Recent Advances in Rapid Antimicrobial Susceptibility Testing.

BACKGROUND: Antimicrobial susceptibility testing (AST) is classically performed using growth-based techniques that essentially require viable bacterial matter to become visible to the naked eye or a sophisticated densitometer. CONTENT: Technologies based on the measurement of bacterial density in suspension have evolved marginally in accuracy and rapidity over the 20th century, but assays expanded for new combinations of bacteria and antimicrobials have been automated, and made amenable to high-throughput turn-around. Over the past 25 years, elevated AST rapidity has been provided by nucleic acid-mediated amplification technologies, proteomic and other "omic" methodologies, and the use of next-generation sequencing. In rare cases, AST at the level of single-cell visualization was developed. This has not yet led to major changes in routine high-throughput clinical microbiological detection of antimicrobial resistance. SUMMARY: We here present a review of the new generation of methods and describe what is still urgently needed for their implementation in day-to-day management of the treatment of infectious diseases.

A role for MED14 and UVH6 in heterochromatin transcription upon destabilization of silencing.

Constitutive heterochromatin is associated with repressive epigenetic modifications of histones and DNA which silence transcription. Yet, particular mutations or environmental changes can destabilize heterochromatin-associated silencing without noticeable changes in repressive epigenetic marks. Factors allowing transcription in this nonpermissive chromatin context remain poorly known. Here, we show that the transcription factor IIH component UVH6 and the mediator subunit MED14 are both required for heat stress-induced transcriptional changes and release of heterochromatin transcriptional silencing in Arabidopsis thaliana. We find that MED14, but not UVH6, is required for transcription when heterochromatin silencing is destabilized in the absence of stress through mutating the MOM1 silencing factor. In this case, our results raise the possibility that transcription dependency over MED14 might require intact patterns of repressive epigenetic marks. We also uncover that MED14 regulates DNA methylation in non-CG contexts at a subset of RNA-directed DNA methylation target loci. These findings provide insight into the control of heterochromatin transcription upon silencing destabilization and identify MED14 as a regulator of DNA methylation.

Loss of CG Methylation in Marchantia polymorpha Causes Disorganization of Cell Division and Reveals Unique DNA Methylation Regulatory Mechanisms of Non-CG Methylation.

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha ortholog of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9) system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Even though numerous TEs were up-regulated, non-CG methylation was generally highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.

sRNAPipe: a Galaxy-based pipeline for bioinformatic in-depth exploration of small RNAseq data.

BACKGROUND: The field of small RNA is one of the most investigated research areas since they were shown to regulate transposable elements and gene expression and play essential roles in fundamental biological processes. Small RNA deep sequencing (sRNA-seq) is now routinely used for large-scale analyses of small RNA. Such high-throughput sequencing typically produces several millions reads. RESULTS: Here we present a computational pipeline (sRNAPipe: small RNA pipeline) based on the Galaxy framework that takes as input a fastq file of small RNA-seq reads and performs successive steps of mapping to categories of genomic sequences: transposable elements, gene transcripts, microRNAs, small nuclear RNAs, ribosomal RNAs and transfer RNAs. It also provides individual mapping and counting for chromosomes, transposable elements and gene transcripts, normalization, small RNA length analysis and plotting of the data along genomic coordinates to build publication-quality graphs and figures. sRNAPipe evaluates 10-nucleotide 5'-overlaps of reads on opposite strands to test ping-pong amplification for putative PIWI-interacting RNAs, providing counts of overlaps and corresponding z-scores. CONCLUSIONS: sRNAPipe is easy to use and does not require command-line or coding knowledge. This pipeline gives quick visual and quantitative results, which are usable for publications. sRNAPipe is freely available as a Galaxy tool and via GitHub.

CLIFinder: identification of LINE-1 chimeric transcripts in RNA-seq data.

Summary: L1 Chimeric Transcripts (LCTs) are initiated by repeated LINE-1 element antisense promoters and include the L1 5'UTR sequence in antisense orientation followed by the adjacent genomic region. LCTs have been characterized mainly using bioinformatics approaches to query dbEST. To take advantage of NGS data to unravel the transcriptome composition, we developed Chimeric LIne Finder (CLIFinder), a new bioinformatics tool. Using stranded paired-end RNA-seq data, we demonstrated that CLIFinder can identify genome-wide transcribed chimera sequences corresponding to potential LCTs. Moreover, CLIFinder can be adapted to study transcription from other repeat types. Availability and implementation: The code is available at: https://github.com/GReD-Clermont/CLIFinder; and for Galaxy users, it is directly accessible in the tool shed at: https://toolshed.g2.bx.psu.edu/view/clifinder/clifinder/. Contact: catherine.barriere@uca.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Arabidopsis proteins with a transposon-related domain act in gene silencing.

Transposable elements (TEs) are prevalent in most eukaryotes, and host genomes have devised silencing strategies to rein in TE activity. One of these, transcriptional silencing, is generally associated with DNA methylation and short interfering RNAs. Here we show that the Arabidopsis genes MAIL1 and MAIN define an alternative silencing pathway independent of DNA methylation and short interfering RNAs. Mutants for MAIL1 or MAIN exhibit release of silencing and appear to show impaired condensation of pericentromeric heterochromatin. Phylogenetic analysis suggests not only that MAIL1 and MAIN encode a retrotransposon-related plant mobile domain, but also that host plant mobile domains were captured by DNA transposons during plant evolution. Our results reveal a role for Arabidopsis proteins with a transposon-related domain in gene silencing.

Epigenome confrontation triggers immediate reprogramming of DNA methylation and transposon silencing in Arabidopsis thaliana F1 epihybrids.

Genes and transposons can exist in variable DNA methylation states, with potentially differential transcription. How these epialleles emerge is poorly understood. Here, we show that crossing an Arabidopsis thaliana plant with a hypomethylated genome and a normally methylated WT individual results, already in the F1 generation, in widespread changes in DNA methylation and transcription patterns. Novel nonparental and heritable epialleles arise at many genic loci, including a locus that itself controls DNA methylation patterns, but with most of the changes affecting pericentromeric transposons. Although a subset of transposons show immediate resilencing, a large number display decreased DNA methylation, which is associated with de novo or enhanced transcriptional activation and can translate into transposon mobilization in the progeny. Our findings reveal that the combination of distinct epigenomes can be viewed as an epigenomic shock, which is characterized by a round of epigenetic variation creating novel patterns of gene and TE regulation.

Increased production of piRNAs from euchromatic clusters and genes in Anopheles gambiae compared with Drosophila melanogaster.

BACKGROUND: Specific genomic loci, termed Piwi-interacting RNA (piRNA) clusters, manufacture piRNAs that serve as guides for the inactivation of complementary transposable elements (TEs). The piRNA pathway has been accurately detailed in Drosophila melanogaster, while it remains poorly examined in other insects. This pathway is increasingly recognized as critical for germline development and reproduction. Understanding of the piRNA functions in mosquitoes could offer an opportunity for disease vector control by the reduction of their reproductive potential. RESULTS: To analyze the similarities and differences in this pathway between Drosophila and mosquito, we performed an in-depth analysis of the genomic loci producing piRNAs and their targets in the African malaria vector Anopheles gambiae. We identified 187 piRNA clusters in the An. gambiae genome and 155 piRNA clusters in the D. melanogaster genome. We demonstrate that many more piRNA clusters in the mosquito compared with the fruit fly are uni-directionally transcribed and are located outside pericentromeric heterochromatin. About 11 % of the An. gambiae piRNA population map to gene transcripts. This is a noticeable increase compared with the ~6 % of the piRNA population mapped to genes in D. melanogaster. A subset of the piRNA-enriched genes in An. gambiae has functions related to reproduction and development. At least 24 and 65 % of the mapped piRNAs correspond to genomic TE sequences in An. gambiae and D. melanogaster, respectively. DNA transposons and non-LTR retrotransposons are more abundant in An. gambiae, while LTR retrotransposons are more abundant in D. melanogaster. Yet, piRNAs predominantly target LTR retrotransposons in both species, which may point to a distinct feature of these elements compared to the other classes of TEs concerning their silencing by the piRNA pathway. CONCLUSIONS: Here, we demonstrate that piRNA-producing loci have more ubiquitous distribution in the An. gambiae genome than in the genome of D. melanogaster. Also, protein-coding genes have an increased role in production of piRNAs in the germline of this mosquito. Genes involved in germline and embryonic development of An. gambiae generate a substantial portion of piRNAs, suggesting a role of the piRNA pathway in the epigenetic regulation of the reproductive processes in the African malaria vector.

Oxidative DNA damage in mouse sperm chromosomes: Size matters.

Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization.

MosaicFinder: identification of fused gene families in sequence similarity networks.

MOTIVATION: Gene fusion is an important evolutionary process. It can yield valuable information to infer the interactions and functions of proteins. Fused genes have been identified as non-transitive patterns of similarity in triplets of genes. To be computationally tractable, this approach usually imposes an a priori distinction between a dataset in which fused genes are searched for, and a dataset that may have provided genetic material for fusion. This reduces the 'genetic space' in which fusion can be discovered, as only a subset of triplets of genes is investigated. Moreover, this approach may have a high-false-positive rate, and it does not identify gene families descending from a common fusion event. RESULTS: We represent similarities between sequences as a network. This leads to an efficient formulation of previous methods of fused gene identification, which we implemented in the Python program FusedTriplets. Furthermore, we propose a new characterization of families of fused genes, as clique minimal separators of the sequence similarity network. This well-studied graph topology provides a robust and fast method of detection, well suited for automatic analyses of big datasets. We implemented this method in the C++ program MosaicFinder, which additionally uses local alignments to discard false-positive candidates and indicates potential fusion points. The grouping into families will help distinguish sequencing or prediction errors from real biological fusions, and it will yield additional insight into the function and history of fused genes. AVAILABILITY: FusedTriplets and MosaicFinder are published under the GPL license and are freely available with their source code at this address: http://sourceforge.net/projects/mosaicfinder. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.