ASU Scholarship Showcase

We designed and evaluated an active sampling device, using as analytical targets a family of pesticides purported to contribute to honeybee colony collapse disorder. Simultaneous sampling of bulk water and pore water was accomplished using a low-flow, multi-channel pump to deliver water to an array of solid-phase extraction cartridges. Analytes were separated using either liquid or gas chromatography, and analysis was performed using tandem mass spectrometry (MS/MS). Achieved recoveries of fipronil and degradates in water spiked to nominal concentrations of 0.1, 1, and 10 ng/L ranged from 77 ± 12 to 110 ± 18%. Method detection limits (MDLs) were as low as 0.040–0.8 ng/L. Extraction and quantitation of total fiproles at a wastewater-receiving wetland yielded concentrations in surface water and pore water ranging from 9.9 ± 4.6 to 18.1 ± 4.6 ng/L and 9.1 ± 3.0 to 12.6 ± 2.1 ng/L, respectively. Detected concentrations were statistically indistinguishable from those determined by conventional, more laborious techniques (p > 0.2 for the three most abundant fiproles). Aside from offering time-averaged sampling capabilities for two phases simultaneously with picogram-per-liter MDLs, the novel methodology eliminates the need for water and sediment transport via in situ solid phase extraction.

Two distinct monocyte (Mo)/macrophage (Mp) subsets (Ly6C^low and Ly6C^hi) orchestrate cardiac recovery process following myocardial infarction (MI). Prostaglandin (PG) E₂ is involved in the Mo/Mp-mediated inflammatory response, however, the role of its receptors in Mos/Mps in cardiac healing remains to be determined. Here we show that pharmacological inhibition or gene ablation of the Ep3 receptor in mice suppresses accumulation of Ly6C^low Mos/Mps in infarcted hearts. Ep3 deletion in Mos/Mps markedly attenuates healing after MI by reducing neovascularization in peri-infarct zones. Ep3 deficiency diminishes CX3C chemokine receptor 1 (CX3CR1) expression and vascular endothelial growth factor (VEGF) secretion in Mos/Mps by suppressing TGFβ1 signaling and subsequently inhibits Ly6C^low Mos/Mps migration and angiogenesis. Targeted overexpression of Ep3 receptors in Mos/Mps improves wound healing by enhancing angiogenesis. Thus, the PGE₂/Ep3 axis promotes cardiac healing after MI by activating reparative Ly6C^low Mos/Mps, indicating that Ep3 receptor activation may be a promising therapeutic target for acute MI.

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers.

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.

Accumulating data from genome-wide association studies (GWAS) have provided a collection of novel candidate genes associated with complex diseases, such as atherosclerosis. We identified an atherosclerosis-associated single-nucleotide polymorphism (SNP) located in the intron of the long noncoding RNA (lncRNA) LINC00305 by searching the GWAS database. Although the function of LINC00305 is unknown, we found that LINC00305 expression is enriched in atherosclerotic plaques and monocytes. Overexpression of LINC00305 promoted the expression of inflammation-associated genes in THP-1 cells and reduced the expression of contractile markers in co-cultured human aortic smooth muscle cells (HASMCs). We showed that overexpression of LINC00305 activated nuclear factor-kappa beta (NF-κB) and that inhibition of NF-κB abolished LINC00305-mediated activation of cytokine expression. Mechanistically, LINC00305 interacted with lipocalin-1 interacting membrane receptor (LIMR), enhanced the interaction of LIMR and aryl-hydrocarbon receptor repressor (AHRR), and promoted protein expression as well as nuclear localization of AHRR. Moreover, LINC00305 activated NF-κB exclusively in the presence of LIMR and AHRR. In light of these findings, we propose that LINC00305 promotes monocyte inflammation by facilitating LIMR and AHRR cooperation and the AHRR activation, which eventually activates NF-κB, thereby inducing HASMC phenotype switching.

The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an ‘epigenetic’ drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat’s differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.

In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett’s esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF). Functional assays revealed both cell types showed repressed proliferation and significant changes in motility (speed, displacement and directionality) as a result of interactions between the two cell types. Cellular interactions appear to be mediated through both direct cell-cell contact and secreted ligands. The findings of this study are important in that they reveal, for the first time, the effects of cellular communication on gene expression and cellular function between premalignant (dysplastic) epithelial cells and their normal counterparts.

Despite increasing interest in the effects of triclosan and triclocarban on human biology, current knowledge is still limited on the impact of these additives to antimicrobial personal care products on the human microbiome. A carefully designed recent study published in mSphere by Poole and colleagues [A. C. Poole et al., mSphere 1(3):e00056-15, 2016, http://dx.doi.org/10.1128/mSphere.00056-15] highlights both the power of novel methodologies for microbiome elucidation and the longstanding challenge of employing small-cohort studies to inform risk assessment for chemicals of ubiquitous use in modern society.

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

Traditionally, hazardous chemicals have been regulated in the U.S. on a one-by-one basis, an approach that is slow, expensive and can be inefficient, as illustrated by a decades-long succession of replacing one type of organohalogen flame retardants (OHFRs) with another one, without addressing the root cause of toxicity and associated public health threats posed. The present article expounds on the need for efficient monitoring strategies and pragmatic steps in reducing environmental pollution and adverse human health impacts. A promising approach is to combine specific bioassays with state-of-the-art chemical screening to identify chemicals and chemical mixtures sharing specific modes of action (MOAs) and pathways of toxicity (PoTs). This approach could be used to identify and regulate hazardous chemicals as classes or compound families, featuring similar biological end-points, such as endocrine disruption and mutagenicity. Opportunities and potential obstacles of implementing this approach are discussed.