How to Sample Dozens of Substitutions per Site with λ Dynamics.

Alchemical free energy methods are useful in computer-aided drug design and computational protein design because they provide rigorous statistical mechanics-based estimates of free energy differences from molecular dynamics simulations. λ dynamics is a free energy method with the ability to characterize combinatorial chemical spaces spanning thousands of related systems within a single simulation, which gives it a distinct advantage over other alchemical free energy methods that are mostly limited to pairwise comparisons. Recently developed methods have improved the scalability of λ dynamics to perturbations at many sites; however, the size of chemical space that can be explored at each individual site has previously been limited to fewer than ten substituents. As the number of substituents increases, the volume of alchemical space corresponding to nonphysical alchemical intermediates grows exponentially relative to the size corresponding to the physical states of interest. Beyond nine substituents, λ dynamics simulations become lost in an alchemical morass of intermediate states. In this work, we introduce new biasing potentials that circumvent excessive sampling of intermediate states by favoring sampling of physical end points relative to alchemical intermediates. Additionally, we present a more scalable adaptive landscape flattening algorithm for these larger alchemical spaces. Finally, we show that this potential enables more efficient sampling in both protein and drug design test systems with up to 24 substituents per site, enabling, for the first time, simultaneous simulation of all 20 amino acids.

Systemic methotrexate (MTX) in early pregnancy: a retrospective study of a tertiary maternity hospital.

BACKGROUND: Methotrexate (MTX) is used in clinical practice as a medical treatment option in patients with early pregnancy complications like ectopic pregnancy. AIMS: To review systemic MTX therapy use in the first trimester of pregnancy in our hospital and to examine subsequent clinical outcomes. METHODS: Retrospective review of all women treated with systemic MTX in early pregnancy identified from electronic prescription records from 1 January 2018 to 31 December 2020 at Cork University Maternity Hospital, Ireland. Relevant data was transcribed from electronic health records. RESULTS: Indications for treatment were tubal ectopic pregnancy (70%, n = 51), persistent pregnancy of unknown location (22%, n = 16) and caesarean scar pregnancy (7%, n = 5). Treatment was successful in 88% (n = 44) of tubal ectopic pregnancies with 73% (n = 37) and 14% (n = 7) of women receiving a single dose and repeated doses, respectively. Only 8% (n = 4) of tubal ectopic pregnancies required emergency surgery for subsequent tubal rupture. In 93% (n = 15) of cases of persistent pregnancy of unknown location, treatment was successful with one patient requiring uterine evacuation. Women with caesarean scar pregnancy were treated with combined MTX and uterine evacuation without complication. CONCLUSIONS: The efficacy of medical treatment with systemic MTX for confirmed tubal ectopic pregnancy in our hospital is in line with national and international standards. Careful consideration should be given to treating caesarean scar pregnancy and persistent pregnancy of unknown location with systemic MTX. Systemic MTX use guided by clinicians specialised in early pregnancy complications and safe medication practices may improve treatment success and reduce adverse events.

Pooled DNA sequencing in hairy vetch (Vicia villosa Roth) reveals QTL for seed dormancy but not pod dehiscence.

Introduction: Hairy vetch (Vicia villosa Roth) is a promising legume cover crop, but its use is limited by high rates of pod dehiscence and seed dormancy. Methods: We used phenotypically contrasting pooled DNA samples (n=24 with 29-74 individuals per sample) from an ongoing cover crop breeding program across four environments (site-year combinations: Maryland 2020, Maryland 2022, Wisconsin 2021, Wisconsin 2022) to find genetic associations and genomic prediction accuracies for pod dehiscence and seed dormancy. We also combined pooled DNA sample genetic association results with the results of a prior genome-wide association study. Results and discussion: Genomic prediction resulted in positive predictive abilities for both traits between environments and with an independent dataset (0.34-0.50), but reduced predictive ability for DNA pools with divergent seed dormancy in the Maryland environments (0.07-0.15). The pooled DNA samples found six significant (false discovery rate q-value<0.01) quantitative trait loci (QTL) for seed dormancy and four significant QTL for pod dehiscence. Unfortunately, the minor alleles of the pod dehiscence QTL increased the rate of pod dehiscence and are not useful for marker-assisted selection. When combined with a prior association study, sixteen seed dormancy QTL and zero pod dehiscence QTL were significant. Combining the association studies did not increase the detection of useful QTL.

Selection pressures on evolution of ribonuclease H explored with rigorous free-energy-based design.

Understanding natural protein evolution and designing novel proteins are motivating interest in development of high-throughput methods to explore large sequence spaces. In this work, we demonstrate the application of multisite λ dynamics (MSλD), a rigorous free energy simulation method, and chemical denaturation experiments to quantify evolutionary selection pressure from sequence-stability relationships and to address questions of design. This study examines a mesophilic phylogenetic clade of ribonuclease H (RNase H), furthering its extensive characterization in earlier studies, focusing on E. coli RNase H (ecRNH) and a more stable consensus sequence (AncCcons) differing at 15 positions. The stabilities of 32,768 chimeras between these two sequences were computed using the MSλD framework. The most stable and least stable chimeras were predicted and tested along with several other sequences, revealing a designed chimera with approximately the same stability increase as AncCcons, but requiring only half the mutations. Comparing the computed stabilities with experiment for 12 sequences reveals a Pearson correlation of 0.86 and root mean squared error of 1.18 kcal/mol, an unprecedented level of accuracy well beyond less rigorous computational design methods. We then quantified selection pressure using a simple evolutionary model in which sequences are selected according to the Boltzmann factor of their stability. Selection temperatures from 110 to 168 K are estimated in three ways by comparing experimental and computational results to evolutionary models. These estimates indicate selection pressure is high, which has implications for evolutionary dynamics and for the accuracy required for design, and suggests accurate high-throughput computational methods like MSλD may enable more effective protein design.

Fast free energy estimates from λ-dynamics with bias-updated Gibbs sampling.

Relative binding free energy calculations have become an integral computational tool for lead optimization in structure-based drug design. Classical alchemical methods, including free energy perturbation or thermodynamic integration, compute relative free energy differences by transforming one molecule into another. However, these methods have high operational costs due to the need to perform many pairwise perturbations independently. To reduce costs and accelerate molecular design workflows, we present a method called λ-dynamics with bias-updated Gibbs sampling. This method uses dynamic biases to continuously sample between multiple ligand analogues collectively within a single simulation. We show that many relative binding free energies can be determined quickly with this approach without compromising accuracy. For five benchmark systems, agreement to experiment is high, with root mean square errors near or below 1.0 kcal mol-1. Free energy results are consistent with other computational approaches and within statistical noise of both methods (0.4 kcal mol-1 or less). Notably, large efficiency gains over thermodynamic integration of 18-66-fold for small perturbations and 100-200-fold for whole aromatic ring substitutions are observed. The rapid determination of relative binding free energies will enable larger chemical spaces to be more readily explored and structure-based drug design to be accelerated.

Genome-wide association mapping in hairy vetch (Vicia villosa) discovers a large effect locus controlling seed dormancy.

Hairy vetch (Vicia villosa Roth), a winter-hardy annual legume, is a promising cover crop. To fully leverage its potential, seed production and field performance of V. villosa must be improved to facilitate producer adoption. Two classic domestication traits, seed dormancy (hard seed) and dehiscence (pod shatter), are selection targets in an ongoing breeding program. This study reports a genome-wide association study of 1,019 V. villosa individuals evaluated at two sites (Knox City, Texas and Corvallis, Oregon) for the proportion of dormant seed, visual pod dehiscence scores, and two dehiscence surrogate measures (force to dehiscence and pod spiraling score). Trait performance varied between sites, but reliability (related to heritability) across sites was strong (dormant seed proportion: 0.68; dehiscence score: 0.61; spiraling score: 0.42; force to dehiscence: 0.41). A major locus controlling seed dormancy was found (q-value: 1.29 × 10-5; chromosome 1: position: 63611165), which can be used by breeding programs to rapidly reduce dormancy in breeding populations. No significant dehiscence score QTL was found, primarily due to the high dehiscence rates in Corvallis, Oregon. Since Oregon is a potentially major V. villosa seed production region, further dehiscence resistance screening is necessary.

Three cases of black pleural effusion.

Black pleural effusions (BPE) are rare, exudative pleural effusions that produce a black fluid on thoracentesis. While the name and definition of this pathology is undeniably simple, the etiologies, outcomes, and treatments for BPE are incredibly complex. Currently, BPE is not well-demonstrated in the literature. This case series reports three patients with different etiologies, past medical histories, presenting symptoms, treatments, and outcomes. BPE caused by pancreatic-pleural fistula and opportunistic infections are demonstrated in this case series. This report shows that early identification and treatment of the underlying cause of BPE is critical to the recovery of the patients.

Audit of Ireland's first manual vacuum aspiration service.

INTRODUCTION: Manual vacuum aspiration (MVA) is a safe and effective alternative option for the management of first-trimester miscarriage, termination of pregnancy, or retained pregnancy tissue. Ireland's first MVA clinic was set up in the Rotunda Hospital in April 2020. OBJECTIVE: To identify the number of women who have undergone MVA since establishing our service, to assess the efficacy and safety of MVA in that service, and to develop local Irish studies that further support the safety of MVA, adding to the international body of evidence. METHODS: With the approval and assistance of the Clinical Audit Committee, we obtained a log of all patients who underwent MVA in the first 18 months of the service. We performed a retrospective electronic chart review using Maternal and Newborn Clinical Management System. We collected the data and preformed a descriptive analysis. RESULTS: In total, 86 women underwent MVA, 85 (98.8%) of which were successfully completed. There were no immediate procedural complications, inter-hospital transfers, or emergency electric vacuum aspiration (EVA) required. We obtained an incomplete evacuation rate of 4.7% (n = 4). CONCLUSION: We have demonstrated that the MVA service in the Rotunda Hospital is a safe, effective management option with advantages for both the patient and the healthcare system. We recommend consideration for provision of funding and resources to enable expansion of this service nationally in order to give women greater autonomy of choice in the management of early pregnancy complications and termination of pregnancy.

COVID-19 Vaccine Provider Access and Vaccination Coverage Among Children Aged 5-11 Years - United States, November 2021-January 2022.

On October 29, 2021, the Pfizer-BioNTech pediatric COVID-19 vaccine received Emergency Use Authorization for children aged 5-11 years in the United States.† For a successful immunization program, both access to and uptake of the vaccine are needed. Fifteen million doses were initially made available to pediatric providers to ensure the broadest possible access for the estimated 28 million eligible children aged 5-11 years, especially those in high social vulnerability index (SVI)§ communities. Initial supply was strategically distributed to maximize vaccination opportunities for U.S. children aged 5-11 years. COVID-19 vaccination coverage among persons aged 12-17 years has lagged (1), and vaccine confidence has been identified as a concern among parents and caregivers (2). Therefore, COVID-19 provider access and early vaccination coverage among children aged 5-11 years in high and low SVI communities were examined during November 1, 2021-January 18, 2022. As of November 29, 2021 (4 weeks after program launch), 38,732 providers were enrolled, and 92% of U.S. children aged 5-11 years lived within 5 miles of an active provider. As of January 18, 2022 (11 weeks after program launch), 39,786 providers had administered 13.3 million doses. First dose coverage at 4 weeks after launch was 15.0% (10.5% and 17.5% in high and low SVI areas, respectively; rate ratio [RR] = 0.68; 95% CI = 0.60-0.78), and at 11 weeks was 27.7% (21.2% and 29.0% in high and low SVI areas, respectively; RR = 0.76; 95% CI = 0.68-0.84). Overall series completion at 11 weeks after launch was 19.1% (13.7% and 21.7% in high and low SVI areas, respectively; RR = 0.67; 95% CI = 0.58-0.77). Pharmacies administered 46.4% of doses to this age group, including 48.7% of doses in high SVI areas and 44.4% in low SVI areas. Although COVID-19 vaccination coverage rates were low, particularly in high SVI areas, first dose coverage improved over time. Additional outreach is critical, especially in high SVI areas, to improve vaccine confidence and increase coverage rates among children aged 5-11 years.

Addressing Intersite Coupling Unlocks Large Combinatorial Chemical Spaces for Alchemical Free Energy Methods.

Alchemical free energy methods are playing a growing role in molecular design, both for computer-aided drug design of small molecules and for computational protein design. Multisite λ dynamics (MSλD) is a uniquely scalable alchemical free energy method that enables more efficient exploration of combinatorial alchemical spaces encountered in molecular design, but simulations have typically been limited to a few hundred ligands or sequences. Here, we focus on coupling between sites to enable scaling to larger alchemical spaces. We first discuss updates to the biasing potentials that facilitate MSλD sampling to include coupling terms and show that this can provide more thorough sampling of alchemical states. We then harness coupling between sites by developing a new free energy estimator based on the Potts models underlying direct coupling analysis, a method for predicting contacts from sequence coevolution, and find it yields more accurate free energies than previous estimators. The sampling requirements of the Potts model estimator scale with the square of the number of sites, a substantial improvement over the exponential scaling of the standard estimator. This opens up exploration of much larger alchemical spaces with MSλD for molecular design.

Optimizing Multisite λ-Dynamics Throughput with Charge Renormalization.

With the ability to sample combinations of alchemical perturbations at multiple sites off a small molecule core, multisite λ-dynamics (MSλD) has become an attractive alternative to conventional alchemical free energy methods for exploring large combinatorial chemical spaces. However, current software implementations dictate that combinatorial sampling with MSλD must be performed with a multiple topology model (MTM), which is nontrivial to create by hand, especially for a series of ligand analogues which may have diverse functional groups attached. This work introduces an automated workflow, referred to as msld_py_prep, to assist in the creation of a MTM for use with MSλD. One approach for partitioning partial atomic charges between ligands to create a MTM, called charge renormalization, is also presented and rigorously evaluated. We find that msld_py_prep greatly accelerates the preparation of MSλD ready-to-use files and that charge renormalization can provide a successful approach for MTM generation, as long as bookending calculations are applied to correct small differences introduced by charge renormalization. Charge renormalization also facilitates the use of many different force field parameters with MSλD, broadening the applicability of MSλD for computer-aided drug design.

Early pregnancy teaching and training amongst obstetric trainees in a tertiary maternity hospital.

Proficiency in early pregnancy assessment and management is a core component of Basic Speciality Training (BST) in Obstetrics & Gynaecology. Performance and interpretation of early pregnancy ultrasound is not formalised in Ireland, resulting in variation in exposure and experience amongst trainees. We aimed to improve trainee's knowledge and confidence in early pregnancy ultrasound. This was though a multimodel teaching program of didactic lectures, tutorials, and case-based discussions. Additionally, we organised structed assessment tests over a six-week period with a repeat revision of teaching to allow all participants attendance. All teaching was provided with facilities already available in our institution. We assessed knowledge and confidence by an anonymised survey comprising both quantitative and qualitative components pre and post training completion. We showed an improvement in knowledge, a non-significant increase was noted in mean test score in multiple choice questions relating to early pregnancy from 78.8% to 83.5% post study completion. Trainees were noted to report an increase in training during the duration of the study with 70% reporting have received little training prior to the study dropping to 13% at the end. Increased exposure to both witnessed and performed transabdominal and transvaginal ultrasound and training on the interpretation of early pregnancy ultrasound was noted. Among trainees 62% reported no formalised training in interpreting early pregnancy ultrasound at commencement reducing to 13% post study (p = 0.04). Improved confidence although non-significant was also reported among trainees. Our study demonstrates that a low-cost multimodel teaching program over a short time frame can improve knowledge, confidence and interpretation in early pregnancy ultrasound. This simple model can be easily reproduced in other institutions. As this was a pilot study, we would envision expanding it across further sites to assess its effectiveness.

A Systematic Review of Echocardiographic Parameters for the Screening of Pulmonary Hypertension: What Are the Odds?

