Identifying infection in chronic wounds in a community setting: A systematic review of diagnostic test accuracy studies.

AIM: To determine the diagnostic accuracy of different methods currently available to identify infection in chronic wounds applicable to adult patients in a community setting. DESIGN: Systematic review of diagnostic test accuracy studies. REVIEW METHODS: Two authors independently completed screening, data extraction and quality and bias assessments (QUADAS2). Eligible studies compared a method (index test) for detecting infection (diagnosis of interest) with microscopy and culture of either deep tissue biopsy or wound swab (reference test) in adult patients with wounds of >4 weeks duration (participants). The results were synthesized narratively. DATA SOURCES: We systematically searched CINAHL, Embase and Medline from 2011 to April 2022. RESULTS: Four studies were included, all recruiting from secondary care wound clinics. Two studies assessed the diagnostic accuracy of Moleculight i:X, a bacterial fluorescence imaging device against deep tissue biopsy culture. One study assessed the diagnostic accuracy of the elevation of various enzymes detected in wound fluid against wound swab microscopy of culture. One study assessed the diagnostic accuracy of bacterial protease activity against wound swab microscopy and culture. Sensitivities of these methods ranged from 50 to 75% and specificities from 47 to 100%. CONCLUSION: Only a small number of studies were included in this systematic review due to our strict inclusion criteria. We have not identified any methods for diagnosing infection in chronic wounds with either a sufficient quality of evidence to recommend their use in community settings at present. Further research is needed to develop and evaluate appropriate diagnostics for this purpose. IMPACT: This study highlights the paucity of research into wound diagnostics in a community setting and should prompt further research in this area. Accurate diagnostic tests have the potential to improve community-based wound care by optimizing antibiotic use and potentially improving healing time. REPORTING METHOD: PRISMA-DTA checklist. PATIENT OR PUBLIC CONTRIBUTION: The PPI group for the NIHR Community Healthcare MIC were supportive of this topic of work.

Parallel evolution and cryptic diversification in a common and widespread Amazonian tree, Protium subserratum.

The lowland Amazon rainforest houses some of the greatest tree diversity on Earth. While the vast majority of these species are rare, a small number are common and widespread and thus considered to play a disproportionate role in many of the global ecosystem services provided by the Amazon. However, the extent to which dominant Amazonian tree species actually include multiple clades, each on their own unique evolutionary trajectory, is unknown. Here we investigate the extent to which lineage divergence may be occurring within Protium subserratum (Burseraceae), a common and widespread tree species that is monophyletic with populations exhibiting genotypic and phenotypic differences associated with soil and geography. Utilizing a combination of phylogenomic and population genomic methods with sampling from across the range, we found that P. subserratum contains at least eight distinct clades. Specialization onto white-sand soils has evolved independently at least twice within the species; however, phenotype is not correlated with soil type. Finally, cryptic diversity at the base of the Andes is associated with elevational shifts. Together these results lend support to the hypothesis that common and widespread Amazon tree species may not represent evolutionary cohesive units. Instead, these dominant species may more commonly represent species complexes, undergoing evolutionary transitions on a trajectory to become multiple range-restricted, specialist species.

Generation of a fertile ask1 mutant uncovers a comprehensive set of SCF-mediated intracellular functions.

Many eukaryotic intracellular processes employ protein ubiquitylation by ubiquitin E3 ligases for functional regulation or protein quality control. In plants, the multi-subunit Skp1-Cullin1-F-box (SCF) complexes compose the largest group of E3 ligases whose specificity is determined by a diverse array of F-box proteins. Although both sequence divergence and polymorphism of F-box genes well support a broad spectrum of SCF functions, experimental evidence is scarce due to the low number of identified SCF substrates. Taking advantage of the bridge role of Skp1 between F-box and Cullin1 in the complex, we systematically analyzed the functional influence of a well-characterized Arabidopsis Skp1-Like1 (ASK1) Ds insertion allele, ask1, in different Arabidopsis accessions. Through 10 generations of backcrossing with Columbia-0 (Col-0), we partially rescued the fertility of this otherwise sterile ask1 allele in Landsberg erecta, thus providing experimental evidence showing the polymorphic roles of SCF complexes. This ask1 mutant produces twisted rosette leaves, a reduced number of petals, fewer viable pollen grains, and larger embryos and seeds compared to Col-0. RNA-Seq-based transcriptome analysis of ask1 uncovered a large spectrum of SCF functions, which is greater than a 10-fold increase compared with previous studies. We also identified its hyposensitive responses to auxin and abscisic acid treatments and enhanced far-red light/phyA-mediated photomorphogenesis. Such diverse roles are consistent with the 20-30% reduction of ubiquitylation events in ask1 estimated by immunoblotting analysis in this work. Collectively, we conclude that ASK1 is a predominant Skp1 protein in Arabidopsis and that the fertile ask1 mutant allowed us to uncover a comprehensive set of SCF functions.

