Draft Genome Sequence of the Murine Bacterial Isolate Lactobacillus murinus EF-1.

Screening for lysogenic lactobacilli in rat fecal samples has identified Lactobacillus murinus EF-1. Whole-genome sequencing revealed a 2.30-Mb draft genome with 39.6% G+C content and 2,196 open reading frames. PHAST analysis identified three intact prophages of 26.1 kb, 25.4 kb, and 49.6 kb in size.

Structural and functional annotation of the porcine immunome.

BACKGROUND: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. RESULTS: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. CONCLUSIONS: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

A comprehensive analysis of the effects of the deaminase AID on the transcriptome and methylome of activated B cells.

Beyond its well-characterized functions in antibody diversification, the cytidine deaminase AID can catalyze off-target DNA damage and has been hypothesized to edit RNA and mediate DNA demethylation. To comprehensively examine the effects of AID on the transcriptome and the pattern of DNA methylation ('methylome'), we analyzed AID-deficient (Aicda(-/-)), wild-type and AID-overexpressing activated B cells by high-throughput RNA sequencing (RNA-Seq) and reduced-representation bisulfite sequencing (RRBS). These analyses confirmed the known role of AID in immunoglobulin isotype switching and also demonstrated few other effects of AID on gene expression. Additionally, we detected no evidence of AID-dependent editing of mRNA or microRNA. Finally, the RRBS data did not support the proposed role for AID in regulating DNA methylation. Thus, despite evidence of its additional activities in other systems, antibody diversification seems to be the sole physiological function of AID in activated B cells.

Prediction of altered 3'- UTR miRNA-binding sites from RNA-Seq data: the swine leukocyte antigen complex (SLA) as a model region.

THE SLA (swine leukocyte antigen, MHC: SLA) genes are the most important determinants of immune, infectious disease and vaccine response in pigs; several genetic associations with immunity and swine production traits have been reported. However, most of the current knowledge on SLA is limited to gene coding regions. MicroRNAs (miRNAs) are small molecules that post-transcriptionally regulate the expression of a large number of protein-coding genes in metazoans, and are suggested to play important roles in fine-tuning immune mechanisms and disease responses. Polymorphisms in either miRNAs or their gene targets may have a significant impact on gene expression by abolishing, weakening or creating miRNA target sites, possibly leading to phenotypic variation. We explored the impact of variants in the 3'-UTR miRNA target sites of genes within the whole SLA region. The combined predictions by TargetScan, PACMIT and TargetSpy, based on different biological parameters, empowered the identification of miRNA target sites and the discovery of polymorphic miRNA target sites (poly-miRTSs). Predictions for three SLA genes characterized by a different range of sequence variation provided proof of principle for the analysis of poly-miRTSs from a total of 144 M RNA-Seq reads collected from different porcine tissues. Twenty-four novel SNPs were predicted to affect miRNA-binding sites in 19 genes of the SLA region. Seven of these genes (SLA-1, SLA-6, SLA-DQA, SLA-DQB1, SLA-DOA, SLA-DOB and TAP1) are linked to antigen processing and presentation functions, which is reminiscent of associations with disease traits reported for altered miRNA binding to MHC genes in humans. An inverse correlation in expression levels was demonstrated between miRNAs and co-expressed SLA targets by exploiting a published dataset (RNA-Seq and small RNA-Seq) of three porcine tissues. Our results support the resource value of RNA-Seq collections to identify SNPs that may lead to altered miRNA regulation patterns.

Probing genetic control of swine responses to PRRSV infection: current progress of the PRRS host genetics consortium.

BACKGROUND: Understanding the role of host genetics in resistance to porcine reproductive and respiratory syndrome virus (PRRSV) infection, and the effects of PRRS on pig health and related growth, are goals of the PRRS Host Genetics Consortium (PHGC). METHODS: The project uses a nursery pig model to assess pig resistance/susceptibility to primary PRRSV infection. To date, 6 groups of 200 crossbred pigs from high health farms were donated by commercial sources. After acclimation, the pigs were infected with PRRSV in a biosecure facility and followed for 42 days post infection (dpi). Blood samples were collected at 0, 4, 7, 10, 14, 21, 28, 35 and 42 dpi for serum and whole blood RNA gene expression analyses; weekly weights were recorded for growth traits. All data have been entered into the PHGC relational database. Genomic DNAs from all PHGC1-6 pigs were prepared and genotyped with the Porcine SNP60 SNPchip. RESULTS: Results have affirmed that all challenged pigs become PRRSV infected with peak viremia being observed between 4-21 dpi. Multivariate statistical analyses of viral load and weight data have identified PHGC pigs in different virus/weight categories. Sera are now being compared for factors involved in recovery from infection, including speed of response and levels of immune cytokines. Genome-wide association studies (GWAS) are underway to identify genes and chromosomal locations that identify PRRS resistant/susceptible pigs and pigs able to maintain growth while infected with PRRSV. CONCLUSIONS: Overall, the PHGC project will enable researchers to discover and verify important genotypes and phenotypes that predict resistance/susceptibility to PRRSV infection. The availability of PHGC samples provides a unique opportunity to continue to develop deeper phenotypes on every PRRSV infected pig.

Cytidine deaminases: AIDing DNA demethylation?

The presence of 5-methylcytosine (5-mC) in DNA is a vital epigenetic mark in vertebrates. While the enzymes responsible for methylating DNA in vertebrates have been identified, the means by which this mark can be removed are still unclear. Recently, it has been shown that activation-induced cytidine deaminase (AID) contributes to the demethylation of DNA in certain systems. This enzyme has been intensely studied in its role as a key driver of antibody diversification in B cells, but recent observations from early development in zebrafish and mice as well as heterokaryons point to a role beyond immunology. This review takes stock of the reports linking AID and related deaminases to DNA demethylation, and describes the many important questions left to be answered in this field.

