Radioactivity as a driver of bacterial community composition in naturally radioactive mineral springs in the French Massif Central.

Some natural environments on Earth are characterised by high levels of radiation, including naturally radioelement enriched mineral springs in the French Massif Central. Therefore, naturally radioactive mineral springs are interesting ecosystems for understanding how bacterial populations in these springs have adapted to high levels of natural and chronic radioactivity over the very long term. The aim of this study was to analyse the bacterial communities of sediments from five naturally radioactive mineral springs in the French Massif Central, sampled in autumn 2019 and spring 2020, and to observe whether radionuclides, compared to other physicochemical parameters, are drivers of the bacterial community structuring in these extreme environments. Physicochemical measurements showed that two springs, Dourioux and Montagne had high radioelement concentrations/activities (uranium, thorium and radon). Analysis of the structure of the bacterial communities, by next generation sequencing based on 16S rRNA gene sequencing, showed that the presence of radionuclides in Dourioux and Montagne, did not lead to a reduction in bacterial diversity and richness compared to the other springs. However, Dourioux and Montagne were characterised by specific bacterial populations, whose presence correlates with the radioelement concentrations/activities measured in these springs. This suggests that radioelements could partly explain the structuring of bacterial communities in these springs. In addition, several of these operational taxonomic units (OTUs) specific to Dourioux and Montagne, mainly affiliated to Proteobacteria, Firmicutes, Acidobacteria, Actinobacteria, and Bacteroidetes, could be involved in the biogeochemistry of radionuclides through different mechanisms (biosorption, biomineralisation, bioaccumulation, and bioreduction), which would allow the development of other bacterial species sensitive to these metals/radioelements. In particular, the co-occurrence of sulphate and/or iron-reducing bacteria, capable of bioreducing uranium, with fermentative bacteria, releasing sources of organic carbons, reflects associations of bacteria with complementary functions that allow them to grow in this peculiar environment and maintain a high diversity in these extreme environments. This study has provided a better understanding of the structuring of bacterial communities exposed to ionising radiation for thousands of years in naturally radioactive environments.

Assessing the chronic effect of the bioavailable fractions of radionuclides and heavy metals on stream microbial communities: A case study at the Rophin mining site.

This study aimed to assess the potential impact of long-term chronic exposure (69 years) to naturally-occurring radionuclides (RNs) and heavy metals on microbial communities in sediment from a stream flowing through a watershed impacted by an ancient mining site (Rophin, France). Four sediment samples were collected along a radioactivity gradient (for 238U368 to 1710 Bq.Kg-1) characterized for the presence of the bioavailable fractions of radionuclides (226Ra, 210Po), and trace metal elements (Th, U, As, Pb, Cu, Zn, Fe). Results revealed that the available fraction of contaminants was significant although it varied considerably from one element to another (0 % for As and Th, 5-59 % for U). Nonetheless, microbial communities appeared significantly affected by such chronic exposure to (radio)toxicities. Several microbial functions carried by bacteria and related with carbon and nitrogen cycling have been impaired. The high values of fungal diversity and richness observed with increasing downstream contamination (H' = 4.4 and Chao1 = 863) suggest that the community had likely shifted toward a more adapted/tolerant one as evidenced, for example, by the presence of the species Thelephora sp. and Tomentella sp. The bacterial composition was also affected by the contaminants with enrichment in Myxococcales, Acidovorax or Nostocales at the most contaminated points. Changes in microbial composition and functional structure were directly related to radionuclide and heavy metal contaminations, but also to organic matter which also significantly affected, directly or indirectly, bacterial and fungal compositions. Although it was not possible to distinguish the specific effects of RNs from heavy metals on microbial communities, it is essential to continue studies considering the available fraction of elements, which is the only one able to interact with microorganisms.

Assessing radiation dosimetry for microorganisms in naturally radioactive mineral springs using GATE and Geant4-DNA Monte Carlo simulations.

Mineral springs in Massif Central, France can be characterized by higher levels of natural radioactivity in comparison to the background. The biota in these waters is constantly under radiation exposure mainly from the α-emitters of the natural decay chains, with 226Ra in sediments ranging from 21 Bq/g to 43 Bq/g and 222Rn activity concentrations in water up to 4600 Bq/L. This study couples for the first time micro- and nanodosimetric approaches to radioecology by combining GATE and Geant4-DNA to assess the dose rates and DNA damages to microorganisms living in these naturally radioactive ecosystems. It focuses on unicellular eukaryotic microalgae (diatoms) which display an exceptional abundance of teratological forms in the most radioactive mineral springs in Auvergne. Using spherical geometries for the microorganisms and based on γ-spectrometric analyses, we evaluate the impact of the external exposure to 1000 Bq/L 222Rn dissolved in the water and 30 Bq/g 226Ra in the sediments. Our results show that the external dose rates for diatoms are significant (9.7 µGy/h) and comparable to the threshold (10 µGy/h) for the protection of the ecosystems suggested by the literature. In a first attempt of simulating the radiation induced DNA damage on this species, the rate of DNA Double Strand Breaks per day is estimated to 1.11E-04. Our study confirms the significant mutational pressure from natural radioactivity to which microbial biodiversity has been exposed since Earth origin in hydrothermal springs.

ConnecSenS, a Versatile IoT Platform for Environment Monitoring: Bring Water to Cloud.

Climate change is having an increasingly rapid impact on ecosystems and particularly on the issue of water resources. The Internet of Things and communication technologies have now reached a level of maturity that allows sensors to be deployed more easily on sites to monitor them. The communicating node based on LoRaWAN technology presented in this article is open and allows the interfacing of numerous sensors for designing long-term environmental monitoring systems of isolated sites. The data integration in the cloud is ensured by a workflow driving the storage and indexing of data, allowing a simple and efficient use of the data for different users (scientists, administration, citizens) through specific dashboards and extractions. This article presents this infrastructure through environmental monitoring use cases related to water resources.

Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities.

Little is still known about the low dose effects of radiation on the microbial communities in the environment. Mineral springs are ecosystems than can be affected by natural radioactivity. These extreme environments are, therefore, observatories for studying the influence of chronic radioactivity on the natural biota. In these ecosystems we find diatoms, unicellular microalgae, playing an essential role in the food chain. The present study aimed to investigate, using DNA metabarcoding, the effect of natural radioactivity in two environmental compartments (i.e. spring sediments and water) on the genetic richness, diversity and structure of diatom communities in 16 mineral springs in the Massif Central, France. Diatom biofilms were collected during October 2019, and a 312 bp region of the chloroplast gene rbcL (coding for the Ribulose Bisphosphate Carboxylase) used as a barcode for taxonomic assignation. A total of 565 amplicon sequence variants (ASV) were found. The dominant ASV were associated with Navicula sanctamargaritae, Gedaniella sp., Planothidium frequentissimum, Navicula veneta, Diploneis vacillans, Amphora copulata, Pinnularia brebissonii, Halamphora coffeaeformis, Gomphonema saprophilum, and Nitzschia vitrea, but some of the ASVs could not be assigned at the species level. Pearson correlation failed to show a correlation between ASV' richness and radioactivity parameters. Non-parametric MANOVA analysis based on ASVs occurrence or abundances revealed that geographical location was the main factor influencing ASVs distribution. Interestingly, 238U was the second factor that explained diatom ASV structure. Among the ASVs in the mineral springs monitored, ASV associated with one of the genetic variants of Planothidium frequentissimum was well represented in the springs and with higher levels of 238U, suggesting its high tolerance to this particular radionuclide. This diatom species may therefore represent a bio-indicator of high natural levels of uranium.

Bedrock radioactivity influences the rate and spectrum of mutation.

All organisms on Earth are exposed to low doses of natural radioactivity but some habitats are more radioactive than others. Yet, documenting the influence of natural radioactivity on the evolution of biodiversity is challenging. Here, we addressed whether organisms living in naturally more radioactive habitats accumulate more mutations across generations using 14 species of waterlice living in subterranean habitats with contrasted levels of radioactivity. We found that the mitochondrial and nuclear mutation rates across a waterlouse species' genome increased on average by 60% and 30%, respectively, when radioactivity increased by a factor of three. We also found a positive correlation between the level of radioactivity and the probability of G to T (and complementary C to A) mutations, a hallmark of oxidative stress. We conclude that even low doses of natural bedrock radioactivity influence the mutation rate possibly through the accumulation of oxidative damage, in particular in the mitochondrial genome.

From Sensor to Cloud: An IoT Network of Radon Outdoor Probes to Monitor Active Volcanoes.