Pulmonary hypertension (PH) is an insidious disease that often presents in late stages due to nonspecific signs and symptoms. Right heart catheterization (RHC) is the gold standard diagnostic test, and echocardiogram (ECHO) is the best screening tool. However, the strength of evidence and diagnostic utility of various echocardiographic parameters to screen for is not well elucidated. This systematic review (SR) is reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Literature searches was performed for the period of January 1, 2016, to June 1, 2021, on seven databases. We included full-text studies with adult patients that used RHC for comparison and provided sensitivity and specificity results. Likelihood ratios (LRs) and diagnostic odds ratios (DORs) were calculated. Risk of bias was assessed using the Quality Assessment Tool for the Observational Cohort and Cross-Sectional Studies. We identified 102 studies, but only 14 satisfied our inclusion criteria. The most significant parameters identified for PH screening based on LRs are, in descending order, tricuspid regurgitation gradient peak >36mmHg, systolic pulmonary artery pressure >41mmHg, and tricuspid regurgitation velocity >2.9 m/s. There is strong correlation between LR and DOR for these parameters. This SR indicates the superiority of some ECHO parameters over others to aid in the screening and severity assessment of PH. Variables with low LR (-) ratios may help to prevent unnecessary invasive assessment for PH. Clinicians should utilize a multi-parameter approach when interpreting echocardiograms for PH assessment.

Womens experience of Manual Vacuum Aspiration: An Irish perspective.

OBJECTIVE: Manual Vacuum Aspiration (MVA) is a well-established management option for early pregnancy loss or early termination of pregnancy. MVA is performed as out-patient surgical procedure using local anaesthetic whereby aspiration of uterine contents is achieved through use of a hand-held negative pressure syringe. Ireland's first MVA service was established at the Rotunda Hospital Dublin in April 2020,. The purpose of this study was to gather feedback from women who had undergone MVA in the unit. STUDY DESIGN: Prospective mixed methods study of women attending for uterine aspiration under local anaesthetic from July to October 2020 in the unit. Consenting women were contacted one week following MVA via telephone. The survey conducted consisted of structured closed questions along with open-ended questions, to assess womens satisfaction in relation to all aspects of the MVA service. RESULTS: Nineteen women took part in the study, a response rate of 86.4%. Participants reported feeling well informed prior to attending for MVA. Prior to the procedure, pain expectation scores were high but actual reported pain scores were much lower. Although some participants did find MVA uncomfortable, the fact the procedure was very quick and the side effects so minimal generally negated this. The location and set up of the clinic scored highly among participants as did the staff of the clinic. Overall satisfaction with the MVA service was high with 84.2% of participants reporting they would opt again for MVA in the future. CONCLUSIONS: Women living in Ireland are interested and agreeable with having Manual Vacuum Aspiration as an available option for management of early pregnancy complications. Consideration should be given to expansion of MVA services nationally.

BLaDE: A Basic Lambda Dynamics Engine for GPU-Accelerated Molecular Dynamics Free Energy Calculations.

There is an accelerating interest in practical applications of alchemical free energy methods to problems in protein design, constant pH simulations, and especially computer-aided drug design. In the present paper, we describe a basic lambda dynamics engine (BLaDE) that enables alchemical free energy simulations, including multisite λ dynamics (MSλD) simulations, on graphical processor units (GPUs). We find that BLaDE is 5 to 8 times faster than the current GPU implementation of MSλD-based free energy calculations in CHARMM. We also demonstrate that BLaDE running standard molecular dynamics attains a performance competitive with and sometimes exceeding that of the highly optimized OpenMM GPU code. BLaDE is available as a standalone program and through an API in CHARMM.

Medical management of first trimester miscarriage: a quality improvement initiative.

INTRODUCTION: Medical management of first trimester pregnancy loss is a safe option that is well tolerated and affords women more autonomy in relation to their care. Recent trials provide robust evidence that mifepristone pretreatment is the optimal approach for women with missed miscarriage who desire medical management. METHODS: Following a change in medical management of first trimester miscarriage in our unit, we conducted a retrospective audit over a 3-month period of all women who had elected medical management as their primary treatment option. We compared the results with a previous audit that had been undertaken prior to the change in practice. RESULTS: The implementation of mifepristone resulted in an increased effectiveness of primary medical treatment for first trimester miscarriage from 53.8% to 85.2% (p=<0.001). DISCUSSION: The results of our study support the introduction of mifepristone into routine clinical practice for medical management of first trimester pregnancy loss across all maternity units.

