Influence of PH3 exposure on silicon substrate morphology in the MOVPE growth of III-V on silicon multijunction solar cells

Dual-junction solar cells formed by a GaAsP or GaInP top cell and a silicon bottom cell seem to be attractive candidates to materialize the long sought-for integration of III-V materials on silicon for photovoltaic applications. One of the first issues to be considered in the development of this structure will be the strategy to create the silicon emitter of the bottom subcell. In this study, we explore the possibility of forming the silicon emitter by phosphorus diffusion (i.e. exposing the wafer to PH3 in a MOVPE reactor) and still obtain good surface morphologies to achieve a successful III-V heteroepitaxy as occurs in conventional III-V on germanium solar cell technology. Consequently, we explore the parameter space (PH3 partial pressure, time and temperature) that is needed to create optimized emitter designs and assess the impact of such treatments on surface morphology using atomic force microscopy. Although a strong degradation of surface morphology caused by prolonged exposure of silicon to PH3 is corroborated, it is also shown that subsequent anneals under H-2 can recover silicon surface morphology and minimize its RMS roughness and the presence of pits and spikes.

Methodology of image analysis for study of the vertisols moisture content

The main problem to study vertical drainage from the moisture distribution, on a vertisol profile, is searching for suitable methods using these procedures. Our aim was to design a digital image processing methodology and its analysis to characterize the moisture content distribution of a vertisol profile. In this research, twelve soil pits were excavated on a ba re Mazic Pellic Vertisols ix of them in May 13/2011 and the rest in May 19 /2011 after a moderate rainfall event. Digital RGB images were taken from each vertisol pit using a Kodak? camera selecting a size of 1600x945 pixels. Each soil image was processed to homogenized brightness and then a spatial filter with several window sizes was applied to select the optimum one. The RGB image obtained were divided in each matrix color selecting the best thresholds for each one, maximum and minimum, to be applied and get a digital binary pattern. This one was analyzed by estimating two fractal scaling exponents box counting dimension D BC) and interface fractal dimension (D) In addition, three pre-fractal scaling coefficients were determinate at maximum resolution: total number of boxes intercepting the foreground pattern (A), fractal lacunarity (?1) and Shannon entropy S1). For all the images processed the spatial filter 9x9 was the optimum based on entropy, cluster and histogram criteria. Thresholds for each color were selected based on bimodal histograms.

Anatomía comparada de la madera de Cupressaceae y su correspondencia con los estudios de filogenia

La familia Cupressaceae incluye un total de 133 especies agrupadas en 30 géneros, 17 de los cuales son monospecíficos. Esta familia se encuentra representada en todos los continentes salvo en la Antártida. Sus especies se distribuyen en distintas regiones climáticas, y en altitudes que varían desde el nivel del mar hasta los 5.000 m. La falta de descripción anatómica de muchos de los géneros y especies de Cupressaceae es notable, así como la contradicción que aparece entre distintas investigaciones sobre las características anatómicas de la madera descritas para cada especie. Este estudio describe la anatomía de la madera de Cupressaceae y analiza las características que podrían representar sinapomorfías de los clados delimitados en los estudios filogenéticos. Siguiendo los métodos tradicionales de preparación y descripción de la madera a nivel microscópico, se ha estudiado la madera de 113 especies de los 30 géneros de Cupressaceae. Para ello se han empleado muestras de madera de origen trazable, procedentes de colecciones de madera de distintas instituciones internacionales. Se ha empleado una robusta filogenia molecular para la reconstrucción de los caracteres ancestrales. La anatomía de la madera de los 30 géneros de Cupressaceae, pone de manifiesto la gran homogeneidad de la familia, caracterizada por la presencia de traqueidas axiales sin engrosamientos helicoidales, parénquima radial con paredes horizontales lisas, punteaduras del campo de cruce de tipo cupresoide y la carencia de canales resiníferos fisiológicos. Además, todos presentan parénquima axial (salvo Neocallitropsis, Thuja y Xanthocyparis), punteaduras radiales areoladas con toro definido (salvo Thuja y Thujopsis), siendo habitual la presencia de punteaduras areoladas en las paredes tangenciales de la madera tardía, y verrugosidades en la cara interna de las traqueidas (salvo Ca. macleayana, Libocedrus, Papuacedrus y Neocallitropsis). Los radios leñosos son homogéneos y están compuestos de parénquima radial (con la presencia de traqueidas radiales en algunas especies de Cupressus, Sequoia, Thujopsis y X. nootkatensis) con paredes finales lisas o lisas y noduladas (exclusivamente noduladas en Cal. macrolepis, C. bakeri y en la mayoría de especies de Juniperus), y el rango de altura de los radios leñosos se encuentra entre 5 y 15 células. Se consideran posibles sinapomorfismos de Cupressaceae la presencia de verrugosidades en la cara interna de las traqueidas, la presencia de traqueidas axiales sin engrosamientos helicoidales, la presencia de parénquima axial, la presencia de radios leñosos homogéneos (compuestos únicamente de parénquima radial), la tipología de las paredes horizontales del parénquima radial, las punteaduras del campo de cruce de tipo cupresoide y la ausencia de canales resiníferos fisiológicos, pero lo que realmente diferencia a este grupo de coníferas es la simultaneidad de todos estos caracteres en sus maderas. Como sinapomorfías específicas por clados se proponen: la ausencia de toro definido y muescas en el borde de las punteaduras en Thuja-Thujopsis, la existencia de extensiones de toro en Diselma-Fitzroya-Widdringtonia; la presencia de engrosamientos callitroides en Callitris-Actinostrobus; la presencia de espacios intercelulares y las muescas en el borde de las punteaduras en el clado formado por el género Juniperus y las especies de Cupressus en la región oriental; la presencia de paredes finales del parénquima radial tanto lisas como noduladas en los clados formados por el género Xanthocyparis y las especies de Cupressus en la región occidental y en Fitzroya-Diselma; y por último, la presencia de punteaduras del campo de cruce de tipo taxodioide en los clados taxodioid y sequoioid. ABSTRACT The Cupressaceae family comprises 133 species grouped into 30 genera, 17 of which are monotypic. The family is represented in all continents except Antarctica. Its species are distributed in various climate zones and at altitudes from sea level to 5,000 m. There is a considerable lack of anatomical descriptions for many genera and species of Cupressaceae and much contradiction between studies about the wood anatomical features described for each species. This study describes the wood anatomy of Cupressaceae and analyses the features that could represent synapomorphies of the clades recovered in phylogenetic studies. Following the traditional methods of preparation and description of wood at microscopic level, a study was made of the wood of 113 species of the 30 Cupressaceae genera. The study samples had traceable origins and came from wood collections held at various international institutions. A robust molecular phylogeny was used for ancestral state reconstruction. The wood anatomy of the 30 genera of the Cupressaceae shows the high homogeneity of the family, which is characterised by the presence of axial tracheids without helical thickenings, smooth horizontal walls of ray parenchyma cells, cupressoid cross-field pits, and the absence of physiological resin canals. In addition, they all have axial parenchyma (except Neocallitropsis, Thuja and Xanthocyparis), a warty layer on the inner wall of the tracheids (except Ca. macleayana, Libocedrus, Papuacedrus and Neocallitropsis) and tracheid pitting in radial walls with a well defined torus (except Thuja and Thujopsis); tracheid pitting in the tangential walls of the latewood is common. Rays are homogeneous and are composed of ray parenchyma (with the presence of ray tracheids in some species of Cupressus, Sequoia, Thujopsis and X. nootkatensis), with smooth end walls or both smooth and nodular end walls (exclusively nodular in Cal. macrolepis, C. bakeri and most Juniperus species), and ray height range is 5 to 15 cells. Possible synapomorphies of Cupressaceae are the presence of a warty layer on the inner layer of the tracheids, axial tracheids without helical thickenings, the presence of axial parenchyma, homogeneous rays (composed exclusively of ray parenchyma), the typology of the horizontal walls of ray parenchyma cells, cupressoid cross-field pits and the absence of physiological resin canals, but what truly differentiates this group of softwoods is the co-occurrence of all these features in their wood. The following are proposed as clade-specific synapomorphies: absence of a well-defined torus and presence of pits with notched borders in Thuja-Thujopsis, torus extensions in Diselma-Fitzroya-Widdringtonia; callitroid thickenings in Callitris-Actinostrobus; intercellular spaces and pits with notched borders in the clade formed by the genus Juniperus and the species of Cupressus in the eastern region; smooth and nodular ray parenchyma end walls in the clades formed by the genus Xanthocyparis and the species of Cupressus in the western region and in Fitzroya-Diselma, and taxodioid cross-field pits in the taxodioid and sequoioid clades.