Interleukin-8, interleukin-1beta, and interferon-gamma levels are linked to PRRS virus clearance.

Infection with porcine reproductive and respiratory syndrome virus (PRRSV) results in a weak antiviral immune response that leads to a persistent infection in a subset of pigs. We investigated the intensity and timing of the early cytokine responses to PRRSV infection to determine their utility as a predictor of persistence. As part of the "Big Pig" project, we evaluated cytokine gene expression in lymphoid tissues collected from pigs for up 202 days post-infection (dpi); serum samples were collected biweekly. Cytokine mRNA levels were compared between pigs that cleared the viral infection from serum and tissues (non-persistent [NP] pigs) to those of persistent (P) pigs, that had viral RNA in their serum for up to 126 dpi. The gene expression studies in the tracheobronchial lymph nodes (TBLN) of all the pigs showed upregulation of interferon-gamma (IFN-gamma)-associated T-helper 1 (Th-1) markers from 14-84 dpi, and of T-regulatory interleukin-10 (IL-10), but no upregulation of innate markers (IFN-A, IL-1B, and IL-8). At later time points (>112 dpi) these genes were no longer differentially expressed and thus were uninformative for persistence studies. Statistical analyses of serum cytokine levels indicated that innate cytokine (IL-1beta and IL-8) levels were upregulated early after infection. Interestingly, serum IL-8 levels in NP pigs were significantly higher than in P pigs at 14 dpi. When analyzed together, variations in all three of the serum cytokines tested (IL-8, IL-1beta, and IFN-gamma) was significantly correlated with virus level, accounting for approximately 84% of the variations observed. These results indicate that while each cytokine individually has minor effects on the length of virus replication, the combination of cytokine activities should be considered when understanding the role of immunity in persistence.

BEAP: The BLAST Extension and Alignment Program- a tool for contig construction and analysis of preliminary genome sequence.

BACKGROUND: Fine-mapping projects require a high density of SNP markers and positional candidate gene sequences. In species with incomplete genomic sequence, the DNA sequences needed to generate markers for fine-mapping within a linkage analysis confidence interval may be available but may not have been assembled. To manually piece these sequences together is laborious and costly. Moreover, annotation and assembly of short, incomplete DNA sequences is time consuming and not always straightforward. FINDINGS: We have created a tool called BEAP that combines BLAST and CAP3 to retrieve sequences and construct contigs for localized genomic regions in species with unfinished sequence drafts. The rational is that a completed genome can be used as a template to query target genomic sequence for closing the gaps or extending contig sequence length in species whose genome is incomplete on the basis that good homology exists. Each user must define what template sequence is appropriate based on comparative mapping data such as radiation hybrid (RH) maps or other evidence linking the gene sequence of the template species to the target species. CONCLUSION: The BEAP software creates contigs suitable for discovery of orthologous genes for positional cloning. The resulting sequence alignments can be viewed graphically with a Java graphical user interface (GUI), allowing users to evaluate contig sequence quality and predict SNPs. We demonstrate the successful use of BEAP to generate genomic template sequence for positional cloning of the Angus dwarfism mutation. The software is available for free online for use on UNIX systems at http://www.animalgenome.org/bioinfo/tools/beap/.

AnimalQTLdb: a livestock QTL database tool set for positional QTL information mining and beyond.

The Animal Quantitative Trait Loci (QTL) database (AnimalQTLdb) is designed to house all publicly available QTL data on livestock animal species from which researchers can easily locate and compare QTL within species. The database tools are also added to link the QTL data to other types of genomic information, such as radiation hybrid (RH) maps, finger printed contig (FPC) physical maps, linkage maps and comparative maps to the human genome, etc. Currently, this database contains data on 1287 pig, 630 cattle and 657 chicken QTL, which are dynamically linked to respective RH, FPC and human comparative maps. We plan to apply the tool to other animal species, and add more structural genome information for alignment, in an attempt to aid comparative structural genome studies (http://www.animalgenome.org/QTLdb/).

Hipertrigliceridemia y embarazo: reporte de un caso / Hypertriglyceridemia and pregnancy: a case report

Both cholesterol and triglycerides become physiologically elevated during pregnancy. This increase is the result of metabolic adaptation of the maternal homeostasis allowing constant transfer of glucose and energy to the fetus. An excessive increase in the levels of triglycerides may lead to pancreatitis as its major consequence. Effective measures for treatment include a combination of diet (nutritional input based on medium-chain triglycerides) and caesarean operation planning to avoid pancreatitis when triglyceride levels are high. We here present a case of hypertriglyceridemis which was managed at the High Risk Polyclinic at the Regional Hospital Clinic of Antofagasta, and we comment on the management process, clinical evolution, type of birth, and perinatal results.

Tanto el colesterol como de los triglicéridos están fisiológicamente aumentados durante el embarazo. Este aumento es el resultado de la adaptación metabólica de la homeostasis materna para permitir un aporte constante de glucosa y energía para el feto. Un incremento excesivo en los niveles de triglicéridos puede llevar a una pancreatitis la cual constituye su principal consecuencia. Una combinación de dieta, un aporte nutricional en base de triglicéridos de cadena media, y la planificación de cesárea para evitar la pancreatitis cuando los niveles de triglicéridos son altos, son medidas efectivas. A continuación presentamos un caso de hipertrigliceridemia que fue manejado en el policlínico de Alto Riesgo del Hospital Clínico Regional de Antofagasta, se comente su manejo, su evolución clínica, el tipo de parto y los resultados perinatales.