The Eye as a Transplantation Site to Monitor Pancreatic Islet Cell Plasticity

The endocrine cells confined in the islets of Langerhans are responsible for the maintenance of blood glucose homeostasis. In particular, beta cells produce and secrete insulin, an essential hormone regulating glucose uptake and metabolism. An insufficient amount of beta cells or defects in the molecular mechanisms leading to glucose-induced insulin secretion trigger the development of diabetes, a severe disease with epidemic spreading throughout the world. A comprehensive appreciation of the diverse adaptive procedures regulating beta cell mass and function is thus of paramount importance for the understanding of diabetes pathogenesis and for the development of effective therapeutic strategies. While significant findings were obtained by the use of islets isolated from the pancreas, in vitro studies are inherently limited since they lack the many factors influencing pancreatic islet cell function in vivo and do not allow for longitudinal monitoring of islet cell plasticity in the living organism. In this respect a number of imaging methodologies have been developed over the years for the study of islets in situ in the pancreas, a challenging task due to the relatively small size of the islets and their location, scattered throughout the organ. To increase imaging resolution and allow for longitudinal studies in individual islets, another strategy is based on the transplantation of islets into other sites that are more accessible for imaging. In this review we present the anterior chamber of the eye as a transplantation and imaging site for the study of pancreatic islet cell plasticity, and summarize the major research outcomes facilitated by this technological platform.Published versionOwn work discussed in this review was supported by funding from Karolinska Institutet, the Strategic Research Program in Diabetes at Karolinska Institutet, the Swedish Research Council, the Novo Nordisk Foundation, the Swedish Diabetes Association, the Family Knut and Alice Wallenberg Foundation, Diabetes Research and Wellness Foundation, the Stichting af Jochnick Foundation, the Family Erling-Persson Foundation, Berth von Kantzow’s Foundation, ERC-2018-AdG 834860 EYELETS, the European Union’s Seventh Framework Programme under grant agreements No 289932 and 613879, and the European Diabetes Research Programme in Cellular Plasticity Underlying the Pathophysiology of Type 2 Diabetes

Scaling quantum-dot light-emitting diodes to submicrometer sizes

We introduce a device structure and a fabrication technique that allow the realization of efficient light-emitting diodes (LEDs) with dimensions of the active area in the 100 nm range. Using optical lithography, selective oxidation, and an active region consisting of InAs quantum dots (QDs), we fabricated LEDs with light–current–voltage characteristics which scale well with nominal device area down to 600 nm diam at room temperature. The scaling behavior provides evidence for strong carrier confinement in the QDs and shows the potential for the realization of high-efficiency single-photon LEDs operating at room temperature. ©2002 American Institute of Physics

Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRN

Monitoring mis‐acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site‐specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run‐off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser‐scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl‐tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell type

In vivo protein labeling for structural and functional investigation of the 5-HT3A neurotransmitter receptor

In this thesis, two different fluorescent labeling techniques for in vivo investigations on the 5-HT3 receptor (5-HT3R) functions are presented. This plasma membrane protein contains five subunits surrounding an ion channel that opens after binding of a 5-HT3-specific neurotransmitter. The first technique, described in the first part of this report, focuses on receptor labeling via genetic fusion to spectral variants of the green fluorescent protein. I found that the resulting chimeras containing one fluorescent protein per subunit exhibit preserved ligand binding and channel activity, opening a wide range of biological research applications. Among these, I present the possibility to follow the 5-HT3R trafficking and localization during its entire life cycle by multicolor imaging in live cells, starting with its cytoplasmic biogenesis and ending with its ligand-induced internalization. The intracellular subunit assembly is shown to occur in the endoplasmic reticulum, and the importance of the cytoskeleton microtubules for proper membrane targeting is unraveled. The utility of bioluminescent 5-HT3R contructs was also demonstrated in another approach using the chemical disruption of cellular actin filaments to produce vesicular fractions of cells, in the order of 0.1 to a few micrometers in diameter. These so-called native vesicles, containing the labeled receptors in their membrane, were shown to be suitable for measurements using fluorescence confocal microscopy of ligand binding and of ion influx, opening new possibilities for miniaturized bioanalytics. In a third approach, I demonstrated that after detergent-solubilization of the receptor, the green fluorescent protein (GFP) inserted into the receptor sequence permitted to monitor ligand binding via fluorescence resonance energy transfer (FRET). Furthermore, I could observe a spatial reorientation of the receptor GFPs upon binding an agonist to the receptor. In the second part of this thesis, I adapted the mis-acylated suppressor tRNA technology to mammalian cells, permitting the introduction of unnatural amino acids at specific positions in the protein of interest. I achieved an efficiency of amino acid incorporation using in vitro aminoacylated suppressor tRNA in the order of 15% in CHO cells. A novel methodology for the quantification of background natural nonsense codon readthrough in different cell lines was also developed, permitting to select the most suitable codon-anticodon pair for this suppressor tRNA technique in various cell lines. Finally, I present a general strategy to increase the aforementioned artificial incorporation efficiency by down-regulating the competing eukaryotic release factor 1 (eRF1) using small interfering RNAs

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chem. aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amts. of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately det. suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control expts. showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential exptl. parameters for expanding the scope of the suppressor tRNA technol. to different cell types. [on SciFinder (R)

Time-resolved optical characterization of InAs/InGaAs quantum dots emitting at 1.3 mu m

We present the time-resolved optical characterization of InAs/InGaAs self-assembledquantum dots emitting at 1.3 μm at room temperature. The photoluminescence decay time varies from 1.2 (5 K) to 1.8 ns (293 K). Evidence of thermalization among dots is seen in both continuous-wave and time-resolved spectra around 150 K. A short rise time of 10±2 ps is measured, indicating a fast capture and relaxation of carriers inside the dots

Muonium as a shallow center in GaN

A paramagnetic muonium (Mu) state with an extremely small hyperfine parameter was observed for the first time in single-crystalline GaN below 25 K. It has a highly anisotropic hyperfine structure with axial symmetry along the [0001] direction, suggesting that it is located either at a nitrogen-antibonding or a bond-centered site oriented parallel to the c-axis. Its small ionization energy (=< 14 meV) and small hyperfine parameter (--10^{-4} times the vacuum value) indicate that muonium in one of its possible sites produces a shallow state, raising the possibility that the analogous hydrogen center could be a source of n-type conductivity in as-grown GaN.Comment: 4 figures, to be published in Phys. Rev. Letter