While radon in soil gases has been identified for decades as a potential precursor of volcanic eruptions, there has been a recent interest for monitoring radon in air on active volcanoes. We present here the first network of outdoor air radon sensors that was installed successfully on Mt. Etna volcano, Sicily, Italy in September 2019. Small radon sensors designed for workers and home dosimetry were tropicalized in order to be operated continuously in harsh volcanic conditions with an autonomy of several months. Two stations have been installed on the south flank of the volcano at ~3000 m of elevation. A private network has been deployed in order to transfer the measurements from the stations directly to a server located in France, using a low-power wide-area transmission technology from Internet of Things (IoT) called LoRaWAN. Data finally feed a data lake, allowing flexibility in data management and sharing. A first analysis of the radon datasets confirms previous observations, while adding temporal information never accessed before. The observed performances confirm IoT solutions are very adapted to active volcano monitoring in terms of range, autonomy, and data loss.

Analyses of pig genomes provide insight into porcine demography and evolution.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

Age should be considered in the decision making of prophylactic splenic angioembolization in nonoperative management of blunt splenic trauma: a study of 208 consecutive civilian trauma patients.

BACKGROUND: A strategy of prophylactic splenic angioembolization using observation failure risk (OFR) computed tomographic (CT) scan criteria has been proposed recently. The main aim of the present study was to evaluate the relevance of the criteria in terms of delayed splenic rupture in patients with blunt splenic injury. METHODS: All patients with blunt splenic injuries admitted consecutively between January 2005 and January 2010 to our institution were included. Clinical, CT scan, and angiographic data, initial management, and outcome were noted. Patients managed expectantly were classified according to OFR CT scan criteria (high OFR was defined by at least one of the following CT scan signs: blush, pseudoaneurysm, Organ Injury Scale [OIS] grade III with a large hemoperitoneum, and OIS grade IV or 5). Initial management success was especially studied. RESULTS: Among the 208 patients included, 161 (77%) were treated by observation (35 OIS grade I, 64 OIS grade II, 33 OIS grade III, 18 OIS grade IV, and 11 OIS grade V) and 129 (80%) were men, with a mean (SD) age of 36.1 (18.7) years and a mean (SD) Injury Severity Score of 20.8 (15.4). Forty-nine patients (30%) had high OFR CT scan criteria. Thirteen patients (8%) experienced observation failure. High OFR CT scan criteria (odds ratio, 11; 95% confidence interval, 2.5-47.5) and patients 50 years and older (odds ratio, 33.9; 95% confidence interval, 6.2-185.5) were independent factors related to observation failure. The positive predictive value of OFR CT scan criteria for observation failure was 18%, and the negative predictive value was 96%. The corresponding values were 67% and 90%, respectively, in patients 50 years and older and 3% and 99%, respectively, in patients younger than 50 years. CONCLUSION: OFR CT scan criteria lack specificity to predict observation failure, mainly in patients younger than 50 years. Age should be considered when identifying patients requiring prophylactic splenic angioembolization. LEVEL OF EVIDENCE: Diagnostic study, level III.

An outbreak of heterogeneous glycopeptide-intermediate Staphylococcus aureus related to a device source in an intensive care unit.

OBJECTIVE: The emergence of Staphylococcus aureus with reduced susceptibility to glycopeptides (glycopeptide-intermediate S. aureus [GISA] and heterogeneous GISA [h-GISA]) leads to intensive care unit (ICU) outbreaks that frequently result in ward closure. We investigated the role of hospital hygiene in the transmission and eradication of an h-GISA outbreak. DESIGN: The study is a description of an original environmental investigation around a series of 12 cases. SETTING AND PATIENTS: The outbreak occurred in a 20-bed polyvalent/trauma ICU in a 2,800-bed tertiary care university hospital in France. INTERVENTIONS: Specimens were obtained for surveillance and diagnostic cultures from all patients in the unit. Surface sampling was also performed. Geographic cohorting, contact isolation, emphasis on adherence to infection control practices, and environmental cleaning were implemented. RESULTS: Twelve patients with h-GISA infection (n = 5) or colonization (n = 7) were identified. The mean interval between admission and h-GISA detection was 23.6 days (range, 10-89 days), with a median of 16.5 days. Environmental investigation identified an unexpected reservoir, namely, SpO(2) sensors. The outbreak was controlled by a combination of measures, including eradication of this reservoir, avoiding total ward closure. CONCLUSIONS: Targeted surface sampling helps to secure the environment through active investigation of various reservoirs while maintaining normal activity on the ward. In our study, this method led to the detection of an unsuspected reservoir, the eradication of which helped control the h-GISA epidemic. Further applications of this original investigative procedure should allow confirmation of its relevance and efficiency.

What are the specific computed tomography scan criteria that can predict or exclude the need for renal angioembolization after high-grade renal trauma in a conservative management strategy?

