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Emergency Department (ED) crowding is defined as a situation wherein the demands of emergency services overcome the ability of a department to provide high-quality care within an appropriate time frame. There is a need for solutions, as the harms of crowding impact patients, staff, and healthcare spending. An overview of ED crowding was previously published by our group, which outlines these global issues. The problem of overcrowding in emergency departments has emerged as a global public health concern, and several healthcare agencies have addressed the issue and proposed possible solutions at each level of emergency care. There is no current literature summarizing the extensive research on interventions and solutions, thus there is a need for data synthesis to inform policymakers in this field. The aim of this overview was to summarize the interventions at each level of emergency care: input, throughput, and output. The methodology was supported by the current PRIOR statement for an overview of reviews. The study summarized twenty-seven full-text systematic reviews, which encompassed three hundred and eight primary studies. The results of the summary displayed a requirement for increasing studies in input and output interventions, as these showed the best outcomes with regard to ED crowding metrics. Moreover, the results displayed heterogeneous results at each level of ED care; these reflected that generally solutions have not been matched to specific problems facing regional centres. Thus, individual factors need to be considered when implementing solutions in Emergency Departments., (© 2023. The Author(s), under exclusive licence to Società Italiana di Medicina Interna (SIMI).)

Background: Recent advances in texture analysis and machine learning offer new opportunities to improve the application of imaging to intervertebral disc biomechanics. This study employed texture analysis and machine learning on MRIs to investigate the lumbar disc's response to loading., Methods: Thirty-five volunteers (30 (SD 11) yrs.) with and without chronic back pain spent 20 min lying in a relaxed unloaded supine position, followed by 20 min loaded in compression, and then 20 min with traction applied. T 2 -weighted MR images were acquired during the last 5 min of each loading condition. Custom image analysis software was used to segment discs from adjacent tissues semi-automatically and segment each disc into the nucleus, anterior and posterior annulus automatically. A grey-level, co-occurrence matrix with one to four pixels offset in four directions (0°, 45°, 90° and 135°) was then constructed (320 feature/tissue). The Random Forest Algorithm was used to select the most promising classifiers. Linear mixed-effect models and Cohen's d compared loading conditions., Findings: All statistically significant differences (p < 0.001) were observed in the nucleus and posterior annulus in the 135° offset direction at the L4-5 level between lumbar compression and traction. Correlation (P 2-Offset , P 4-Offset ) and information measure of correlation 1 (P 3-Offset , P 4-Offset ) detected significant changes in the nucleus. Statistically significant changes were also observed for homogeneity (P 2-Offset , P 3-Offset ), contrast (P 2-Offset ), and difference variance (P 4-Offset ) of the posterior annulus., Interpretation: MRI textural features may have the potential of identifying the disc's response to loading, particularly in the nucleus and posterior annulus, which appear most sensitive to loading., Level of Evidence: Diagnostic: individual cross-sectional studies with consistently applied reference standard and blinding., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: In this clinical focus article, we discuss the nature of critical thinking, its importance for adolescents, and its interaction with later language development. We also introduce a language arts program, Philosophy for Adolescents . The program aims to support critical thinking, narrative and expository discourse, and the use of complex syntax and literate vocabulary., Method: In designing Philosophy for Adolescents , we examined research from education, psychology, philosophy, and speech-language pathology in the areas of critical thinking and narrative discourse. The resulting program encourages students to think independently, listen to others, offer reasons and evidence to support their views, and express themselves with accuracy, clarity, and efficiency. Implementation is illustrated with a case study of a 12-year-old girl., Results: Although critical thinking improves during adolescence, many students struggle in this area, particularly in the ability to offer reasons and evidence to support their views. This suggests that these adolescents could benefit from instruction in critical thinking. Research also indicates that competence in critical thinking is associated with narrative and expository discourse, and with the use of complex syntax and literate words such as metacognitive verbs. Instruction using Philosophy for Adolescents may be carried out in small groups or individually by a speech-language pathologist working collaboratively with a teacher, teaching assistant, or other professional. Designed for flexible application, the program may be used with adolescents who have language disorders and those who have typical language development., Conclusion: Research is necessary to evaluate the program with students from diverse backgrounds, having differing levels of language competence and academic achievement., Supplemental Material: https://doi.org/10.23641/asha.19736059.

