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1.  A Combination Microbicide Gel Protects Macaques Against Vaginal Simian Human Immunodeficiency Virus-Reverse Transcriptase Infection, But Only Partially Reduces Herpes Simplex Virus-2 Infection After a Single High-Dose Cochallenge 
Abstract
Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.
doi:10.1089/aid.2013.0165
PMCID: PMC3910668  PMID: 24117013
2.  Effects of worker size on the dynamics of fire ant tunnel construction 
Social insects work together to complete tasks. However, different individuals within a colony may vary in task proficiency. We investigated if fire ant (Solenopsis invicta) worker body size influenced the ability to construct tunnels—a key component of subterranean nests. We monitored excavation by worker groups in a substrate of small wetted glass particles in quasi-two-dimensional arenas. Morphological and network features of the tunnel system were measured. Total tunnel area did not differ significantly between groups of large and small workers, although the tunnel area of control sized workers was significantly larger than that of large workers. Moreover, large workers created wider but shorter tunnels, with slower growth rate of tunnel number. However, edge–vertex scaling and degree distribution of the tunnel network were similar across all treatments. In all cases, the amount of excavated material was correlated with the number of active workers. Our study reveals that morphological features of excavated tunnels show modest variation when constructed by workers of varying sizes, but topological features associated with the tunnel network are conserved. These results suggest that important behavioural aspects of tunnel construction—and thus nest building—are similar among morphologically distinct members of fire ant societies.
doi:10.1098/rsif.2012.0423
PMCID: PMC3481571  PMID: 22915634
adaptive demography; division of labour; nest construction; social insects; network; granular media
3.  A Computational Model of a Microfluidic Device to Measure the Dynamics of Oxygen-Dependent ATP Release from Erythrocytes 
PLoS ONE  2013;8(11):e81537.
Erythrocytes are proposed to be involved in blood flow regulation through both shear- and oxygen-dependent mechanisms for the release of adenosine triphosphate (ATP), a potent vasodilator. In a recent study, the dynamics of shear-dependent ATP release from erythrocytes was measured using a microfluidic device with a constriction in the channel to increase shear stress. The brief period of increased shear stress resulted in ATP release within 25 to 75 milliseconds downstream of the constriction. The long-term goal of our research is to apply a similar approach to determine the dynamics of oxygen-dependent ATP release. In the place of the constriction, an oxygen permeable membrane would be used to decrease the hemoglobin oxygen saturation of erythrocytes flowing through the channel. This paper describes the first stage in achieving that goal, the development of a computational model of the proposed experimental system to determine the feasibility of altering oxygen saturation rapidly enough to measure ATP release dynamics. The computational model was constructed based on hemodynamics, molecular transport of oxygen and ATP, kinetics of luciferin/luciferase reaction for reporting ATP concentrations, light absorption by hemoglobin, and sensor characteristics. A linear model of oxygen saturation-dependent ATP release with variable time delay was used in this study. The computational results demonstrate that a microfluidic device with a 100 µm deep channel will cause a rapid decrease in oxygen saturation over the oxygen permeable membrane that yields a measurable light intensity profile for a change in rate of ATP release from erythrocytes on a timescale as short as 25 milliseconds. The simulation also demonstrates that the complex dynamics of ATP release from erythrocytes combined with the consumption by luciferin/luciferase in a flowing system results in light intensity values that do not simply correlate with ATP concentrations. A computational model is required for proper interpretation of experimental data.
doi:10.1371/journal.pone.0081537
PMCID: PMC3842322  PMID: 24312316
4.  Using Computational and Mechanical Models to Study Animal Locomotion 
Recent advances in computational methods have made realistic large-scale simulations of animal locomotion possible. This has resulted in numerous mathematical and computational studies of animal movement through fluids and over substrates with the purpose of better understanding organisms’ performance and improving the design of vehicles moving through air and water and on land. This work has also motivated the development of improved numerical methods and modeling techniques for animal locomotion that is characterized by the interactions of fluids, substrates, and structures. Despite the large body of recent work in this area, the application of mathematical and numerical methods to improve our understanding of organisms in the context of their environment and physiology has remained relatively unexplored. Nature has evolved a wide variety of fascinating mechanisms of locomotion that exploit the properties of complex materials and fluids, but only recently are the mathematical, computational, and robotic tools available to rigorously compare the relative advantages and disadvantages of different methods of locomotion in variable environments. Similarly, advances in computational physiology have only recently allowed investigators to explore how changes at the molecular, cellular, and tissue levels might lead to changes in performance at the organismal level. In this article, we highlight recent examples of how computational, mathematical, and experimental tools can be combined to ultimately answer the questions posed in one of the grand challenges in organismal biology: “Integrating living and physical systems.”
doi:10.1093/icb/ics115
PMCID: PMC3475976  PMID: 22988026
5.  Modeling steady state SO2-dependent changes in capillary ATP concentration using novel O2 micro-delivery methods 
Adenosine triphosphate (ATP) is known to be released from the erythrocyte in an oxygen (O2) dependent manner. Since ATP is a potent vasodilator, it is proposed to be a key regulator in the pathway that mediates micro-vascular response to varying tissue O2 demand. We propose that ATP signaling mainly originates in the capillaries due to the relatively long erythrocyte transit times in the capillary and the short ATP diffusion distance to the electrically coupled endothelium. We have developed a computational model to investigate the effect of delivering or removing O2 to limited areas at the surface of a tissue with an idealized parallel capillary array on total ATP concentration. Simulations were conducted when exposing full surface to perturbations in tissue O2 tension (PO2) or locally using a circular micro-outlet (~100 μm in diameter), a square micro-slit (200 × 200 μm), or a rectangular micro-slit (1000 μm wide × 200 μm long). Results indicated the rectangular micro-slit has the optimal dimensions for altering hemoglobin saturations (SO2) in sufficient number capillaries to generate effective changes in total [ATP]. This suggests a threshold for the minimum number of capillaries that need to be stimulated in vivo by imposed tissue hypoxia to induce a conducted micro-vascular response. SO2 and corresponding [ATP] changes were also modeled in a terminal arteriole (9 μm in diameter) that replaces 4 surface capillaries in the idealized network geometry. Based on the results, the contribution of terminal arterioles to the net change in [ATP] in the micro-vascular network is minimal although they would participate as O2 sources thus influencing the O2 distribution. The modeling data presented here provide important insights into designing a novel micro-delivery device for studying micro-vascular O2 regulation in the capillaries in vivo.
doi:10.3389/fphys.2013.00260
PMCID: PMC3781332  PMID: 24069001
adenosine triphosphate (ATP); microcirculation; capillaries; computational model; simulation; local PO2 perturbation; O2 regulation; micro-delivery device
6.  Microvascular Flow Modeling using In Vivo Hemodynamic Measurements in Reconstructed 3D Capillary Networks 
We describe a systematic approach to modeling blood flow using reconstructed capillary networks and in vivo hemodynamic measurements. Our objective was to produce flow solutions that represent convective O2 delivery in vivo. Two capillary networks, I & II, (84×168×342 & 70×157×268 μm3) were mapped using custom software. Total network red blood cell supply rate (SR) was calculated from in vivo data and used as a target metric for the flow model. To obtain inlet hematocrits, mass balances were applied recursively from downstream vessels. Pressure differences across the networks were adjusted to achieve target SR. Baseline flow solutions were used as inputs to existing O2 transport models. To test the impact of flow redistribution, asymmetric flow solutions (Asym) were generated by applying a ±20% pressure change to network outlets. Asym solutions produced a mean absolute difference in SR per capillary of 27.6 ± 33.3% in network I & 33.2 ± 40.1% in network II vs. baseline. The O2 transport model calculated mean tissue PO2 of 28.2 ± 4.8 & 28.1 ± 3.5 mmHg for baseline and 27.6 ± 5.2 & 27.7 ± 3.7 mmHg for Asym. This illustrates that moderate changes in flow distribution within a capillary network have little impact on tissue PO2 provided that total SR remains unchanged.
doi:10.1111/j.1549-8719.2012.00178.x
PMCID: PMC3393846  PMID: 22429386
blood flow; capillary networks; oxygen transport modeling; red blood cell supply rate
7.  Insm1a-mediated gene repression is essential for the formation and differentiation of Müller glia-derived progenitors in the injured retina 
Nature cell biology  2012;14(10):1013-1023.
