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Homologous recombination in ES cells was employed to generate mice with targeted deletion of the first three exons of the γ-synuclein gene. Complete inactivation of gene expression in null mutant mice was confirmed on the mRNA and protein levels. Null mutant mice are viable, are fertile, and do not display evident phenotypical abnormalities. The effects of γ-synuclein deficiency on motor and peripheral sensory neurons were studied by various methods in vivo and in vitro. These two types of neurons were selected because they both express high levels of γ-synuclein from the early stages of mouse embryonic development but later in the development they display different patterns of intracellular compartmentalization of the protein. We found no difference in the number of neurons between wild-type and null mutant animals in several brain stem motor nuclei, in lumbar dorsal root ganglia, and in the trigeminal ganglion. The survival of γ-synuclein-deficient trigeminal neurons in various culture conditions was not different from that of wild-type neurons. There was no difference in the numbers of myelinated and nonmyelinated fibers in the saphenous nerves of these animals, and sensory reflex thresholds were also intact in γ-synuclein null mutant mice. Nerve injury led to similar changes in sensory function in wild-type and mutant mice. Taken together, our data suggest that like α-synuclein, γ-synuclein is dispensable for the development and function of the nervous system.
Several neurodegenerative diseases have been recently coalesced into a distinct group named synucleinopathies (12, 16, 20, 53). Although they are diverse in symptoms and clinical signs, these diseases share a common histopathological feature, i.e., formation of large intracellular inclusions whose principal component is an aggregated small protein, α-synuclein. Neither the normal cellular function of α-synuclein nor the exact mechanism of its involvement in neurodegeneration is clearly understood; possible scenarios are discussed in many recent reviews (see, for example, references 10, 28, 33, 34, and 43). Even less clear are the normal functions and roles in neurodegeneration of the other two members of the synuclein family. Both β-synuclein/PNP14 (24, 35) and γ-synuclein/BCSG1/persyn (7, 26, 29) have a very high degree of amino acid similarity with α-synuclein within the N-terminal KTK repeat region of the protein molecule, and this is reflected in such common features of synucleins as a native unfolded state in physiological solutions, reversible binding to lipid vesicles, and localization in presynaptic terminals (13, 25, 31). However, the C-terminal regions of synucleins, although all highly acidic, are rather different (7, 29, 52). It is perhaps this structural diversity that leads to differences in the behavior of synucleins in vitro and in various in vivo model systems. Consistent with the finding that β-synuclein and γ-synuclein are much less fibrillogenic than α-synuclein (4, 47, 55), aggregates of these two proteins are not constituents of Lewy bodies or other histopathological hallmarks of synucleinopathies, although abnormal β- and γ-synuclein-positive structures have been observed in several cases (15, 17, 49). Recent in vitro studies have also shown that both β- and γ-synuclein are able to inhibit fibrillation of α-synuclein (40, 55). In transgenic mice overexpression of β-synuclein reduces the severity of neurodegenerative alterations and the number of α-synuclein-positive interneuronal inclusions caused by α-synuclein overexpression (23). Changes of expression of all three synucleins in brain areas affected in neurodegenerative diseases have been reported (44). Previously we demonstrated that overexpression of α-synuclein, but not γ-synuclein, kills sensory neurons in primary cultures (6, 45). Moreover, it has been shown that γ-synuclein is able to block JNK signaling, a pathway whose activation is commonly associated with induction of apoptosis (39). These observations suggest that the correct balance of synucleins might be important for survival of at least some populations of neurons and that decreased expression of γ-synuclein might have a proapoptotic effect. The obvious way to investigate this is to assess whether the absence of γ-synuclein affects neurons that normally express these two proteins. In different vertebrate species, high levels of γ-synuclein mRNA are detected from the early stages of embryonic development in two neuronal populations, motoneurons and peripheral sensory neurons (7, 52), and α-synuclein is also expressed in these neurons (18, 32, 52; our unpublished observations). Moreover, in transgenic mice overexpression of α-synuclein leads to pathological changes in spinal and brain stem motoneurons (19, 30, 56), suggesting that these neurons are susceptible to changes in the metabolism of synucleins. Therefore, we studied populations of motoneurons and peripheral sensory neurons in γ-synuclein null mutant mice, which we produced.
