A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells.

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina.

Daily mitochondrial dynamics in cone photoreceptors.

Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover.

Mitochondrial Calcium Uniporter (MCU) deficiency reveals an alternate path for Ca uptake in photoreceptor mitochondria.

Rods and cones use intracellular Ca to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu zebrafish and a rod photoreceptor-specific Mcu mouse.

Increasing Ca in photoreceptor mitochondria alters metabolites, accelerates photoresponse recovery, and reveals adaptations to mitochondrial stress.

Photoreceptors are specialized neurons that rely on Ca to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca homeostasis is disrupted. Ca homeostasis is maintained partly by mitochondrial Ca uptake through the mitochondrial Ca uniporter (MCU), which can influence cytosolic Ca signals, stimulate energy production, and trigger apoptosis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments.

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu.

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs.

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology.

Mitochondria Maintain Distinct Ca Pools in Cone Photoreceptors.

Ca ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca domains are maintained by Ca uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment.

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption.

Arrestins regulate cell spreading and motility via focal adhesion dynamics.

Focal adhesions (FAs) play a key role in cell attachment, and their timely disassembly is required for cell motility. Both microtubule-dependent targeting and recruitment of clathrin are critical for FA disassembly. Here we identify nonvisual arrestins as molecular links between microtubules and clathrin.

Priorities in lower limb prosthetic service delivery based on an international survey of prosthetists in low- and high-income countries.

Background: Prosthetic services, including the provision of an appropriate prosthesis, are a crucial part of the rehabilitation process for individuals with lower limb amputations. However, globally there exist unique challenges in the delivery of prosthetic services that are limiting rehabilitation outcomes and consequently the well-being and socio-economic status of individuals with lower limb amputations.

Objectives: The objective of this work was to explore the issues related to the provision of appropriate prosthetic technologies and to compare these across different economies of the world.

Selective roles of E2Fs for ErbB2- and Myc-mediated mammary tumorigenesis.

Previous studies have demonstrated that cyclin D1, an upstream regulator of the Rb/E2F pathway, is an essential component of the ErbB2/Ras (but not the Wnt/Myc) oncogenic pathway in the mammary epithelium. However, the role of specific E2fs for ErbB2/Ras-mediated mammary tumorigenesis remains unknown. Here, we show that in the majority of mouse and human primary mammary carcinomas with ErbB2/HER2 overexpression, E2f3a is up-regulated, raising the possibility that E2F3a is a critical effector of the ErbB2 oncogenic signaling pathway in the mammary gland.

Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina.

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain.

Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death.

Apoptosis is programmed cell death triggered by activation of death receptors or cellular stress. Activation of caspases is the hallmark of apoptosis. Arrestins are best known for their role in homologous desensitization of G protein-coupled receptors (GPCRs).

Cytosolic reducing power preserves glutamate in retina.

Glutamate in neurons is an important excitatory neurotransmitter, but it also is a key metabolite. We investigated how glutamate in a neural tissue is protected from catabolism. Flux analysis using (13)C-labeled fuels revealed that retinas use activities of the malate aspartate shuttle to protect >98% of their glutamate from oxidation in mitochondria.

Conformation of receptor-bound visual arrestin.

Arrestin-1 (visual arrestin) binds to light-activated phosphorylated rhodopsin (P-Rh*) to terminate G-protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin-1 and double electron-electron resonance was used to monitor interspin distance changes upon P-Rh* binding. The results indicate that the relative position of the N and C domains remains largely unchanged, contrary to expectations of a "clam-shell" model.

Progressive reduction of its expression in rods reveals two pools of arrestin-1 in the outer segment with different roles in photoresponse recovery.

Light-induced rhodopsin signaling is turned off with sub-second kinetics by rhodopsin phosphorylation followed by arrestin-1 binding. To test the availability of the arrestin-1 pool in dark-adapted outer segment (OS) for rhodopsin shutoff, we measured photoresponse recovery rates of mice with arrestin-1 content in the OS of 2.5%, 5%, 60%, and 100% of wild type (WT) level by two-flash ERG with the first (desensitizing) flash at 160, 400, 1000, and 2500 photons/rod.

Improving the gait performance of non-fluid-based swing-phase control mechanisms in transfemoral prostheses.

A prosthetic swing-phase control mechanism simulates the action of leg musculature, aiding gait function by controlling the duration of swing, extent of heel-rise, and by allowing the shank to smoothly decelerate into full knee extension without excessive impact. Non-fluid-based mechanisms have the potential to provide a durable and affordable solution as required in many parts of the world, but the design variables that lead to improved performance of non-fluid-based swing-phase control technologies are not well established. Seven transfemoral amputees were fitted with a prosthetic knee joint and different non-fluid-based swing-phase setups were systematically assessed.

Robust self-association is a common feature of mammalian visual arrestin-1.

Arrestin-1 binds light-activated phosphorhodopsin and ensures rapid signal termination. Its deficiency in humans and mice results in prolonged signaling and rod degeneration. However, most of the biochemical studies were performed on bovine arrestin-1, which was shown to self-associate forming dimers and tetramers, although only the monomer binds rhodopsin.

Arrestins as multi-functional signaling adaptors.

Arrestins are versatile regulators of cellular signaling expressed in every cell in the body. Arrestins bind active phosphorylated forms of their cognate G-protein-coupled receptors, shutting down G-protein activation and linking receptors to alternative signaling pathways. Arrestins directly interact with more than 20 surprisingly diverse proteins, such as several Src family kinases, ubiquitin ligases, protein phosphatases, microtubules, etc.

Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development.

The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. Here, we have analyzed the consequences of inactivating E2f7 and E2f8 in mice and show that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.

Arrestin mobilizes signaling proteins to the cytoskeleton and redirects their activity.

Arrestins regulate the activity and subcellular localization of G protein-coupled receptors and other signaling molecules. Here, we demonstrate that arrestins bind microtubules (MTs) in vitro and in vivo. The MT-binding site on arrestins overlaps significantly with the receptor-binding site, but the conformations of MT-bound and receptor-bound arrestin are different.

Design and quantitative evaluation of a stance-phase controlled prosthetic knee joint for children.

The aims of this study were to demonstrate a theoretical basis for the design of a stance-phase controlled paediatric prosthetic knee joint, clinically test prototypes of the knee, and use a questionnaire to evaluate its efficacy. Biomechanical models were used to analyze the stance-phase control characteristics of the proposed knee, and those of three other commonly prescribed paediatric knee joint mechanisms, which were also the conventional knee joints used by the six participants of this study (mean age 10.8 years).

Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation.

The E2F transcription factor family plays a crucial and well established role in cell cycle progression. Deregulation of E2F activities in vivo leads to developmental defects and cancer. Based on current evidence in the field, mammalian E2Fs can be functionally categorized into either transcriptional activators (E2F1, E2F2, and E2F3a) or repressors (E2F3b, E2F4, E2F5, E2F6, and E2F7).

Design characteristics of pediatric prosthetic knees.

We examined whether pediatric prosthetic single-axis knees can theoretically provide the beneficial functional characteristics of polycentric knees and the design considerations needed to realize this. Five children and their parents provided subjective opinions of the relative importance of functional requirements (FRs) for the knee. FRs related to comfort, fatigue, stability, and falling were found to be of high importance, while sitting appearance and adequate knee flexion were of lower importance.