In zebrafish, retinal injury stimulates Müller glia (MG) reprograming; allowing them to generate multipotent progenitors that regenerate damaged cells and restore vision. Recent studies suggest transcriptional repression may underlie these events. To identify these repressors, we compared the transcriptomes of MG and MG-derived progenitors and identified insm1a, a transcriptional repressor exhibiting a biphasic pattern of expression that is essential for retina regeneration. Insm1a was found to suppress ascl1a and its own expression and link injury-dependent ascl1a induction with dickkopf (dkk) suppression, which is necessary for MG dedifferentiation. We also found that Insm1a was responsible for sculpting the zone of injury-responsive MG by suppressing hb-egfa expression. Finally, we provide evidence that Insm1a stimulates progenitor cell cycle exit by suppressing a genetic program driving progenitor proliferation. Our studies identify Insm1a as a key regulator of retina regeneration and provide a mechanistic understanding of how it contributes to multiple phases of this process.
doi:10.1038/ncb2586
PMCID: PMC3463712  PMID: 23000964
8.  HB-EGF is necessary and sufficient for Müller glia dedifferentiation and retina regeneration 
Developmental Cell  2012;22(2):334-347.
Summary
Müller glia (MG) dedifferentiation into a cycling population of multipotent progenitors is crucial to zebrafish retina regeneration. The mechanisms underlying MG dedifferentiation are unknown. Here we report that heparin-binding epidermal-like growth factor (HB-EGF) is rapidly induced in MG residing at the injury site and that proHB-EGF ectodomain shedding is necessary for retina regeneration. Remarkably, HB-EGF stimulates the formation of multipotent MG-derived progenitors in the uninjured retina. We show that HB-EGF mediates its effects via an EGFR/MAPK signal transduction cascade that regulates the expression of regeneration-associated genes, like ascl1a and pax6b. We also uncover an HB-EGF/Ascl1a/Notch/hb-egfa signaling loop that helps define the zone of injury-responsive MG. Finally, we show that HB-EGF acts upstream of the Wnt/β-catenin signaling cascade that controls progenitor proliferation. These data provide a link between extracellular signaling and regeneration-associated gene expression in the injured retina and suggest strategies for stimulating retina regeneration in mammals.
doi:10.1016/j.devcel.2011.11.020
PMCID: PMC3285435  PMID: 22340497
9.  Mechanics of Undulatory Swimming in a Frictional Fluid 
PLoS Computational Biology  2012;8(12):e1002810.
The sandfish lizard (Scincus scincus) swims within granular media (sand) using axial body undulations to propel itself without the use of limbs. In previous work we predicted average swimming speed by developing a numerical simulation that incorporated experimentally measured biological kinematics into a multibody sandfish model. The model was coupled to an experimentally validated soft sphere discrete element method simulation of the granular medium. In this paper, we use the simulation to study the detailed mechanics of undulatory swimming in a “granular frictional fluid” and compare the predictions to our previously developed resistive force theory (RFT) which models sand-swimming using empirically determined granular drag laws. The simulation reveals that the forward speed of the center of mass (CoM) oscillates about its average speed in antiphase with head drag. The coupling between overall body motion and body deformation results in a non-trivial pattern in the magnitude of lateral displacement of the segments along the body. The actuator torque and segment power are maximal near the center of the body and decrease to zero toward the head and the tail. Approximately 30% of the net swimming power is dissipated in head drag. The power consumption is proportional to the frequency in the biologically relevant range, which confirms that frictional forces dominate during sand-swimming by the sandfish. Comparison of the segmental forces measured in simulation with the force on a laterally oscillating rod reveals that a granular hysteresis effect causes the overestimation of the body thrust forces in the RFT. Our models provide detailed testable predictions for biological locomotion in a granular environment.
Author Summary
The sandfish lizard uses body undulation to propel itself within granular media (sand). Previously we developed a numerical simulation model consisting of an experimentally validated multi-particle model of the granular medium, and a sandfish model with prescribed body deformation (a traveling sinusoidal wave with parameters measured from biological experiment). We used the simulation to capture average swimming speed and compared predictions to our previously developed resistive force theory (RFT) for granular media. In this paper, we use the numerical model to perform more detailed analysis of the mechanics of sand-swimming in a so-called “granular frictional fluid”. These include center-of-mass kinematics, force distributions along the body, effects of body and head shape, power generation and dissipation. We discuss how these aspects of sand-swimming compare to those for swimmers (like nematodes and eels) in true fluids. We use the numerical model to reveal how transients during start-up in granular drag generates discrepancies between the simulation and the RFT predictions. The predictions from our models can give insight into locomotor capabilities, musculoskeletal structure and morphological features of sand-swimming animals. These results may also provide guidance for the design and control of sand-swimming robots.
doi:10.1371/journal.pcbi.1002810
PMCID: PMC3531286  PMID: 23300407
10.  A Micro-delivery Approach for Studying Microvascular Responses to Localized Oxygen Delivery 
In vivo video microscopy has been used to study blood flow regulation as a function of varying oxygen concentration in microcirculatory networks. However, previous studies have measured the collective response of stimulating large areas of the microvascular network at the tissue surface.