Generation of γ-synuclein null mutant mice.To generate a targeting vector, a 1.2-kb XbaI-XbaI fragment from lambda genomic clone PS92 (2) was cloned as a short arm into an XbaI site of the pPNT vector (54) between PGK-TK and PGK-neo cassettes. The resulting plasmid was digested with Sse8387I, blunted with the Klenow fragment of DNA polymerase I, further digested with XhoI, and used as a vector for ligation with a long arm. DNA of lambda genomic clone PS91 (2) was digested with BglII, blunted with Klenow fragment of DNA polymerase I, and further digested with SalI, and a 4-kb fragment was used as a long arm in our targeting vector (Fig. 1a). For electroporation of 129Ola mouse ES cells (clone E14Tg2a; a gift of A. Smith, Centre for Genome Research, University of Edinburgh), DNA of the resulting targeting vector was linearized by digestion with NotI. After electroporation and selection, G418- and ganciclovir-resistant ES cell clones were checked for correct homologous recombination by Southern hybridization analysis of EcoRI-digested (for probes a and b in Fig. 1a) or BamHI-digested (for probe c) DNA. Suitable clones were injected into blastocysts of C57Bl6J mice (Charles River). All procedures involved in generation of mutant mice were carried out according to the United Kingdom Animals (Scientific Procedures) Act (1986) and other Home Office regulations under specific-pathogen-free conditions. The presence of a mutant allele in animals was checked by PCR analysis of DNA extracted from mouse tail biopsies. A common upstream primer (5′-AGTCCTGGCACCTCTAAGCA-3′) and primers specific for the wild-type allele (5′-GGGCTGATGTGTGGCTATCT-3′) and the PGK-neo cassette in the mutant allele (5′-GAAGAACGAGATCAGCAGCC-3′) were used for amplification. Forty cycles of 45 s at 95°C, 30 s at 56°C, and 60 s at 72°C were carried out. The presence of 480-bp (for the wild-type allele) and 397-bp (for the mutant allele) amplification products in the reaction mixture was checked by electrophoresis in a 1.5% agarose gel. Heterozygous animals were used for at least six further generations of backcrosses with C57BL6J mice before null mutant, wild-type, and heterozygous littermates were produced for further studies by breeding heterozygous males and females. The mutant mouse strain was registered in the Mouse Genome Information database under the official name (B6-TgHSNCGtm1VLB).
α-synuclein null mutant mice.Generation of α-synuclein mutant mice was described previously (1). We backcrossed these mice with C57Bl6J mice for at least six generations before setting up intercrosses to produce null mutant, wild-type, and heterozygous littermates.
Expression studies.Extraction of RNA from mouse tissues, Northern blotting, and preparation of labeled probes were carried out as described previously (7, 36).
Affinity-purified polyclonal rabbit SK23 antibody generated against a C-terminal peptide of mouse γ-synuclein (persyn) was used at a 1:500 dilution for Western blotting and enhanced chemiluminescence detection of this protein in total cell lysates as described earlier (7, 36). For immunohistochemistry, adult mouse brains and embryonic day 12 (E12), E15, and E18 embryos were fixed in 4% paraformaldehyde-phosphate-buffered saline (PBS) or Carnoy's fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) at 4°C overnight following dehydration in alcohol series and embedding in paraffin blocks. Eight-micrometer-thick sections were cut with a Leica or Microm microtome and mounted on SuperFrost slides (BDH, Poole, United Kingdom) for conventional staining or on Gold Seal slides (Gold Seal Products, Portsmouth, N.H.) for immunostaining. The paraffin sections were cleared in xylene and rehydrated through a graded alcohol series. Endogenous peroxidase activity was quenched by incubating the slides in 3% H2O in methanol for 20 min. After being washed with PBS, the tissues were blocked in 10% horse or goat serum and 0.4% Triton X-100 in PBS for 1 h at room temperature. Incubation with a 1:40 dilution of SK26 anti-mouse γ-synuclein antibody was carried out at 4°C overnight. Detection of immune complexes with biotinylated anti-rabbit antibody and avidin-peroxidase complex from the Vectastain ABC kit (Vector Laboratories, Peterborough, United Kingdom) and diaminobenzidine (Fast 3,3′-diaminobenzidine tablet sets; Sigma, St. Louis, Mo.) as a substrate was carried out according to the manufacturer's instructions. Motor nuclei were identified by staining of alternate sections with hematoxylin-eosin (Raymond A Lamb, London, United Kingdom) and a goat antibody against choline acetyltransferase (Chemicon, Temecula, Calif.) at a 1:100 dilution. In the latter case, anti-goat secondary antibody was used for detection.
Whole-mount immunofluorescence.Mouse triangularis sterni muscle was dissected in PBS and fixed in 4% paraformaldehyde-PBS for 20 min, followed by incubation with AlexaFluor 647-conjugated μ-bungarotoxin (5 μg/ml in PBS) for 30 min. After washing in PBS and blocking and permeabilization for 1 h in 1% bovine serum albumin-0.4% lysine-0.5% Triton X-100 in PBS, samples were incubated overnight with primary antibodies diluted in the same buffer at 4°C. Mouse monoclonal anti-neurofilament M (clone 2H3; Developmental Studies Hybridoma Bank, Iowa City, Iowa) and anti-synaptic vesicles (clone SV2; Developmental Studies Hybridoma Bank) antibodies were used at a 1:200 dilution, and rabbit polyclonal SK23 antibody was used at a 1:40 dilution. After several washes, incubation with 1:200-diluted secondary antibodies (fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulins and tetramethyl rhodamine isocyanate-conjugated goat anti-mouse immunoglobulins, both from Jackson ImmunoResearch Laboratories, West Grove, Pa.) was carried out for 3 h at room temperature, followed by extensive washing and mounting in Vectashield (Vector Laboratories, Burlingame, Calif.). Images were obtained with a Zeiss LSM 510/Axioplan 2 confocal microscope.
DRG neuron counts.The spinal columns from P2 mice were fixed and embedded as described above. Serial 8-μm-thick longitudinal sections were cut, mounted on SuperFrost slides (BDH), cleared in xylene, rehydrated through a graded alcohol series, and stained with 0.1% cresyl violet acetate (Sigma). Lumbar L6 dorsal root ganglia (DRG) were identified by using characteristic tissue landmarks on sections. Neurons were identified by virtue of the Nissl substance and their large, round, pale-stained nuclei (27). Neurons in the L6 DRG displaying a prominent nucleolus were counted on every eighth section. The neuronal number was quantified by using a digital stereology system that employs a combination of the optical dissector and volume fraction-Cavalieri methods (Kinetics Imaging, Bromborough, United Kingdom). All counts were carried out blindly by a person unaware of animal genotypes.
Motoneuron counts.Adult mouse brains were fixed and processed as described above for expression studies. The whole brain stem was sectioned into 8-μm-thick sections, which were mounted on SuperFrost slides. Rehydrated sections were stained in a 1% solution of neutral red (Raymond A Lamb) in distilled water for 30 min, followed by dehydration and mounting as described above. A stereological fractionator method (22), which allows estimation of the number of particles independent of the volume of the structure that the particles are part of and is not affected by tissue shrinkage during processing, was used to assess the number of motoneurons in five motor nuclei of the brain stem. Large (∼20 μm in diameter) neurons displaying a prominent nucleolus were counted in total on every fifth section throughout the nucleus; the first section for counting was randomly chosen from the first five sections that included this nucleus.
Nerve fibers counts.Anesthetized animals were transcardially perfused with heparinized saline and then 2.5% glutaraldehyde-2% paraformaldehyde-0.1 M sodium cacodylate buffer (pH 7.3)-1 mM CaCl2 for 5 min. The saphenous nerve was removed and fixed for 2 h in the same fixative, postfixed in OsO4, and embedded in araldite. One-micrometer resin sections were prepared and stained with toluidine blue for light microscopy to measure the area of the nerve cross-section. Ultrathin 80-nm transverse sections were stained with uranyl acetate and lead citrate, mounted onto copper slot grids, and examined on a transmission electron microscope (Phillips BioTwin; FEI, Cambridge, United Kingdom). Myelinated A-fibers and unmyelinated C-fibers were identified and counted as described previously (57). The densities of both types of fibers were calculated after counting of 12 to 15 randomly chosen electron microscope images and multiplied by the area of the nerve cross-section.
Primary neuronal cultures.Cultures from trigeminal and superior cervical ganglia were prepared as described previously (42) and maintained in neurobasal medium with B27 complement (Invitrogen, Carlsbad, Calif.). The number of neurons attached to each culture dish within a 12- by 12-mm square was counted 3 h after plating and was taken as the initial number of neurons (100%). In experiments with proteasome inhibitors and metal ions, the initial count was carried out 24 h after plating. In all cases drugs were added to cultures immediately after the initial count. The number of surviving neurons in the same area was counted 24 and 48 h later and was expressed as a percentage of the initial count.
Behavioral tests.All experiments were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986). Behavioral testing was carried out prior to surgery to establish a baseline for comparison to postsurgical values, as described in detail previously (5, 11). The procedure of chronic constriction injury (CCI) to the sciatic nerve was modified for mice from a procedure described previously for rats (3). In brief, adult male mice (90 to 120 g) were anaesthetized with sodium pentobarbital (Sagatal; Rhône Merieux, Essex, United Kingdom) (0.06 ml/100 g, intraperitoneally) supplemented with halothane-O2 (Zeneca, Cheshire, United Kingdom). Under aseptic conditions, the right sciatic nerve was exposed proximal to the trifurcation, at mid-thigh level, and three chromic cat gut ligatures were tied to loosely constrict the nerve. The overlying muscle and skin were closed with sutures, and the animals were allowed to recover before reflex testing recommenced. Thermal hyperalgesia was monitored by using noxious radiant heat (30 to 55°C) (Hargreaves' thermal device; Linton Instruments, Diss, United Kingdom) applied to the mid-plantar glabrous surface of the hind paw. The withdrawal response latency was characterized as a brief paw flick, and a standard cutoff latency of 20 s prevented tissue damage. Mechanical allodynia was measured as the threshold for paw withdrawal in response to graded mechanical stimuli applied to the mid-plantar glabrous surface of the hind paw with calibrated von Frey filaments (Stoelting, Wood Dale, Ill.). The threshold was defined as the pressure (force per unit area) that caused foot withdrawal five times in every 10 applications, repeated at 1- to 2-s intervals. The pressure applied to the hind limb by the von Frey filaments is calibrated as force (millinewtons) divided by the area over which it is applied (square millimeters).
Generation of γ-synuclein null mutant mice.Mouse strains with a targeted deletion of the first three exons of the mouse γ-synuclein gene (Fig. 1) were generated from three independent ES cell clones as described in Materials and Methods. Two of these strains were used in this study, and because the results were identical, the experimental data obtained with both strains were combined. Mice lacking the γ-synuclein gene were viable and fertile and did not show any obvious abnormalities in development, behavior, or gross morphology of the nervous system (data not shown). Western blotting (Fig. 1e) and immunohistochemistry (not shown) with an antibody specific to the C-terminal peptide of mouse γ-synuclein showed a complete absence of the protein in all tissues of null mutant mice. Northern hybridization of RNAs from various tissues confirmed complete inactivation of γ-synuclein gene function in null mutant mice, which was not accompanied by compensatory increases of α- or β-synuclein mRNA levels (Fig. 1d and data not shown).