BACKGROUND: The indications of renal angioembolization for patients with high-grade renal trauma (HGRT) are based on angiographic criteria to reduce the failure rate of conservative management (CM). There is no consensus to predict or exclude an indication of renal angioembolization with a computed tomography (CT) scan. The aim of this study was to evaluate CT-specific criteria to predict or exclude the need for renal embolization. METHODS: All traumatized patients admitted with renal injury were considered between 2005 and 2009. We included all patients who had an HGRT (classified by American Association for the Surgery of Trauma Organ Injury Scale grade≥3) treated by CM. We collected the demographic, CT, angiographic, management, and outcome data for these patients. CT criteria were retrospectively studied to define their predictive values for renal embolization. RESULTS: Among 101 patients with renal injury, 58 were HGRT, and 53 of them were treated by CM. Ten patients (19%) received renal embolization because of an ongoing renal hemorrhage. There was no significant difference for urologic interventions (2 [20%] vs. 7 [16%]), CM failure rate (1 [10%] vs. 2 [5%]), and during hospital stay between these patients and those who did not received embolization. None of the CT criteria had a negative predictive value for renal embolization to 100%, only the absence of intravascular contrast extravasation associated with a perirenal hematoma rim distance<25 mm excludes an indication for embolization. CONCLUSIONS: In patients with HGRT who had bleeding, a strategy of targeted angiography can be realized safely in using specific CT scan criteria that can predict with high accuracy and exclude the need for embolization, without reducing the success rate of CM.

Antibody repertoire development in fetal and neonatal piglets. XI. The relationship of variable heavy chain gene usage and the genomic organization of the variable heavy chain locus.

In this study, we have mapped the 3' H chain V region (V(H)) genes and those in the H chain diversity, H chain joining, and 5' portion of the H chain constant locus. We show that swine possess only two functional H chain diversity segments and only one functional H chain joining segment. These data help to explain more than a decade of observations on the preimmune repertoire of this species and reveal the vulnerability of swine to natural or designed mutational events. The results are consistent with earlier studies on the region containing Enh, Cmu, and Cdelta while revealing that the ancestral IgG3 is the most 5' Cgamma gene. We also observed a recent duplication ( approximately 1.6 million years ago) in the V(H) locus that contains six of the seven V(H) genes that comprise 75% of the preimmune repertoire. Because there are no known transfers of immune regulators or Ags that cross the placenta as in mice and humans, fetal V(H) usage must be intrinsically regulated. Therefore, we quantified V(H) usage in fetal piglets and demonstrated that usage is independent of the position of V(H) genes in the genome; the most 3' functional V(H) gene (IGHV2) is rarely used, whereas certain upstream genes (IGHV14 and IGHV15) are predominately used early in fetal liver but seldom thereafter. Similar to previous studies, three V(H) genes account for 40% of the repertoire and six for approximately 70%. This limited combinatorial diversity of the porcine V(H) repertoire further emphasizes the dependence on CDR3 diversity for generating the preimmune Ab repertoire of this species.

A new index derived from the cerebrovascular pressure transmission and correlated with consciousness recovery in severely head-injured intensive care patients.

BACKGROUND: In patients with serious head trauma, a moderate (20-25 mm Hg) mean level of intracranial pressure (ICP) may fail to distinguish patients with a real deteriorated intracranial status from those who are stable or improving. Because of these limitations, we analyzed the ICP curve in search of other relevant information regarding cerebrovascular pressure transmission. We looked for parameters with physiological meaning extracted from spectral analysis of cerebrovascular pressure transmission and correlated with consciousness recovery in patients with severe head injuries. METHODS: A prospective cohort study was conducted in an intensive care unit of the University Hospital, Montpellier, France, from December 2003 to December 2005. Thirty consecutive patients admitted for severe head trauma were subjected to sedatives, mechanical ventilation, and intraparenchymatous recording of ICP and were evaluated with Glasgow Outcome Scale score. Simultaneous 60-s recordings of ICP and arterial blood pressure (BP) signals, beginning as soon as possible after head trauma, were repeated until death or clinical stabilization, every 15 min, with physicians blinded to the patients' data. Spectra of ICP and BP waveforms were computed with Fourier transform. Amplitudes of cardiac and respiratory harmonics were analyzed. Cardiac (or respiratory) gain was defined as the ratio of amplitudes of cardiac (or respiratory) harmonic of ICP to BP signals and referred to as Gc and Gr, respectively. RESULTS: Twenty of the 30 enrolled patients recovered consciousness (Glasgow Outcome Scale score = 3, 4, or 5). Gr/Gc averaged over the whole recording period performed better in discriminating consciousness recovery (area under receiver operating characteristic [ROC] curve: 0.98; 95% confidence interval [CI]: 0.91-1) than ICP (0.76; 95% CI: 0.54-0.97), cerebral perfusion pressure (0.75; 95% CI: 0.53-0.97) and Gc (0.77; 95% CI: 0.57-0.99) (P < 0.001 for each comparison). When considering the recording period 30 h posttrauma (hpt), 162 hpt, a value of Gr/Gc > or =4 was always associated with consciousness recovery, and the relative risk was equal to 9 (95% CI: 1.42-57.12). CONCLUSIONS: Gr/Gc, which characterizes the cerebrovascular transmission, better discriminates bad evolution than high values of ICP or low values of cerebral perfusion pressure in patients with severe head trauma. A reduction in Gr/Gc ratio might be an early alarm signaling worsening intracranial hemodynamic conditions.

Difference in number of loci of swine leukocyte antigen classical class I genes among haplotypes.

The structure of the entire genomic region of swine leukocyte antigen (SLA)-the porcine major histocompatibility complex--was recently elucidated in a particular haplotype named Hp-1.0 (H01). However, it has been suggested that there are differences in the number of loci of SLA genes, particularly classical class I genes, among haplotypes. To clarify the between-haplotype copy number variance in genes of the SLA region, we sequenced the genomic region carrying SLA classical class I genes on two different haplotypes, revealing increments of up to six in the number of classical class I genes in a single haplotype. All of the SLA-1(-like) (SLA-1 and newly designated SLA-12) and SLA-3 genes detected in the haplotypes thus analyzed were transcribed in the individual. The process by which duplication of SLA classical class I genes was likely to have occurred was interpreted from an analysis of repetitive sequences adjacent to the duplicated class I genes.

Quantification of urinary F2-isoprostanes with 4(RS)-F4t-neuroprostane as an internal standard using gas chromatography-mass spectrometry Application to polytraumatized patients.

Isoprostanes are a family of prostaglandin isomers produced from oxidation of polyunsaturated fatty acids through a non-enzymatic free radical-catalyzed mechanism. Quantification of F(2)-isoprostanes (F(2)-IsoPs) provides a good index of oxidative stress and allows non-invasive assessment of lipid peroxidation in vivo. Since "interferences peaks" at m/z 573 co-elute with d(4)-15-F(2t)-IsoP preferentially used, we propose a new GC-NICI-MS approach to quantify urinary F(2)-IsoPs by using 4(RS)-F(4t)-neuroprostane as the internal standard. This method was applied to quantify urinary F(2)-IsoPs excretion in healthy volunteers and polytraumatized patients. Our results showed a significant increase (p<0.0001) in urinary F(2)-IsoPs in polytraumatized patients compared with healthy volunteers (4.73+/-2.75 ng/mg vs. 0.811+/-0.359 ng/mg creatinine).

Early tracheotomy versus prolonged endotracheal intubation in unselected severely ill ICU patients.

OBJECTIVE: Although several advantages are attributed to tracheotomy in ICU patients requiring mechanical ventilation (MV), true benefits and the optimal timing of tracheotomy remain controversial. In this study, we compared early tracheotomy (ET) with prolonged intubation (PI) in severely ill patients requiring prolonged MV. DESIGN: Prospective, randomized study. SETTING: Twenty-five medical and surgical ICUs in France. PATIENTS: Patients expected to require MV > 7 days. MEASUREMENTS AND RESULTS: Patients were randomised to either (open or percutaneous) ET within 4 days or PI. The primary end-point was 28-day mortality. Secondary end-points were: the incidence of ICU-acquired pneumonia, number of d1-d28 ventilator-free days, time spent in the ICU, 60-day mortality, number of septic episodes, amount of sedation, comfort and laryngeal and tracheal complications. A sample size of 470 patients was considered necessary to obtain a reduction from 45 to 32% in 28-day mortality. After 30 months, 123 patients had been included (ET = 61, PI = 62) in 25 centres and the study was prematurely closed. All group characteristics were similar upon admission to ICU. No difference was found between the two groups for any of the primary or secondary end-points. Greater comfort was the sole benefit afforded by tracheotomy after subjective self-assessment by patients. CONCLUSIONS: The trial did not demonstrate any major benefit of tracheotomy in a general population of ICU patients, as suggested in a previous meta-analysis, but was underpowered to draw any firm conclusions. The potential advantage of ET may be restricted to selected groups of patients.

