Design principles for riboswitch function

Scientific and technological advances that enable the tuning of integrated regulatory components to match network and system requirements are critical to reliably control the function of biological systems. RNA provides a promising building block for the construction of tunable regulatory components based on its rich regulatory capacity and our current understanding of the sequence–function relationship. One prominent example of RNA-based regulatory components is riboswitches, genetic elements that mediate ligand control of gene expression through diverse regulatory mechanisms. While characterization of natural and synthetic riboswitches has revealed that riboswitch function can be modulated through sequence alteration, no quantitative frameworks exist to investigate or guide riboswitch tuning. Here, we combined mathematical modeling and experimental approaches to investigate the relationship between riboswitch function and performance. Model results demonstrated that the competition between reversible and irreversible rate constants dictates performance for different regulatory mechanisms. We also found that practical system restrictions, such as an upper limit on ligand concentration, can significantly alter the requirements for riboswitch performance, necessitating alternative tuning strategies. Previous experimental data for natural and synthetic riboswitches as well as experiments conducted in this work support model predictions. From our results, we developed a set of general design principles for synthetic riboswitches. Our results also provide a foundation from which to investigate how natural riboswitches are tuned to meet systems-level regulatory demands

Maximum expected accuracy structural neighbors of an RNA secondary structure

International audienceBACKGROUND: Since RNA molecules regulate genes and control alternative splicing by allostery, it is important to develop algorithms to predict RNA conformational switches. Some tools, such as paRNAss, RNAshapes and RNAbor, can be used to predict potential conformational switches; nevertheless, no existent tool can detect general (i.e., not family specific) entire riboswitches (both aptamer and expression platform) with accuracy. Thus, the development of additional algorithms to detect conformational switches seems important, especially since the difference in free energy between the two metastable secondary structures may be as large as 15-20 kcal/mol. It has recently emerged that RNA secondary structure can be more accurately predicted by computing the maximum expected accuracy (MEA) structure, rather than the minimum free energy (MFE) structure. RESULTS: Given an arbitrary RNA secondary structure S₀ for an RNA nucleotide sequence a = a₁,..., a(n), we say that another secondary structure S of a is a k-neighbor of S₀, if the base pair distance between S₀ and S is k. In this paper, we prove that the Boltzmann probability of all k-neighbors of the minimum free energy structure S₀ can be approximated with accuracy ε and confidence 1 - p, simultaneously for all 0 ≤ k N(ε,p,K)=Φ⁻¹(p/2K)²/4ε², where Φ(z) is the cumulative distribution function (CDF) for the standard normal distribution. We go on to describe the algorithm RNAborMEA, which for an arbitrary initial structure S₀ and for all values 0 ≤ k < K, computes the secondary structure MEA(k), having maximum expected accuracy over all k-neighbors of S₀. Computation time is O(n³ * K²), and memory requirements are O(n² * K). We analyze a sample TPP riboswitch, and apply our algorithm to the class of purine riboswitches. CONCLUSIONS: The approximation of RNAbor by sampling, with rigorous bound on accuracy, together with the computation of maximum expected accuracy k-neighbors by RNAborMEA, provide additional tools toward conformational switch detection. Results from RNAborMEA are quite distinct from other tools, such as RNAbor, RNAshapes and paRNAss, hence may provide orthogonal information when looking for suboptimal structures or conformational switches. Source code for RNAborMEA can be downloaded from http://sourceforge.net/projects/rnabormea/ or http://bioinformatics.bc.edu/clotelab/RNAborMEA/