In the absence of an obvious phenotypic manifestation of the null mutant genotype in γ-synuclein knockout mice, we carried out comparative studies of several neuronal populations with wild-type and null mutant mice. Motoneurons and peripheral sensory neurons were chosen because both populations normally express high levels of γ-synuclein mRNA (7) but, as shown below, display different intracellular compartmentalization of γ-synuclein.
Developmental changes of γ-synuclein compartmentalization in motoneurons.Previously we have demonstrated by in situ hybridization high levels of γ-synuclein mRNA expression in spinal and cranial motoneurons from early stages of mouse and rat embryonic development (7). However, neither the abundance nor the intracellular localization of γ-synuclein protein in these neurons has been studied. Using a specific antibody which recognizes the C-terminal peptide of mouse γ-synuclein, we now demonstrated that the protein expression pattern follows the mRNA expression pattern but that dramatic changes in compartmentalization of γ-synuclein within motoneurons take place during postnatal development. Immunohistochemistry was performed on sagittal as well as coronal paraffin sections of mouse embryos of different developmental stages and adult mouse brains. To identify oculomotor, trochlear, facial, hypoglossal, and trigeminal motor nuclei, adjacent sections were stained with hematoxylin-eosin and an antibody against choline acetyltransferase, a motoneuron marker (see Materials and Methods). From the early stages (E12) and throughout the embryonic development, γ-synuclein is localized in both motoneuron cell bodies and axons (Fig. 2 and data not shown). However, at E18, in addition to cytoplasmic staining, a dotted neuropil staining became evident in all studied motor nuclei (Fig. 2c to g). The pattern is completely different in motor nuclei of postnatal, particularly adult, mouse brain. At postnatal day 2 (P2), only neuropil staining (Fig. 3c) or very few neurons with γ-synuclein-positive cytoplasm on the background of neuropil staining (Fig. 3d) are evident in these nuclei. In adult brain all motor nuclei (with the exception of the facial nucleus, where a few neurons still display positive cytoplasmic staining) show a complete absence of γ-synuclein in the cytoplasm of neuronal cell bodies, but axons in all studied motor nerve roots display intense γ-synuclein immunoreactivity (Fig. 4). Using immunofluorescence, we clearly demonstrated that γ-synuclein is present not only in axons but also in presynaptic terminals of motoneurons in neuromuscular junctions (Fig. 4A).
Intracellular localization of γ-synuclein in mouse sensory neurons.As in motoneurons, in sensory neurons of embryonic peripheral ganglia γ-synuclein is distributed throughout the cytoplasm of cell bodies and axons (Fig. 2 and data not shown). However, in contrast to the case for motoneurons, γ-synuclein compartmentalization in sensory neurons does not change during late embryonic and postnatal development. Postnatal neurons of DRG and cranial sensory ganglia display intensive immunostaining of their cell bodies as well as nerve fibers with anti-γ-synuclein antibody (Fig. 3a and b and data not shown).
Numbers of motoneurons in cranial nuclei of wild-type and γ-synuclein null mutant mice.Serial coronal sections of adult mouse brain were prepared and stained, and motoneurons were counted in five cranial nuclei as described in Materials and Methods. In neither of these ganglia were statistically significant differences in the number of neurons between wild-type and γ-synuclein null mutant mice found (Fig. 5).