Strategies to unblock the n-GaAs surface when electrodepositing Bi from acidic solutions

Bismuth ultra-thin films grown on n-GaAs electrodes via electrodeposition are porous due to a blockade of the electrode surface caused by adsorbed hydrogen when using acidic electrolytes. In this study, we discuss the existence of two sources of hydrogen adsorption and we propose different routes to unblock the n-GaAs surface in order to improve Bi films compactness. Firstly, we demonstrate that increasing the electrolyte temperature provides compact yet polycrystalline Bi films. Cyclic voltammetry scans indicate that this low crystal quality might be a result of the incorporation of Bi hydroxides within the Bi film as a result of the temperature increase. Secondly, we have illuminated the semiconductor surface to take advantage of photogenerated holes. These photocarriers oxidize the adsorbed hydrogen unblocking the surface, but also create pits at the substrate surface that degrade the Bi/GaAs interface and prevent an epitaxial growth. Finally, we show that performing a cyclic voltammetry scan before electrodeposition enables the growth of compact Bi ultra-thin films of high crystallinity on semiconductor substrates with a doping level low enough to perform transport measurements.

The β2-adrenergic receptor/βarrestin complex recruits the clathrin adaptor AP-2 during endocytosis

βarrestins mediate the desensitization of the β2-adrenergic receptor (β2AR) and many other G protein-coupled receptors (GPCRs). Additionally, βarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that βarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, βarrestin 2, and the β2-adaptin subunit of AP-2. β2-Adaptin binds βarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with βarrestins and β2AR in an agonist-dependent manner in HEK-293 cells. Moreover, β2-adaptin translocates from the cytosol to the plasma membrane in response to the β2AR agonist isoproterenol and colocalizes with β2AR in clathrin-coated pits. Finally, expression of βarrestin 2 minigene constructs containing the β2-adaptin interacting region inhibits β2AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.

Dynamin-dependent endocytosis of ionotropic glutamate receptors

Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.

Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56lck, undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56lck, we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

RGS-GAIP, a GTPase-activating Protein for Gαi Heterotrimeric G Proteins, Is Located on Clathrin-coated Vesicles

RGS-GAIP (Gα-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Gαi/Gαq-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Gαi3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

Differential Distribution of Dynamin Isoforms in Mammalian Cells

Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.

Amphiphysin Heterodimers: Potential Role in Clathrin-mediated Endocytosis

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1–Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin’s GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

Ubiquitously Expressed Dynamin-II Has a Higher Intrinsic GTPase Activity and a Greater Propensity for Self-assembly Than Neuronal Dynamin-I

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a ∼threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.