Global 5'-UTR RNA structure regulates translation of a SERPINA1 mRNA.

SERPINA1 mRNAs encode the protease inhibitor α-1-antitrypsin and are regulated through post-transcriptional mechanisms. α-1-antitrypsin deficiency leads to chronic obstructive pulmonary disease (COPD) and liver cirrhosis, and specific variants in the 5'-untranslated region (5'-UTR) are associated with COPD. The NM_000295.4 transcript is well expressed and translated in lung and blood and features an extended 5'-UTR that does not contain a competing upstream open reading frame (uORF). We show that the 5'-UTR of NM_000295.4 folds into a well-defined multi-helix structural domain. We systematically destabilized mRNA structure across the NM_000295.4 5'-UTR, and measured changes in (SHAPE quantified) RNA structure and cap-dependent translation relative to a native-sequence reporter. Surprisingly, despite destabilizing local RNA structure, most mutations either had no effect on or decreased translation. Most structure-destabilizing mutations retained native, global 5'-UTR structure. However, those mutations that disrupted the helix that anchors the 5'-UTR domain yielded three groups of non-native structures. Two of these non-native structure groups refolded to create a stable helix near the translation initiation site that decreases translation. Thus, in contrast to the conventional model that RNA structure in 5'-UTRs primarily inhibits translation, complex folding of the NM_000295.4 5'-UTR creates a translation-optimized message by promoting accessibility at the translation initiation site.

Implementing the WHO Labour Care Guide to reduce the use of Caesarean section in four hospitals in India: protocol and statistical analysis plan for a pragmatic, stepped-wedge, cluster-randomized pilot trial.

BACKGROUND: The World Health Organization (WHO) Labour Care Guide (LCG) is a paper-based labour monitoring tool designed to facilitate the implementation of WHO's latest guidelines for effective, respectful care during labour and childbirth. Implementing the LCG into routine intrapartum care requires a strategy that improves healthcare provider practices during labour and childbirth. Such a strategy might optimize the use of Caesarean section (CS), along with potential benefits on the use of other obstetric interventions, maternal and perinatal health outcomes, and women's experience of care. However, the effects of a strategy to implement the LCG have not been evaluated in a randomised trial. This study aims to: (1) develop and optimise a strategy for implementing the LCG (formative phase); and (2) To evaluate the implementation of the LCG strategy compared with usual care (trial phase). METHODS: In the formative phase, we will co-design the LCG strategy with key stakeholders informed by facility assessments and provider surveys, which will be field tested in one hospital. The LCG strategy includes a LCG training program, ongoing supportive supervision from senior clinical staff, and audit and feedback using the Robson Classification. We will then conduct a stepped-wedge, cluster-randomized pilot trial in four public hospitals in India, to evaluate the effect of the LCG strategy intervention compared to usual care (simplified WHO partograph). The primary outcome is the CS rate in nulliparous women with singleton, term, cephalic pregnancies in spontaneous labour (Robson Group 1). Secondary outcomes include clinical and process of care outcomes, as well as women's experience of care outcomes. We will also conduct a process evaluation during the trial, using standardized facility assessments, in-depth interviews and surveys with providers, audits of completed LCGs, labour ward observations and document reviews. An economic evaluation will consider implementation costs and cost-effectiveness. DISCUSSION: Findings of this trial will guide clinicians, administrators and policymakers on how to effectively implement the LCG, and what (if any) effects the LCG strategy has on process of care, health and experience outcomes. The trial findings will inform the rollout of LCG internationally. TRIAL REGISTRATION: CTRI/2021/01/030695 (Protocol version 1.4, 25 April 2022).

The new WHO Labour Care Guide (LCG) is an innovative partograph that emphasises women-centred, evidence-based care during labour and childbirth. Together with clinicians working at four hospitals in India, we will develop and test a strategy to implement the LCG into routine care in labour wards of these hospitals. We will use a randomised trial design where this LCG strategy is introduced sequentially in each of the four hospitals, in a random order. We will collect data on all women giving birth and their newborns during this period and analyse whether the LCG strategy has any effects on the use of Caesarean section, women's and newborn's health outcomes, and women's experiences during labour and childbirth. While the trial is being conducted, we will also collect qualitative and quantitative data from doctors, nurses and midwives working in these hospitals, to understand their perspectives and experiences of using the LCG in their day-to-day work. In addition, we will collect economic data to understand how much the LCG strategy costs, and how much money it might save if it is effective. Through this study, our international collaboration will generate critical evidence and innovative tools to support implementation of the LCG in other countries.

