Efficacy of single and double SiNx interlayers on defect reduction in GaN overlayers grown by organometallic vapor-phase epitaxy

We report on the growth of and evolution of defects in GaN epilayers having single- and double-layer SiNx nanoporous insertion layers. The SiNx was formed in situ in the growth chamber of an organometallic vapor-phase epitaxy system by simultaneous flow of diluted silane and ammonia. The GaN epilayers and SiNx interlayers were grown on 6H-SiC substrates using three different nucleation layers, namely, low-temperature GaN, high-temperature GaN, and high-temperature AlN nucleation layers. X-ray-diffraction rocking curves and cross-sectional and plan-view transmission electron microscope analyses indicated that a nanoporous SiNx layer can reduce the dislocations density in the GaN overgrown layer to ∼3×108cm−2 range; below this level the defect blocking effect of SiNx would saturate. Therefore the insertion of a second SiNx layer becomes much less effective in reducing dislocations, although it continues to reduce the point defects, as suggested by time-resolved photoluminescence measurements. The insertion of SiNx interlayers was found to improve significantly the mechanical strength of the GaN epilayers resulting in a much lower crack line density

Effectiveness of TiN porous templates on the reduction of threading dislocations in GaN overgrowth by organometallic vapor-phase epitaxy

We report on the reduction of threading dislocations in GaN overlayers grown by organometallic vapor phase epitaxy on micro-porous TiN networks. These networks were obtained by in situannealing of thin Ti layers deposited in a metalization chamber, on the (0001) face of GaN templates. Observations by transmission electron microscopy indicate dislocation reduction by factors of up to 10 in GaN layers grown on TiN networks compared with the control GaN.X-ray diffraction shows that GaNgrown on the TiN network has a smaller (102) plane peak width (4.6 arcmin) than the control GaN (7.8 arcmin). In low temperature photoluminescence spectra, a narrow excitonic full-width-at-half-maximum of 2.4 meV was obtained, as compared to 3.0 meV for the control GaN, confirming the improved crystalline quality of the overgrown GaN layers

Low dislocation densities and long carrier lifetimes in GaN thin films grown on a SiNx nanonetwork

Significant improvement of structural and optical qualities of GaNthin films on sapphire substrates was achieved by metal organic chemical vapor deposition with in situ SiNxnanonetwork. Transmission electron microscope (TEM) studies revealed that screw- and edge-type dislocations were reduced to 4.4×107 and 1.7×107cm−2, respectively, for a ∼5.5-μm-thick layer. Furthermore, room temperature carrier lifetimes of 2.22 and 2.49ns were measured by time-resolved photoluminescence(TRPL) for samples containing single and double SiNx network layers, respectively, representing a significant improvement over the previous studies. The consistent trends among the TEM,x-ray diffraction, and TRPL measurements suggest that in situ SiNx network reduces line defects effectively as well as the point-defect-related nonradiative centers

Dislocation reduction in GaN grown on porous TiN networks by metal-organic vapor-phase epitaxy

We report on the effectiveness of porous TiN nanonetworks on the reduction of threading dislocations (TDs) in GaN grown by metal-organic vapor-phase epitaxy (MOVPE). The porous TiN networks were formed by in situ annealing of thin-deposited Ti films deposited ex situ on GaN templates within the MOVPE growth chamber. Different annealing parameters in relation to surface porosity of TiN networks were investigated. Transmission electron micrographs indicated dislocation reduction by factors of up to 10 in GaN layers grown on the TiN nanonetwork, compared with a control sample. TiN prevented many dislocations present in the GaN templates from penetrating into the upper layer. Microscale epitaxial lateral overgrowth of GaN above TiN also contributed to TD reduction. The surface porosity of the TiN network had a strong impact on the efficiency of TD reduction. X-ray-diffraction and time-resolved photoluminescence measurements further confirmed the improved GaN quality

The Association of AMPK with ULK1 Regulates Autophagy

Autophagy is a highly orchestrated intracellular bulk degradation process that is activated by various environmental stresses. The serine/threonine kinase ULK1, like its yeast homologue Atg1, is a key initiator of autophagy that is negatively regulated by the mTOR kinase. However, the molecular mechanism that controls the inhibitory effect of mTOR on ULK1-mediated autophagy is not fully understood. Here we identified AMPK, a central energy sensor, as a new ULK1-binding partner. We found that AMPK binds to the PS domain of ULK1 and this interaction is required for ULK1-mediated autophagy. Interestingly, activation of AMPK by AICAR induces 14-3-3 binding to the AMPK-ULK1-mTORC1 complex, which coincides with raptor Ser792 phosphorylation and mTOR inactivation. Consistently, AICAR induces autophagy in TSC2-deficient cells expressing wild-type raptor but not the mutant raptor that lacks the AMPK phosphorylation sites (Ser722 and Ser792). Taken together, these results suggest that AMPK association with ULK1 plays an important role in autophagy induction, at least in part, by phosphorylation of raptor to lift the inhibitory effect of mTOR on the ULK1 autophagic complex

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium

mTOR signaling: implications for cancer and anticancer therapy

Mounting evidence links deregulated protein synthesis to tumorigenesis via the translation initiation factor complex eIF4F. Components of this complex are often overexpressed in a large number of cancers and promote malignant transformation in experimental systems. mTOR affects the activity of the eIF4F complex by phosphorylating repressors of the eIF4F complex, the eIF4E binding proteins. The immunosuppressant rapamycin specifically inhibits mTOR activity and retards cancer growth. Importantly, mutations in upstream negative regulators of mTOR cause hamartomas, haemangiomas, and cancers that are sensitive to rapamycin treatment. Such mutations lead to increased eIF4F formation and consequently to enhanced translation initiation and cell growth. Thus, inhibition of translation initiation through targeting the mTOR-signalling pathway is emerging as a promising therapeutic option

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G⇔A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology