Rapid and label-free identification of single leukemia cells from blood in a high-density microfluidic trapping array by fluorescence lifetime imaging microscopy.

The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization

‘Trust me, we can sort this out’: a theory-testing case study of the role of epistemic trust in fostering relationships

Novel psychological theories are often conceived in a general or heuristic form that can benefit from development and granulation through context-specific theory testing. Here, a theory-testing single case study methodology, adapted from an approach developed in the field of psychoanalysis, is presented. The study exemplifies this methodology though an interrogation of the explanatory value of a relatively new child development theory, the theory of epistemic trust, in the context of the relationship between a foster carer (“John”) and a young person in their care (“Buster”). Using in-depth interview material, the ways and extent to which the theory of epistemic trust could aid understanding of this fostering relationship are examined. We discuss the implications for the development of the theory of epistemic trust and the applications of these findings to social work contexts. The strengths and limitations of this theory-testing case study approach are explored

Chasing the thrill or just passing the time? Trialing a new mixed methods approach to understanding heterogeneity amongst recreational fishers based on motivations

Human dimensions researchers and fisheries managers have long recognized the value of exploring the heterogeneity that exists amongst recreational fishers. Understanding the differences between fishers has the potential to assist managers in developing targeted communication strategies, direct resources to active management more efficiently and improve understanding of how fishers will respond to changes in regulations or new management interventions. Human dimensions research has traditionally explored fisher heterogeneity through research into the different reasons why people choose to fish, as well as attempts to categorize or segment fishers using variable based approaches. These studies have, to date, relied primarily on large scale, quantitative survey techniques with a particular focus on fisher avidity and commitment. They are therefore limited in their ability to explain how different fishing motivations might interact within an individual, why particular motivations are prioritized, and how this might influence fisher behavior and attitudes. This study trialed a mixed methods approach to understanding fisher heterogeneity based primarily on motivations using a case study in NSW, Australia. This trial involved utilizing a person-centered approach known as Latent Class Analysis (LCA), followed by qualitative, in depth focus group discussions. This revealed five distinct fisher classes; Social fishers, Trophy Fishers, Outdoor Enthusiasts, Generalists and Hunter-Gatherers, each with distinct and significantly different combinations of catch and non-catch-related motivations. The qualitative analysis sought to explore the intersection of motivations and attitudes towards management within and across the different fisher classes. The results highlighted the importance of more detailed examination of the intersection between motivations and attitudes in future LCA, with a particular focus on the potential influence of mastery (or challenge/experience) motivations on fisher attitudes to wards marine and fisheries management approaches

Live calcium imaging of Aedes aegypti neuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies.

BackgroundThe mosquito Aedes aegypti has a wide variety of sensory pathways that have supported its success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as responding motor outputs, there has been a lack of studies able to monitor both concurrently.ResultsTo begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced microscopy, we simultaneously measured live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution.ConclusionsBy coupling in vivo live calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies

Assessing the utility of multiplexed liquid chromatography-mass spectrometry for gluten detection in Australian breakfast food products

Coeliac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten that is associated with gastrointestinal issues, including diarrhea, abdominal pain, and malabsorption. Gluten is a general name for a class of cereal storage proteins of wheat, barley, and rye that are notably resistant to gastrointestinal digestion. After ingestion, immunogenic peptides are subsequently recognized by T cells in the gastrointestinal tract. The only treatment for CD is a life-long gluten-free diet. As such, it is critical to detect gluten in diverse food types, including those where one would not expect to find gluten. The utility of liquid chromatography-mass spectrometry (LC-MS) using cereal-specific peptide markers to detect gluten in heavily processed food types was assessed. A range of breakfast products, including breakfast cereals, breakfast bars, milk-based breakfast drinks, powdered drinks, and a savory spread, were tested. No gluten was detected by LC-MS in the food products labeled gluten-free, yet enzyme-linked immunosorbent assay (ELISA) measurement revealed inconsistencies in barley-containing products. In products containing wheat, rye, barley, and oats as labeled ingredients, gluten proteins were readily detected using discovery proteomics. Panels comprising ten cereal-specific peptide markers were analyzed by targeted proteomics, providing evidence that LC-MS could detect and differentiate gluten in complex matrices, including baked goods and milk-based products

The effect of IL-13 and IL-13R130Q, a naturally occurring IL-13 polymorphism, on the gene expression of human airway smooth muscle cells

BACKGROUND: Growing evidence shows that interleukin 13 (IL-13) may play an essential role in the development of airway inflammation and bronchial hyper-responsiveness (BHR), two defining features of asthma. Although the underlying mechanisms remain unknown, a number of reports have shown that IL-13 may exert its deleterious effects in asthma by directly acting on airway resident cells, including epithelial cells and airway smooth muscle cells. In this report, we hypothesize that IL-13 may participate in the pathogenesis of asthma by activating a set of "pro-asthmatic" genes in airway smooth muscle (ASM) cells. METHODS: Microarray technology was used to study the modulation of gene expression of airway smooth muscle by IL-13 and IL-13R130Q. TaqMan™ Real Time PCR and flow cytometry was used to validate the gene array data. RESULTS: IL-13 and the IL-13 polymorphism IL-13R130Q (Arg130Gln), recently associated with allergic asthma, seem to modulate the same set of genes, which encode many potentially interesting proteins including vascular cellular adhesion molecule (VCAM)-1, IL-13Rα2, Tenascin C and Histamine Receptor H1, that may be relevant for the pathogenesis of asthma. CONCLUSIONS: The data supports the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma