Architectural control of metabolic plasticity in epithelial cancer cells.

Metabolic plasticity enables cancer cells to switch between glycolysis and oxidative phosphorylation to adapt to changing conditions during cancer progression, whereas metabolic dependencies limit plasticity. To understand a role for the architectural environment in these processes we examined metabolic dependencies of cancer cells cultured in flat (2D) and organotypic (3D) environments. Here we show that cancer cells in flat cultures exist in a high energy state (oxidative phosphorylation), are glycolytic, and depend on glucose and glutamine for growth. In contrast, cells in organotypic culture exhibit lower energy and glycolysis, with extensive metabolic plasticity to maintain growth during glucose or amino acid deprivation. Expression of KRASG12V in organotypic cells drives glucose dependence, however cells retain metabolic plasticity to glutamine deprivation. Finally, our data reveal that mechanical properties control metabolic plasticity, which correlates with canonical Wnt signaling. In summary, our work highlights that the architectural and mechanical properties influence cells to permit or restrict metabolic plasticity.

Ventilatory responses to constant load exercise following the inhalation of a short-acting ß2-agonist in a laboratory-controlled diesel exhaust exposure study in individuals with exercise-induced bronchoconstriction.

OBJECTIVE: Individuals with exercise-induced bronchoconstriction (EIB) use ß2-agonists to reduce respiratory symptoms during acute exercise. The resultingbronchodilation could increase the dose of inhaled pollutants and impair respiratory function when exercise is performedin air pollution. We aimed to assess respiratory responses in individuals with EIB when completing a cycling bout while being exposed to diesel exhaust (DE) or filtered air (FA) with and without the inhalation of salbutamol (SAL), a short-acting ß2-agonist. METHODS: In a double-blind, repeated-measures design, 19 participants with EIB (22-33 years of age) completed four visits: FA-placebo (FA-PLA), FA-SAL, DE-PLA, DE-SAL. After the inhalation of either 400 µg of SAL or PLA, participants sat in the exposure chamber for 60 min, breathing either FA or DE (PM2.5 = 300 µg/m3). Participants then cycled for 30 min at 50 % of peak work rate while breathing FA or DE. Respiratory responses were assessed via spirometry, work of breathing (WOB), fractional use of ventilatory capacity (V̇E/V̇E,CAP), area under the maximal expiratory flow-volume curve (MEFVAUC), and dyspnea during and following cycling. RESULTS: Bronchodilation in response to SAL and acute cycling was observed, independent of FA/DE exposure. Specifically, FEV1 was increased by 7.7 % (confidence interval (CI): 7.2-8.2 %; p < 0.01) in response to SAL, and MEFVAUC was increased after cycling by 1.1 % (0.9-1.3 %; p = 0.03). Despite a significant decrease in total WOB by 6.2 J/min (4.7-7.5 J/min; p = 0.049) and a reduction in V̇E/V̇E,CAP by 5.8 % (5-6 %, p < 0.01) in the SAL exposures, no changes were observed in dyspnea. The DE exposure significantly increased V̇E/V̇E,CAP by 2.4 % (0.9-3.9 %; p < 0.01), but this did not affect dyspnea. DISCUSSION: Our findings suggest that the use of SAL prior to moderate-intensity exercise when breathing high levels of DE, does not reduce respiratory function or exercise ventilatory responses for up to 60 min following exercise.

Accessible dynamic micropatterns in monolayer cultures via modified desktop xurography.

Micropatterned cell cultures provide an important tool to understand dynamic biological processes, but often require specialized equipment and expertise. Here we present subtractive bioscribing (SuBscribing), a readily accessible and inexpensive technique to generate dynamic micropatterns in biomaterial monolayers on-the-fly. We first describe our modifications to a commercially available desktop xurographer and demonstrate the utility and limits of this system in creating micropatterned cultures by mechanically scribing patterns into a brittle, non-adhesive biomaterial layer. Patterns are sufficiently small to influence cell morphology and orientation and can be extended to pattern large areas with complex reproducible shapes. We also demonstrate the use of this system as a dynamic patterning tool for cocultures. Finally, we use this technique to explore and improve upon the well-established epithelial scratch assay, and demonstrate that robotic control of the scratching tool can be used to create custom-shaped wounds in epithelial monolayers, and that the scribing direction leaves trace remnants of matrix molecules that may significantly affect conventional implementations of this common assay.

Developmentally-Inspired Biomimetic Culture Models to Produce Functional Islet-Like Cells From Pluripotent Precursors.

Insulin-producing beta cells sourced from pluripotent stem cells hold great potential as a virtually unlimited cell source to treat diabetes. Directed pancreatic differentiation protocols aim to mimic various stimuli present during embryonic development through sequential changes of in vitro culture conditions. This is commonly accomplished by the timed addition of soluble signaling factors, in conjunction with cell-handling steps such as the formation of 3D cell aggregates. Interestingly, when stem cells at the pancreatic progenitor stage are transplanted, they form functional insulin-producing cells, suggesting that in vivo microenvironmental cues promote beta cell specification. Among these cues, biophysical stimuli have only recently emerged in the context of optimizing pancreatic differentiation protocols. This review focuses on studies of cell-microenvironment interactions and their impact on differentiating pancreatic cells when considering cell signaling, cell-cell and cell-ECM interactions. We highlight the development of in vitro cell culture models that allow systematic studies of pancreatic cell mechanobiology in response to extracellular matrix proteins, biomechanical effects, soluble factor modulation of biomechanics, substrate stiffness, fluid flow and topography. Finally, we explore how these new mechanical insights could lead to novel pancreatic differentiation protocols that improve efficiency, maturity, and throughput.

Vascular effects of physical activity are not modified by short-term inhaled diesel exhaust: Results of a controlled human exposure study.

BACKGROUND: The combined effects of physical activity and air pollution exposure on vascular function are insufficiently understood, particularly after the inhalation of a ß2-agonist, a vasodilating agent. OBJECTIVE: To assess the micro- and macrovascular response to physical activity after ß2-agonist use while breathing diesel exhaust (DE) in individuals with exercise-induced bronchoconstriction. METHODS: On four exposure visits, eighteen adults inhaled either 400 µg of the ß2-agonist salbutamol or placebo before resting for 60 min, followed by a 30-min cycling bout. During rest and cycling, participants inhaled filtered air (FA) or DE (300 µg/m3 of PM2.5). Microvascular (central retinal arteriolar and venular equivalents, CRAE and CRVE, respectively) and macrovascular parameters (blood pressure (BP)) and heart rate (HR)) were assessed at baseline (T1), 10 min (T2) and 70 min (T3) after cycling. RESULTS: The cycling bout increased CRAE (T2-T1 difference (95th % confidence interval): 4.88 µm (4.73, 5.00 µm), p < 0.001; T3-T1 difference: 2.10 µm (1.62, 2.58 µm), p = 0.031) and CRVE (T2-T1 difference: 3.78 µm (3.63, 3.92 µm), p < 0.001; T3-T1 difference: 3.73 µm (3.63, 3.92 µm), p < 0.001). The exposure to DE had no effect on CRAE (FA-DE difference at T2: 0.46 µm (-0.02, 0.92 µm); p = 0.790; FA-DE difference at T3: 1.76 µm (1.36, 2.16 µm), p = 0.213) and CRVE (FA-DE difference at T2: 0.26 µm (-0.35, 0.88 µm), p = 0.906; FA-DE difference at T3: 0.55 µm (0.05, 1.06 µm), p = 0.750). Compared to T1, systolic BP was decreased at T2 by 2.5 mmHg (2.8, 2.3 mmHg, p = 0.047), independent of inhaled exposure. Heart rate at T2 was significantly increased by 3 bpm (2, 3 bpm, p = 0.025) after the DE-exposure when compared to FA. DISCUSSION: Acute physical activity induces a vasodilatory response in the micro- and macrovasculature in healthy adults by increasing CRAE and CRVE, and by reducing systolic BP post exercise, despite breathing DE. The DE-associated increase in HR might be indicative of an increased sympathetic response to physical activity while breathing DE.

Controlled clustering enhances PDX1 and NKX6.1 expression in pancreatic endoderm cells derived from pluripotent stem cells.

Pluripotent stem cell (PSC)-derived insulin-producing cells are a promising cell source for diabetes cellular therapy. However, the efficiency of the multi-step process required to differentiate PSCs towards pancreatic beta cells is variable between cell lines, batches and even within cultures. In adherent pancreatic differentiation protocols, we observed spontaneous local clustering of cells expressing elevated nuclear expression of pancreatic endocrine transcription factors, PDX1 and NKX6.1. Since aggregation has previously been shown to promote downstream differentiation, this local clustering may contribute to the variability in differentiation efficiencies observed within and between cultures. We therefore hypothesized that controlling and directing the spontaneous clustering process would lead to more efficient and consistent induction of pancreatic endocrine fate. Micropatterning cells in adherent microwells prompted clustering, local cell density increases, and increased nuclear accumulation of PDX1 and NKX6.1. Improved differentiation profiles were associated with distinct filamentous actin architectures, suggesting a previously overlooked role for cell-driven morphogenetic changes in supporting pancreatic differentiation. This work demonstrates that confined differentiation in cell-adhesive micropatterns may provide a facile, scalable, and more reproducible manufacturing route to drive morphogenesis and produce well-differentiated pancreatic cell clusters.

Biomimetic Micropatterned Adhesive Surfaces To Mechanobiologically Regulate Placental Trophoblast Fusion.

The placental syncytiotrophoblast is a giant multinucleated cell that forms a tree-like structure and regulates transport between mother and baby during development. It is maintained throughout pregnancy by continuous fusion of trophoblast cells, and disruptions in fusion are associated with considerable adverse health effects including diseases such as preeclampsia. Developing predictive control over cell fusion in culture models is hence of critical importance in placental drug discovery and transport studies, but this can currently be only partially achieved with biochemical factors. Here, we investigate whether biophysical signals associated with budding morphogenesis during development of the placental villous tree can synergistically direct and enhance trophoblast fusion. We use micropatterning techniques to manipulate physical stresses in engineered microtissues and demonstrate that biomimetic geometries simulating budding robustly enhance fusion and alter spatial patterns of synthesis of pregnancy-related hormones. These findings indicate that biophysical signals play a previously unrecognized and significant role in regulating placental fusion and function, in synergy with established soluble signals. More broadly, our studies demonstrate that biomimetic strategies focusing on tissue mechanics can be important approaches to design, build, and test placental tissue cultures for future studies of pregnancy-related drug safety, efficacy, and discovery.

Meta-Analysis of Time in Therapeutic Range in Continuous-Flow Left Ventricular Assist Device Patients Receiving Warfarin.

Continuous-flow left ventricular assist devices (CF-LVADs) prolong survival in advanced heart failure patients. Anticoagulation control is critical in CF-LVAD patients due to increased thromboembolic and bleeding risk. We assessed the quality of INR control in CF-LVAD patients measured by time in therapeutic range (TTR). We performed a systematic literature search of MEDLINE and SCOPUS through July 2017 to identify studies evaluating TTR in anticoagulated adult CF-LVAD patients. Data on key characteristics and the TTR end point were then extracted from each study by two investigators using a standardized tool. Using a Hartung-Knapp random effects model, a weighted mean TTR estimate with accompanying 95% confidence interval (CI) was calculated. Statistical heterogeneity was estimated using the I2 statistic. Five published studies were included. All studies were single-center, retrospective investigations that calculated TTR using the Rosendaal method. Sample sizes ranged from 11 to 115 patients (total of 270 patients) with durations of follow-up ranging from 9 to 76 person-years. On meta-analysis, CF-LVAD patients had a weighted mean TTR of 46.6% (95% CI: 36.0-57.3%, I2 = 94%). This suggests that warfarin is difficult to manage in CF-LVAD patients, which may contribute to high rates of bleeding and thromboembolic complications.

Endocrine network essential for reproductive success in Drosophila melanogaster.

Ecdysis-triggering hormone (ETH) was originally discovered and characterized as a molt termination signal in insects through its regulation of the ecdysis sequence. Here we report that ETH persists in adult Drosophila melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction. ETH signaling deficits lead to sharply reduced JH levels and consequent reductions of ovary size, egg production, and yolk deposition in mature oocytes. Expression of ETH and ETH receptor genes is in turn dependent on ecdysone (20E). Furthermore, 20E receptor knockdown specifically in Inka cells reduces fecundity. Our findings indicate that the canonical developmental roles of 20E, ETH, and JH during juvenile stages are repurposed to function as an endocrine network essential for reproductive success.

Stem cells: to be born great, achieve greatness, or have greatness thrust upon them?

Induced pluripotent stem cells (iPSCs) have opened new doors in providing an ethical, patient-specific cell source towards tissue engineering. Developing these therapies involves the production of reprogrammed iPSCs, expanding them while maintaining pluripotency, then differentiating them into functional tissues. To bring these therapies to the clinic, efficient and GMP-compliant manufacturing methods are required. In this Research Highlight, we describe recent innovations to several aspects of the pluripotent cell therapy pipeline.

Repurposing the Clinically Efficacious Antifungal Agent Itraconazole as an Anticancer Chemotherapeutic.

Itraconazole (ITZ) is an FDA-approved member of the triazole class of antifungal agents. Two recent drug repurposing screens identified ITZ as a promising anticancer chemotherapeutic that inhibits both the angiogenesis and hedgehog (Hh) signaling pathways. We have synthesized and evaluated first- and second-generation ITZ analogues for their anti-Hh and antiangiogenic activities to probe more fully the structural requirements for these anticancer properties. Our overall results suggest that the triazole functionality is required for ITZ-mediated inhibition of angiogenesis but that it is not essential for inhibition of Hh signaling. The synthesis and evaluation of stereochemically defined des-triazole ITZ analogues also provides key information as to the optimal configuration around the dioxolane ring of the ITZ scaffold. Finally, the results from our studies suggest that two distinct cellular mechanisms of action govern the anticancer properties of the ITZ scaffold.

Cerebral metabolism after forced or voluntary physical exercise.

The pathophysiology of stroke, a leading cause of morbidity and mortality, is still in the process of being understood. Pre-ischemic exercise has been known to be beneficial in reducing the severity of stroke-induced brain injury in animal models. Forced exercise with a stressful component, rather than voluntary exercise, was better able to induce neuroprotection. This study further determined the changes in cerebral metabolism resulting from the two methods of exercise (forced versus voluntary). Adult male Sprague-Dawley rats were randomly assigned to 3 groups: the control group (no exercise), the forced treadmill exercise group, and the voluntary running wheel exercise group. In order to measure the extent of cerebral metabolism in animals with different exercise regimens, mRNA levels and protein expression of glucose transporter 1 and glucose transporter 3 (GLUT-1 and GLUT-3), phosphofructokinase (PFK), lactate dehydrogenase (LDH), and adenosine monophosphate kinase (AMPK) were measured utilizing real-time reverse transcription polymerase chain reaction (PCR) analysis as well as Western blot analysis. Phosphorylated AMPK activity was also measured using an ELISA activity kit, and hypoxic inducible factor (HIF)-1α was measured at transcription and translation levels. The data show that the forced exercise group had a significant (p < 0.05) increase in cerebral glycolysis, including expressions of GLUT-1, GLUT-3, PFK, LDH, phosphorylated AMPK activity and HIF-1α, when compared to the voluntary exercise and the control groups. Our results suggest that the effects of different exercise on HIF-1α expression and cerebral glycolysis may provide a possible reason for the discrepancy in neuroprotection, with forced exercise faring better than voluntary exercise through increased cerebral metabolism.