Placental growth factor in assessment of women with suspected pre-eclampsia to reduce maternal morbidity: a stepped wedge cluster randomised control trial (PARROT Ireland).

OBJECTIVE: To determine whether the addition of placental growth factor (PlGF) measurement to current clinical assessment of women with suspected pre-eclampsia before 37 weeks' gestation would reduce maternal morbidity without increasing neonatal morbidity. DESIGN: Stepped wedge cluster randomised control trial from 29 June 2017 to 26 April 2019. SETTING: National multisite trial in seven maternity hospitals throughout the island of Ireland PARTICIPANTS: Women with a singleton pregnancy between 20+0 to 36+6 weeks' gestation, with signs or symptoms suggestive of evolving pre-eclampsia. Of the 5718 women screened, 2583 were eligible and 2313 elected to participate. INTERVENTION: Participants were assigned randomly to either usual care or to usual care plus the addition of point-of-care PlGF testing based on the randomisation status of their maternity hospital at the time point of enrolment. MAIN OUTCOMES MEASURES: Co-primary outcomes of composite maternal morbidity and composite neonatal morbidity. Analysis was on an individual participant level using mixed-effects Poisson regression adjusted for time effects (with robust standard errors) by intention-to-treat. RESULTS: Of the 4000 anticipated recruitment target, 2313 eligible participants (57%) were enrolled, of whom 2219 (96%) were included in the primary analysis. Of these, 1202 (54%) participants were assigned to the usual care group, and 1017 (46%) were assigned the intervention of additional point-of-care PlGF testing. The results demonstrate that the integration of point-of-care PlGF testing resulted in no evidence of a difference in maternal morbidity-457/1202 (38%) of women in the control group versus 330/1017 (32%) of women in the intervention group (adjusted risk ratio (RR) 1.01 (95% CI 0.76 to 1.36), P=0.92)-or in neonatal morbidity-527/1202 (43%) of neonates in the control group versus 484/1017 (47%) in the intervention group (adjusted RR 1.03 (0.89 to 1.21), P=0.67). CONCLUSIONS: This was a pragmatic evaluation of an interventional diagnostic test, conducted nationally across multiple sites. These results do not support the incorporation of PlGF testing into routine clinical investigations for women presenting with suspected preterm pre-eclampsia, but nor do they exclude its potential benefit. TRIAL REGISTRATION: ClinicalTrials.gov NCT02881073.

Mapping and identification of genetic loci affecting earliness of bolting and flowering in lettuce.

KEY MESSAGE: Photoperiod and temperature conditions elicit different genetic regulation over lettuce bolting and flowering. This study identifies environment-specific QTLs and putative genes and provides information for genetic marker assay. Bolting, defined as stem elongation, marks the plant life cycle transition from vegetative to reproductive stage. Lettuce is grown for its leaf rosettes, and premature bolting may reduce crop quality resulting in economic losses. The transition to reproductive stage is a complex process that involves many genetic and environmental factors. In this study, the effects of photoperiod and ambient temperature on bolting and flowering regulation were studied by utilizing a lettuce mapping population to identify quantitative trait loci (QTL) and by gene expression analyses of genotypes with contrasting phenotypes. A recombinant inbred line (RIL) population, derived from a cross between PI 251246 (early bolting) and cv. Salinas (late bolting), was grown in four combinations of short (8 h) and long (16 h) days and low (20 °C) and high (35 °C) temperature. QTL models revealed both genetic (G) and environmental (E) effects, and GxE interactions. A major QTL for bolting and flowering time was found on chromosome 7 (qFLT7.2), and two candidate genes were identified by fine mapping, homology, and gene expression studies. In short days and high temperature conditions, qFLT7.2 had no effect on plant development, while several small-effect loci on chromosomes 2, 3, 6, 8, and 9 were associated with bolting and flowering. Of these, the QTL on chromosome 2, qBFr2.1, co-located with the Flowering Locus T (LsFT) gene. Polymorphisms between parent genotypes in the promotor region may explain identified gene expression differences and were used to design a genetic marker which may be used to identify the late bolting trait.