Cas9 and a guide RNA (gRNA) function to target specific genomic loci for generation of a double-stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double-stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase (CD) to induce C > T (or G>A) mutations at targeted sites. In this study, we test base editing in Drosophila by expressing a transgenic Drosophila base editor (based on the mammalian BE2) that consists of a fusion protein of CD, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the Drosophila base editor ( Actin5C-BE2 ) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C > T were found in positions 3-9 of the gRNA-targeted site, with conversion reaching ∼100% of targeted DNA in somatic tissues. Surprisingly, the simultaneous use of two gRNAs targeting a genomic region spaced ∼50 bp apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila , and could be used to induce point mutations at select loci.

Magnetic resonance imaging findings often do not distinguish between people with and without low back pain (LBP). However, there are still a large number of people who undergo magnetic resonance imaging to help determine the etiology of their back pain. Texture analysis shows promise for the classification of tissues that look similar, and machine learning can minimize the number of comparisons. This study aimed to determine if texture features from lumbar spine magnetic resonance imaging differ between people with and without LBP. In total, 14 participants with chronic LBP were matched for age, weight, and gender with 14 healthy volunteers. A custom texture analysis software was used to construct a gray-level co-occurrence matrix with one to four pixels offset in 0° direction for the disc and superior and inferior endplate regions. The Random Forests Algorithm was used to select the most promising classifiers. The linear mixed-effect model analysis was used to compare groups (pain vs. pain-free) at each level controlling for age. The Random Forest Algorithm recommended focusing on intervertebral discs and endplate zones at L4-5 and L5-S1. Differences were observed between groups for L5-S1 superior endplate contrast, homogeneity, and energy (p = .02). Differences were observed for L5-S1 disc contrast and homogeneity (p < .01), as well as for the inferior endplates contrast, homogeneity, and energy (p < .03). Magnetic resonance imaging textural features may have potential in identifying structures that may be the target of further investigations about the reasons for LBP., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Aims: Selection for optimal root system architecture (RSA) is important to ensure genetic gains in the sustainable production of wheat ( Triticum aestivum L.). Here we examine the hypothesis that past wheat breeding has led to changes in RSA and that future breeding efforts can focus directly on RSA to improve adaptation to target environments., Methods: We conducted field trials using diverse wheat varieties, including modern and historic UK varieties and non-UK landraces, tested under contrasting tillage regimes (non-inversion tillage versus conventional ploughing) for two trial years or different seeding rates (standard versus high rate) for one trial year. We used field excavation, washing and measurement of root crowns ('shovelomics') to characterise RSA traits, including: numbers of seminal, crown and nodal roots per plant, and crown root growth angle., Results: We found differences among genotypes for all root traits. Modern varieties generally had fewer roots per plant than historic varieties. On average, there were fewer crown roots and root angles were wider under shallow non-inversion tillage compared with conventional ploughing. Crown root numbers per plant also tended to be smaller at a high seeding rate compared with the standard. There were significant genotype-by-year, genotype-by-tillage and genotype-by-seeding-rate interactions for many root traits., Conclusions: Smaller root systems are likely to be a result of past selection that facilitated historical yield increases by reducing below-ground competition within the crop. The effects of crop management practices on RSA depend on genotype, suggesting that future breeding could select for improved RSA traits in resource-efficient farming systems., (© The Author(s) 2020.)

There has been an increase in opioid analgesic prescribing in general practice (GP). This is causing some concern around this contributing to dependency. NHS Lanarkshire have attempted to reduce the prescribing from GP surgeries through the development of specialised Pharmacist Independent Prescriber clinics being delivered from the practices. This article looks at the development of these services with pharmacist independent prescribers and the results from developing the services. The article aims to provide advice and recommendations for the development of other services and strategies to minimise the risks associated with Opioid Analgesic Dependence for patients.

An autonomously bioluminescent Saccharomyces cerevisiae BLYAhS bioreporter was developed in this study for the simple and rapid detection of dioxin-like compounds (DLCs) and aryl hydrocarbon receptor (AhR) agonists. This recombinant yeast reporter was based on a synthetic bacterial luciferase reporter gene cassette (lux) that can produce the luciferase as well as the enzymes capable of self-synthesizing the requisite substrates for bioluminescent production from endogenous cellular metabolites. As a result, bioluminescent signal production is generated continuously and autonomously without cell lysis or exogenous reagent addition. By linking the expression of the autobioluminescent lux reporter cassette to AhR activation via the use of a dioxin-responsive promoter, the S. cerevisiae BLYAhS bioreporter emitted a bioluminescent signal in response to DLC exposure in a dose-responsive manner. The model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), could be detected within 4 h with a half maximal effective concentration (EC 50 ) of ~ 8.1 nM and a lower detection limit of 500 pM. The autobioluminescent response of BLYAhS to other AhR agonists, including 2,3,7,8-tetrachlorodibenzofuran (TCDF), polychlorinated bisphenyl congener 126 (PCB-126) and 169 (PCB-169), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), benzo[a]pyrene (BaP), and β-naphthoflavone (bNF), were also characterized in this study. The non-destructive and reagent-free nature of the BLYAhS reporter assay facilitated near-continuous, automated signal acquisition without additional hands-on effort and cost, providing a simple and cost-effective method for rapid DLC detection.

