Genetic factors predict hybrid formation in the British flora.

Natural hybridization can have a profound evolutionary impact, with consequences ranging from the extinction of rare taxa to the origin of new species. Natural hybridization is particularly common in plants; however, our understanding of the general factors that promote or prevent hybridization is hampered by the highly variable outcomes in different lineages. Here, we quantify the influence of different predictors on hybrid formation across species from an entire flora. We combine estimates of hybridization with ecological attributes and a new species-level phylogeny for over 1,100 UK flowering plant species. Our results show that genetic factors, particularly parental genetic distance, as well as phylogenetic position and ploidy, are key determinants of hybrid formation, whereas many other factors such as range overlap and genus size explain much less variation in hybrid formation. Overall, intrinsic genetic factors shape the evolutionary and ecological consequences of natural hybridization across species in a flora.

Sphingosine-1-Phosphate (S1P) Lyase Inhibition Causes Increased Cardiac S1P Levels and Bradycardia in Rats.

Inhibition of the sphingosine-1-phosphate (S1P)-catabolizing enzyme S1P lyase (S1PL) elevates the native ligand of S1P receptors and provides an alternative mechanism for immune suppression to synthetic S1P receptor agonists. S1PL inhibition is reported to preferentially elevate S1P in lymphoid organs. Tissue selectivity could potentially differentiate S1PL inhibitors from S1P receptor agonists, the use of which also results in bradycardia, atrioventricular block, and hypertension. But it is unknown if S1PL inhibition would also modulate cardiac S1P levels or cardiovascular function. The S1PL inhibitor 6-[(2R)-4-(4-benzyl-7-chlorophthalazin-1-yl)-2-methylpiperazin-1-yl]pyridine-3-carbonitrile was used to determine the relationship in rats between drug concentration, S1P levels in select tissues, and circulating lymphocytes. Repeated oral doses of the S1PL inhibitor fully depleted circulating lymphocytes after 3 to 4 days of treatment in rats. Full lymphopenia corresponded to increased levels of S1P of 100- to 1000-fold in lymph nodes, 3-fold in blood (but with no change in plasma), and 9-fold in cardiac tissue. Repeated oral dosing of the S1PL inhibitor in telemeterized, conscious rats resulted in significant bradycardia within 48 hours of drug treatment, comparable in magnitude to the bradycardia induced by 3 mg/kg fingolimod. These results suggest that S1PL inhibition modulates cardiac function and does not provide immune suppression with an improved cardiovascular safety profile over fingolimod in rats.

Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate.

We present a complete generic-level phylogeny of the complex thalloid liverworts, a lineage that includes the model system Marchantia polymorpha. The complex thalloids are remarkable for their slow rate of molecular evolution and for being the only extant plant lineage to differentiate gas exchange tissues in the gametophyte generation. We estimated the divergence times and analyzed the evolutionary trends of morphological traits, including air chambers, rhizoids and specialized reproductive structures. A multilocus dataset was analyzed using maximum likelihood and Bayesian approaches. Relative rates were estimated using local clocks. Our phylogeny cements the early branching in complex thalloids. Marchantia is supported in one of the earliest divergent lineages. The rate of evolution in organellar loci is slower than for other liverwort lineages, except for two annual lineages. Most genera diverged in the Cretaceous. Marchantia polymorpha diversified in the Late Miocene, giving a minimum age estimate for the evolution of its sex chromosomes. The complex thalloid ancestor, excluding Blasiales, is reconstructed as a plant with a carpocephalum, with filament-less air chambers opening via compound pores, and without pegged rhizoids. Our comprehensive study of the group provides a temporal framework for the analysis of the evolution of critical traits essential for plants during land colonization.

East Meets West: Effect of Acupuncture on Lactation and Maternal Quality of Life.

