Dissemination of blaNDM-5-carrying IncX3-type plasmid among non-clonal Escherichia coli strains colonising a dog with a skin infection caused by a carbapenem-resistant Klebsiella pneumoniae, United Kingdom.

A successful outcome of a post-surgical wound infection management by a carbapenem-resistant Klebsiella pneumoniae is described in a dog. Four multidrug-resistant and carbapenem-resistant Escherichia coli strains belonging to ST410 (n = 1) and ST648 (n = 3) were isolated from faecal samples and nasal swabs of this dog at admission to a veterinary hospital in the United Kingdom, and one month after discharge. Whole-genome sequencing analysis suggests dissemination of a 46,161-bp IncX3 blaNDM-5-carrying plasmid among E. coli strains from the different lineages. In this study, the E. coli ST648 strains were virtually identical to each other (5 SNPs difference) indicating dissemination and persistence of this clone over time and across different anatomical sites in the same dog maybe due to the prolonged antimicrobial therapy. The carbapenemase carrying plasmid also showed homology with other publicly available plasmid sequences from Asian countries. These results suggests that plasmids may be a major vehicle in mediating the dissemination of carbapenem-resistance. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst companion animals are needed as it may further contaminate other environments posing a threat to public health.

Longitudinal study of ESBL/AmpC-producing Enterobacterales strains sharing between cohabiting healthy companion animals and humans in Portugal and in the United Kingdom.

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales (ESBL/AmpC-E) are an increasing healthcare problem in both human and veterinary medicine. The aim of this study was to investigate the possible sharing of ESBL/AmpC-E strains between healthy companion animals and humans of the same household in Portugal (PT) and the United Kingdom (UK). In a prospective longitudinal study, between 2018 and 2020, faecal samples were collected from healthy dogs (n=90), cats (n=20) and their cohabiting humans (n=119) belonging to 41 PT and 44 UK households. Samples were screened for the presence of ESBL/AmpC-E and carbapenemase-producing bacteria. Clonal relatedness between animal and human strains was established by using REP-PCR fingerprinting method, followed by whole-genome sequencing (WGS) of selected strains. ESBL/AmpC-E strains were detected in companion animals (PT=12.7%, n=8/63; UK=8.5%, n=4/47) and humans (PT=20.7%, n=12/58; UK=6.6%, n=4/61) in at least one timepoint. REP-PCR identified paired multidrug-resistant ESBL/AmpC-producing Escherichia coli strains from companion animals and owners in two Portuguese households (4.8%) and one UK household (2.3%). WGS analysis of nine E. coli strains from these three households confirmed that interhost sharing occurred only between the two animal-human pairs from Portugal. Three shared strains were identified: one CTX-M-15-producing E. coli strain in a cat-human pair (O15-H33-ST93) and two CTX-M-15- and CTX-M-55/CMY-2-producing E. coli strains, in a dog-human pair (O8:H9-ST410 and O11:H25-ST457, respectively) at different timepoints. These E. coli clonal lineages are human pandemic, highlighting the role of companion animals living in close contact with humans in the dissemination and persistence of antimicrobial resistance in the household environment.

Lost pigs of Angola: Whole genome sequencing reveals unique regions of selection with emphasis on metabolism and feed efficiency.

Angola, in the western coast of Africa, has been through dramatic social events that have led to the near-disappearance of native swine populations, and the recent introduction of European exotic breeds has also contributed to the erosion of this native swine repertoire. In an effort to investigate the genetic basis of native pigs in Angola (ANG) we have generated whole genomes from animals of a remote local pig population in Huambo province, which we have compared with 78 genomes of European and Asian pig breeds as well as European and Asian wild boars that are currently in public domain. Analyses of population structure showed that ANG pigs grouped within the European cluster and were clearly separated from Asian pig breeds. Pairwise F ST ranged from 0.14 to 0.26, ANG pigs display lower levels of genetic differentiation towards European breeds. Finally, we have identified candidate regions for selection using a complementary approach based on various methods. All results suggest that selection towards feed efficiency and metabolism has occurred. Moreover, all analysis identified CDKAL1 gene, which is related with insulin and cholesterol metabolism, as a candidate gene overlapping signatures of selection unique to ANG pigs. This study presents the first assessment of the genetic relationship between ANG pigs and other world breeds and uncovers selection signatures that may indicate adaptation features unique to this important genetic resource.

IsomiR_Window: a system for analyzing small-RNA-seq data in an integrative and user-friendly manner.

BACKGROUND: IsomiRs are miRNA variants that vary in length and/or sequence when compared to their canonical forms. These variants display differences in length and/or sequence, including additions or deletions of one or more nucleotides (nts) at the 5' and/or 3' end, internal editings or untemplated 3' end additions. Most available tools for small RNA-seq data analysis do not allow the identification of isomiRs and often require advanced knowledge of bioinformatics. To overcome this, we have developed IsomiR Window, a platform that supports the systematic identification, quantification and functional exploration of isomiR expression in small RNA-seq datasets, accessible to users with no computational skills. METHODS: IsomiR Window enables the discovery of isomiRs and identification of all annotated non-coding RNAs in RNA-seq datasets from animals and plants. It comprises two main components: the IsomiR Window pipeline for data processing; and the IsomiR Window Browser interface. It integrates over ten third-party softwares for the analysis of small-RNA-seq data and holds a new algorithm that allows the detection of all possible types of isomiRs. These include 3' and 5'end isomiRs, 3' end tailings, isomiRs with single nucleotide polymorphisms (SNPs) or potential RNA editings, as well as all possible fuzzy combinations. IsomiR Window includes all required databases for analysis and annotation, and is freely distributed as a Linux virtual machine, including all required software. RESULTS: IsomiR Window processes several datasets in an automated manner, without restrictions of input file size. It generates high quality interactive figures and tables which can be exported into different formats. The performance of isomiR detection and quantification was assessed using simulated small-RNA-seq data. For correctly mapped reads, it identified different types of isomiRs with high confidence and 100% accuracy. The analysis of a small RNA-seq data from Basal Cell Carcinomas (BCCs) using isomiR Window confirmed that miR-183-5p is up-regulated in Nodular BCCs, but revealed that this effect was predominantly due to a novel 5'end variant. This variant displays a different seed region motif and 1756 isoform-exclusive mRNA targets that are significantly associated with disease pathways, underscoring the biological relevance of isomiR-focused analysis. IsomiR Window is available at https://isomir.fc.ul.pt/ .

Genomic Tools for the Conservation and Genetic Improvement of a Highly Fragmented Breed-The Ramo Grande Cattle from the Azores.

Ramo Grande is a local cattle breed raised in the archipelago of Azores, with a small and dispersed census, where inbreeding control is of utmost importance. A single nucleotide polymorphism (SNP) Beadchip array was used to assess inbreeding, by analysis of genomic regions harboring contiguous homozygous genotypes named runs of homozygosity (ROH), and to estimate past effective population size by analysis of linkage disequilibrium (LD). Genetic markers associated with production traits were also investigated, exploiting the unique genetic and adaptation features of this breed. A total of 639 ROH with length >4 Mb were identified, with mean length of 14.96 Mb. The mean genomic inbreeding was 0.09, and long segments of ROH were common, indicating recent inbred matings. The LD pattern indicates a large effective population size, suggesting the inflow of exotic germplasm in the past. The genome-wide association study identified novel markers significantly affecting longevity, age at first calving and direct genetic effects on calf weight. These results provide the first evidence of the association of longevity with genes related with DNA recognition and repair, and the association of age at first calving with aquaporin proteins, which are known to have a crucial role in reproduction.

Autoimmunity and allergy control in adults submitted to complete thymectomy early in infancy.

The contribution of the decline in thymic activity for the emergence of autoimmunity is still debatable. Immune-competent adults submitted to complete thymectomy early in life provide a unique model to address this question. We applied here strict criteria to identify adults lacking thymic activity based on sjTREC levels, to exclude thymic rebound and/or ectopic thymuses. In agreement, they featured severe naïve CD4 T-cell depletion and contraction of T-cell receptor diversity. Notwithstanding this, there was neither increased incidence of autoimmune disease in comparison with age-matched controls nor significant changes in their IgG/IgA/IgM/IgE autoreactivity profiles, as assessed through extensive arrays. We reasoned that the observed relative preservation of the regulatory T-cell compartment, including maintenance of naïve regulatory CD4 T-cells, may contribute to limit the emergence of autoimmunity upon thymectomy. Our findings have implications in other clinical settings with impaired thymic activity, and are particularly relevant to studies of autoimmunity in ageing.

miRNA profiling of human naive CD4 T cells links miR-34c-5p to cell activation and HIV replication.

