Simon Blouin

Comparative Study between Transcriptionally- and Translationally-Acting Adenine Riboswitches Reveals Key Differences in Riboswitch Regulatory Mechanisms

Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms.

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

Riboswitches: Ancient and Promising Genetic Regulators

Bait and switch: Metabolite‐sensing riboswitches make use of RNA structural modulation to regulate gene expression, as illustrated in the scheme, in response to subtle changes in metabolite concentrations. This review describes the current knowledge about naturally occurring riboswitches and their growing potential as antibacterial cellular targets and as molecular biosensors.

Folding of the lysine riboswitch: importance of peripheral elements for transcriptional regulation

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2–loop L3 and helix P2–loop L4)—all of this for the creation of a specific lysine binding site. We have determined that the interaction P2–L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2–L3 and P2–L4 allows both interactions to fold at lower magnesium ion concentrations. The P2–L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem–loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.

Functional Studies of DNA-Protein Interactions Using FRET Techniques

Protein-DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein-DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual Protein-DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of Protein-DNA interactions.

Maternal and paternal satisfaction in the delivery room: a cross-sectional comparative study

Objectives Maternal satisfaction during the birthing process has been well documented, whereas little is known about the fathers’ birth experiences. Our objective was to evaluate and compare the birth satisfaction of mothers and fathers. Design Comparative cross-sectional study. Setting Number of participating centres: one level III maternity centre (2813 births in 2011) in Sherbrooke, Quebec, Canada. Participants 200 mothers and 200 accompanying fathers/mothers partner recruited 12–24 h after the birth over a 6-week period. Primary and secondary outcome measures The Birth Satisfaction Scale (BSS) was used for the mother, and it was adapted to the fathers perspective. Paired-samples t tests were used for comparing mothers and fathers for the BSS global and thematic scores. Multiple linear regressions (forward stepwise method) were made to identify predicting factors of mothers’ and fathers’ satisfaction. Results Global satisfaction scores for mothers (115.5/150) and fathers (114.4/150) were relatively high and similar (p=0.116). The analysis of subthemes showed that more distress during childbirth was reported by mothers (p<0.001), while less support (p<0.001) and care satisfaction (p<0.001) were reported by fathers. The use of epidural anaesthesia during vaginal birth was the sole concordant lower satisfaction predictor. For mothers, other satisfaction predictors were labour length, tearing and type of anaesthesia used in caesarean section. For fathers, lower satisfaction predictors were instrumental delivery, primary caesarean delivery and infants distress factors after caesarean section. Conclusions This study highlights differences in mothers’ and fathers’ birth satisfaction and in their predictors. It is thus important to take into account the birth experience of each parent and to support parents accordingly by adapting care provision surrounding childbirth. More research on this topic from the prenatal to the postnatal period is suggested, as it might have an impact on parents’ satisfaction and on early parenthood experience.

Neuroprotective effects of antenatal magnesium sulfate under inflammatory conditions in a Sprague–Dawley pregnant rat model*

Abstract Objective: Antenatal magnesium sulfate (MgSO4) is recommended for fetal neuroprotection. The aim of this animal study was to assess the neuroprotective effect of in utero exposure to MgSO4, under inflammatory conditions. Methods: Timed pregnant Sprague–Dawley (SD) rats (n = 29) received four intra-peritoneal (IP) injections of lipopolysaccharides (LPS; 200 μg/kg), combined with increasing concentrations of MgSO4 (25, 50 or 100 mg/kg, n = 19) or saline solution (SS; n = 10). In the second set of experiments, animals (n = 8) received a single IP injection of i) LPS (500 μg/kg), MgSO4 (50 mg/kg) and SS (n = 4) or ii) LPS (500 μg/kg), MgSO4 (50 mg/kg) and IL-6 (12 μg/kg) (n = 4). Neurodevelopmental outcomes of surviving pups (n = 212) were assessed by the open field and the rotarod tests. Results: Pups’ average weight at postnatal day (P) 25 was 75.77 g and 89.08 g in MgSO4 and control groups, respectively (p = 0.02). Pups in MgSO4 group have traveled a shorter distance and have shown reduced motor balance and coordination (p < 0.01). Average weight of pups receiving (LPS + MgSO4+ IL-6) was 92.26 g at P25, compared to 75.86 g in (LPS + MgSO4+SS) group (p < 0.05). Conclusions: In our model, MgSO4 induces pup’s growth retardation and motor deficits, which may partly be related to a lower IL-6 circulating concentration.

Leukotriene receptor antagonist as a novel tocolytic in an in vitro model of human uterine contractility

OBJECTIVE This study analyzed the ability of montelukast, a cysteinyl-leukotrienes receptor antagonist and anti-inflammatory agent, to produce a consistent tocolytic effect alone or in combination with nifedipine, a calcium (Ca(2+)) channel blocker currently used in clinical practice. STUDY DESIGN Uterine biopsies were obtained from consenting women undergoing elective cesarean sections at term (n=20). Myometrial microsomal fractions were analyzed by immunoblotting to quantify relative cysteinyl leukotrienes receptor 1 (CysLTR1) levels. Isometric tension measurements were performed in vitro on human myometrial strips (n=120) in isolated organ baths in order to establish concentration-response curves to montelukast and to quantify changes in Ca(2+) sensitivity on β-escin permeabilized tissues. RESULTS Immunodetection analysis revealed the presence of CysLTR1 receptor in uterine tissues, fetal membranes and placenta. A significant increase in area under the curve (AUC) was quantified following the addition of leukotriene D4 (LTD4) (0.01-0.3 μM), an end-product of the lipoxygenase pathway. Conversely, addition of montelukast produced a significant tocolytic effect by decreasing the frequency and AUC (IC₅₀=1 μM). Moreover, addition of montelukast also resulted in a reduced Ca(2+) sensitivity as compared to control tissues (EC₅₀ values of 654 and 403 nM; p=0.02 at pCa 6), while an additive effect was observed in combination with 0.1 nM nifedipine (p=0.004). CONCLUSION This original study demonstrates the potency of montelukast as a tocolytic agent in an in vitro human uterine model. Montelukast, in combination with nifedipine, could represent a therapeutic approach to reduce inflammation associated with prematurity while facilitating the inhibition of preterm labor.

That was intense! Spirituality during childbirth: a mixed-method comparative study of mothers' and fathers' experiences in a public hospital.

BackgroundWhile spirituality is well described in end-of-life care literature, research on its place in the delivery room remains largely limited to mother-oriented qualitative studies focusing on life-threatening situations (e.g., high-risk pregnancies). Our aim was to compare mothers’ and fathers’ spirituality during childbirth.MethodsA mixed methods questionnaire was developed from our childbirth-related spirituality categorization and distributed to all parents of newborns, 12–24 h postpartum, over 45 consecutive days. Paired-sample t-tests and qualitative thematic analysis were used to compare mothers and fathers. Multiple linear regressions identified factors associated with their respective global scores (vaginal and cesarean deliveries separately).ResultsThe global scores for mothers (38.6/50) and fathers (37.2/50) were similarly high (N = 197; p = 0.001). Highest-ranked (“respect”, “moral responsibility”, “beauty of life”, “gratitude”) and lowest-ranked spiritual themes (“prayer”, “greater than self”) were in agreement. Fathers scored higher on “fragility of life” (p = 0.006) and mothers on “self-accomplishment” (p‹0.001), “letting go” (p‹0.001), and “meaningfulness” (p = 0.003). “Admission of baby in neonatal unit” was associated with higher global score for both mothers and fathers. Other factors also increased fathers’ (witnessing a severe tear) and mothers’ scores (birthplace outside Canada; for vaginal deliveries, religious belonging and longer pushing stage).ConclusionThese first quantitative data on the prevalence of spirituality during childbirth highlight a high score for both parents, among a non-selected public hospital population. Spirituality emerges not only from unordinary situations but from any childbirth as an “intensification of the human experience”. Significant differences for some spiritual themes indicate the need to consider the spirituality of both parents.