Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells-a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality

ADHD symptomatology in eating disorders : a secondary psychopathological measure of severity?

Background: Attention-deficit/hyperactivity disorder (ADHD) has commonly been described in psychiatric disorders. Although several studies have found positive associations between abnormal eating patterns during childhood and ADHD, there is a lack of studies on ADHD and Eating Disorders (ED). The aims of this exploratory study were 1) to assess the ADHD symptoms level in ED and to ascertain whether there are differences among ED subtypes; 2) to analyze whether the presence of ADHD symptoms is associated with more severe eating disorder symptoms and greater general psychopathology; and 3) to assess whether the ADHD symptoms level is associated with specific temperament and character traits. Methods: 191 female ED patients were included. Assessment was carried out with the EDI-2, ASRS-v1.1, the SCL-90-R and the TCI-R. Results: The ADHD symptoms level was similar in bulimia, eating disorder not otherwise specified and binge eating subtypes, and lower in anorexic patients. Obsessiveness and Hostility were significantly positively associated with ADHD symptoms. A path model showed that ADHD was associated with high Novelty Seeking and low Self-Directedness, whereas ED severity was influenced by ADHD severity and low Self-Directedness. Conclusions: Bingeing/purging ED subtypes have a high ADHD symptoms level, also related with more severe eating, general and personality psychopathology

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis

Changes in Body Composition in Anorexia Nervosa: Predictors of Recovery and Treatment Outcome

The restoration of body composition (BC) parameters is considered to be one of the most important goals in the treatment of patients with anorexia nervosa (AN). However, little is known about differences between AN diagnostic subtypes [restricting (AN-R) and binge/purging (AN-BP)] and weekly changes in BC during refeeding treatment. Therefore, the main objectives of our study were twofold: 1) to assess the changes in BC throughout nutritional treatment in an AN sample and 2) to analyze predictors of BC changes during treatment, as well as predictors of treatment outcome. The whole sample comprised 261 participants [118 adult females with AN (70 AN-R vs. 48 AN-BP), and 143 healthy controls]. BC was measured weekly during 15 weeks of day-hospital treatment using bioelectrical impedance analysis (BIA). Assessment measures also included the Eating Disorders Inventory-2, as well as a number of other clinical indices. Overall, the results showed that AN-R and AN-BP patients statistically differed in all BC measures at admission. However, no significant time×group interaction was found for almost all BC parameters. Significant time×group interactions were only found for basal metabolic rate (p = .041) and body mass index (BMI) (p = .035). Multiple regression models showed that the best predictors of pre-post changes in BC parameters (namely fat-free mass, muscular mass, total body water and BMI) were the baseline values of BC parameters. Stepwise predictive logistic regressions showed that only BMI and age were significantly associated with outcome, but not with the percentage of body fat. In conclusion, these data suggest that although AN patients tended to restore all BC parameters during nutritional treatment, only AN-BP patients obtained the same fat mass values as healthy controls. Put succinctly, the best predictors of changes in BC were baseline BC values, which did not, however, seem to influence treatment outcome

Rate and duration of hospitalisation for acute pulmonary embolism in the real-world clinical practice of different countries : Analysis from the RIETE registry

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Mapping of 3’ termini of DrrS variants.

<p>The panels illustrate how the DrrS 3’ termini vary as the 3’ region is modified. The top panel shows the 3’ RACE results of wildtype DrrS sequence with two ATCCTC repeats, which in <i>M</i>. <i>tuberculosis</i> leads to the majority of transcripts ending in U or C within the first repeat (indicated with red, vertical lines). Panels 2–4 show the results of DrrS variants expressed in <i>M</i>. <i>smegmatis</i>; panel 2 represents 3’ RACE results of DrrS cloned with two ATCCTC repeats before the SynB terminator, similar to wildtype DrrS; panel 3 shows the results when an extra repeat has been inserted, and panel 4 the results when only one repeat is included.</p

Turnover of DrrS in <i>M</i>. <i>tuberculosis</i>.

<p>A stationary phase culture of <i>M</i>. <i>tuberculosis</i> OD₆₀₀ ~6 was diluted into fresh, pre-warmed medium to OD₆₀₀ = 0.4 either without rifampicin (panel A and C) or with 200 μg/ml rifampicin (panel B and D) and samples removed for RNA extraction and Northern blotting at the indicated time points. A and B: Northerns probed for DrrS; C and D: Northerns re-probed for MTS2823.</p

DrrS promoter activity.

<p>A: ß-galactosidase assays of DrrS promoter-reporter fusions containing either core or ‘full-length’ promoter, including DBS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174079#pone.0174079.g002" target="_blank">Fig 2</a>) were transformed into <i>M</i>. <i>bovis</i> BCG. Stationary phase cultures (Sta) were grown one week past OD₆₀₀ = 1.0 before harvest and ß-galactosidase assays performed on cell extracts. Results represent mean ± standard deviation of three biological replicates normalised to total protein and values for empty vector subtracted; p<0.05. B: ß-galactosidase assays of DrrS promoter-reporter fusions expressed in <i>M</i>. <i>smegmatis</i> mc²155 or C: Δ<i>dosR</i>_<i>sm</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174079#pone.0174079.ref045" target="_blank">45</a>] with and without <i>dosR</i>_<i>tb</i>. The assays were performed on exponential phase cultures and the Results represent mean ± standard deviation of three biological replicates normalised to total protein and values for empty vector subtracted; p<0.05.</p

Turnover of DrrS and its 5’ derivatives in <i>M</i>. <i>smegmatis</i>.

<p>A: Northern blot of total RNA from <i>M</i>. <i>smegmatis</i> expressing DrrS from a heterologous promoter. Rifampicin (200 μg/ml rifampicin) was added at time zero and RNA was isolated at the indicated time point and probed for DrrS as previously described. B: Northern blot of 1–20 μg of total RNA from <i>M</i>. <i>smegmatis</i> expressing wildtype and 5’ variants of DrrS. RNA from the strain expressing wildtype DrrS was loaded in the first and last wells to ensure proper alignment. C: Turnover of DrrS 5’ variants. Each DrrS 5’ variant was expressed in <i>M</i>. <i>smegmatis</i>, cultures were grown to OD₆₀₀ = 0.6 and rifampicin added to a final concentration of 200 μg/ml. Samples were withdrawn at the indicated time points and subjected to Northern blotting. As the relative amount of DrrS varied significantly between the wildtype and the derivatives (see panel B), different amounts of total RNA were loaded on the gels, i.e. 1 μg each wildtype and A₁DrrS; 2 μg of A₂DrrS; 10 μg A₃DrrS and 20 μg A₄DrrS. Cartoon on the right illustrate the 5’ additions (red asterisks) to the DrrS structure.</p