Cell activation is a vital step for T-cell memory/effector differentiation as well as for productive HIV infection. To identify novel regulators of this process, we used next-generation sequencing to profile changes in microRNA expression occurring in purified human naive CD4 T cells in response to TCR stimulation and/or HIV infection. Our results demonstrate, for the first time, the transcriptional up-regulation of miR-34c-5p in response to TCR stimulation in naive CD4 T cells. The induction of this miR was further consistently found to be reduced by both HIV-1 and HIV-2 infections. Overexpression of miR-34c-5p led to changes in the expression of several genes involved in TCR signaling and cell activation, confirming its role as a novel regulator of naive CD4 T-cell activation. We additionally show that miR-34c-5p promotes HIV-1 replication, suggesting that its down-regulation during HIV infection may be part of an anti-viral host response.

Prospects and challenges for the conservation of farm animal genomic resources, 2015-2025.

Livestock conservation practice is changing rapidly in light of policy developments, climate change and diversifying market demands. The last decade has seen a step change in technology and analytical approaches available to define, manage and conserve Farm Animal Genomic Resources (FAnGR). However, these rapid changes pose challenges for FAnGR conservation in terms of technological continuity, analytical capacity and integrative methodologies needed to fully exploit new, multidimensional data. The final conference of the ESF Genomic Resources program aimed to address these interdisciplinary problems in an attempt to contribute to the agenda for research and policy development directions during the coming decade. By 2020, according to the Convention on Biodiversity's Aichi Target 13, signatories should ensure that "…the genetic diversity of …farmed and domesticated animals and of wild relatives …is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity." However, the real extent of genetic erosion is very difficult to measure using current data. Therefore, this challenging target demands better coverage, understanding and utilization of genomic and environmental data, the development of optimized ways to integrate these data with social and other sciences and policy analysis to enable more flexible, evidence-based models to underpin FAnGR conservation. At the conference, we attempted to identify the most important problems for effective livestock genomic resource conservation during the next decade. Twenty priority questions were identified that could be broadly categorized into challenges related to methodology, analytical approaches, data management and conservation. It should be acknowledged here that while the focus of our meeting was predominantly around genetics, genomics and animal science, many of the practical challenges facing conservation of genomic resources are societal in origin and are predicated on the value (e.g., socio-economic and cultural) of these resources to farmers, rural communities and society as a whole. The overall conclusion is that despite the fact that the livestock sector has been relatively well-organized in the application of genetic methodologies to date, there is still a large gap between the current state-of-the-art in the use of tools to characterize genomic resources and its application to many non-commercial and local breeds, hampering the consistent utilization of genetic and genomic data as indicators of genetic erosion and diversity. The livestock genomic sector therefore needs to make a concerted effort in the coming decade to enable to the democratization of the powerful tools that are now at its disposal, and to ensure that they are applied in the context of breed conservation as well as development.

Quality assessment and control of tissue specific RNA-seq libraries of Drosophila transgenic RNAi models.

RNA-sequencing (RNA-seq) is rapidly emerging as the technology of choice for whole-transcriptome studies. However, RNA-seq is not a bias free technique. It requires large amounts of RNA and library preparation can introduce multiple artifacts, compounded by problems from later stages in the process. Nevertheless, RNA-seq is increasingly used in multiple studies, including the characterization of tissue-specific transcriptomes from invertebrate models of human disease. The generation of samples in this context is complex, involving the establishment of mutant strains and the delicate contamination prone process of dissecting the target tissue. Moreover, in order to achieve the required amount of RNA, multiple samples need to be pooled. Such datasets pose extra challenges due to the large variability that may occur between similar pools, mostly due to the presence of cells from surrounding tissues. Therefore, in addition to standard quality control of RNA-seq data, analytical procedures for control of "biological quality" are critical for successful comparison of gene expression profiles. In this study, the transcriptome of the central nervous system (CNS) of a Drosophila transgenic strain with neuronal-specific RNAi of an ubiquitous gene was profiled using RNA-seq. After observing the existence of an unusual variance in our dataset, we showed that the expression profile of a small panel of marker genes, including GAL4 under control of a tissue specific driver, can identify libraries with low levels of contamination from neighboring tissues, enabling the selection of a robust dataset for differential expression analysis. We further analyzed the potential of profiling a complex tissue to identify cell-type specific changes in response to target gene down-regulation. Finally, we showed that trimming 5' ends of reads decreases nucleotide frequency biases, increasing the coverage of protein coding genes with a potential positive impact in the incurrence of systematic technical errors.

A long journey to effective obesity treatments: is there light at the end of the tunnel?

As the obesity epidemic continues, more Americans are getting fatter, having more weight-related problems such as cardiovascular disease, and are experiencing new metabolic dysfunctions. For over 50 years, the adipose tissue (AT), commonly referred to as fat, has been of interest to academic and clinical scientists, public health officials and individuals interested in body composition and image including much of the average public, athletes, parents, etc. On one hand, efforts to alter body shape, weight and body fat percentage still include bizarre and scientifically unfounded methods. On the other hand, significant new scientific strides have been made in understanding the growth, function and regulation of anatomical and systemic AT. Markers of transition/conversion of precursor cells that mature to form lipid assimilating adipocytes have been identified. Molecular 'master' regulators such as peroxisome proliferator-activated receptor gamma and CCAAT-enhancer-binding proteins were uncovered and regulatory mechanisms behind variables of adiposity defined and refined. Interventions including pharmaceutical compounds, surgical, psychosocial interventions have also been tested. Has all of the preceding research helped alleviate the adverse physiologies of overweight and/or obese people? Does research to date point to new modalities that should be the focus of efforts to rid the world of obesity-related problems in the 21st century? This review provides a general overview of scientific efforts to date and a provocative view of the future for adiposity.

Genome-wide footprints of pig domestication and selection revealed through massive parallel sequencing of pooled DNA.

BACKGROUND: Artificial selection has caused rapid evolution in domesticated species. The identification of selection footprints across domesticated genomes can contribute to uncover the genetic basis of phenotypic diversity. METHODOLOGY/MAIN FINDINGS: Genome wide footprints of pig domestication and selection were identified using massive parallel sequencing of pooled reduced representation libraries (RRL) representing ∼2% of the genome from wild boar and four domestic pig breeds (Large White, Landrace, Duroc and Pietrain) which have been under strong selection for muscle development, growth, behavior and coat color. Using specifically developed statistical methods that account for DNA pooling, low mean sequencing depth, and sequencing errors, we provide genome-wide estimates of nucleotide diversity and genetic differentiation in pig. Widespread signals suggestive of positive and balancing selection were found and the strongest signals were observed in Pietrain, one of the breeds most intensively selected for muscle development. Most signals were population-specific but affected genomic regions which harbored genes for common biological categories including coat color, brain development, muscle development, growth, metabolism, olfaction and immunity. Genetic differentiation in regions harboring genes related to muscle development and growth was higher between breeds than between a given breed and the wild boar. CONCLUSIONS/SIGNIFICANCE: These results, suggest that although domesticated breeds have experienced similar selective pressures, selection has acted upon different genes. This might reflect the multiple domestication events of European breeds or could be the result of subsequent introgression of Asian alleles. Overall, it was estimated that approximately 7% of the porcine genome has been affected by selection events. This study illustrates that the massive parallel sequencing of genomic pools is a cost-effective approach to identify footprints of selection.

Application of massive parallel sequencing to whole genome SNP discovery in the porcine genome.