Objective
We aim to limit the area being stimulated by controlling oxygen availability to highly localized regions of the microvascular bed within intact muscle.
Design and Method
Gas of varying O2 levels was delivered to specific locations on the surface of the Extensor Digitorum Longus muscle of rat through a set of micro-outlets (100 μm diameter) patterned in ultrathin glass using state-of-the-art microfabrication techniques. O2 levels were oscillated and digitized video sequences were processed for changes in capillary hemodynamics and erythrocyte O2 saturation.
Results and Conclusions
Oxygen saturations in capillaries positioned directly above the micro-outlets were closely associated with the controlled local O2 oscillations. Radial diffusion from the micro-outlet is limited to ~75 μm from the center as predicted by computational modelling and as measured in vivo. These results delineate a key step in the design of a novel micro-delivery device for controlled oxygen delivery to the microvasculature to understand fundamental mechanisms of microvascular regulation of O2 supply.
doi:10.1111/j.1549-8719.2011.00132.x
PMCID: PMC3221889  PMID: 21914035
In vivo video-microscopy; microcirculation; microfabrication; oxygen delivery; micro-delivery; hemodynamic parameters
11.  The Nonnucleoside Reverse Transcription Inhibitor MIV-160 Delivered from an Intravaginal Ring, But Not from a Carrageenan Gel, Protects Against Simian/Human Immunodeficiency Virus-RT Infection 
AIDS Research and Human Retroviruses  2012;28(11):1467-1475.
Abstract
We previously showed that a carrageenan (CG) gel containing 50 μM MIV-150 (MIV-150/CG) reduced vaginal simian/human immunodeficiency virus (SHIV)-RT infection of macaques (56%, p>0.05) when administered daily for 2 weeks with the last dose given 8 h before challenge. Additionally, when 100 mg of MIV-150 was loaded into an intravaginal ring (IVR) inserted 24 h before challenge and removed 2 weeks after challenge, >80% protection was observed (p<0.03). MIV-160 is a related NNRTI with a similar IC50, greater aqueous solubility, and a shorter synthesis. To objectively compare MIV-160 with MIV-150, herein we evaluated the antiviral effects of unformulated MIV-160 in vitro as well as the in vivo protection afforded by MIV-160 delivered in CG (MIV-160/CG gel) and in an IVR under regimens used with MIV-150 in earlier studies. Like MIV-150, MIV-160 exhibited potent antiviral activity against SHIV-RT in macaque vaginal explants. However, formulated MIV-160 exhibited divergent effects in vivo. The MIV-160/CG gel offered no protection compared to CG alone, whereas the MIV-160 IVRs protected significantly. Importantly, the results of in vitro release studies of the MIV-160/CG gel and the MIV-160 IVR suggested that in vivo efficacy paralleled the amount of MIV-160 released in vitro. Hundreds of micrograms of MIV-160 were released daily from IVRs while undetectable amounts of MIV-160 were released from the CG gel. Our findings highlight the importance of testing different modalities of microbicide delivery to identify the optimal formulation for efficacy in vivo.
doi:10.1089/aid.2012.0080
PMCID: PMC3484820  PMID: 22816564
12.  Pre-Diabetes Augments Neuropeptide Y1- and α1-Receptor Control of Basal Hindlimb Vascular Tone in Young ZDF Rats 
PLoS ONE  2012;7(10):e46659.
Background
Peripheral vascular disease in pre-diabetes may involve altered sympathetically-mediated vascular control. Thus, we investigated if pre-diabetes modifies baseline sympathetic Y1-receptor (Y1R) and α1-receptor (α1R) control of hindlimb blood flow (Qfem) and vascular conductance (VC).
Methods
Qfem and VC were measured in pre-diabetic ZDF rats (PD) and lean controls (CTRL) under infusion of BIBP3226 (Y1R antagonist), prazosin (α1R antagonist) and BIBP3226+prazosin. Neuropeptide Y (NPY) concentration and Y1R and α1R expression were determined from hindlimb skeletal muscle samples.
Results
Baseline Qfem and VC were similar between groups. Independent infusions of BIBP3226 and prazosin led to increases in Qfem and VC in CTRL and PD, where responses were greater in PD (p<0.05). The percent change in VC following both drugs was also greater in PD compared to CTRL (p<0.05). As well, Qfem and VC responses to combined blockade (BIBP3226+prazosin) were greater in PD compared to CTRL (p<0.05). Interestingly, an absence of synergistic effects was observed within groups, as the sum of the VC responses to independent drug infusions was similar to responses following combined blockade. Finally, white and red vastus skeletal muscle NPY concentration, Y1R expression and α1R expression were greater in PD compared to CTRL.
Conclusions
For the first time, we report heightened baseline Y1R and α1R sympathetic control of Qfem and VC in pre-diabetic ZDF rats. In support, our data suggest that augmented sympathetic ligand and receptor expression in pre-diabetes may contribute to vascular dysregulation.
doi:10.1371/journal.pone.0046659
PMCID: PMC3465334  PMID: 23071607
13.  Mechanical models of sandfish locomotion reveal principles of high performance subsurface sand-swimming 
We integrate biological experiment, empirical theory, numerical simulation and a physical model to reveal principles of undulatory locomotion in granular media. High-speed X-ray imaging of the sandfish lizard, Scincus scincus, in 3 mm glass particles shows that it swims within the medium without using its limbs by propagating a single-period travelling sinusoidal wave down its body, resulting in a wave efficiency, η, the ratio of its average forward speed to the wave speed, of approximately 0.5. A resistive force theory (RFT) that balances granular thrust and drag forces along the body predicts η close to the observed value. We test this prediction against two other more detailed modelling approaches: a numerical model of the sandfish coupled to a discrete particle simulation of the granular medium, and an undulatory robot that swims within granular media. Using these models and analytical solutions of the RFT, we vary the ratio of undulation amplitude to wavelength (A/λ) and demonstrate an optimal condition for sand-swimming, which for a given A results from the competition between η and λ. The RFT, in agreement with the simulated and physical models, predicts that for a single-period sinusoidal wave, maximal speed occurs for A/λ ≈ 0.2, the same kinematics used by the sandfish.
doi:10.1098/rsif.2010.0678
PMCID: PMC3140717  PMID: 21378020
locomotion; granular; modelling; robot; lizard; swimming
14.  Myogenin Regulates Denervation-Dependent Muscle Atrophy in Mouse Soleus Muscle 
Journal of cellular biochemistry  2011;112(8):2149-2159.
Muscle inactivity due to injury or disease results in muscle atrophy. The molecular mechanisms contributing to muscle atrophy are poorly understood. However, it is clear that expression of atrophy-related genes, like Atrogin-1 and MuRF-1, are intimately tied to loss of muscle mass. When these atrophy-related genes are knocked out, inactive muscles retain mass. Muscle denervation stimulates muscle atrophy and Myogenin (Myog) is a muscle-specific transcription factor that is highly induced following muscle denervation. To investigate if Myog contributes to muscle atrophy, we have taken advantage of conditional Myog null mice. We show that in the denervated soleus muscle Myog expression contributes to reduced muscle force, mass and cross sectional area. We found that Myog mediates these effects, at least in part, by regulating expression of the Atrogin-1 and MuRF-1 genes. Indeed Myog over-expression in innervated muscle stimulates Atrogin-1 gene expression and Myog over-expression stimulates Atrogin-1 promoter activity. Thus Myog and the signaling cascades regulating its induction following muscle denervation may represent novel targets for therapies aimed at reducing denervation-induced muscle atrophy.
doi:10.1002/jcb.23136
PMCID: PMC3135771  PMID: 21465538
Myogenin; Atrogin-1; MuRF-1; muscle mass; muscle force; muscle cross sectional area; muscle denervation
15.  Injury-dependent Müller glia and ganglion cell reprogramming during tissue regeneration requires Apobec2a and Apobec2b 
The Journal of Neuroscience  2012;32(3):1096-1109.
