Faculty Bibliography

Certain tumor phenomena, like metabolic heterogeneity and local stable regions of chronic hypoxia, signify a tumor's resistance to therapy. Although recent research has shed light on the intracellular mechanisms of cancer metabolic reprogramming, little is known about how tumors become metabolically heterogeneous or chronically hypoxic, namely the initial conditions and spatiotemporal dynamics that drive these cell population conditions. To study these aspects, we developed a minimal, spatially-resolved simulation framework for modeling tissue-scale mixed populations of cells based on diffusible particles the cells consume and release, the concentrations of which determine their behavior in arbitrarily complex ways, and on stochastic reproduction. We simulate cell populations that self-sort to facilitate metabolic symbiosis, that grow according to tumor-stroma signaling patterns, and that give rise to stable local regions of chronic hypoxia near blood vessels. We raise two novel questions in the context of these results: (1) How will two metabolically symbiotic cell subpopulations self-sort in the presence of glucose, oxygen, and lactate gradients? We observe a robust pattern of alternating striations. (2) What is the proper time scale to observe stable local regions of chronic hypoxia? We observe the stability is a function of the balance of three factors related to O2-diffusion rate, local vessel release rate, and viable and hypoxic tumor cell consumption rate. We anticipate our simulation framework will help researchers design better experiments and generate novel hypotheses to better understand dynamic, emergent whole-tumor behavior.

Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application.

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2's alpha subunit (eIF2alpha) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear. Using CRISPR-Cas9 genome editing we introduced the most severe known VWM mutation, EIF2B4A391D, into CHO cells. Compared to isogenic wildtype cells, GEF activity of cells with the VWM mutation was impaired and the mutant cells experienced modest enhancement of the ISR. However, despite their enhanced ISR, imposed by the intrinsic defect in eIF2B, disrupting the inhibitory effect of phosphorylated eIF2alpha on GEF by a contravening EIF2S1/eIF2alphaS51A mutation that functions upstream of eIF2B, selectively enfeebled both EIF2B4A391D and the related severe VWM EIF2B4R483W cells. The basis for paradoxical dependence of cells with the VWM mutations on an intact eIF2alpha genotype remains unclear, as both translation rates and survival from stressors that normally activate the ISR were not reproducibly affected by the VWM mutations. Nonetheless, our findings support an additional layer of complexity in the development of VWM, beyond a hyperactive ISR.

BACKGROUND/AIMS/OBJECTIVE:The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. METHODS:We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. RESULTS:Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. CONCLUSION/CONCLUSIONS:Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.

The amygdala and the dorsolateral prefrontal cortex (DLPFC) play important roles in "emotion dysregulation," which has a profound impact on etiologic research of generalized anxiety disorder (GAD). The present study analyzed both eyes-open and eyes-closed resting state functional MRI (rs-fMRI) of 43 subjects (21 GAD patients with medicine free and 22 matched healthy controls). The amygdala and the DLPFC were defined as regions of interest (ROI) to analyze functional connectivity (FC) in GAD patients compared with healthy controls. The main findings revealed GAD patients had increased FC between the amygdala and the temporal pole compared to healthy controls, which was found in both eyes-open and eyes-closed rs-fMRI. And altered FC between the ROIs and brain regions that mainly belonged to the default mode network (DMN) were found. These findings suggest that the abnormal FC between the amygdala and the temporal pole may contribute to the pathophysiology of GAD, and provide insights into the current understanding of the emotion dysregulation of anxiety disorders.

Studies on the yolk and albumen content in bird eggs, and the effects of variations in their relative loads in the phenotype of the birds, have revealed multiple consequences at different levels of biological organization, from biochemical traits to behavior. However, little is known about the effect of albumen variation on energetics performance during development and early ontogeny, despite the fact that variation in energy expenditure may have consequences in terms of fitness for both feral and domestic species. In this work, we evaluated experimentally whether variations in the content of albumen of Gallus gallus eggs could generate differences in metabolic rates during embryonic development. Additionally, we assessed changes in the activity of mitochondrial enzymes (cytochrome c oxidase and citrate synthase) in skeletal muscles and liver. Finally, we evaluated the success of hatching of these embryos and their metabolic rates (MR) post-hatching. The results revealed a significant reduction in MR in the last fifth of embryonic life, and reduced catabolic activities in the skeletal muscle of chicks hatched from albumen-removed eggs. However, the same group demonstrated an increase in catabolic activity in the liver, suggesting the existence of changes in energy allocation between tissues. Besides, we found a decrease in hatching success in the albumen-removed group, suggesting a negative effect of the lower albumen content on eggs, possibly due to lower catabolic activities in skeletal muscle. We also found a compensatory phenomenon in the first week after hatching, i.e., birds from albumen-removed eggs did not show a decrease in MR either at thermoneutral temperatures or at 10 degrees C, compared to the control group. Collectively, our data suggest that a reduction in albumen may generate a trade-off between tissue metabolic activities, and may explain the differences in metabolic rates and hatching success, supporting the immediate adaptive response (IAR) hypothesis.