Objective: To evaluate the impact of acupuncture as part of a traditional Chinese medicine (TCM) treatment plan on lactation and maternal well-being in pump-dependent mothers of hospitalized neonates during the first 30 days after delivery. Study Design: This single-center study was conducted in a level IV regional neonatal intensive care unit with access to integrative health services. Sixty-six mothers were prospectively enrolled in two nonparallel groups: (1) Standard lactation support and (2) standard lactation support augmented with acupuncture. Daily pump volumes were documented. Participants completed a quality-of-life (QOL) survey at baseline and neonatal day of life 30. A linear model was constructed, adjusting for increased milk production over time. Results: Acupuncture was associated with increased milk production at all time points: Day 10 (p = 0.0002), day 14 (p < 0.0001), day 21 (p < 0.0001), and day 30 (p < 0.0001). Acupuncture was associated with an increase in three of five QOL components: psychological/child's health (p = 0.0006), family/friend relationship (p = 0.0006), and health/functioning (p = 0.02). Conclusion: Mothers receiving acupuncture reported enhanced milk supply and improved QOL. The limited sample size restricts the broad applicability of the results; nonetheless, this study paves the way for further research on the advantages of merging Eastern and Western treatments to enhance human lactation.

Pharmacological modulation of brain activity in a preclinical model of osteoarthritis.

The earliest stages of osteoarthritis are characterized by peripheral pathology; however, during disease progression chronic pain emerges-a major symptom of osteoarthritis linked to neuroplasticity. Recent clinical imaging studies involving chronic pain patients, including osteoarthritis patients, have demonstrated that functional properties of the brain are altered, and these functional changes are correlated with subjective behavioral pain measures. Currently, preclinical osteoarthritis studies have not assessed if functional properties of supraspinal pain circuitry are altered, and if these functional properties can be modulated by pharmacological therapy either by direct or indirect action on brain systems. In the current study, functional connectivity was first assessed in order to characterize the functional neuroplasticity occurring in the rodent medial meniscus tear (MMT) model of osteoarthritis-a surgical model of osteoarthritis possessing peripheral joint trauma and a hypersensitive pain state. In addition to knee joint trauma at week 3 post-MMT surgery, we observed that supraspinal networks have increased functional connectivity relative to sham animals. Importantly, we observed that early and sustained treatment with a novel, peripherally acting broad-spectrum matrix metalloproteinase (MMP) inhibitor (MMPi) significantly attenuates knee joint trauma (cartilage degradation) as well as supraspinal functional connectivity increases in MMT animals. At week 5 post-MMT surgery, the acute pharmacodynamic effects of celecoxib (selective cyclooxygenase-2 inhibitor) on brain function were evaluated using pharmacological magnetic resonance imaging (phMRI) and functional connectivity analysis. Celecoxib was chosen as a comparator, given its clinical efficacy for alleviating pain in osteoarthritis patients and its peripheral and central pharmacological action. Relative to the vehicle condition, acute celecoxib treatment in MMT animals yielded decreased phMRI infusion responses and decreased functional connectivity, the latter observation being similar to what was detected following chronic MMPi treatment. These findings demonstrate that an assessment of brain function may provide an objective means by which to further evaluate the pathology of an osteoarthritis state as well as measure the pharmacodynamic effects of therapies with peripheral or peripheral and central pharmacological action.

Target capture and genome skimming for plant diversity studies.

Recent technological advances in long-read high-throughput sequencing and assembly methods have facilitated the generation of annotated chromosome-scale whole-genome sequence data for evolutionary studies; however, generating such data can still be difficult for many plant species. For example, obtaining high-molecular-weight DNA is typically impossible for samples in historical herbarium collections, which often have degraded DNA. The need to fast-freeze newly collected living samples to conserve high-quality DNA can be complicated when plants are only found in remote areas. Therefore, short-read reduced-genome representations, such as target capture and genome skimming, remain important for evolutionary studies. Here, we review the pros and cons of each technique for non-model plant taxa. We provide guidance related to logistics, budget, the genomic resources previously available for the target clade, and the nature of the study. Furthermore, we assess the available bioinformatic analyses, detailing best practices and pitfalls, and suggest pathways to combine newly generated data with legacy data. Finally, we explore the possible downstream analyses allowed by the type of data generated using each technique. We provide a practical guide to help researchers make the best-informed choice regarding reduced genome representation for evolutionary studies of non-model plants in cases where whole-genome sequencing remains impractical.