Intravenous doxycycline, azithromycin, or both for severe scrub typhus

BACKGROUND: The appropriate antibiotic treatment for severe scrub typhus, a neglected but widespread reemerging zoonotic infection, is unclear. METHODS: In this multicenter, double-blind, randomized, controlled trial, we compared the efficacy of intravenous doxycycline, azithromycin, or a combination of both in treating severe scrub typhus. Patients who were 15 years of age or older with severe scrub typhus with at least one organ involvement were enrolled. The patients were assigned to receive a 7-day course of intravenous doxycycline, azithromycin, or both (combination therapy). The primary outcome was a composite of death from any cause at day 28, persistent complications at day 7, and persistent fever at day 5. RESULTS: Among 794 patients (median age, 48 years) who were included in the modified intention-to-treat analysis, complications included those that were respiratory (in 62%), hepatic (in 54%), cardiovascular (in 42%), renal (in 30%), and neurologic (in 20%). The use of combination therapy resulted in a lower incidence of the composite primary outcome than the use of doxycycline (33% and 47%, respectively), for a risk difference of −13.3 percentage points (95% confidence interval [CI], (21.6 to −5.1; P=0.002). The incidence with combination therapy was also lower than that with azithromycin (48%), for a risk difference of −14.8 percentage points (95% CI, −23.1 to −6.5; P<0.001). No significant difference was seen between the azithromycin and doxycycline groups (risk difference, 1.5 percentage points; 95% CI, −7.0 to 10.0; P=0.73). The results in the per-protocol analysis were similar to those in the primary analysis. Adverse events and 28-day mortality were similar in the three groups. CONCLUSIONS: Combination therapy with intravenous doxycycline and azithromycin was a better therapeutic option for the treatment of severe scrub typhus than monotherapy with either drug alone. (Funded by the India Alliance and Wellcome Trust; INTREST Clinical Trials Registry–India number, CTRI/2018/08/015159.

Efficient Algorithms for Probing the RNA Mutation Landscape

The diversity and importance of the role played by RNAs in the regulation and development of the cell are now well-known and well-documented. This broad range of functions is achieved through specific structures that have been (presumably) optimized through evolution. State-of-the-art methods, such as McCaskill's algorithm, use a statistical mechanics framework based on the computation of the partition function over the canonical ensemble of all possible secondary structures on a given sequence. Although secondary structure predictions from thermodynamics-based algorithms are not as accurate as methods employing comparative genomics, the former methods are the only available tools to investigate novel RNAs, such as the many RNAs of unknown function recently reported by the ENCODE consortium. In this paper, we generalize the McCaskill partition function algorithm to sum over the grand canonical ensemble of all secondary structures of all mutants of the given sequence. Specifically, our new program, RNAmutants, simultaneously computes for each integer k the minimum free energy structure MFE(k) and the partition function Z(k) over all secondary structures of all k-point mutants, even allowing the user to specify certain positions required not to mutate and certain positions required to base-pair or remain unpaired. This technically important extension allows us to study the resilience of an RNA molecule to pointwise mutations. By computing the mutation profile of a sequence, a novel graphical representation of the mutational tendency of nucleotide positions, we analyze the deleterious nature of mutating specific nucleotide positions or groups of positions. We have successfully applied RNAmutants to investigate deleterious mutations (mutations that radically modify the secondary structure) in the Hepatitis C virus cis-acting replication element and to evaluate the evolutionary pressure applied on different regions of the HIV trans-activation response element. In particular, we show qualitative agreement between published Hepatitis C and HIV experimental mutagenesis studies and our analysis of deleterious mutations using RNAmutants. Our work also predicts other deleterious mutations, which could be verified experimentally. Finally, we provide evidence that the 3′ UTR of the GB RNA virus C has been optimized to preserve evolutionarily conserved stem regions from a deleterious effect of pointwise mutations. We hope that there will be long-term potential applications of RNAmutants in de novo RNA design and drug design against RNA viruses. This work also suggests potential applications for large-scale exploration of the RNA sequence-structure network. Binary distributions are available at http://RNAmutants.csail.mit.edu/

