Transcript tinkering: RNA modifications in protozoan parasites.

Apicomplexan and trypanosomatid parasites have evolved a wide range of post-transcriptional processes that allow them to replicate, differentiate, and transmit within and among multiple different tissue, host, and vector environments. In this review, we highlight the recent advances that point toward the regulatory potential of RNA modifications in mediating these processes on the coding and noncoding transcriptome throughout the life cycle of protozoan parasites. We discuss the recent technical advancements enabling the study of the 'epitranscriptome' and how parasites evolved RNA modification-mediated mechanisms adapted to their unique lifestyles.

Optimization of highly inclined illumination for diffraction-limited and super-resolution microscopy.

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and of how this impacts on the image quality complicates the setting of HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the effects of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 µm). Based on this, we present a simple optical solution that exploits a rectangular slit to reduce the inclined beam thickness down to 2.6 µm while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

Characterization of substituted piperazines able to reverse MDR in Escherichia coli strains overexpressing resistance-nodulation-cell division (RND) efflux pumps.

BACKGROUND: MDR in bacteria is threatening to public health. Overexpression of efflux pumps is an important cause of MDR. The co-administration of antimicrobial drugs and efflux pump inhibitors (EPIs) is a promising approach to address the problem of MDR. OBJECTIVES: To identify new putative EPIs and to characterize their mechanisms of action. METHODS: The effects of four selected piperazine derivatives on resistance-nodulation-cell division (RND) pumps was evaluated in Escherichia coli strains overexpressing or not expressing RND pumps by assays aimed at evaluating antibiotic potentiation, membrane functionality, ethidium bromide accumulation and AcrB expression. The cytotoxicity of selected piperazines towards primary cultures of human dermal fibroblasts was also investigated. RESULTS: Four molecules enhanced levofloxacin activity against strains overexpressing RND efflux pumps (AcrAB-TolC and AcrEF-TolC), but not against RND pump-deficient strains. They had little effects on membrane potential. Molecule 4 decreased, whereas the other three increased, membrane permeability compared with untreated control cells. The four molecules showed differences in the specificity of interaction with RND efflux pumps, by inactivating the transport of one or more antibiotics, and in the levels of ethidium bromide accumulation and of acrB expression inhibition. CONCLUSIONS: Piperazine derivatives are good candidates as inhibitors of RND efflux pumps. They decreased the activity of RND pumps by mixed mechanisms of action. Small structural differences among the molecules can be critical in defining their behaviour.

1-benzyl-1,4-diazepane reduces the efflux of resistance-nodulation-cell division pumps in Escherichia coli.

Aim: To investigate the action mechanism of 1-benzyl-1,4-diazepane (1-BD) as efflux pump inhibitor (EPI) in Escherichia coli mutants: ΔacrAB or overexpressing AcrAB and AcrEF efflux pumps. Materials & methods: Effect of 1-BD on: antibiotic potentiation, by microdilution method; membrane functionality, by fluorimetric assays; ethidium bromide accumulation, by fluorometric real-time efflux assay; AcrB expression, by quantitative photoactivated localization microscopy. Results: 1-BD decreases the minimal inhibitory concentration of levofloxacin and other antibiotics and increase ethidium bromide accumulation in E. coli overexpressing efflux pumps but not in the ΔacrAB strain. 1-BD increases membranes permeability, without sensibly affecting inner membrane polarity and decreases acrAB transcription. Conclusion: 1-BD acts as an EPI in E. coli with a mixed mechanism, different from that of major reference EPIs.

Creation and Characterization of a Genomically Hybrid Strain in the Nitrogen-Fixing Symbiotic Bacterium Sinorhizobium meliloti.

Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbor a multipartite genome composed of more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti. We term this strain as cis-hybrid, since it is produced by genomic material coming from the same species' pangenome. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S. meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extracellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modeling were employed for in silico prediction of metabolic flux reorganization. Phenotypes of the cis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with the cis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.

Template-Assisted Metabolic Reconstruction and Assembly of Hybrid Bacterial Models.

Intraspecific genomic exchanges happen frequently between bacteria living in the same natural environment and can also be performed artificially in the laboratory for basic research or genetic/metabolic engineering purposes. In silico metabolic reconstruction and simulation of the metabolism of the hybrid strains that result from these processes can be used to predict the phenotypic outcome of such genomic rearrangements; this can be especially helpful as a designing tool in the purview of synthetic biology. However, reconstructing the metabolism of a bacterium with a hybrid genome through in silico approaches is not a trivial task, as it requires taking into account the complex relationships existing between metabolic genes and how they change (or remain unchanged) when new genes are placed in a different genomic context. Furthermore, in order to "mix" the metabolic models of different bacterial strains one needs at least two different metabolic models to begin with, and reconstructing a genome-scale model from the ground up is a challenging task itself, requiring an intensive manual effort and a great deal of information. In this chapter, we propose two general protocols to address the aforementioned issues of: (1) quickly generating strain-specific metabolic models, given the relevant genomic sequence and an already existing, high-quality metabolic model of a different strain belonging to the same species, and (2) reconstructing the metabolic model of a hybrid strain containing genomic elements from two different parental strains.

Investigation on torquetenovirus (TTV) microRNA transcriptome in vivo.

Torquetenovirus (TTV) is a widespread anellovirus that establishes persistent infections in human showing an increased viremia in immunosuppressed patients. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Here, the presence of TTV DNA and miRNAs expression was investigated in plasma samples of 77 diseased (20 infected with human immunodeficiency virus (HIV), 18 infected with hepatitis B (HBV) virus, 18 infected with hepatitis C (HCV) virus, 21 solid organ transplanted) patients, and 25 healthy controls. TTV prevalence was significantly different in healthy controls (60%, 15/25) versus diseased patients (80%, 62/77), showing the highest TTV loads in transplant recipients. Genetic TTV analysis showed the highest prevalence of group 1, followed by groups 3, 4 and 5, and a lack of isolates of group 2. The expression of at least one TTV miRNAs of group 1, 3 and 5 was found in exosomes of plasma of the great majority of individuals (96%, 98/102 subjects) showing the higher prevalence of miRNAs of TTV group 3 (90%, 92/102), followed by miRNAs of group 1 (66%, 67/102), and miRNA of group 5 (49%, 50/102). TTV miRNAs expression and TTV viremia were not always directly correlated, and significant differences appeared in production of some TTV miRNAs between healthy controls and diseased patients. The reported TTV miRNAs status in exosomes encourages further investigation to understand their potential role in the expansion of anelloviruses upon immunosuppression.