Disease-associated human genetic variation through the lens of precursor and mature RNA structure.

Disease-associated variants (DAVs) are commonly considered either through a genomic lens that describes variant function at the DNA level, or at the protein function level if the variant is translated. Although the genomic and proteomic effects of variation are well-characterized, genetic variants disrupting post-transcriptional regulation is another mechanism of disease that remains understudied. Specific RNA sequence motifs mediate post-transcriptional regulation both in the nucleus and cytoplasm of eukaryotic cells, often by binding to RNA-binding proteins or other RNAs. However, many DAVs map far from these motifs, which suggests deeper layers of post-transcriptional mechanistic control. Here, we consider a transcriptomic framework to outline the importance of post-transcriptional regulation as a mechanism of disease-causing single-nucleotide variation in the human genome. We first describe the composition of the human transcriptome and the importance of abundant yet overlooked components such as introns and untranslated regions (UTRs) of messenger RNAs (mRNAs). We present an analysis of Human Gene Mutation Database variants mapping to mRNAs and examine the distribution of causative disease-associated variation across the transcriptome. Although our analysis confirms the importance of post-transcriptional regulatory motifs, a majority of DAVs do not directly map to known regulatory motifs. Therefore, we review evidence that regions outside these well-characterized motifs can regulate function by RNA structure-mediated mechanisms in all four elements of an mRNA: exons, introns, 5' and 3' UTRs. To this end, we review published examples of riboSNitches, which are single-nucleotide variants that result in a change in RNA structure that is causative of the disease phenotype. In this review, we present the current state of knowledge of how DAVs act at the transcriptome level, both through altering post-transcriptional regulatory motifs and by the effects of RNA structure.

Effects of the WHO Labour Care Guide on cesarean section in India: a pragmatic, stepped-wedge, cluster-randomized pilot trial.

Cesarean section rates worldwide are rising, driven by medically unnecessary cesarean use. The new World Health Organization Labour Care Guide (LCG) aims to improve the quality of care for women during labor and childbirth. Using the LCG might reduce overuse of cesarean; however, its effects have not been evaluated in randomized trials. We conducted a stepped-wedge, cluster-randomized pilot trial in four hospitals in India to evaluate the implementation of an LCG strategy intervention, compared with routine care. We performed this trial to pilot the intervention and obtain preliminary effectiveness data, informing future research. Eligible clusters were four hospitals with >4,000 births annually and cesarean rates ≥30%. Eligible women were those giving birth at ≥20 weeks' gestation. One hospital transitioned to intervention every 2 months, according to a random sequence. The primary outcome was the cesarean rate among women in Robson Group 1 (that is, those who were nulliparous and gave birth to a singleton, term pregnancy in cephalic presentation and in spontaneous labor). A total of 26,331 participants gave birth. A 5.5% crude absolute reduction in the primary outcome was observed (45.2% versus 39.7%; relative risk 0.85, 95% confidence interval 0.54-1.33). Maternal process-of-care outcomes were not significantly different, though labor augmentation with oxytocin was 18.0% lower with the LCG strategy. No differences were observed for other health outcomes or women's birth experiences. These findings can guide future definitive effectiveness trials, particularly in settings where urgent reversal of rising cesarean section rates is needed. Clinical Trials Registry India number: CTRI/2021/01/030695 .

Remi-fent 1-A pragmatic randomised controlled study to evaluate the feasibility of using remifentanil or fentanyl as sedation adjuncts in mechanically ventilated patients.

Objective: To evaluate the feasibility of conducting a prospective randomised controlled trial (pRCT) comparing remifentanil and fentanyl as adjuncts to sedate mechanically ventilated patients. Design: Single-center, open-labelled, pRCT with blinded analysis. Setting: Australian tertiary intensive care unit (ICU). Participants: Consecutive adults between June 2020 and August 2021 expected to receive invasive ventilation beyond the next day and requiring opioid infusion were included. Exclusion criteria were pregnant/lactating women, intubation >12 h, or study-drug hypersensitivity. Interventions: Open-label fentanyl and remifentanil infusions per existing ICU protocols. Outcomes: Primary outcomes were feasibility of recruiting ≥1 patient/week and >90 % compliance, namely no other opioid infusion used during the study period. Secondary outcomes included complications, ICU-, ventilator- and hospital-free days, and mortality (ICU, hospital). Blinded intention-to-treat analysis was performed concealing the allocation group. Results: 208 patients were enrolled (mean 3.7 patients/week). Compliance was 80.6 %. More patients developed complications with fentanyl than remifentanil: bradycardia (n = 44 versus n = 21; p < 0.001); hypotension (n = 78 versus n = 53; p < 0.01); delirium (n = 28 versus n = 15; p = 0.001). No differences were seen in ICU (24.3 % versus 27.6 %,p = 0.60) and hospital mortalities (26.2 % versus 30.5 %; p = 0.50). Ventilator-free days were higher with remifentanil (p = 0.01). Conclusions: We demonstrated the feasibility of enrolling patients for a pRCT comparing remifentanil and fentanyl as sedation adjuncts in mechanically ventilated patients. We failed to attain the study-opioid compliance target, likely because of patients with complex sedative/analgesic requirements. Secondary outcomes suggest that remifentanil may reduce mechanical ventilation duration and decrease the incidence of complications. An adequately powered multicentric phase 2 study is required to evaluate these results.

Quantitative prediction of variant effects on alternative splicing in MAPT using endogenous pre-messenger RNA structure probing.

Splicing is highly regulated and is modulated by numerous factors. Quantitative predictions for how a mutation will affect precursor mRNA (pre-mRNA) structure and downstream function are particularly challenging. Here, we use a novel chemical probing strategy to visualize endogenous precursor and mature MAPT mRNA structures in cells. We used these data to estimate Boltzmann suboptimal structural ensembles, which were then analyzed to predict consequences of mutations on pre-mRNA structure. Further analysis of recent cryo-EM structures of the spliceosome at different stages of the splicing cycle revealed that the footprint of the Bact complex with pre-mRNA best predicted alternative splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene, achieving 74% accuracy. We further developed a ß-regression weighting framework that incorporates splice site strength, RNA structure, and exonic/intronic splicing regulatory elements capable of predicting, with 90% accuracy, the effects of 47 known and 6 newly discovered mutations on inclusion of exon 10 of MAPT. This combined experimental and computational framework represents a path forward for accurate prediction of splicing-related disease-causing variants.

The Cell Cycle Browser: An Interactive Tool for Visualizing, Simulating, and Perturbing Cell-Cycle Progression.

The cell cycle is driven by precise temporal coordination among many molecular activities. To understand and explore this process, we developed the Cell Cycle Browser (CCB), an interactive web interface based on real-time reporter data collected in proliferating human cells. This tool facilitates visualizing, organizing, simulating, and predicting the outcomes of perturbing cell-cycle parameters. Time-series traces from individual cells can be combined to build a multi-layered timeline of molecular activities. Users can simulate the cell cycle using computational models that capture the dynamics of molecular activities and phase transitions. By adjusting individual expression levels and strengths of molecular relationships, users can predict effects on the cell cycle. Virtual assays, such as growth curves and flow cytometry, provide familiar outputs to compare cell-cycle behaviors for data and simulations. The CCB serves to unify our understanding of cell-cycle dynamics and provides a platform for generating hypotheses through virtual experiments.

Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes.

Saccharomyces cerevisiae expresses two Pif1-family helicases-Pif1 and Rrm3-which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterized the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulates strand displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication-fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes. In agreement with this observation, we found that head-on collisions between tRNA transcription and replication are under-represented in the S. cerevisiae genome. We demonstrate that tRNA-mediated arrest is R-loop independent and propose that replisome arrest and DNA damage are mechanistically separable.

Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells.

During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing.

Usability evaluation of low-cost virtual reality hand and arm rehabilitation games.

The emergence of lower-cost motion tracking devices enables home-based virtual reality rehabilitation activities and increased accessibility to patients. Currently, little documentation on patients' expectations for virtual reality rehabilitation is available. This study surveyed 10 people with stroke for their expectations of virtual reality rehabilitation games. This study also evaluated the usability of three lower-cost virtual reality rehabilitation games using a survey and House of Quality analysis. The games (kitchen, archery, and puzzle) were developed in the laboratory to encourage coordinated finger and arm movements. Lower-cost motion tracking devices, the P5 Glove and Microsoft Kinect, were used to record the movements. People with stroke were found to desire motivating and easy-to-use games with clinical insights and encouragement from therapists. The House of Quality analysis revealed that the games should be improved by obtaining evidence for clinical effectiveness, including clinical feedback regarding improving functional abilities, adapting the games to the user's changing functional ability, and improving usability of the motion-tracking devices. This study reports the expectations of people with stroke for rehabilitation games and usability analysis that can help guide development of future games.