Unlike mammals, adult zebrafish are able to regenerate multiple tissues including those of the CNS. In the zebrafish retina, injury stimulates Müller glia dedifferentiation into a multipotent retinal progenitor that is capable of regenerating all lost cell types. This dedifferentiation is driven by the reactivation of gene expression programs that share many characteristics with those that operate during early development. Although the mechanisms underlying the reactivation of these programs remain unknown, it is likely that changes in DNA methylation play a significant role. To begin investigating whether DNA demethylation may contribute to retina regeneration, we characterized the expression of genes associated with DNA demethylation in the uninjured and injured retina. We found that two cytidine deaminases (apobec2a and apobec2b) were expressed basally in the uninjured retina and that they were induced in proliferating, dedifferentiated Müller glia. The maximal induction of apobec2b required Ascl1a, but was independent of Lin28, and therefore defines an independent signaling pathway stemming from Ascl1a. Strikingly, when Apobec2a or Apobec2b was knocked down by antisense morpholino oligonucleotides, the proliferative response of Müller glia following injury was significantly reduced and injury-dependent induction of ascl1a and its target genes were inhibited, suggesting the presence of a regulatory feedback loop between Apobec proteins and ascl1a. Finally, Ascl1a, Apobec2a and Apobec2b were found to be essential for optic nerve regeneration. These data identify an essential role for Apobec proteins during retina and optic nerve regeneration and suggest DNA demethylation may underlie the reprogramming of cells to mount a regenerative response.
doi:10.1523/JNEUROSCI.5603-11.2012
PMCID: PMC3306176  PMID: 22262907
16.  Toward a Multiscale Description of Microvascular Flow Regulation: O2-Dependent Release of ATP from Human Erythrocytes and the Distribution of ATP in Capillary Networks 
Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100 ms) that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2). The model further predicts how insulin, at concentrations found in pre-diabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by erythrocyte-derived ATP.
doi:10.3389/fphys.2012.00246
PMCID: PMC3429024  PMID: 22934004
oxygen supply regulation; signal pathway modeling; ATP transport model; O2 transport model
17.  A Zebrafish Model of Axenfeld-Rieger Syndrome Reveals That pitx2 Regulation by Retinoic Acid Is Essential for Ocular and Craniofacial Development 
The homeobox transcription factor pitx2 is a regulator of ocular development, and human PITX2 mutations are associated with Axenfeld-Rieger syndrome (ARS). The authors describe a zebrafish model of ARS that reveals remarkable functional homology of pitx2 across vertebrate classes.
Purpose.
The homeobox transcription factor PITX2 is a known regulator of mammalian ocular development, and human PITX2 mutations are associated with Axenfeld-Rieger syndrome (ARS). However, the treatment of patients with ARS remains mostly supportive and palliative.
Methods.
The authors used molecular genetic, pharmacologic, and embryologic techniques to study the biology of ARS in a zebrafish model that uses transgenes to mark neural crest and muscle cells in the head.
Results.
The authors demonstrated in vivo that pitx2 is a key downstream target of retinoic acid (RA) in craniofacial development, and this pathway is required for coordinating neural crest, mesoderm, and ocular development. pitx2a knockdown using morpholino oligonucleotides disrupts jaw and pharyngeal arch formation and recapitulates ocular characteristics of ARS, including corneal and iris stroma maldevelopment. These phenotypes could be rescued with human PITX2A mRNA, demonstrating the specificity of the knockdown and evolutionary conservation of pitx2a function. Expression of the ARS dominant negative human PITX2A K50E allele also caused ARS-like phenotypes. Similarly, inhibition of RA synthesis in the developing eye (genetic or pharmacologic) disrupted craniofacial and ocular development, and human PITX2A mRNA partially rescued these defects.
Conclusions.
RA regulation of pitx2 is essential for coordinating interactions among neural crest, mesoderm, and developing eye. The marked evolutionary conservation of Pitx2 function in eye and craniofacial development makes zebrafish a potentially powerful model of ARS, amenable to in vivo experimentation and development of potential therapies.
doi:10.1167/iovs.11-8494
PMCID: PMC3292384  PMID: 22125274
18.  Deletion of a remote enhancer near ATOH7 disrupts retinal neurogenesis, causing NCRNA disease 
Nature neuroscience  2011;14(5):578-586.
Individuals with nonsyndromic congenital retinal nonattachment (NCRNA) are totally blind from birth. The disease afflicts ~1% of Kurdish people living in a group of neighboring villages in North Khorasan, Iran. We show NCRNA is caused by a 6523bp deletion that spans a remote cis regulatory element 20 kb upstream from ATOH7 (Math5), a bHLH transcription factor gene required for retinal ganglion cell (RGC) and optic nerve development. In humans, the absence of RGCs stimulates massive neovascular growth of fetal blood vessels within the vitreous, and early retinal detachment. The remote ATOH7 element appears to act as a secondary or ‘shadow’ transcriptional enhancer. It has minimal sequence similarity to the primary enhancer, which is close to the Atoh7 promoter, but drives transgene expression with an identical spatiotemporal pattern in the mouse retina. The human transgene also functions in zebrafish, reflecting deep evolutionary conservation. These dual enhancers may reinforce Atoh7 expression during early critical stages of eye development when retinal neurogenesis is initiated.
