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.

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.

We investigated the contribution of blood vessel formation and neuronal excitability to the development of functional neural circuitry in larval zebrafish by analyzing oculomotor performance in response to visual and vestibular stimuli. To address the dependence of neuronal function on the presence of blood vessels, we compared wild type embryos to reck and cloche mutants that lacked intracerebral blood vessels. To test how neuronal excitability impacts neuronal development and intracerebral vascularization, we blocked neural activity using Tetraodotoxin (TTX) and Tricaine. In reck mutants, we found both slow phase horizontal tracking and fast phase resets with only a slightly reduced amplitude and bandwidth. Spontaneous saccades, eye position holding and vestibular gravitoinertial induced eye rotation were also present. All of these behaviors except for visual tracking were observed in cloche mutants that lacked any head vasculature. Thus, numerous oculomotor neuronal circuits spanning the forebrain, midbrain and hindbrain compartments, ending in motor innervations of the eye muscles, were correctly formed and generated appropriate oculomotor behaviors without blood vessels. However, our observations indicate that beginning at approximately six days, circulation was required for sustained behavioral performance. We further found that blocking neuronal excitability with either TTX or Tricaine up to 4-5 days post fertilization did not noticeably interfere with intracerebral blood vessel formation in wild type larvae. After removal of drug treatments, the oculomotor behaviors returned within hours. Thus, development of neuronal circuits that drive oculomotor performance does not require neuronal spiking or activity. Together these findings demonstrate that neither vascularization nor neuronal excitability are essential for the formation of numerous oculomotor nuclei with intricately designed connectivity and signal processing. We conclude that a genetic blueprint specifies early larval structural and physiological features, and this developmental strategy may be viewed as a unique adaptation required for early survival.

[This corrects the article on p. 67 in vol. 10, PMID: 27013978.].

BACKGROUND: Voluntary medical male circumcision (VMMC) for HIV prevention has been a priority for Swaziland since 2009. Initially focusing on men ages 15-49, the Ministry of Health reduced the minimum age for VMMC from 15 to 10 years in 2012, given the existing demand among 10- to 15-year-olds. To understand the implications of focusing VMMC service delivery on specific age groups, the MOH undertook a modeling exercise to inform policy and implementation in 2013-2014. METHODS AND FINDINGS: The impact and cost of circumcising specific age groups were assessed using the Decision Makers' Program Planning Tool, Version 2.0 (DMPPT 2.0), a simple compartmental model. We used age-specific HIV incidence from the Swaziland HIV Incidence Measurement Survey (SHIMS). Population, mortality, births, and HIV prevalence were imported from a national Spectrum/Goals model recently updated in consultation with country stakeholders. Baseline male circumcision prevalence was derived from the most recent Swaziland Demographic and Health Survey. The lowest numbers of VMMCs per HIV infection averted are achieved when males ages 15-19, 20-24, 25-29, and 30-34 are circumcised, although the uncertainty bounds for the estimates overlap. Circumcising males ages 25-29 and 20-24 provides the most immediate reduction in HIV incidence. Circumcising males ages 15-19, 20-24, and 25-29 provides the greatest magnitude incidence reduction within 15 years. The lowest cost per HIV infection averted is achieved by circumcising males ages 15-34: $870 U.S. dollars (USD). CONCLUSIONS: The potential impact, cost, and cost-effectiveness of VMMC scale-up in Swaziland are not uniform. They vary by the age group of males circumcised. Based on the results of this modeling exercise, the Ministry of Health's Swaziland Male Circumcision Strategic and Operational Plan 2014-2018 adopted an implementation strategy that calls for circumcision to be scaled up to 50% coverage for neonates, 80% among males ages 10-29, and 55% among males ages 30-34.

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.

Atopic dermatitis (AD) is a chronic, inflammatory skin condition caused by a complex interaction of genetic and environment factors. Inherited defects within the stratum corneum are increasingly recognised as a key causative factor in the development of AD. Patients with AD also demonstrate a high rate of colonisation by microbes, notably Staphylococcus aureus. Controversy exists regarding the use of antimicrobial agents in the management of AD. We briefly review the role of stratum corneum dysfunction in AD, the influence of cutaneous colonisation and infections, and provide an update on the utility of anti-staphylococcal treatment in AD.