with regular blood and faecal sampling as well as blood and liver samples collected at slaughterhouse. Virological and serological analyses were performed to detect HEV, PCV2 and PRRSV genome and antibodies. The links between 12 explanatory variables and four outcomes describing HEV dynamics were assessed using cox-proportional hazard models and logistic regression. HEV infection dynamics was found highly variable between farms and in a lower magnitude between batches. HEV positive livers were more likely related to short time-intervals between HEV infection and slaughter time (<40 days, OR = 4.1 [3.7-4.5]). In addition to an influence of piglets' sex and sows' parity, the sequence of co-infections was strongly associated with different HEV dynamics: a PRRSV or PCV2/PRRSV pre- or co-infection was associated with a higher age at HEV shedding (Hazard Ratio = 0.3 [0.2-0.5]), as well as a higher age at HEV seroconversion (HR = 0.5 [0.3-0.9] and HR = 0.4 [0.2-0.7] respectively). A PCV2/PRRSV pre- or co-infection was associated with a longer duration of shedding (HR = 0.5 [0.3-0.8]). Consequently, a PRRSV or PCV2/PRRSV pre- or co-infection was strongly associated with a higher risk of having positive livers at slaughter (OR = 4.1 [1.9-8.9] and OR = 6.5 [3.2-13.2] respectively). In conclusion, co-infections with immunomodulating viruses were found to affect HEV dynamics in the farrow-to-finish pig farms that were followed in this study.

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.

While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data.

The Human Anatomic Gene Expression Library (H-ANGEL), the H-Inv integrative display of human gene expression across disparate technologies and platforms.

The Human Anatomic Gene Expression Library (H-ANGEL) is a resource for information concerning the anatomical distribution and expression of human gene transcripts. The tool contains protein expression data from multiple platforms that has been associated with both manually annotated full-length cDNAs from H-InvDB and RefSeq sequences. Of the H-Inv predicted genes, 18 897 have associated expression data generated by at least one platform. H-ANGEL utilizes categorized mRNA expression data from both publicly available and proprietary sources. It incorporates data generated by three types of methods from seven different platforms. The data are provided to the user in the form of a web-based viewer with numerous query options. H-ANGEL is updated with each new release of cDNA and genome sequence build. In future editions, we will incorporate the capability for expression data updates from existing and new platforms. H-ANGEL is accessible at http://www.jbirc.aist.go.jp/hinv/h-angel/.

Individual and pen-based oral fluid sampling: A welfare-friendly sampling method for group-housed gestating sows.

The aims of this study were to assess the feasibility of individual and pen-based oral fluid sampling (OFS) in 35 pig herds with group-housed sows, compare these methods to blood sampling, and assess the factors influencing the success of sampling. Individual samples were collected from at least 30 sows per herd. Pen-based OFS was performed using devices placed in at least three pens for 45min. Information related to the farm, the sows, and their living conditions were collected. Factors significantly associated with the duration of sampling and the chewing behaviour of sows were identified by logistic regression. Individual OFS took 2min 42s on average; the type of floor, swab size, and operator were associated with a sampling time >2min. Pen-based OFS was obtained from 112 devices (62.2%). The type of floor, parity, pen-level activity, and type of feeding were associated with chewing behaviour. Pen activity was associated with the latency to interact with the device. The type of floor, gestation stage, parity, group size, and latency to interact with the device were associated with a chewing time >10min. After 15, 30 and 45min of pen-based OFS, 48%, 60% and 65% of the sows were lying down, respectively. The time spent after the beginning of sampling, genetic type, and time elapsed since the last meal were associated with 50% of the sows lying down at one time point. The mean time to blood sample the sows was 1min 16s and 2min 52s if the number of operators required was considered in the sampling time estimation. The genetic type, parity, and type of floor were significantly associated with a sampling time higher than 1min 30s. This study shows that individual OFS is easy to perform in group-housed sows by a single operator, even though straw-bedded animals take longer to sample than animals housed on slatted floors, and suggests some guidelines to optimise pen-based OFS success.

Differential repair of the two major UV-induced photolesions in trichothiodystrophy fibroblasts.

Defects in nucleotide excision repair have been shown to be associated with the photosensitive form of the disorder trichothiodystrophy (TTD). Most repair-deficient TTD patients are mutated in the XPD gene, a subunit of the transcription factor TFIIH. Knowledge of the kinetics and efficiency of repair of the two major UV-induced photolesions in TTD is critical to understand the role of unrepaired lesions in the process of carcinogenesis and explain the absence of enhanced skin cancer incidence in TTD patients contrarily to the xeroderma pigmentosum D patients. In this study, we used different approaches to quantify repair of UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) at the gene and the genome overall level. In cells of two TTD patients, repair of CPD and 6-4PP was reduced compared with normal human cells, but the reduction was more severe in confluent cells than in exponentially growing cells. Moreover, the impairment of repair was more drastic for CPD than 6-4PP. Most notably, exponentially growing TTD cells displayed complete repair 6-4PP over a broad dose range, albeit at a reduced rate compared with normal cells. Strand-specific analysis of CPD repair in a transcriptional active gene revealed that TTD cells were capable to perform transcription-coupled repair. Taken together, the data suggest that efficient repair of 6-4PP in dividing TTD cells in concert with transcription-coupled repair might account for the absence of increased skin carcinogenesis in TTD patients.

Modifications in the myogenic program induced by in vivo and in vitro aging.

In this study, we have used high density cDNA arrays to assess age-related changes in gene expression in the myogenic program of human satellite cells and to elucidate modifications in differentiation capacity that could occur throughout in vitro cellular aging. We have screened a collection of 2016 clones from a human skeletal muscle 3'-end cDNA library in order to investigate variations in the myogenic program of myotubes formed by the differentiation of myoblasts of individuals with different ages (5 days old, 52 years old and 79 years old) and induced to differentiate at different stages of their lifespan (early proliferation, presenescence and senescence). Although our analysis has not been able to underline specific changes in the expression of genes encoding proteins involved in muscle structure and/or function, we have demonstrated an age-related induction of genes involved in stress response and a down-regulation of genes involved both in mitochondrial electron transport/ATP synthase and in glycolysis/TCA cycle. From this global approach of post-mitotic cell aging, we have identified 2 potential new markers of presenescence for human myotubes, both strongly linked to carbohydrate metabolism, which could be useful in developing therapeutic strategies.

Matching gene expression with hypometabolism after cerebral ischemia in the nonhuman primate.

To correlate brain metabolic status with the molecular events during cerebral ischemia, a cDNA array was performed after positron emission tomography scanning in a model of focal cerebral ischemia in baboons. Cluster analysis for the expression of 74 genes allowed the identification of 4 groups of genes. In each of the distinct groups, the authors observed a marked inflection in the pattern of gene expression when the CMRo was reduced by 48% to 66%. These patterns of coordinated modifications in gene expression could define molecular checkpoints for the development of an ischemic infarct and a molecular definition of the penumbra.

Gene expression profiling of human satellite cells during muscular aging using cDNA arrays.

It is well established that biological aging is associated with functional deficits at the cellular, tissue, organ and system levels, but the molecular mechanisms that control lifespan and age-related phenotypes are still not well understood. In order to investigate the molecular mechanisms underlying myoblast aging, we have used quantitative hybridization of a cDNA array of 2016 clones from a human skeletal muscle 3'-end cDNA library to monitor gene expression patterns of myoblasts of individuals with different ages (5 days old, 52 years old and 79 years old) and at different stages of proliferation (early, presenescent and senescent). We have shown that expression profiles in satellite cells vary with donor age, with an up-regulation of genes involved in muscle structure, muscle differentiation and in metabolism in the newborn, and a down-regulation of genes involved in protein renewal in adults. We have also observed that myoblasts isolated from subjects of different ages have typical expression profiles at the beginning of their proliferative lifespan. However, this phenomenon progressively disappears as the cells approach senescence. In addition, even though some of the modifications are similar to those observed in other cell types, we have observed that many changes in gene expression are characteristic of the myoblasts, confirming the hypothesis that the program of replicative senescence is specific for each cell type. Finally, we have identified four potential new markers of presenescence for human myoblasts, which could be useful in developing therapeutic strategies.

M19 modulates skeletal muscle differentiation and insulin secretion in pancreatic ß-cells through modulation of respiratory chain activity.

Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic ß-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic ß-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.

A functional and regulatory network associated with PIP expression in human breast cancer.

BACKGROUND: The PIP (prolactin-inducible protein) gene has been shown to be expressed in breast cancers, with contradictory results concerning its implication. As both the physiological role and the molecular pathways in which PIP is involved are poorly understood, we conducted combined gene expression profiling and network analysis studies on selected breast cancer cell lines presenting distinct PIP expression levels and hormonal receptor status, to explore the functional and regulatory network of PIP co-modulated genes. PRINCIPAL FINDINGS: Microarray analysis allowed identification of genes co-modulated with PIP independently of modulations resulting from hormonal treatment or cell line heterogeneity. Relevant clusters of genes that can discriminate between [PIP+] and [PIP-] cells were identified. Functional and regulatory network analyses based on a knowledge database revealed a master network of PIP co-modulated genes, including many interconnecting oncogenes and tumor suppressor genes, half of which were detected as differentially expressed through high-precision measurements. The network identified appears associated with an inhibition of proliferation coupled with an increase of apoptosis and an enhancement of cell adhesion in breast cancer cell lines, and contains many genes with a STAT5 regulatory motif in their promoters. CONCLUSIONS: Our global exploratory approach identified biological pathways modulated along with PIP expression, providing further support for its good prognostic value of disease-free survival in breast cancer. Moreover, our data pointed to the importance of a regulatory subnetwork associated with PIP expression in which STAT5 appears as a potential transcriptional regulator.