Mutations in Hnrnpa1 cause congenital heart defects

Incomplete penetrance of congenital heart defects (CHDs) was observed in a mouse model. We hypothesized that the contribution of a major genetic locus modulates the manifestation of the CHDs. After genome-wide linkage mapping, fine mapping, and high-throughput targeted sequencing, a recessive frameshift mutation of the heterogeneous nuclear ribonucleoprotein A1 (Hnrnpa1) gene was confirmed (Hnrnpa1ct). Hnrnpa1 was expressed in both the first heart field (FHF) and second heart field (SHF) at the cardiac crescent stage but was only maintained in SHF progenitors after heart tube formation. Hnrnpa1ct/ct homozygous mutants displayed complete CHD penetrance, including truncated and incomplete looped heart tube at E9.5, ventricular septal defect (VSD) and persistent truncus arteriosus (PTA) at E13.5, and VSD and double outlet right ventricle at P0. Impaired development of the dorsal mesocardium and sinoatrial node progenitors was also observed. Loss of Hnrnpa1 expression leads to dysregulation of cardiac transcription networks and multiple signaling pathways, including BMP, FGF, and Notch in the SHF. Finally, two rare heterozygous mutations of HNRNPA1 were detected in human CHDs. These findings suggest a role of Hnrnpa1 in embryonic heart development in mice and humans.published_or_final_versio

Pooled analysis of who surgical safety checklist use and mortality after emergency laparotomy

Background: The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods: In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results: Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89⋅6 per cent) compared with that in countries with a middle (753 of 1242, 60⋅6 per cent; odds ratio (OR) 0⋅17, 95 per cent c.i. 0⋅14 to 0⋅21, P &lt; 0⋅001) or low (363 of 860, 42⋅2 percent; OR 0⋅08, 0⋅07 to 0⋅10, P &lt; 0⋅001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference −9⋅4 (95 per cent c.i. −11⋅9 to −6⋅9) per cent; P &lt; 0⋅001), but the relationship was reversed in low-HDI countries (+12⋅1 (+7⋅0 to +17⋅3) per cent; P &lt; 0⋅001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0⋅60, 0⋅50 to 0⋅73; P &lt; 0⋅001). The greatest absolute benefit was seen for emergency surgery in low-and middle-HDI countries. Conclusion: Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries

Evaluation of prognostic risk models for postoperative pulmonary complications in adult patients undergoing major abdominal surgery: a systematic review and international external validation cohort study

Background Stratifying risk of postoperative pulmonary complications after major abdominal surgery allows clinicians to modify risk through targeted interventions and enhanced monitoring. In this study, we aimed to identify and validate prognostic models against a new consensus definition of postoperative pulmonary complications. Methods We did a systematic review and international external validation cohort study. The systematic review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched MEDLINE and Embase on March 1, 2020, for articles published in English that reported on risk prediction models for postoperative pulmonary complications following abdominal surgery. External validation of existing models was done within a prospective international cohort study of adult patients (≥18 years) undergoing major abdominal surgery. Data were collected between Jan 1, 2019, and April 30, 2019, in the UK, Ireland, and Australia. Discriminative ability and prognostic accuracy summary statistics were compared between models for the 30-day postoperative pulmonary complication rate as defined by the Standardised Endpoints in Perioperative Medicine Core Outcome Measures in Perioperative and Anaesthetic Care (StEP-COMPAC). Model performance was compared using the area under the receiver operating characteristic curve (AUROCC). Findings In total, we identified 2903 records from our literature search; of which, 2514 (86·6%) unique records were screened, 121 (4·8%) of 2514 full texts were assessed for eligibility, and 29 unique prognostic models were identified. Nine (31·0%) of 29 models had score development reported only, 19 (65·5%) had undergone internal validation, and only four (13·8%) had been externally validated. Data to validate six eligible models were collected in the international external validation cohort study. Data from 11 591 patients were available, with an overall postoperative pulmonary complication rate of 7·8% (n=903). None of the six models showed good discrimination (defined as AUROCC ≥0·70) for identifying postoperative pulmonary complications, with the Assess Respiratory Risk in Surgical Patients in Catalonia score showing the best discrimination (AUROCC 0·700 [95% CI 0·683–0·717]). Interpretation In the pre-COVID-19 pandemic data, variability in the risk of pulmonary complications (StEP-COMPAC definition) following major abdominal surgery was poorly described by existing prognostication tools. To improve surgical safety during the COVID-19 pandemic recovery and beyond, novel risk stratification tools are required. Funding British Journal of Surgery Society

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone