Vitamin E-inspired multi-scale imaging agent

© 2018 Elsevier Ltd The production and use of multi-modal imaging agents is on the rise. The vast majority of these imaging agents are limited to a single length scale for the agent (e.g. tissues only), which is typically at the organ or tissue scale. This work explores the synthesis of such an imaging agent and discusses the applications of our vitamin E-inspired multi-modal and multi-length scale imaging agents TB-Toc ((S,E)-5,5-difluoro-7-(2-(5-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl) methyl) thiophen-2-yl) vinyl)-9-methyl-5H-dipyrrolo-[1,2-c:2’,1’-f][1,3,2]diazaborinin-4-ium-5-uide). We investigate the toxicity of TB-Toc along with the starting materials and lipid based delivery vehicle in mouse myoblasts and fibroblasts. Further we investigate the uptake of TB-Toc delivered to cultured cells in both solvent and liposomes. TB-Toc has low toxicity, and no change in cell viability was observed up to concentrations of 10 mM. TB-Toc shows time-dependent cellular uptake that is complete in about 30 min. This work is the first step in demonstrating our vitamin E derivatives are viable multi-modal and length scale diagnostic tools

How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions.

Most mammalian tissue cells experience oxygen partial pressures in vivo equivalent to 1-6% O2 (i.e., physioxia). In standard cell culture, however, headspace O2 levels are usually not actively regulated and under these conditions are ~18%. This drives hyperoxia in cell culture media that can affect a wide variety of cellular activities and may compromise the ability of in vitro models to reproduce in vivo biology. Here, we review and discuss some specific O2-consuming organelles and enzymes, including mitochondria, NADPH oxidases, the transplasma membrane redox system, nitric oxide synthases, xanthine oxidase, and monoamine oxidase with respect to their sensitivities to O2 levels. Many of these produce reactive oxygen and/or nitrogen species (ROS/RNS) as either primary end products or byproducts and are acutely sensitive to O2 levels in the range from 1% to 18%. Interestingly, many of them are also transcriptional targets of hypoxia-inducible factors (HIFs) and chronic cell growth at physioxia versus 18% O2 may alter their expression. Aquaporins, which facilitate hydrogen peroxide diffusion into and out of cells, are also regulated by HIFs, indicating that O2 levels may affect intercellular communication via hydrogen peroxide. The O2 sensitivities of these important activities emphasize the importance of maintaining physioxia in culture.Peer Reviewe

Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress.

BACKGROUND: Tissue hypoxia is a key feature of several endemic hepatic diseases, including alcoholic and non-alcoholic fatty liver disease, and organ failure. Hypoxia imposes a severe metabolic challenge on the liver, potentially disrupting its capacity to carry out essential functions including fuel storage and the integration of lipid metabolism at the whole-body level. Mitochondrial respiratory function is understood to be critical in mediating the hepatic hypoxic response, yet the time-dependent nature of this response and the role of the respiratory chain in this remain unclear. RESULTS: Here, we report that hepatic respiratory capacity is enhanced following short-term exposure to hypoxia (2 days, 10% O2) and is associated with increased abundance of the respiratory chain supercomplex III2+IV and increased cardiolipin levels. Suppression of this enhanced respiratory capacity, achieved via mild inhibition of mitochondrial complex III, disrupted metabolic homeostasis. Hypoxic exposure for 2 days led to accumulation of plasma and hepatic long chain acyl-carnitines. This was observed alongside depletion of hepatic triacylglycerol species with total chain lengths of 39-53 carbons, containing palmitic, palmitoleic, stearic, and oleic acids, which are associated with de novo lipogenesis. The changes to hepatic respiratory capacity and lipid metabolism following 2 days hypoxic exposure were transient, becoming resolved after 14 days in line with systemic acclimation to hypoxia and elevated circulating haemoglobin concentrations. CONCLUSIONS: The liver maintains metabolic homeostasis in response to shorter term hypoxic exposure through transient enhancement of respiratory chain capacity and alterations to lipid metabolism. These findings may have implications in understanding and treating hepatic pathologies associated with hypoxia.GlaxoSmithKlin

A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells

Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/β-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3β) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5–1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3β (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2–2.5 fold, p < 0.05), a result associated with an increase in total β-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (−86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditionsBrock University Library Open Access Publishing Fun

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells

Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

[This corrects the article DOI: 10.3389/fonc.2018.00388.]

Carbon budget and carbon chemistry in Photon Dominated Regions

We present a study of small carbon chains and rings in Photon Dominated Regions (PDRs) performed at millimetre wavelengths. Our sample consists of the Horsehead nebula (B33), the rho,Oph L1688 cloud interface, and the cometary-shaped cloud IC63. Using the IRAM 30-m telescope, the SEST and the Effelsberg 100-m teles cope at Effelsberg., we mapped the emission of \cch, c-C3H2 and C4H, and searched for heavy hydrocarbons such as c-C3H, l-C3H, l-C3H2, l-C4H2 and C6H. The large scale maps show that small hydrocarbons are present until the edge of all PDRs, which is surprising as they are expected to be easily destroyed by UV radiation. Their spatial distribution reasonably agrees with the aromatic emission mapped in mid-IR wavelength bands. Their abundances relative to H2 are relatively high and comparable to the ones derived in dark clouds such as L134N or TMC-1, known as efficient carbon factories. In particular, we report the first detection of C6H in a PDR. We have run steady-state PDR models using several gas-phase chemical networks (UMIST95 and the New Standard Model) and conclude that both networks fail in reproducing the high abundances of some of these hydrocarbons by an order of magnitude. The high abundance of hydrocarbons in the PDR may suggest that the photo-erosion of UV-irradiated large carbonaceous compounds could efficiently feed the ISM with small carbon clusters or molecules. This new production mechanism of carbon chains and rings could overcome their destruction by the UV radiation field. Dedicated theoretical and laboratory measurements are required in order to understand and implement these additional chemical routes.Comment: 18 pages, 12 figure

Impact of cross-section uncertainties on supernova neutrino spectral parameter fitting in the Deep Underground Neutrino Experiment

A primary goal of the upcoming Deep Underground Neutrino Experiment (DUNE) is to measure the $\mathcal{O}(10)$ MeV neutrinos produced by a Galactic core-collapse supernova if one should occur during the lifetime of the experiment. The liquid-argon-based detectors planned for DUNE are expected to be uniquely sensitive to the $\nu_e$ component of the supernova flux, enabling a wide variety of physics and astrophysics measurements. A key requirement for a correct interpretation of these measurements is a good understanding of the energy-dependent total cross section $\sigma(E_\nu)$ for charged-current $\nu_e$ absorption on argon. In the context of a simulated extraction of supernova $\nu_e$ spectral parameters from a toy analysis, we investigate the impact of $\sigma(E_\nu)$ modeling uncertainties on DUNE's supernova neutrino physics sensitivity for the first time. We find that the currently large theoretical uncertainties on $\sigma(E_\nu)$ must be substantially reduced before the $\nu_e$ flux parameters can be extracted reliably: in the absence of external constraints, a measurement of the integrated neutrino luminosity with less than 10\% bias with DUNE requires $\sigma(E_\nu)$ to be known to about 5%. The neutrino spectral shape parameters can be known to better than 10% for a 20% uncertainty on the cross-section scale, although they will be sensitive to uncertainties on the shape of $\sigma(E_\nu)$. A direct measurement of low-energy $\nu_e$-argon scattering would be invaluable for improving the theoretical precision to the needed level.Comment: 25 pages, 21 figure

The DUNE far detector vertical drift technology. Technical design report

DUNE is an international experiment dedicated to addressing some of the questions at the forefront of particle physics and astrophysics, including the mystifying preponderance of matter over antimatter in the early universe. The dual-site experiment will employ an intense neutrino beam focused on a near and a far detector as it aims to determine the neutrino mass hierarchy and to make high-precision measurements of the PMNS matrix parameters, including the CP-violating phase. It will also stand ready to observe supernova neutrino bursts, and seeks to observe nucleon decay as a signature of a grand unified theory underlying the standard model. The DUNE far detector implements liquid argon time-projection chamber (LArTPC) technology, and combines the many tens-of-kiloton fiducial mass necessary for rare event searches with the sub-centimeter spatial resolution required to image those events with high precision. The addition of a photon detection system enhances physics capabilities for all DUNE physics drivers and opens prospects for further physics explorations. Given its size, the far detector will be implemented as a set of modules, with LArTPC designs that differ from one another as newer technologies arise. In the vertical drift LArTPC design, a horizontal cathode bisects the detector, creating two stacked drift volumes in which ionization charges drift towards anodes at either the top or bottom. The anodes are composed of perforated PCB layers with conductive strips, enabling reconstruction in 3D. Light-trap-style photon detection modules are placed both on the cryostat's side walls and on the central cathode where they are optically powered. This Technical Design Report describes in detail the technical implementations of each subsystem of this LArTPC that, together with the other far detector modules and the near detector, will enable DUNE to achieve its physics goals

Performance of a modular ton-scale pixel-readout liquid argon time projection chamber

The Module-0 Demonstrator is a single-phase 600 kg liquid argon time projection chamber operated as a prototype for the DUNE liquid argon near detector. Based on the ArgonCube design concept, Module-0 features a novel 80k-channel pixelated charge readout and advanced high-coverage photon detection system. In this paper, we present an analysis of an eight-day data set consisting of 25 million cosmic ray events collected in the spring of 2021. We use this sample to demonstrate the imaging performance of the charge and light readout systems as well as the signal correlations between the two. We also report argon purity and detector uniformity measurements and provide comparisons to detector simulations