Single base mismatches in the mRNA target site allow specific seed region-mediated off-target binding of siRNA targeting human coagulation factor 7

We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3′-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3′-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts

Functional analysis and transcriptional output of the Göttingen minipig genome

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.; Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.; Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed

Patterns of foraging activity and fidelity in a Southeast Asian flying fox

Background: Improved understanding of the foraging ecology of bats in the face of ongoing habitat loss and modification worldwide is essential to their conservation and maintaining the substantial ecosystem services they provide. It is also fundamental to assessing potential transmission risks of zoonotic pathogens in human-wildlife interfaces. We evaluated the influence of environmental and behavioral variables on the foraging patterns of Pteropus lylei (a reservoir of Nipah virus) in a heterogeneous landscape in Cambodia. Methods: We employed an approach based on animal-movement modeling, which comprised a path-segmentation method (hidden Markov model) to identify individual foraging-behavior sequences in GPS data generated by eight P. lylei. We characterized foraging localities, foraging activity, and probability of returning to a given foraging locality over consecutive nights. Generalized linear mixed models were also applied to assess the influence of several variables including proxies for energetic costs and quality of foraging areas. Results: Bats performed few foraging bouts (area-restricted searches) during a given night, mainly in residential areas, and the duration of these decreased during the night. The probability of a bat revisiting a given foraging area within 48 h varied according to the duration previously spent there, its distance to the roost site, and the corresponding habitat type. We interpret these fine-scale patterns in relation to global habitat quality (including food-resource quality and predictability), habitat-familiarity and experience of each individual. Conclusions: Our study provides evidence that heterogeneous human-made environments may promote complex patterns of foraging-behavior and short-term re-visitation in fruit bat species that occur in such landscapes. This highlights the need for similarly detailed studies to understand the processes that maintain biodiversity in these environments and assess the potential for pathogen transmission in human-wildlife interfaces

Slow erosion of a quantitative apple resistance to Venturia inaequalis based on an isolate-specific Quantitative Trait Locus

Quantitative plant resistance affects the aggressiveness of pathogens and is usually considered more durable than qualitative resistance. However, the efficiency of a quantitative resistance based on an isolate-specific Quantitative Trait Locus (QTL) is expected to decrease over time due to the selection of isolates with a high level of aggressiveness on resistant plants. To test this hypothesis, we surveyed scab incidence over an eight-year period in an orchard planted with susceptible and quantitatively resistant apple genotypes. We sampled 79 Venturia inaequalis isolates from this orchard at three dates and we tested their level of aggressiveness under controlled conditions. Isolates sampled on resistant genotypes triggered higher lesion density and exhibited a higher sporulation rate on apple carrying the resistance allele of the QTL T1 compared to isolates sampled on susceptible genotypes. Due to this ability to select aggressive isolates, we expected the QTL T1 to be non-durable. However, our results showed that the quantitative resistance based on the QTL T1 remained efficient in orchard over an eight-year period, with only a slow decrease in efficiency and no detectable increase of the aggressiveness of fungal isolates over time. We conclude that knowledge on the specificity of a QTL is not sufficient to evaluate its durability. Deciphering molecular mechanisms associated with resistance QTLs, genetic determinants of aggressiveness and putative trade-offs within pathogen populations is needed to help in understanding the erosion processe

Additional file 7: Figure S3. of Functional analysis and transcriptional output of the GĂśttingen minipig genome

Schematic description of the in silico workflow for selection of Sus scrofa specific lncRNAs. * 16 genomes: cow, horse, dog, human, orang utan, chimpanzee, cynomolgus monkey, rhesus monkey, marmoset, rabbit, guinea pig, hamster, mouse, rat, opossum, chicken. See text for more detailed description. (JPEG 854 kb

Additional file 2: Table S2. of Functional analysis and transcriptional output of the GĂśttingen minipig genome

Contig assembly statistics. Roche-454 reads that were not incorporated into the minipig genome were assembled de-novo with Roche-Newbler software. (DOCX 13 kb