Development of an In Vitro Chloroplast Splicing System: Sequences Required for Correct pre-mRNA Splicing.

Chloroplast genomes in land plants include approximately 20 intron-containing genes. Most of the introns are similar to the group II introns found in fungi, algae and some bacteria, but no self-splicing has been reported. To analyze splicing reactions in chloroplasts, we developed a tobacco (Nicotiana tabacum) chloroplast-based in vitro system. We optimized the splicing reaction using atpF precursor messenger RNA (pre-mRNA). Our system requires a high ATP concentration, whereas ATP is not necessary for self-splicing group II introns. Self-splicing group II introns possess two exon-binding sites (EBS1 and 2) complementary to two intron-binding sites (IBS1 and 2) in the 3' end of 5' exons, which are involved in 5' splice-site selection. Using our in vitro system and atpF pre-mRNA, we analyzed short sequences corresponding to the above EBSs and IBSs. Mutation analyses revealed that EBS1-IBS1 pairing is essential, while EBS2-IBS2 pairing is important but not crucial for splicing. The first 3' exon nucleotide determines the 3' splice sites of self-splicing introns. However, mutations to this nucleotide in atpF pre-mRNA did not affect splicing. This result suggests that the mechanism underlying chloroplast pre-mRNA splicing differs partly from that mediating the self-splicing of group II introns.

Updating of in vitro antifungal susceptibility tests. / Actualización de los métodos de estudio de sensibilidad in vitro a los antifúngicos.

Fungal diseases, including those caused by (multi)drug-resistant fungi, still represent a global public health concern. Information on the susceptibility of these microorganisms to antifungal agents must be quickly produced to help clinicians initiate appropriate antifungal therapies. Unfortunately, antifungal susceptibility tests are not as developed or widely implemented as antibacterial tests, being similar in design, accuracy and reproducibility, but also laborious and slow. In this article, we review the methods of in vitro susceptibility testing, both reference (CLSI and EUCAST), commercial and new methods based on proteomics (MALDI-TOF MS) and in the detection of resistance genes by nucleic acid amplification techniques. In addi-tion, we discuss the newly established clinical breakpoints, as well as the epidemiological cut-off points, which constitute a new category that can help in the early identification of isolates that have acquired resistance mechanisms. We also discuss the advantages and limitations of each of the methods studied. Therefore, we can conclude that, although there has been much progress in studies of in vitro susceptibility testing to antifungals, there are still limitations in its application in the daily routine of microbiology labo-ratories, although it seems that the future is promising with the new technologies based on proteomics and nucleic acid amplification. Supplement information: This article is part of a supplement entitled «SEIMC External Quality Control Programme. Year 2016¼, which is sponsored by Roche, Vircell Microbiologists, Abbott Molecular and Francisco Soria Melguizo, S.A. © 2019 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosasy Microbiología Clínica. All rights reserved.