Probiotic treatment for women with gestational diabetes to improve maternal and infant health and well-being.

BACKGROUND: Gestational diabetes mellitus (GDM) is carbohydrate intolerance first recognised during pregnancy and associated with complications for mothers and babies. Probiotics are naturally occurring micro-organisms, which when ingested in adequate amounts, may confer health benefits. Evidence of the role of probiotics as treatment for GDM is limited. OBJECTIVES: To evaluate the safety and effectiveness of probiotics in treating women with GDM on maternal and infant outcomes. SEARCH METHODS: We searched the Cochrane Pregnancy and Childbirth's Trials Register ClinicalTrials.gov, WHO International Clinical Trials Registry Platform (ICTRP) (24 July 2019), and reference lists of retrieved studies. SELECTION CRITERIA: Randomised controlled trials (RCTs) comparing the use of probiotics versus placebo/standard care for the treatment of GDM. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed study eligibility, extracted data, checked data accuracy, and assessed risk of bias of included trials. The certainty of evidence for selected maternal and infant/child outcomes was assessed using GRADE. MAIN RESULTS: Nine RCTs (695 pregnant women with GDM) comparing probiotics versus placebo were identified. The overall risk of bias in the nine RCTs was low to unclear and the evidence was downgraded for imprecision due to the small numbers of women participating in the trials. The trials were carried out in hospitals and universities in Iran (seven trials), Thailand (one trial) and Ireland (one trial). All trials compared probiotics with placebo. Maternal outcomes We are uncertain if probiotics have any effect compared with placebo on hypertensive disorders of pregnancy, (risk ratio (RR) 1.50, 95% confidence interval (CI) 0.64 to 3.53; participants = 256; studies = 3; low-certainty evidence) and mode of birth as caesareans (average RR 0.64, 95% CI 0.30 to 1.35; participants = 267; studies = 3; low-certainty evidence) because the certainty of evidence is low and the 95% CIs span possible benefit and possible harm. No trials reported primary outcomes of: mode of birth as vaginal/assisted and subsequent development of type 2 diabetes. We are uncertain if probiotics have any effect compared with placebo on induction of labour (RR 1.33, 95% CI 0.74 to 2.37; participants = 127; studies = 1; very low-certainty evidence). For other secondary maternal outcomes, we are uncertain if there are differences between probiotics and placebo for: postpartum haemorrhage; weight gain during pregnancy intervention and total gestational weight gain; fasting plasma glucose and need for extra pharmacotherapy (insulin). Probiotics may be associated with a slight reduction in triglycerides and total cholesterol. In probiotics compared with placebo, there was evidence of reduction in markers for insulin resistance (HOMA-IR) and HOMA-B; and insulin secretion. There was also an increase in quantitative insulin sensitivity check index (QUICKI). Probiotics were associated with minor benefits in relevant bio-markers with evidence of a reduction in inflammatory markers high-sensitivity C-reactive protein (hs-CRP), interleukin 6 (IL-6), and marker of oxidative stress malondialdehyde; and an increase in antioxidant total glutathione, but we are uncertain if there is any difference in total antioxidant capacity. No trials reported secondary outcomes: perineal trauma, postnatal weight retention or return to pre-pregnancy weight and postnatal depression. Infant/child/adult outcomes We are uncertain if probiotics have any effect, compared with placebo, on the risk of large-for-gestational-age babies (RR 0.73, 95% CI 0.35 to 1.52; participants = 174; studies = 2; low-certainty evidence) or infant hypoglycaemia (RR 0.85, 95% CI 0.39 to 1.84; participants = 177; studies = 3; low-certainty evidence) because the certainty of evidence is low and the 95% CIs span possible benefit and possible harm. No trials reported primary outcomes of: perinatal (fetal/neonatal) mortality; or neurosensory disability. For other secondary outcomes, we are uncertain if there is any difference between probiotics and placebo in gestational age at birth, preterm birth, macrosomia, birthweight, head circumference, length, infant hypoglycaemia, and neonatal intensive care unit (NICU) admissions. There was evidence of a reduction in infant hyperbilirubinaemia with probiotics compared with placebo. No trials reported secondary outcomes: infant adiposity, and later childhood adiposity. There were no adverse events reported by any of the trials. AUTHORS' CONCLUSIONS: Low-certainty evidence means we are not certain if there is any difference between probiotic and placebo groups in maternal hypertensive disorders of pregnancy, caesareans; and large-for-gestational-age babies. There were no adverse events reported by the trials. Due to the variability of probiotics used and small sample sizes of trials, evidence from this review has limited ability to inform practice. Well-designed adequately-powered trials are needed to identify whether probiotics may improve maternal blood glucose levels and/or infant/child/adult outcomes; and whether they can be used to treat GDM.