BACKGROUND: Although the Illumina 1 G Genome Analyzer generates billions of base pairs of sequence data, challenges arise in sequence selection due to the varying sequence quality. Therefore, in the framework of the International Porcine SNP Chip Consortium, this pilot study aimed to evaluate the impact of the quality level of the sequenced bases on mapping quality and identification of true SNPs on a large scale. RESULTS: DNA pooled from five animals from a commercial boar line was digested with DraI; 150-250-bp fragments were isolated and end-sequenced using the Illumina 1 G Genome Analyzer, yielding 70,348,064 sequences 36-bp long. Rules were developed to select sequences, which were then aligned to unique positions in a reference genome. Sequences were selected based on quality, and three thresholds of sequence quality (SQ) were compared. The highest threshold of SQ allowed identification of a larger number of SNPs (17,489), distributed widely across the pig genome. In total, 3,142 SNPs were validated with a success rate of 96%. The correlation between estimated minor allele frequency (MAF) and genotyped MAF was moderate, and SNPs were highly polymorphic in other pig breeds. Lowering the SQ threshold and maintaining the same criteria for SNP identification resulted in the discovery of fewer SNPs (16,768), of which 259 were not identified using higher SQ levels. Validation of SNPs found exclusively in the lower SQ threshold had a success rate of 94% and a low correlation between estimated MAF and genotyped MAF. Base change analysis suggested that the rate of transitions in the pig genome is likely to be similar to that observed in humans. Chromosome X showed reduced nucleotide diversity relative to autosomes, as observed for other species. CONCLUSION: Large numbers of SNPs can be identified reliably by creating strict rules for sequence selection, which simultaneously decreases sequence ambiguity. Selection of sequences using a higher SQ threshold leads to more reliable identification of SNPs. Lower SQ thresholds can be used to guarantee sufficient sequence coverage, resulting in high success rate but less reliable MAF estimation. Nucleotide diversity varies between porcine chromosomes, with the X chromosome showing less variation as observed in other species.

Design of a high density SNP genotyping assay in the pig using SNPs identified and characterized by next generation sequencing technology.

BACKGROUND: The dissection of complex traits of economic importance to the pig industry requires the availability of a significant number of genetic markers, such as single nucleotide polymorphisms (SNPs). This study was conducted to discover several hundreds of thousands of porcine SNPs using next generation sequencing technologies and use these SNPs, as well as others from different public sources, to design a high-density SNP genotyping assay. METHODOLOGY/PRINCIPAL FINDINGS: A total of 19 reduced representation libraries derived from four swine breeds (Duroc, Landrace, Large White, Pietrain) and a Wild Boar population and three restriction enzymes (AluI, HaeIII and MspI) were sequenced using Illumina's Genome Analyzer (GA). The SNP discovery effort resulted in the de novo identification of over 372K SNPs. More than 549K SNPs were used to design the Illumina Porcine 60K+SNP iSelect Beadchip, now commercially available as the PorcineSNP60. A total of 64,232 SNPs were included on the Beadchip. Results from genotyping the 158 individuals used for sequencing showed a high overall SNP call rate (97.5%). Of the 62,621 loci that could be reliably scored, 58,994 were polymorphic yielding a SNP conversion success rate of 94%. The average minor allele frequency (MAF) for all scorable SNPs was 0.274. CONCLUSIONS/SIGNIFICANCE: Overall, the results of this study indicate the utility of using next generation sequencing technologies to identify large numbers of reliable SNPs. In addition, the validation of the PorcineSNP60 Beadchip demonstrated that the assay is an excellent tool that will likely be used in a variety of future studies in pigs.

Linkage disequilibrium decay and haplotype block structure in the pig.

Linkage disequilibrium (LD) may reveal much about domestication and breed history. An investigation was conducted, to analyze the extent of LD, haploblock partitioning, and haplotype diversity within haploblocks across several pig breeds from China and Europe and in European wild boar. In total, 371 single-nucleotide-polymorphisms located in three genomic regions were genotyped. The extent of LD differed significantly between European and Chinese breeds, extending up to 2 cM in Europe and up to 0.05 cM in China. In European breeds, LD extended over large haploblocks up to 400 kb, whereas in Chinese breeds the extent of LD was smaller and generally did not exceed 10 kb. The European wild boar showed an intermediate level of LD between Chinese and European breeds. In Europe, the extent of LD also differed according to genomic region. Chinese breeds showed a higher level of haplotype diversity and shared high levels of frequent haplotypes with Large White, Landrace, and Duroc. The extent of LD differs between both centers of pig domestication, being higher in Europe. Two hypotheses can explain these findings. First, the European ancestral stock had a higher level of LD. Second, modern breeding programs increased the extent of LD in Europe and caused differences of LD between genomic regions. Large White, Landrace, and Duroc showed evidence of past introgression from Chinese breeds.