Mosquitoes are vectors for multiple infectious human diseases and use a variety of sensory cues (olfactory, temperature, humidity and visual) to locate a human host. A comprehensive understanding of the circuitry underlying sensory signalling in the mosquito brain is lacking. Here we used the Q-system of binary gene expression to develop transgenic lines of Anopheles gambiae in which olfactory receptor neurons expressing the odorant receptor co-receptor (Orco) gene are labelled with GFP. These neurons project from the antennae and maxillary palps to the antennal lobe (AL) and from the labella on the proboscis to the suboesophageal zone (SEZ), suggesting integration of olfactory and gustatory signals occurs in this brain region. We present detailed anatomical maps of olfactory innervations in the AL and the SEZ, identifying glomeruli that may respond to human body odours or carbon dioxide. Our results pave the way for anatomical and functional neurogenetic studies of sensory processing in mosquitoes.

In vivo bioluminescent imaging (BLI) permits the visualization of engineered bioluminescence from living cells and tissues to provide a unique perspective toward the understanding of biological processes as they occur within the framework of an authentic in vivo environment. The toolbox of in vivo BLI includes an inventory of luciferase compounds capable of generating bioluminescent light signals along with sophisticated and powerful instrumentation designed to detect and quantify these light signals non-invasively as they emit from the living subject. The information acquired reveals the dynamics of a wide range of biological functions that play key roles in the physiological and pathological control of disease and its therapeutic management. This mini review provides an overview of the tools and applications central to the evolution of in vivo BLI as a core technology in the preclinical imaging disciplines.

Escherichia coli O157:H7 is a significant human pathogen that is continually responsible for sickness, and even death, on a worldwide scale. While the pathology of E. coli O157:H7 infection has been well studied, the effect of it's multiple resulting cytotoxic mechanisms on host metabolic activity has not been well characterized. To develop a more thorough understanding of these effects, several bioluminescence assays were evaluated for their ability to track both toxicity and host metabolic activity levels in real-time. The use of continuously autobioluminescent human cells was determined to be the most favorable method for tracking these metrics, as its self-sufficient autobioluminescent phenotype was unaffected by the presence of the infecting bacteria and its signal could be measured without cellular destruction. Using this approach, it was determined that infection with as few as 10 CFU of E. coli O157:H7 could elicit cytotoxic effects. Regardless of the initial infective dose, an impact on metabolic expression was not observed until bacterial populations reached levels between 5 × 10(5) and 1 × 10(6) (R(2) = 0.933), indicating that a critical bacterial infection level must be reached prior to the onset of cytotoxic effects. Supporting this hypothesis, it was found that cells displaying infection-mediated metabolic activity reductions could recover to wild type metabolic activity levels if the infecting bacteria were removed prior to cell death. These results indicate that rapid treatment of E. coli O157:H7 infection could serve to limit host metabolic impact and reduce overall host cell death.

Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of "coding introns" (i.e., introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted "plug-and-play" cassettes (called "Trojan exons") that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette) transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The Q system is a repressible binary expression system for transgenic manipulations in living organisms. Through protein engineering and in vivo functional tests, we report here variants of the Q-system transcriptional activator, including QF2, for driving strong and ubiquitous expression in all Drosophila tissues. Our QF2, Gal4QF and LexAQF chimeric transcriptional activators substantially enrich the toolkit available for transgenic regulation in Drosophila melanogaster.

Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.

Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype., (2010 Elsevier Ltd. All rights reserved.)

By use of chemical enablement and prospective design, a novel series of selective, brain penetrant PDE9A inhibitors have been identified that are capable of producing in vivo elevations of brain cGMP.

Cancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC(50) of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cell-based assay measuring phospho-FAK with an IC(50) of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100x selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC(50) of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATP-competitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity.

Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5 A resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity.

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation., (Copyright 1999 Academic Press.)