Microsatellite diversity and crossover regions within homozygous and heterozygous SLA haplotypes of different pig breeds.

Our aim was to investigate microsatellite (MS) diversity and find crossover regions at 42 polymorphic MS loci in the swine leukocyte antigen (SLA) genomic region of 72 pigs with different well-defined homozygous and heterozygous SLA haplotypes. We analyzed the genetic polymorphisms of 42 MS markers in 23 SLA homozygous-heterozygous, common pig breeds with 12 SLA serological haplotypes and 49 National Institutes of Health (NIH) and Clawn homozygous-heterozygous miniature pigs with nine SLA serological or genotyped haplotypes including four recombinant haplotypes. In comparing the same and different haplotypes, both haplospecific patterns and allelic variations were observed at the MS loci. Some of the shared haplotype blocks extended over 2 Mb suggesting the existence of strong linkage disequilibrium (LD) in the entire SLA region. Crossover regions were easily defined by the MS markers within the class I and/or III region in the NIH and Clawn recombinant haplotypes. The present haplotype comparison shows that our set of MS markers provides a fast and cost-efficient alternative, or complementary, method to the serological or sequence-based determination of the SLA alleles for the characterization of SLA haplotypes and/or the crossover regions between different haplotypes.

Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection.

BACKGROUND: Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry. RESULTS: An increase in the number of differentially expressed cellular genes and PrV genes especially from 4 h post-infection (pi) was observed concomitantly with the onset of viral progeny while no early global cellular shutoff was recorded. Many cellular genes were down-regulated from 4 h pi and their number increased until 12 h pi. UL41 transcripts encoding the virion host shutoff protein were first detected as differentially expressed at 8 h pi. The viral gene UL49.5 encoding a TAP inhibitor protein was differentially expressed as soon as 2 h pi, indicating that viral evasion via TAP inhibition may start earlier than the cellular gene shutoff. We found that many biological processes are altered during PrV infection. Indeed, several genes involved in the SLA class I antigenic presentation pathway (SLA-Ia, TAP1, TAP2, PSMB8 and PSMB9), were down-regulated, thus contributing to viral immune escape from this pathway and other genes involved in apoptosis, nucleic acid metabolism, cytoskeleton signaling as well as interferon-mediated antiviral response were also modulated during PrV infection. CONCLUSION: Our results show that the gene expression of both PrV and porcine cells can be analyzed simultaneously with microarrays, providing a chronology of PrV gene transcription, which has never been described before, and a global picture of transcription with a direct temporal link between viral and host gene expression.

A high utility integrated map of the pig genome.

BACKGROUND: The domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction. RESULTS: Here we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found. CONCLUSION: The map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.

Regulation of porcine classical and nonclassical MHC class I expression.

Major histocompatibility complex (MHC) class I molecules comprise a family of polymorphic cell surface receptors consisting of classical 1 a molecules that present antigenic peptides and nonclassical 1 b molecules. Gene expression for human classical and nonclassical MHC class I molecules has been shown to be differentially regulated by interferon, with variation in the nucleotide sequence of promoter regions, resulting in differences in interferon inducibility and basal levels of gene transcription. In this study on porcine classical and nonclassical swine leukocyte Ag (SLA) class I molecules, we show alignments of putative regulatory elements in the promoters of the three functional classical class I genes, SLA-1, SLA-2, and SLA-3; two nonclassical 1 b genes, SLA-6 and SLA-7; and a MIC-2 gene. Promoter elements were cloned upstream from a luciferase reporter gene, and the basal and inducible activities of each were characterized by expression in Max cells, an immortalized pig cell line that responds to interferon and tumor necrosis factor alpha (TNF-alpha). All three classical class I but not nonclassical promoters responded to interferon. This was confirmed by the transactivation of SLA-1, but not SLA-7, after the co expression with interferon regulatory factors (IRFs), IRF-1, IRF-2, IRF-3, IRF-7, and IRF-9. Classical class I genes were activated by cotransfection with nuclear factor kappa B (NF-kappaB) p65 and by treatment of cells with TNF-alpha, although, unlike human promoter there was no synergistic effect with interferon. The greatest effect on classical class I promoters was coexpression with the class II transactivator (CIITA), important for constitutive transactivation. These results determine the differential regulation of porcine classical and nonclassical MHC class I and reflects their importance in antigen presentation during infection.