2,025 research outputs found
Optimization of Optical and Mechanical Properties of Real Architecture for 3-Dimensional Tissue Equivalents: Towards Treatment of Limbal Epithelial Stem Cell Deficiency
Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimize the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The ‘optimal’ RAFT TE was thin, transparent but still handleable and was produced using 0.6 ml of 3 mg/ml collagen. Degradation rates of the ‘optimal’ RAFT TE and HAM were similar. hLE achieved confluency on ‘optimal’ RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63α. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency
Miro1 – the Missing Link to Peroxisome Motility
This is the author accepted manuscript. The final version is available from Taylor & Francis via the DOI in this recordPeroxisomes are ubiquitous, highly dynamic, multifunctional compartments in eukaryotic
cells, which perform key roles in cellular lipid metabolism and redox balance. Like other
membrane-bound organelles, peroxisomes must move in the cellular landscape to
perform localized functions, interact with other organelles and to properly distribute
during cell division. However, our current knowledge of peroxisome motility in
mammalian cells is still very limited. Recently, three independent studies have identified
Miro1 as a regulator of peroxisome motility in mammalian cells. In these studies, the
authors show that Miro1 is targeted to peroxisomes in several cell lines, in a process that
relies on its interaction with the peroxisomal chaperone Pex19. Interestingly, however,
different conclusions are drawn about which Miro1 isoforms are targeted to peroxisomes,
how it interacts with Pex19 and most importantly, the type of motility Miro1 is regulating.This work was supported by the Biotechnology and Biological Sciences Research
Council (BB/K006231/1, BB/N01541X/1) (to M.S.). I.G.C. is supported by an EMBO
Long-term Fellowship ALTF-580-2017
Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations
This is the author accepted manuscript. The final version is available from Taylor & Francis via the DOI in this record.Cooperation between cellular organelles such as mitochondria, peroxisomes and the ER is
essential for a variety of important and diverse metabolic processes. Effective communication
and metabolite exchange requires physical linkages between the organelles, predominantly in
the form of organelle contact sites. At such contact sites organelle membranes are brought
into close proximity by the action of molecular tethers, which often consist of specific protein
pairs anchored in the membrane of the opposing organelles. Currently numerous tethering
components have been identified which link the ER with multiple other organelles but
knowledge of the factors linking the ER with peroxisomes is limited. Peroxisome-ER
interplay is important because it is required for the biosynthesis of unsaturated fatty acids,
ether-phospholipids and sterols with defects in these functions leading to severe diseases.
Here we characterise acyl-CoA binding domain protein 4 (ACBD4) as a tail-anchored
peroxisomal membrane protein which interacts with the ER protein, vesicle-associated
membrane protein-associated protein–B (VAPB) to promote peroxisome-ER associations.We thank all colleagues who provided plasmids and antibodies, and T Levine for sharing
data. This work was supported by BBSRC (BB/K006231/1, BB/N01541X/1). MS is
supported by the Marie Curie Initial Training Network action PerFuMe (316723). The
authors declare no competing financial interests
Peroxisome Motility Measurement and Quantification Assay
This is the final version of the article. Available from Bio-protocol via the DOI in this record.Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and
polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al., 1996; Wiemer et al., 1997; Schrader et al., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with
ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/K006231/1 and BB/N01541X/1 to M. Schrader). J. Metz and M. Schrader are supported by a Wellcome Trust Institutional Strategic Support Award (WT097835MF and WT105618MA). M. Schrader is supported by Marie Curie Initial Training Network action PerFuMe (316723)
A neutral atom quantum register
We demonstrate the realization of a quantum register using a string of single
neutral atoms which are trapped in an optical dipole trap. The atoms are
selectively and coherently manipulated in a magnetic field gradient using
microwave radiation. Our addressing scheme operates with a high spatial
resolution and qubit rotations on individual atoms are performed with 99%
contrast. In a final read-out operation we analyze each individual atomic
state. Finally, we have measured the coherence time and identified the
predominant dephasing mechanism for our register.Comment: 4 pages, 4 figure
Adiabatic Quantum State Manipulation of Single Trapped Atoms
We use microwave induced adiabatic passages for selective spin flips within a
string of optically trapped individual neutral Cs atoms. We
position-dependently shift the atomic transition frequency with a magnetic
field gradient. To flip the spin of a selected atom, we optically measure its
position and sweep the microwave frequency across its respective resonance
frequency. We analyze the addressing resolution and the experimental robustness
of this scheme. Furthermore, we show that adiabatic spin flips can also be
induced with a fixed microwave frequency by deterministically transporting the
atoms across the position of resonance.Comment: 4 pages, 4 figure
Coherence properties and quantum state transportation in an optical conveyor belt
We have prepared and detected quantum coherences with long dephasing times at
the level of single trapped cesium atoms. Controlled transport by an "optical
conveyor belt" over macroscopic distances preserves the atomic coherence with
slight reduction of coherence time. The limiting dephasing effects are
experimentally identified and are of technical rather than fundamental nature.
We present an analytical model of the reversible and irreversible dephasing
mechanisms. Coherent quantum bit operations along with quantum state transport
open the route towards a "quantum shift register" of individual neutral atoms.Comment: 4 pages, 3 figure
Positronic lithium, an electronically stable Li-e ground state
Calculations of the positron-Li system were performed using the Stochastic
Variational Method and yielded a minimum energy of -7.53208 Hartree for the L=0
ground state. Unlike previous calculations of this system, the system was found
to be stable against dissociation into the Ps + Li channel with a binding
energy of 0.00217 Hartree and is therefore electronically stable. This is the
first instance of a rigorous calculation predicting that it is possible to
combine a positron with a neutral atom and form an electronically stable bound
state.Comment: 11 pages, 2 tables. To be published in Phys.Rev.Let
The Density of States and the Spectral Shift Density of Random Schroedinger Operators
In this article we continue our analysis of Schroedinger operators with a
random potential using scattering theory. In particular the theory of Krein's
spectral shift function leads to an alternative construction of the density of
states in arbitrary dimensions. For arbitrary dimension we show existence of
the spectral shift density, which is defined as the bulk limit of the spectral
shift function per unit interaction volume. This density equals the difference
of the density of states for the free and the interaction theory. This extends
the results previously obtained by the authors in one dimension. Also we
consider the case where the interaction is concentrated near a hyperplane.Comment: 1 figur
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