ABSTRACT
Purpose: Rapid and accurate diagnosis of microbial keratitis (MK) could greatly improve patient outcomes. Here, we present the development of a rapid, accessible multicolour fluorescence imaging device (FluoroPi) and evaluate its performance in combination with fluorescent optical reporters (SmartProbes) to distinguish bacterial Gram status. Furthermore, we show feasibility by imaging samples obtained by corneal scrape and minimally invasive corneal impression membrane (CIM) from ex vivo porcine corneal MK models. Methods: FluoroPi was built using a Raspberry Pi single-board computer and camera, light-emitting-diodes (LEDs), and filters for white-light and fluorescent imaging, with excitation and detection of bacterial optical SmartProbes: Gram-negative, NBD-PMX (exmax 488 nm); Gram positive, Merocy-Van (exmax 590 nm). We evaluated FluoroPi with bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) isolated from ex vivo porcine corneal models of MK by scrape (needle) and CIM with the SmartProbes. Results: FluoroPi provides <1 µm resolution and was able to readily distinguish bacteria isolated from ex vivo models of MK from tissue debris when combined with SmartProbes, retrieved by both scrape and CIM. Single bacteria could be resolved within the field of view, with limits of detection demonstrated as 103 to 104 CFU/mL. Sample preparation prior to imaging was minimal (wash-free), and imaging and postprocessing with FluoroPi were straightforward, confirming ease of use. Conclusions: FluoroPi coupled with SmartProbes provides effective, low-cost bacterial imaging, delineating Gram-negative and Gram-positive bacteria directly sampled from a preclinical model of MK. Translational Relevance: This study provides a crucial stepping stone toward clinical translation of a rapid, minimally invasive diagnostic approach for MK.
Subject(s)
Eye Infections, Bacterial , Keratitis , Animals , Swine , Point-of-Care Systems , Eye Infections, Bacterial/diagnosis , Eye Infections, Bacterial/microbiology , Keratitis/diagnosis , Keratitis/microbiology , Bacteria , Cornea/diagnostic imaging , Cornea/microbiologyABSTRACT
DNA aptamers were selected for their ability to bind specifically and quickly to crystalline hydroxyapatite (Ca10(PO4)6(OH)2; HAP), the primary mineral component of enamel and bone. Aptamers were found to have an enhanced percent of G-nucleotides and a propensity for forming a G-quadruplex secondary structure. One aptamer was studied in comparison to control sequences and was found to bind with high affinity and at high loading capacity, with enhanced binding kinetics, and with specificity for crystalline HAP material over amorphous calcium phosphate (ACP) and ß-tricalcium phosphate (TCP). The fluorescently-functionalized aptamer was demonstrated to specifically label HAP in a surface binding experiment and suggests the usefulness of this selected aptamer in biomedical or biotechnology fields where the labeling of specific calcium phosphate materials is required.
Subject(s)
Aptamers, Nucleotide/chemistry , Durapatite/chemistry , Biocompatible Materials/analysis , Calcium Phosphates/analysis , Particle Size , Surface PropertiesABSTRACT
The challenging nature of change is well documented and adequate preparation before implementing a change initiative is critical to reducing resistance and increasing its chances of success. This article describes a service review conducted in preparation for possible change and improvement at an internationally ranked school of nursing and midwifery in an Irish tertiary education institution. The purpose of the review was to examine organisational structures and work activities, including pressures facing the administrative team that supports the school, and to establish whether operations were fit for purpose and capable of continuing to support the international ranking. The article outlines the importance of change to maintain a competitive edge and the critical role of leadership during change. The background and rationale for the service review are discussed along with the methods used to prepare staff. The article also reports outcomes of the surveys and focus groups undertaken and discusses the main recommendations and changes implemented so far. It also suggests how the outcomes are relevant to clinical practice.
Subject(s)
Leadership , Schools, Nursing/organization & administration , Forecasting , Humans , Ireland , Nursing Education Research , Nursing Evaluation Research , Schools, Nursing/standards , Surveys and QuestionnairesABSTRACT
Autotaxin (ATX) is a secreted enzyme responsible for the hydrolysis of lysophosphatidylcholine (LPC) to the bioactive lysophosphatidic acid (LPA) and choline. The ATX-LPA signaling pathway is implicated in cell survival, migration, and proliferation; thus, the inhibition of ATX is a recognized therapeutic target for a number of diseases including fibrotic diseases, cancer, and inflammation, among others. Many of the developed synthetic inhibitors for ATX have resembled the lipid chemotype of the native ligand; however, a small number of inhibitors have been described that deviate from this common scaffold. Herein, we report the structure-activity relationships (SAR) of a previously reported small molecule ATX inhibitor. We show through enzyme kinetics studies that analogues of this chemotype are noncompetitive inhibitors, and by using a crystal structure with ATX we confirm the discrete binding mode.
Subject(s)
Indoles/chemistry , Phosphodiesterase Inhibitors/chemistry , Phosphoric Diester Hydrolases/chemistry , Picolinic Acids/chemistry , Binding Sites , Crystallography, X-Ray , Indoles/chemical synthesis , Kinetics , Models, Chemical , Molecular Docking Simulation , Phosphodiesterase Inhibitors/chemical synthesis , Picolinic Acids/chemical synthesis , Structure-Activity RelationshipABSTRACT
The effect of the variation of the experimental parameters on the conversion of precursor to products in a typical flash vacuum pyrolysis (FVP) experiment was investigated empirically. Temperature-conversion plots can be used to optimise FVP conditions and their mechanistic significance is exemplified. At a given temperature, the conversion can be increased by an increase in the background pressure, or by packing a section of the furnace tube with inert material (particularly when placed at the trap end of the furnace tube) or by employing a catalyst. Despite the prevailing view that only intramolecular reactions take place by FVP, it has been shown by a 'dual-FVP' cross-over experiment that the dimerisation of benzyl radicals occurs in the gas-phase, before the cold trap, under standard conditions. However, reduction in through-put rate, increase in furnace temperature and reduction in background pressure all reduce the amount of gas-phase coupling.