A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants.

BACKGROUND: PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing. RESULTS: We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively. CONCLUSIONS: DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

The genome sequence of the European crab apple, Malus sylvestris (L.) Mill., 1768.

We present a genome assembly from an individual Malus sylvestris (the European or 'wild' crab apple; Streptophyta; Magnoliopsida; Rosales; Rosaceae). The genome sequence is 642 megabases in span. Most of the assembly (99.98%) is scaffolded into 17 chromosomal pseudomolecules. The mitochondrial and chloroplast genomes were also assembled, with respective lengths of 396.9 kilobases and 160.0 kilobases.

Development of an Advance Care Planning Policy within an Evidenced-Based Evaluation Framework.

Background: As the population is aging and medical advancements enable people to live longer, advance care planning (ACP) becomes increasingly important in guiding future care decisions; however, they are often incomplete or absent from the patient chart. This study describes the development and implementation of an ACP policy in a post-acute care and long-term care setting using a systematic implementation framework. Methods: A process evaluation that parallels the Replicating Effective Programs (REP) framework was used to understand stakeholder experiences with ACP and identify gaps in practice. Physicians, multidisciplinary staff, patients, and substitute decision makers engaged in focus groups and interviews, and completed surveys. A retrospective chart review determined Plan for Life Sustaining Treatment (PLST) form completion rates. Results: Stakeholder feedback identified barriers and facilitators to ACP including a need for staff training, user-friendly resources, and standardization of ACP practice. The PLST form was developed and embedded in the electronic medical record, and had a 92% and an 87% PLST completion rate on 2 pilot units. Conclusion: The study showed the usefulness of the REP model in guiding the evaluation as an effective tool to enhance implementation practices and inform ACP policy development that can be replicated in other organizations.

The first genome for the Cape Primrose Streptocarpus rexii (Gesneriaceae), a model plant for studying meristem-driven shoot diversity.

Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929-Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii. This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.

'There was no preamble': Comparing the Transition from Hospital to Home in Different Care Settings.

Our qualitative descriptive study compared how older patients and their informal caregivers experienced the care transition from acute care or rehabilitation to home. We recruited patients 65 years of age or older, or their informal caregivers, from in-patient units within acute care hospitals and rehabilitation facilities to participate in semi-structured interviews. We identified emergent themes via thematic analysis. In all, 16 patients and four patient caregivers participated. Across all care settings, caregivers were integral in facilitating the transition as well as experiencing variable discharge preparation, health care providers' optimizing transitions, and missed care and medication discrepancies at transition points. Orthopedic and rehabilitation patients more commonly voiced prior transition experiences in discharge preparation, including having to unexpectedly coordinate and wait for outpatient services. Differing responses between acute care and orthopedic settings suggest that transitional care practices and policies favor an individualized approach that considers patients' previous experiences, needs, and care expectations.

Barcode UK: A complete DNA barcoding resource for the flowering plants and conifers of the United Kingdom.

DNA barcoding and metabarcoding provide new avenues for investigating biological systems. These techniques require well-curated reference libraries with extensive coverage. Generating an exhaustive national DNA barcode reference library can open up new avenues of research in ecology, evolution and conservation, yet few studies to date have created such a resource. In plant DNA barcoding, herbarium collections provide taxonomically robust material but also pose challenges in lab processing. Here, we present a national DNA barcoding resource covering all of the native flowering plants and conifers of the United Kingdom. This represents 1,482 plant species, with the majority of specimens (81%) sourced from herbaria. Using Sanger sequencing of the plant DNA barcode markers, rbcL, matK, and ITS2, at least one DNA barcode was retrieved from 98% of the UK flora. We sampled from multiple individuals, resulting in a species coverage for rbcL of 96% (4,477 sequences), 90% for matK (3,259 sequences) and 75% for ITS2 (2,585 sequences). Sequence recovery was lower for herbarium material compared to fresh collections, with the age of the specimen having a significant effect on the success of sequence recovery. Species level discrimination was highest with ITS2, however, the ability to successfully retrieve a sequence was lowest for this region. Analyses of the genetic distinctiveness of species across a complete flora showed DNA barcoding to be informative for all but the most taxonomically complex groups. The UK flora DNA barcode reference library provides an important resource for many applications that require plant identification from DNA.

NRF2 activator A-1396076 ameliorates inflammation in autoimmune disease models by inhibiting antigen dependent T cell activation.

Nuclear factor (erythroid-derived 2) like 2 (NRF2) is a nuclear transcription factor activated in response to oxidative stress that induces a gene program that dampens inflammation and can limit cell damage that perpetuates the inflammatory response. We have identified A-1396076, a potent and selective NRF2 activator with demonstrated KEAP1 binding and modulation of cellular NRF2 mediated effects. In vivo administration of A-1396076 inhibits inflammation across several rodent models of autoimmunity when administered at or before the time of antigen challenge while also inducing NRF2 modulated gene transcription in the liver of the animals. It was not effective when administered after the time of antigen challenge or in a T cell independent model of arthritis induced by passive transfer of anti-collagen antibodies. A-1396076 inhibited antigen dependent T cell activation as measured by IFN-γ production in an ex vivo re-stimulation assay and following anti-CD3 challenge of MOG-sensitized mice. A-1396076 reduced costimulatory molecule expression on dendritic cells in the lungs of OVA LPS challenged mice suggesting that the mechanism of T cell inhibition was mediated at least partially by interfering with antigen presentation. These data suggest that NRF2 activation may be an effective strategy to dampen inflammation for treatment of autoimmune disease.

Comparative phylogeography of five widespread tree species: Insights into the history of western Amazonia.

Various historical processes have been put forth as drivers of patterns in the spatial distribution of Amazonian trees and their population genetic variation. We tested whether five widespread tree species show congruent phylogeographic breaks and similar patterns of demographic expansion, which could be related to proposed Pleistocene refugia or the presence of geological arches in western Amazonia. We sampled Otoba parvifolia/glycycarpa (Myristicaceae), Clarisia biflora, Poulsenia armata, Ficus insipida (all Moraceae), and Jacaratia digitata (Caricaceae) across the western Amazon Basin. Plastid DNA (trnH-psbA; 674 individuals from 34 populations) and nuclear ribosomal internal transcribed spacers (ITS; 214 individuals from 30 populations) were sequenced to assess genetic diversity, genetic differentiation, population genetic structure, and demographic patterns. Overall genetic diversity for both markers varied among species, with higher values in populations of shade-tolerant species than in pioneer species. Spatial analysis of molecular variance (SAMOVA) identified three genetically differentiated groups for the plastid marker for each species, but the areas of genetic differentiation were not concordant among species. Fewer SAMOVA groups were found for ITS, with no detectable genetic differentiation among populations in pioneers. The lack of spatially congruent phylogeographic breaks across species suggests no common biogeographic history of these Amazonian tree species. The idiosyncratic phylogeographic patterns of species could be due instead to species-specific responses to geological and climatic changes. Population genetic patterns were similar among species with similar biological features, indicating that the ecological characteristics of species impact large-scale phylogeography.

Patients' and caregivers' perspectives on factors that influence understanding of and adherence to hospital discharge instructions: a qualitative study.

BACKGROUND: Many patients have difficulty understanding and adhering to discharge instructions once home from hospital. We assessed patient and family caregiver perspectives on factors that influence understanding of and adherence to discharge instructions. METHODS: We conducted a qualitative study using semistructured interviews of participants aged 18 years or more enrolled in a multicentre mixed-methods study who were discharged from 3 acute care hospitals across Ontario with a diagnosis of congestive heart failure, chronic obstructive pulmonary disease or pneumonia. Patients were recruited between March and November 2016. We used directed content analysis to derive themes and subthemes. RESULTS: Twenty-seven participants (16 patients and 11 family members) described 5 themes that affected their understanding of and adherence to discharge instructions: 1) the role of caregivers, 2) relationships with inpatient and outpatient health care providers, 3) previous hospital stay, 4) barriers to accessing postdischarge care and 5) system-level processes. Subthemes highlighted the importance participants attributed to who provides the instructions, the development of resilience and advocacy through previous admissions, the benefits of addressing language and physical disability barriers, reviewing instructions in a unhurried manner, and ensuring that written instructions are meaningful and actionable. INTERPRETATION: Care transition interventions targeting improved communication are unlikely to improve understanding of and adherence to discharge instructions on their own. A patient-centred framework that promotes positive relationships with a patient's circle of care, reflects previous experiences with discharge, addresses equity barriers, and enhances strategies for patient and caregiver engagement at the time of discharge may optimize understanding and adherence once the patient is home.

Pulmonary thrombosis in adults with Eisenmenger syndrome.

OBJECTIVES: We sought to determine the prevalence of pulmonary artery thrombosis in patients with Eisenmenger syndrome and to identify individuals at highest risk. BACKGROUND: Eisenmenger syndrome is associated with pulmonary arterial thrombus formation. Both the prevalence and the determinants of pulmonary arterial thrombosis are unknown. METHODS: This is a review of patients with Eisenmenger syndrome seen at the Toronto Congenital Cardiac Centre for Adults, Canada. Patients underwent a contrast-enhanced computed tomographic (CT) scan of the thorax. RESULTS: Forty-nine consecutive patients with Eisenmenger syndrome were seen in our hospital. Fifteen patients did not undergo CT angiograms; therefore, 34 patients (mean age 42 +/- 10 years) were included in the study. Responsible shunts included ventricular septal defect (65%), atrial septal defect (15%), patent ductus arteriosus (9%), and other (11%). The prevalence of proximal pulmonary artery thrombus was 21% (7/34) of patients. Evidence of more distal vessel thrombosis was observed in 43% (3/7) of the patients who had visible thrombus in the proximal pulmonary arteries. Patients with thrombus were more likely to be female (86% vs. 37%, p = 0.04) and to have lower oxygen saturations (72% +/- 9% vs. 85% +/- 6%, p = 0.01). Differences in functional status did not identify patients at highest risk for thrombosis. CONCLUSIONS: Patients with Eisenmenger syndrome have a substantial risk of pulmonary artery thrombus formation. Women and patients with lower oxygen saturations are at the highest risk of developing thrombosis. In the context of an increased bleeding tendency in these patients, the role of anticoagulation treatment needs to be determined.

A-770041, a novel and selective small-molecule inhibitor of Lck, prevents heart allograft rejection.

Lck, one of eight members of the Src family of tyrosine kinases, is activated after T cell stimulation and is required for T-cell proliferation and interleukin (IL)-2 production. Inhibition of Lck has been a target to prevent lymphocyte activation and acute rejection. Here, we report the pharmacologic characterization of 1-methyl-1H-indole-2-carboxylic acid (4-{1-[4-(4-acetyl-piperazin-l-yl)-cyclohexyl]-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl}-2-methoxy-phenyl)-amide (A-770041), an orally bioavailable pyrazolo[3,4-d]pyrimidine with increased selectivity for Lck compared with previously reported compounds. A-770041 is a 147 nM inhibitor of Lck (1 mM ATP) and is 300-fold selective against Fyn, the other Src family kinase involved in T-cell signaling. Concanavalin A-stimulated IL-2 production in whole blood is inhibited by A-770041 with an EC50 of approximately 80 nM. A-770041 is orally bioavailable (F = 34.1 +/- 7.2% at 10 mg/kg) and has a t(1/2) of 4.1 +/- 0.1 h. Concanavalin A-induced IL-2 production in vivo is inhibited by oral administration of A-770041 (in vivo EC50 = 78 +/- 28 nM). Doses of A-770041 at or above 10 mg/kg/day prevent rejection of hearts transplanted heterotopically in rats from Brown Norway donors to Lewis recipients across a major histocompatibility barrier for least 65 days. Grafts from animals treated with 20 mg/kg/day A-770041 or 10 mg/day Cyclosporin A had minimal microvascular changes or multifocal mononuclear infiltrates. However, mineralization in myocytes from the grafts from A-770041-treated animals was less than animals treated with Cyclosporin A. Lck inhibition is an attractive target to prevent acute rejection.