doi:10.1038/nn.2798
PMCID: PMC3083485  PMID: 21441919
nonsyndromic congenital retinal nonattachment (NCRNA); persistent hyperplastic primary vitreous (PHPV); retinal ganglion cell (RGC); optic nerve hypoplasia; retinopathy of prematurity (ROP); Math5; retinal cell fate specification; corneal blood staining; conserved noncoding element (CNE); shadow enhancer; Iranian Kurds; basic helix-loop-helix (bHLH); retinal dysgenesis; blindness; ATOH7; neurogenesis; hyaloid vasculature; falciform fold; genomic evolution; RNANC
19.  Conditional Gene Expression and Lineage Tracing of tuba1a Expressing Cells during Zebrafish Development and Retina Regeneration 
The Journal of comparative neurology  2010;518(20):4196-4212.
The tuba1a gene encodes a neural-specific alpha-tubulin isoform whose expression is restricted to the developing and regenerating nervous system. Using zebrafish as a model system for studying CNS regeneration we recently showed that retinal injury induces tuba1a gene expression in Müller glia that reentered the cell cycle. However, due to the transient nature of tuba1a gene expression during development and regeneration, it was not possible to trace the lineage of the tuba1a-expressing cells with a reporter directly under the control of the tuba1a promoter. To overcome this limitation, we generated tuba1a:CreERT2 and β-actin2:loxP-mCherrry-loxP-GFP double transgenic fish that allowed us to conditionally and permanently label tuba1a-expressing cells via ligand-induced recombination. During development, recombination revealed transient tuba1a expression in not only neural progenitors, but also cells that contribute to skeletal muscle, heart and intestine. In the adult, recombination revealed tuba1a expression in brain, olfactory neurons and sensory cells of the lateral line, but not in the retina. Following retinal injury, recombination showed tuba1a expression in Müller glia that had reentered the cell cycle and lineage tracing indicated these cells are responsible for regenerating retinal neurons and glia. These results suggest that tuba1a-expressing progenitors contribute to multiple cell lineages during development and that tuba1a-expressing Müller glia are retinal progenitors in the adult.
doi:10.1002/cne.22448
PMCID: PMC2948409  PMID: 20878783
Cre; recombination; neural development; regeneration; retina; Müller glia
20.  Ascl1a regulates Müller glia dedifferentiation and retina regeneration via a Lin-28-dependent, let-7 miRNA signaling pathway 
Nature cell biology  2010;12(11):1101-1107.
Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function1, 2. This regenerative response relies on Müller glia (MG) dedifferentiation into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. Here we report that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor Ascl1a and the pluripotency factor Lin-28 are induced in MG within 6 hrs following retinal injury and are necessary for MG dedifferentiation. We demonstrate that Ascl1a is necessary for lin-28 expression and that Lin-28 suppresses let-7 miRNA expression. Furthermore we show that let-7 represses expression of regeneration-associated genes like, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. Interestingly, hspd1, oct4 and c-myca exhibit basal expression in the uninjured retina and let-7 may inhibit this expression to prevent premature MG dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on MG differentiation/dedifferentiation are similar to that of embryonic stem cells3 and suggest novel targets for stimulating MG dedifferentiation and retina regeneration in mammals.
doi:10.1038/ncb2115
PMCID: PMC2972404  PMID: 20935637
21.  Tuba1a Gene Expression is Regulated by KLF6/7 and is Necessary for CNS Development and Regeneration in Zebrafish 
We report that knockdown of the α1 tubulin isoform Tuba1a, but not the highly related Tuba1b, dramatically impedes nervous system formation during development and RGC axon regeneration following optic nerve injury in adults. Within the tuba1a promoter, a G/C-rich element was identified that is necessary for tuba1a induction during RGC differentiation and optic axon regeneration. KLF6a and 7a, which we previously reported are essential for optic axon regeneration (Veldman et al., 2007), bind this G/C-rich element and transactivate the tuba1a promoter. In vivo knockdown of KLF6a and 7a attenuate regeneration-dependent activation of the endogenous tuba1a and p27 genes. These results suggest tuba1a expression is necessary for CNS development and regeneration and that KLF6a and 7a mediate their effects, at least in part, via transcriptional control of tuba1a promoter activity.