Interventions to prevent women from developing gestational diabetes mellitus: an overview of Cochrane Reviews.

BACKGROUND: The prevalence of gestational diabetes mellitus (GDM) is increasing, with approximately 15% of pregnant women affected worldwide, varying by country, ethnicity and diagnostic thresholds. There are associated short- and long-term health risks for women and their babies. OBJECTIVES: We aimed to summarise the evidence from Cochrane systematic reviews on the effects of interventions for preventing GDM. METHODS: We searched the Cochrane Database of Systematic Reviews (6 August 2019) with key words 'gestational diabetes' OR 'GDM' to identify reviews pre-specifying GDM as an outcome. We included reviews of interventions in women who were pregnant or planning a pregnancy, irrespective of their GDM risk status. Two overview authors independently assessed eligibility, extracted data and assessed quality of evidence using ROBIS and GRADE tools. We assigned interventions to categories with graphic icons to classify the effectiveness of interventions as: clear evidence of benefit or harm (GRADE moderate- or high-quality evidence with a confidence interval (CI) that did not cross the line of no effect); clear evidence of no effect or equivalence (GRADE moderate- or high-quality evidence with a narrow CI crossing the line of no effect); possible benefit or harm (low-quality evidence with a CI that did not cross the line of no effect or GRADE moderate- or high-quality evidence with a wide CI); or unknown benefit or harm (GRADE low-quality evidence with a wide CI or very low-quality evidence). MAIN RESULTS: We included 11 Cochrane Reviews (71 trials, 23,154 women) with data on GDM. Nine additional reviews pre-specified GDM as an outcome, but did not identify GDM data in included trials. Ten of the 11 reviews were judged to be at low risk of bias and one review at unclear risk of bias. Interventions assessed included diet, exercise, a combination of diet and exercise, dietary supplements, pharmaceuticals, and management of other health problems in pregnancy. The quality of evidence ranged from high to very low. Diet Unknown benefit or harm: there was unknown benefit or harm of dietary advice versus standard care, on the risk of GDM: risk ratio (RR) 0.60, 95% CI 0.35 to 1.04; 5 trials; 1279 women; very low-quality evidence. There was unknown benefit or harm of a low glycaemic index diet versus a moderate-high glycaemic index diet on the risk of GDM: RR 0.91, 95% CI 0.63 to 1.31; 4 trials; 912 women; low-quality evidence. Exercise Unknown benefit or harm: there was unknown benefit or harm for exercise interventions versus standard antenatal care on the risk of GDM: RR 1.10, 95% CI 0.66 to 1.84; 3 trials; 826 women; low-quality evidence. Diet and exercise combined Possible benefit: combined diet and exercise interventions during pregnancy versus standard care possibly reduced the risk of GDM: RR 0.85, 95% CI 0.71 to 1.01; 19 trials; 6633 women; moderate-quality evidence. Dietary supplements Clear evidence of no effect: omega-3 fatty acid supplementation versus none in pregnancy had no effect on the risk of GDM: RR 1.02, 95% CI 0.83 to 1.26; 12 trials; 5235 women; high-quality evidence. Possible benefit: myo-inositol supplementation during pregnancy versus control possibly reduced the risk of GDM: RR 0.43, 95% CI 0.29 to 0.64; 3 trials; 502 women; low-quality evidence. Possible benefit: vitamin D supplementation versus placebo or control in pregnancy possibly reduced the risk of GDM: RR 0.51, 95% CI 0.27 to 0.97; 4 trials; 446 women; low-quality evidence. Unknown benefit or harm: there was unknown benefit or harm of probiotic with dietary intervention versus placebo with dietary intervention (RR 0.37, 95% CI 0.15 to 0.89; 1 trial; 114 women; very low-quality evidence), or probiotic with dietary intervention versus control (RR 0.38, 95% CI 0.16 to 0.92; 1 trial; 111 women; very low-quality evidence) on the risk of GDM. There was unknown benefit or harm of vitamin D + calcium supplementation versus placebo (RR 0.33, 95% CI 0.01 to 7.84; 1 trial; 54 women; very low-quality evidence) or vitamin D + calcium + other minerals versus calcium + other minerals (RR 0.42, 95% CI 0.10 to 1.73; 1 trial; 1298 women; very low-quality evidence) on the risk of GDM. Pharmaceutical Possible benefit: metformin versus placebo given to obese pregnant women possibly reduced the risk of GDM: RR 0.85, 95% CI 0.61 to 1.19; 3 trials; 892 women; moderate-quality evidence. Unknown benefit or harm:eight small trials with low- to very low-quality evidence showed unknown benefit or harm for heparin, aspirin, leukocyte immunisation or IgG given to women with a previous stillbirth on the risk of GDM. Management of other health issues Clear evidence of no effect: universal versus risk based screening of pregnant women for thyroid dysfunction had no effect on the risk of GDM: RR 0.93, 95% CI 0.70 to 1.25; 1 trial; 4516 women; moderate-quality evidence. Unknown benefit or harm: there was unknown benefit or harm of using fractional exhaled nitrogen oxide versus a clinical algorithm to adjust asthma therapy on the risk of GDM: RR 0.74, 95% CI 0.31 to 1.77; 1 trial; 210 women; low-quality evidence. There was unknown benefit or harm of pharmacist led multidisciplinary approach to management of maternal asthma versus standard care on the risk of GDM: RR 5.00, 95% CI 0.25 to 99.82; 1 trial; 58 women; low-quality evidence. AUTHORS' CONCLUSIONS: No interventions to prevent GDM in 11 systematic reviews were of clear benefit or harm. A combination of exercise and diet, supplementation with myo-inositol, supplementation with vitamin D and metformin were of possible benefit in reducing the risk of GDM, but further high-quality evidence is needed. Omega-3-fatty acid supplementation and universal screening for thyroid dysfunction did not alter the risk of GDM. There was insufficient high-quality evidence to establish the effect on the risk of GDM of diet or exercise alone, probiotics, vitamin D with calcium or other vitamins and minerals, interventions in pregnancy after a previous stillbirth, and different asthma management strategies in pregnancy. There is a lack of trials investigating the effect of interventions prior to or between pregnancies on risk of GDM.

Targeted Enrichment of Large Gene Families for Phylogenetic Inference: Phylogeny and Molecular Evolution of Photosynthesis Genes in the Portullugo Clade (Caryophyllales).

Hybrid enrichment is an increasingly popular approach for obtaining hundreds of loci for phylogenetic analysis across many taxa quickly and cheaply. The genes targeted for sequencing are typically single-copy loci, which facilitate a more straightforward sequence assembly and homology assignment process. However, this approach limits the inclusion of most genes of functional interest, which often belong to multi-gene families. Here, we demonstrate the feasibility of including large gene families in hybrid enrichment protocols for phylogeny reconstruction and subsequent analyses of molecular evolution, using a new set of bait sequences designed for the "portullugo" (Caryophyllales), a moderately sized lineage of flowering plants (~ 2200 species) that includes the cacti and harbors many evolutionary transitions to C$_{\mathrm{4}}$ and CAM photosynthesis. Including multi-gene families allowed us to simultaneously infer a robust phylogeny and construct a dense sampling of sequences for a major enzyme of C$_{\mathrm{4}}$ and CAM photosynthesis, which revealed the accumulation of adaptive amino acid substitutions associated with C$_{\mathrm{4}}$ and CAM origins in particular paralogs. Our final set of matrices for phylogenetic analyses included 75-218 loci across 74 taxa, with ~ 50% matrix completeness across data sets. Phylogenetic resolution was greatly improved across the tree, at both shallow and deep levels. Concatenation and coalescent-based approaches both resolve the sister lineage of the cacti with strong support: Anacampserotaceae $+$ Portulacaceae, two lineages of mostly diminutive succulent herbs of warm, arid regions. In spite of this congruence, BUCKy concordance analyses demonstrated strong and conflicting signals across gene trees. Our results add to the growing number of examples illustrating the complexity of phylogenetic signals in genomic-scale data.

General practitioners' attitudes and decision making regarding admission for older adults with infection: a UK qualitative interview study.

BACKGROUND: The world has an ageing population. Infection is common in older adults; serious infection has a high mortality rate and is associated with unplanned admissions. In the UK, general practitioners (GPs) must identify which older patients require admission to hospital and provide appropriate care and support for those staying at home. OBJECTIVES: To explore attitudes of UK GPs towards referring older patients with suspected infection to hospital, how they weigh up the decision to admit against the alternatives and how alternatives to admission could be made more effective.Methods. Qualitative study using semi-structured interviews. GPs were purposively sampled from across the UK to achieve maximum variation in terms of GP role, experience and practice population. Interview transcripts were coded and analysed using a modified framework approach. RESULTS: GPs' key influences on decision making were grouped into patient, GP and system factors. Patient factors included clinical factors, social factors and shared decision making. GP factors included gut instinct, risk management and acknowledging an associated personal emotional burden. System factors involved weighing up the pressure on secondary care beds against increasing GP workload. GPs described that finding an alternative to admission could be more time consuming, complex to arrange or were restricted by lack of capacity. CONCLUSION: GPs need to be empowered to make safe decisions about place of care for older adults with suspected infection. This may mean developing strategies to support decision making as well as improving the ease of access to, and capacity of, any alternatives to admission.

Challenges and strategies for general practitioners diagnosing serious infections in older adults: a UK qualitative interview study.

BACKGROUND: Serious infections in older people are associated with unplanned hospital admissions and high mortality. Recognising the presence of a serious infection and making an accurate diagnosis are important challenges for General Practice. This study aimed to explore the issues UK GPs face when diagnosing serious infections in older patients. METHODS: Qualitative study using semi-structured interviews. 28 GPs from 27 practices were purposively sampled from across the UK to achieve maximum variation in terms of GP role, experience and practice population. Interviews began by asking participants to describe recent or memorable cases where they had assessed older patients with suspected serious infections. Additional questions from the topic guide were used to explore the challenges further. Interview transcripts were coded and analysed using a modified framework approach. RESULTS: Diagnosing serious infection in older adults was perceived to be challenging by participating GPs and the diagnosis was often uncertain. Contributing factors included patient complexity, atypical presentations, as well as a lack of knowledge of patients due to a loss in continuity. Diagnostic challenges were present at each stage of the patient assessment. Scoring systems were mainly used as communication tools. Investigations were sometimes used to resolve diagnostic uncertainty, but availability and speed of result limited their practical use. Clear safety-net plans shared with patients and their families helped GPs manage ongoing uncertainty. CONCLUSIONS: Diagnostic challenges are present throughout the assessment of an older adult with a serious infection in primary care. Supporting GPs to provide continuity of care may improve the recognition and developing point of care testing for use in community settings may reduce diagnostic uncertainty.

Testing Evidence-Based Strategies for Clinical Ladder Program Refinement.

BACKGROUND: Institutional objectives for clinical ladder program (CLP) implementation often focus on improving job satisfaction, retention rates, and patient outcomes. Local inconsistency in CLP implementation seemed to be contributing to decreased job satisfaction and nurse retention rates. PURPOSE: The aim of this study was to use current evidence and prevailing perceptions of bedside nurses and nurse managers to refine an existing CLP. METHODS: Pretest/posttest design was used to evaluate differences in perceptions of existing CLP (baseline) and perceptions 6 months after CLP refinements were initiated (post). RESULTS: Baseline perceptions of CLP implementation were significantly different for bedside nurses and managers. Bedside nurses were more dissatisfied than nurse managers. Perceptions became more similar across groups as initiatives were implemented to improve CLP understanding and as CLP implementation became more consistent across the organization. CONCLUSIONS: Targeted strategies tailored to increase understanding and consistent CLP implementation can be effective in improving CLP satisfaction and increasing bedside nurse professional behaviors.

The Impact of Leadership Responses to Sexual Harassment and Gender Discrimination Reports on Emotional Distress and Retention Intentions in Military Members.

Reporting instances of sexual harassment and gender discrimination in the workplace is an integral part of the prevention and response efforts of the United States Department of Defense (DoD). Any military member who comes forward to report their experience should be provided appropriate intervention, support, and restorative care. While much research has focused on prevention efforts and encouraging reporting, few researchers have systematically examined what happens when military members come forward. In the military, leaders (the chain of command) are primarily responsible for receiving and investigating reports of potential sex-based military equal opportunity violations (SBMEO) involving sexual harassment or gender discrimination. This study used results from the congressionally-mandated 2016 Workplace and Gender Relations Survey of Active Duty Members (2016 WGRA) to identify military members who indicated experiencing a potential SBMEO violation and reported to their chain of command (n = 3,982) in order to examine the relationships among the actions as a result of reporting, reporting satisfaction, emotional distress, and retention intentions. Findings revealed experiencing positive actions as a result of reporting was associated with increased satisfaction with reporting, which in turn, was associated with decreased emotional distress and increased retention intentions. Conversely, experiencing negative actions as a result of reporting was associated with decreased satisfaction with reporting, increased emotional distress, and decreased retention intentions. These findings highlight the crucial role military leaders serve in supporting those who come forward in mitigating potential negative outcomes associated with SBMEO violations that could impact military readiness.

Attributes of a Successful Clinical Ladder Program for Nurses: An Integrative Review.

BACKGROUND: Clinical ladder programs (CLPs) are often utilized by healthcare organizations to monitor and incentivize staff nurse development. Few studies exist related to this topic, yet implementation of CLPs can be an important factor in staff nurse retention and satisfaction, and therefore requires better understanding. AIM: To identify and examine the factors that contribute to a successful CLP. METHODS: A systematic search of the literature was conducted in PubMed and CINAHL. Studies were not limited by year and were included if the focus was CLP attributes. PRISMA and PICOT were used to guide the process. A matrix of the existing studies was used, and interrater reliability was established at 90% with consensus building for inclusion of studies by the research team. RESULTS: Twenty-nine studies were identified for inclusion. Most were program evaluation. Institution's organizational culture was identified as the overarching attribute contributing to a successful CLP. Common themes identified as important to a supportive organizational culture were (a) education and experience; (b) competence and critical thinking; (c) job satisfaction and retention; and (d) compensation and institutional cost. Each of these components can play a significant role in the overall success of a CLP. LINKING EVIDENCE TO ACTION: While further research of a higher caliber is needed, some recommendations for practice can be made: (a) CLP description and terminology needs to be consistent with intent, (b) mandatory CLP engagement has positive implications, and (c) implementation needs to be driven by bedside nurses and includes both continuous education and refinement of program requirements and incentives.

Molecular evolution of key metabolic genes during transitions to C4 and CAM photosynthesis.

PREMISE OF THE STUDY: Next-generation sequencing facilitates rapid production of well-sampled phylogenies built from very large genetic data sets, which can then be subsequently exploited to examine the molecular evolution of the genes themselves. We present an evolutionary analysis of 83 gene families (19 containing carbon-concentrating mechanism (CCM) genes, 64 containing non-CCM genes) in the portullugo clade (Caryophyllales), a diverse lineage of mostly arid-adapted plants that contains multiple evolutionary origins of all known photosynthesis types in land plants (C3 , C4 , CAM, C4 -CAM, and various intermediates). METHODS: We inferred a phylogeny of 197 individuals from 167 taxa using coalescent-based approaches and individual gene family trees using maximum likelihood. Positive selection analyses were conducted on individual gene family trees with a mixed effects model of evolution (MEME). We devised new indices to compare levels of convergence and prevalence of particular residues between CCM and non-CCM genes and between species with different photosynthetic pathways. KEY RESULTS: Contrary to expectations, there were no significant differences in the levels of positive selection detected in CCM versus non-CCM genes. However, we documented a significantly higher level of convergent amino acid substitutions in CCM genes, especially in C4 taxa. CONCLUSIONS: Our analyses reveal a new suite of amino acid residues putatively important for C4 and CAM function. We discuss both the advantages and challenges of using targeted enrichment sequence data for exploratory studies of molecular evolution.

Phylogeny, evolution, and biogeographic history of Calandrinia (Montiaceae).

PREMISE OF THE STUDY: Calandrinia are small, succulent herbs that vary broadly in habitat, morphology, life history, and photosynthetic metabolism. The lineage is placed within the Montiaceae, which in turn is sister to the rest of the Portulacineae (Caryophyllales). Calandrinia occupy two distinct biogeographic regions, one in the Americas (~14 species), and one in Australia (~74 species). Past analyses of the Montiaceae present conflicting hypotheses for the phylogenetic placement and monophyly of Calandrinia, and to date, there has been no molecular phylogenetic analysis of the Australian species. METHODS: Using a targeted gene enrichment approach, we sequenced 297 loci from multiple gene families across the Montiaceae, including all named and 16 putative new species of Australian Calandrinia, and the enigmatic monotypic genus Rumicastrum. KEY RESULTS: All data sets and analyses reject the monophyly of Calandrinia, with Australian and New World Calandrinia each comprising distinct and well-supported clades, and Rumicastrum nested within Australian Calandrinia. We provide the first well-supported phylogeny for Australian Calandrinia, which includes all named species and several phrase-named taxa. CONCLUSIONS: This study brings much needed clarity to relationships within Montiaceae and confirms that New World and Australian Calandrinia do not form a clade. Australian Calandrinia is a longtime resident of the continent, having diverged from its sister lineage ~30 Ma, concurrent with separation of Australia from Antarctica. Most diversification occurred during the middle Miocene, with lowered speciation and/or higher extinction rates coincident with the establishment of severe aridity by the late Miocene.

Parallel Pleistocene amphitropical disjunctions of a parasitic plant and its host.

PREMISE OF THE STUDY: Aphyllon is a clade of holoparasites that includes closely related North American and South American species parasitic on Grindelia. Both Aphyllon (Orobanchaceae) and Grindelia (Asteraceae) have amphitropical disjunctions between North America and South America; however, the timing of these patterns and the processes to explain them are unknown. METHODS: Chronograms for the Orobanchaceae and Grindelia and their relatives were constructed using fossil and secondary calibration points, one of which was based on the inferred timing of horizontal gene transfer from a papilionoid legume into the common ancestor of Orobanche and Phelipanche. Elevated rates of molecular evolution in the Orobanchaceae have hindered efforts to determine reliable divergence time estimates in the absence of a fossil record. However, using a horizontal gene transfer event as a secondary calibration overcomes this limitation. These chronograms were used to reconstruct the biogeography of Aphyllon, Grindelia, and relatives using a DEC+J model implemented in RevBayes. KEY RESULTS: Aphyllon had two amphitropical dispersals from North America to South America, while Grindelia had a single dispersal. The dispersal of the Aphyllon lineage that is parasitic on Grindelia (0.40 Ma) took place somewhat after Grindelia began to diversify in South America (0.93 Ma). Using a secondary calibration based on horizontal gene transfer, we infer more recent divergence dates of holoparasitic Orobancheae than previous studies. CONCLUSIONS: Parallel host-parasite amphitropical disjunctions in Grindelia and Aphyllon illustrate one means by which ecological specialization may result in nonindependent patterns of diversity in distantly related lineages. Although Grindelia and Aphyllon both dispersed to South America recently, Grindelia appears to have diversified more extensively following colonization. More broadly, recent Pleistocene glaciations probably have also contributed to patterns of diversity and biogeography of temperate northern hemisphere Orobancheae. We also demonstrate the utility of using horizontal gene transfer events from well-dated clades to calibrate parasite phylogenies in the absence of a fossil record.

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells.

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.

Role of Lymphovascular Invasion in Oral Cavity Squamous Cell Carcinoma Regional Metastasis and Prognosis.

OBJECTIVE: The overall 5-year survival for oral squamous cell carcinoma (OSCC) has not changed in the last 20 years despite advances in treatment. Lymphovascular invasion (LVI) has been shown to be a negative prognostic factor in other cancers, however its role in the prognosis of OSCC remains unclear. This study aims to determine if LVI is a predictor of cervical lymph node metastasis and/or recurrence in OSCC. METHODS: We conducted a retrospective cohort review of patients from our institutional cancer registry who were treated for OSCC between 2004 and 2018. Patient demographics, surgical pathology results, and clinical outcome data were collected. A multivariable logistic regression analysis was performed to determine if LVI was an independent predictor of cervical lymph node metastasis and/or recurrence. RESULTS: 442 patients were included, 32.8% were female and median age at time of diagnosis was 61.2 years. LVI was present in 32.8% of patients. When controlled for age, sex, t-classification, perineural invasion, depth of invasion (DOI), and margin status, LVI was a significant predictor of the presence of cervical node metastasis (OR: 3.42, CI: 2.17-5.39, P < .001). There was no significant association found between LVI and local recurrence (OR: 1.03, CI: 0.57-1.84, P = .92), regional recurrence (OR: 1.10, CI: 0.57-2.11, P = .78), or distant recurrence (OR: 1.59, CI: 0.87-2.94, P = .13). CONCLUSION: The results of this study suggest that LVI is a significant predictor of the presence of cervical lymph node metastasis at presentation independent of other known prognostic factors. LVI, however, was not found to be a significant independent predictor of locoregional or distant recurrence.Level of Evidence: Level III.

Molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancers: a Beat Childhood Cancer Research Consortium trial.

BACKGROUND: Children with relapsed central nervous system (CNS tumors), neuroblastoma, sarcomas, and other rare solid tumors face poor outcomes. This prospective clinical trial examined the feasibility of combining genomic and transcriptomic profiling of tumor samples with a molecular tumor board (MTB) approach to make real­time treatment decisions for children with relapsed/refractory solid tumors. METHODS: Subjects were divided into three strata: stratum 1-relapsed/refractory neuroblastoma; stratum 2-relapsed/refractory CNS tumors; and stratum 3-relapsed/refractory rare solid tumors. Tumor samples were sent for tumor/normal whole-exome (WES) and tumor whole-transcriptome (WTS) sequencing, and the genomic data were used in a multi-institutional MTB to make real­time treatment decisions. The MTB recommended plan allowed for a combination of up to 4 agents. Feasibility was measured by time to completion of genomic sequencing, MTB review and initiation of treatment. Response was assessed after every two cycles using Response Evaluation Criteria in Solid Tumors (RECIST). Patient clinical benefit was calculated by the sum of the CR, PR, SD, and NED subjects divided by the sum of complete response (CR), partial response (PR), stable disease (SD), no evidence of disease (NED), and progressive disease (PD) subjects. Grade 3 and higher related and unexpected adverse events (AEs) were tabulated for safety evaluation. RESULTS: A total of 186 eligible patients were enrolled with 144 evaluable for safety and 124 evaluable for response. The average number of days from biopsy to initiation of the MTB-recommended combination therapy was 38 days. Patient benefit was exhibited in 65% of all subjects, 67% of neuroblastoma subjects, 73% of CNS tumor subjects, and 60% of rare tumor subjects. There was little associated toxicity above that expected for the MGT drugs used during this trial, suggestive of the safety of utilizing this method of selecting combination targeted therapy. CONCLUSIONS: This trial demonstrated the feasibility, safety, and efficacy of a comprehensive sequencing model to guide personalized therapy for patients with any relapsed/refractory solid malignancy. Personalized therapy was well tolerated, and the clinical benefit rate of 65% in these heavily pretreated populations suggests that this treatment strategy could be an effective option for relapsed and refractory pediatric cancers. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02162732. Prospectively registered on June 11, 2014.

The origin of the serpentine endemic Minuartia laricifolia subsp. ophiolitica by vicariance and competitive exclusion.

Serpentine soils harbour a unique flora that is rich in endemics. We examined the evolution of serpentine endemism in Minuartia laricifolia, which has two ecologically distinct subspecies with disjunct distributions: subsp. laricifolia on siliceous rocks in the western Alps and eastern Pyrenees and subsp. ophiolitica on serpentine in the northern Apennines. We analysed AFLPs and chloroplast sequences from 30 populations to examine their relationships and how their current distributions and ecologies were influenced by Quaternary climatic changes. Minuartia laricifolia was divided into four groups with a BAPS cluster analysis of the AFLP data, one group consisted only of subsp. ophiolitica, while three groups were found within subsp. laricifolia: Maritime Alps, north-western Alps and central Alps. The same groups were recovered in a neighbour-joining tree, although subsp. ophiolitica was nested within the Maritime Alps group of subsp. laricifolia. Subspecies ophiolitica contained three different chloroplast haplotypes, which were also found in the Maritime Alps group of subsp. laricifolia. Given its high genetic diversity, subsp. ophiolitica appears to have arisen from subsp. laricifolia by vicariance instead of by long-distance dispersal. Genetic and geographic evidence point to the Maritime Alps populations of subsp. laricifolia as the closest relatives of subsp. ophiolitica. We hypothesize that M. laricifolia was also able to grow on nonserpentine rocks in the northern Apennines during glacial periods when the vegetation was more open, but that only the serpentine-adapted populations were able to persist until the present due to their competitive exclusion from more favourable habitats.

The evolution of substrate differentiation in Minuartia series Laricifoliae (Caryophyllaceae) in the European Alps: In situ origin or repeated colonization?

PREMISE OF THE STUDY: Substrate specialization is often considered an important factor in evolutionary diversification. A classic example of divergence related to different substrate types is the dichotomy between calcicole and calcifuge plants on calcareous and siliceous substrates as found in the European Alps. When closely related species with contrasting substrate preferences are found in the same area, it is generally hypothesized that they diverged where they now occur. However, it is possible that Alpine edaphic diversity instead allows the coexistence of related species whose edaphic differentiation took place deeper in the phylogeny, in some other part of the range of their clades. METHODS: We used sequences of the nuclear internal and external transcribed spacer regions to examine the origin of substrate differentiation in Minuartia series Laricifoliae, which contains many edaphic endemics, including a pair of Alpine taxa with contrasting substrate preferences: Minuartia langii (calcicole) and M. laricifolia (calcifuge). KEY RESULTS: MINUARTIA LANGII and M. laricifolia are each more closely related to Balkan species than they are to each other and reached the Alps independently. The clade to which they belong is ancestrally calcicole. Minuartia langii inherited the ancestral ecology, while M. laricifolia is part of a subclade with serpentine endemics and one substrate generalist. CONCLUSIONS: In the study group, taxa with contrasting substrate preferences did not diverge in the Alps. Instead, taxa whose substrate differentiation arose elsewhere, likely on the Balkan Peninsula, were preadapted to take advantage of Alpine substrate diversity.

Endoscopic Versus Open Surgical Intervention for Congenital Laryngeal Webs: A Systematic Review and Meta-Analysis.

OBJECTIVES: To examine and compare the outcomes of various surgical interventions for congenital laryngeal webs in terms of avoidance of tracheostomy, rate of decannulation, web recurrence, revision surgery, and mortality in children. DATA SOURCES: Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic review was conducted on December 10, 2021, using a comprehensive search in PubMed, Web of Science, Cochrane library, and Embase with no date restriction. REVIEW METHODS: Articles on surgical intervention for congenital laryngeal webs in pediatric (<18 years) patients were included in the analysis. Articles including acquired laryngeal webs, no surgical intervention, or exclusively adult population were excluded. RESULTS: 9027 articles were reviewed, 24 articles met the inclusion criteria and 126 patients were included. In patients with Grades I and II webs, there was no significant difference in rates of tracheostomy or decannulation, between endoscopic (100%) versus open approach (100%). For Grades III and IV webs, 96% of patients who received open surgery were decannulated or avoided tracheostomy compared to 84% of those managed endoscopically (p = 0.081). There were significantly lower rates of revision surgery in the open group compared to the endoscopic group (77.8% vs 30.9%, p = 0.008). CONCLUSION: This study showed no difference in rates of tracheostomy, decannulation, web recurrence, revision, or mortality between endoscopic and open approaches for the treatment of Grades I and II webs. For Grades III and IV, open surgical techniques achieved a lower revision rate. Results should be interpreted in light of associated increased morbidity with open procedures.