Gene model annotations are important community resources that ensure comparability and reproducibility of analyses and are typically the first step for functional annotation of genomic regions. Without up-to-date genome annotations, genome sequences cannot be used to maximum advantage. It is therefore essential to regularly update gene annotations by integrating the latest information to guarantee that reference annotations can remain a common basis for various types of analyses. Here, we report an improvement of the Arabidopsis lyrata gene annotation using extensive RNA-seq data. This new annotation consists of 31,132 protein coding gene models in addition to 2,089 genes with high similarity to transposable elements. Overall, ~87% of the gene models are corroborated by evidence of expression and 2,235 of these models feature multiple transcripts. Our updated gene annotation corrects hundreds of incorrectly split or merged gene models in the original annotation, and as a result the identification of alternative splicing events and differential isoform usage are vastly improved.

Duplications allow for gene functional diversification and accelerate genome evolution. Occasionally, the transposon amplification machinery reverse transcribes the mRNA of a gene, integrates it into the genome, and forms an RNA-duplicated copy: the retrogene. Although retrogenes have been found in plants, their biology and evolution are poorly understood. Here, we identified 251 (216 novel) retrogenes in Arabidopsis thaliana, corresponding to 1% of protein-coding genes. Arabidopsis retrogenes are derived from ubiquitously transcribed parents and reside in gene-rich chromosomal regions. Approximately 25% of retrogenes are cotranscribed with their parents and 3% with head-to-head oriented neighbors. This suggests transcription by novel promoters for 72% of Arabidopsis retrogenes. Many retrogenes reach their transcription maximum in pollen, the tissue analogous to animal spermatocytes, where upregulation of retrogenes has been found previously. This implies an evolutionarily conserved mechanism leading to this transcription pattern of RNA-duplicated genes. During transcriptional repression, retrogenes are depleted of permissive chromatin marks without an obvious enrichment for repressive modifications. However, this pattern is common to many other pollen-transcribed genes independent of their evolutionary origin. Hence, retroposition plays a role in plant genome evolution, and the developmental transcription pattern of retrogenes suggests an analogous regulation of RNA-duplicated genes in plants and animals.

Ciprofloxacin (CIP) is used to treat Pseudomonas aeruginosa biofilm infections. We showed that the pathways of CIP-resistance development during exposure of biofilms and planktonic P. aeruginosa populations to subinhibitory levels of CIP depend on the mode of growth. In the present study, we analyzed CIP-resistant isolates obtained from previous evolution experiments, and we report a variety of evolved phenotypic and genotypic changes that occurred in parallel with the evolution of CIP-resistance. Cross-resistance to beta-lactam antibiotics was associated with mutations in genes involved in cell-wall recycling ( ftsZ , murG ); and could also be explained by mutations in the TCA cycle ( sdhA ) genes and in genes involved in arginine catabolism. We found that CIP-exposed isolates that lacked mutations in quorum-sensing genes and acquired mutations in type IV pili genes maintained swarming motility and lost twitching motility, respectively. Evolved CIP-resistant isolates showed high fitness cost in planktonic competition experiments, yet persisted in the biofilm under control conditions, compared with ancestor isolates and had an advantage when exposed to CIP. Their persistence in biofilm competition experiments in spite of their fitness cost in planktonic growth could be explained by their prolonged lag-phase. Interestingly, the set of mutated genes that we identified in these in vitro-evolved CIP-resistant colonies, overlap with a large number of patho-adaptive genes previously reported in P. aeruginosa isolates from cystic fibrosis (CF) patients. This suggests that the antibiotic stress is contributing to the bacterial evolution in vivo, and that adaptive laboratory evolution can be used to predict the in vivo evolutionary trajectories.

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During chronic biofilm infections, Pseudomonas aeruginosa bacteria are exposed to increased oxidative stress as a result of the inflammatory response. As reactive oxygen species (ROS) are mutagenic, the evolution of resistance to ciprofloxacin (CIP) in biofilms under oxidative stress conditions was investigated. We experimentally evolved six replicate populations of P. aeruginosa lacking the major catalase KatA in colony biofilms and stationary-phase cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of CIP or without CIP (eight replicate lineages for controls) under aerobic conditions. In CIP-evolved biofilms, a larger CIP-resistant subpopulation was isolated in the ΔkatA strain than in the wild-type (WT) PAO1 population, suggesting oxidative stress as a promoter of the development of antibiotic resistance. A higher number of mutations identified by population sequencing were observed in evolved ΔkatA biofilm populations (CIP and control) than in WT PAO1 populations evolved under the same conditions. Genes involved in iron assimilation were found to be exclusively mutated in CIP-evolved ΔkatA biofilm populations, probably as a defense mechanism against ROS formation resulting from Fenton reactions. Furthermore, a hypermutable lineage due to mutL inactivation developed in one CIP-evolved ΔkatA biofilm lineage. In CIP-evolved biofilms of both the ΔkatA strain and WT PAO1, mutations in nfxB, the negative regulator of the MexCD-OprJ efflux pump, were observed while in CIP-evolved planktonic cultures of both the ΔkatA strain and WT PAO1, mutations in mexR and nalD, regulators of the MexAB-OprM efflux pump, were repeatedly found. In conclusion, these results emphasize the role of oxidative stress as an environmental factor that might increase the development of antibiotic resistance in in vivo biofilms.

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Background:In cone beam computed tomography (CBCT), metallic structures can cause artifacts in the images, since most teeth suspected to have vertical root fractures (VRF) are endodontically treated and have a metallic post in the root canal, fracture detection may pose a challenge. Aim: This study aimed to determine the diagnostic accuracy of CBCT using metal artifact reduction (MAR) algorithm in detection of VRF of teeth with metallic posts. Materials and Methods: A total of 120 single-rooted extracted human teeth were obtained and endodontically treated then placed in an acrylic block, and metallic posts were inserted. After post insertion, the teeth roots were divided into two groups one with induced VRF and the other having intact roots with the posts inserted. Then, each tooth was coded and imaged by CBCT with and without using MAR algorithm. Results: Using the MAR algorithm improved the diagnostic accuracy but with no statistically significant difference. Conclusion:In case of presence of metallic posts, CBCT with MAR algorithm can be used to detect VRF.