doi:10.1016/j.mcn.2010.01.004
PMCID: PMC2837137  PMID: 20123021
retina; retinal ganglion cell; tubulin; KLF; optic nerve; p27; promoter
22.  Theoretical Models of Microvascular Oxygen Transport to Tissue 
To improve understanding of microvascular O2 transport, theoretical modeling has been pursued for many years. The large number of studies in this area attests to the complexities (biochemical, structural, hemodynamic) involved. This article focuses on theoretical studies from the last two decades and, in particular, on models of O2 transport to tissue by discrete microvessels. A brief discussion of intravascular O2 transport is first given, highlighting the physiological importance of intravascular resistance to blood-tissue O2 transfer. This is followed by a description of the Krogh tissue cylinder model of O2 transport by a single capillary, which is shown to remain relevant in modified forms that relax many of the original biophysical assumptions. However, there are many geometric and hemodynamic complexities that require the consideration of microvascular arrays and networks. Multi-vessel models are discussed which have shown the physiological importance of heterogeneities in vessel spacing, O2 supply, red blood cell flow path, as well as interactions between capillaries and arterioles. These realistic models require sophisticated methods for solving the governing partial differential equations, and a range of solution techniques are described. Finally, the issue of experimental validation of microvascular O2 delivery models is discussed, and new directions in O2 transport modeling are outlined.
doi:10.1080/10739680801938289
PMCID: PMC3057578  PMID: 18608981
capillary network; heterogeneity; computational model; spatially distributed; mathematical model
23.  Zebrafish dou yan Mutation Causes Patterning Defects and Extensive Cell Death in the Retina 
The size of an organ is largely determined by the number of cells it contains, which in turn is regulated by two opposing processes: cell proliferation and cell death, but it is generally not clear how cell proliferation and cell death are coordinated during development. Here we characterize the zebrafish dou yanmi234 mutation that results in a dramatic reduction of retinal size and a disruption of retinal differentiation and lamination. The retinal size reduction is caused by increased retinal cell death in a non-cell-autonomous manner during early development. The phenotypic defect in dou yanmi234 arises coincident with the onset of retinal neurogenesis and differentiation. Interestingly, unlike many other small eye mutations, the mutation does not increase the level of cell death in the brain, implying the brain and retina utilize different mechanisms to maintain cell survival. Identification and further study of the dou yan gene will enhance our understanding of the molecular mechanisms regulating retinal cellular homeostasis, i.e. the balance between cell proliferation and cell death.
doi:10.1002/dvdy.21148
PMCID: PMC2922982  PMID: 17436278
retina; dou yan; retinal lamination; cell death; small eye
24.  14,15-Epoxyeicosa-5,8,11-trienoic acid (14,15-EET) Surrogates Containing Epoxide Bioisosteres: Influence upon Vascular Relaxation and Soluble Epoxide Hydrolase Inhibition 
Journal of medicinal chemistry  2009;52(16):5069-5075.
All-cis-14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a labile, vasodilatory eicosanoid generated from arachidonic acid by cytochrome P450 epoxygenases. A series of robust, partially saturated analogs containing epoxide bioisosteres were synthesized and evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of soluble epoxide hydrolase (sEH). Depending upon the bioisostere and its position along the carbon chain, varying levels of vascular relaxation and/or sEH inhibition were observed. For example, oxamide 16 and N-iPr-amide 20 were comparable (ED50 1.7 μM) to 14,15-EET as vasorelaxants, but were approx. 10–35 times less potent as sEH inhibitors (IC50 59 and 19 μM, respectively); unsubstituted urea 12 showed useful activity in both assays (ED50 3.5 μM, IC50 16 nM). These data reveal differential structural parameters for the two pharmacophores that could assist the development of potent and specific in vivo drug candidates.
doi:10.1021/jm900634w
PMCID: PMC2888647  PMID: 19653681
25.  Erythrocytes: Oxygen Sensors and Modulators of Vascular Tone in Regions of Low PO2 
Physiology (Bethesda, Md.)  2009;24:107-116.
Through oxygen-dependent release of the vasodilator ATP, the mobile erythrocyte plays a fundamental role in matching microvascular oxygen supply with local tissue oxygen demand. Signal transduction within the erythrocyte and microvessels as well as feedback mechanisms controlling ATP release have been described. Our understanding of the impact of this novel control mechanism will rely on the integration of in vivo experiments and computational models.
Summary
Erythrocytes play a fundamental role in tissue oxygen supply via the controlled release of ATP in areas of increased oxygen need.
doi:10.1152/physiol.00038.2008
PMCID: PMC2725440  PMID: 19364913

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