Numbers of sensory neurons in DRG and trigeminal ganglia of wild-type and null mutant mice.The number of neurons in L6 lumbar DRG and trigeminal ganglia was assessed in P2 mice, after the period of physiological cell death in these ganglia. Serial longitudinal sections of the lumbar part of the spinal column and transverse sections of the head were stained and neurons were counted as described in Materials and Methods. Figure 6a and b show that the absence of γ-synuclein does not have an effect on the number of neurons in both sensory ganglia. Similarly, no effect of the absence of α-synuclein on the neuronal complement in these ganglia was found. Mice with a targeted inactivation of the α-synuclein gene have been described previously (1), but for this work they were further backcrossed with C57BL6J mice (as described in Materials and Methods) to obtain a colony with a genetic background similar to that of the γ-synuclein null mutant mice.
Numbers of nerve fibers in saphenous nerves of wild-type and null mutant mice.To determine whether the absence of synucleins affected growth and myelinization of axons of sensory neurons, we examined the mouse saphenous nerve, which contains mostly afferent sensory nerve fibers, and counted the numbers of myelinated A-fibers and unmyelinated C-fibers. No differences in the general ultrastructural morphology of the fibers (not shown) or in their numbers (Fig. 6c and d) in γ-synuclein and α-synuclein null mutant mice compared to the wild-type mice were found.
Survival in culture of peripheral nervous system neurons of wild-type and null mutant mice.Although no differences in neuron numbers in DRG or trigeminal ganglia of wild-type, γ-synuclein, and α-synuclein null mutant mice were found, it was feasible to check whether neurons with an incorrect balance of synucleins are more sensitive to various stresses than wild-type neurons. For this we compared the survival of neurons from peripheral ganglia of null mutant and wild-type mice in dissociated primary cultures. Dissociated cultures of P2 trigeminal ganglion neurons were prepared and neuronal survival was assessed as described previously (42). Survival characteristics of γ-synuclein- and α-synuclein-deficient neurons were indistinguishable from those of wild-type neurons; they survived similarly well in the presence of nerve growth factor and failed to survive in its absence (Fig. 7a). Similar results were obtained for P2 superior cervical ganglion neurons (data not shown). It has been shown previously that certain types of cells with modified expression of synucleins, particularly dopaminergic neurons overexpressing α-synuclein, have normal survival characteristics under optimal culture conditions but are substantially more susceptible to stresses (37, 38, 41, 50, 51). To determine whether the absence of γ-synuclein renders neurons more sensitive to various toxic insults, we cultivated P2 trigeminal neurons in the absence of antioxidants or in the presence of DNA-damaging agents (Fig. 7c), proteasome inhibitors, heavy metal ions (Fig. 7b), and inhibitors of major intracellular signaling pathways (Fig. 7d) in the medium. None of these treatments revealed differences in the survival of γ-synuclein-deficient neurons compared to wild-type neurons.
Sensory reflexes and effects of CCI in wild-type and γ-synuclein null mutant mice.The behavioral reflex responses of adult male γ-synuclein null mutant and wild-type littermate mice to noxious radiant heat and graded mechanical stimuli were studied as described in Materials and Methods. In both tests the behaviors of mice from these two groups were indistinguishable (Fig. 8). To examine the effects of sciatic nerve injury on behavioral reflex responses, we used the CCI model of neuropathic pain. Over 7 to 10 days following CCI, wild-type and mutant mice progressively developed marked ipsilateral thermal hyperalgesia (reduced paw withdrawal latency) (Fig. 8a and b) and mechanical allodynia (reduced paw withdrawal threshold) (Fig. 8c and d). All responses from the contralateral hind limb remained unaltered. The time courses of recovery from the consequences of CCI were very similar for mice of both genotypes (Fig. 8). These results suggest that degeneration and regeneration of the injured nerve and neuronal plasticity in the spinal cord are not compromised in γ-synuclein null mutant mice.
We have shown that in embryonic motoneurons γ-synuclein is uniformly distributed through the cytoplasm of cell bodies and axons. However, with the exception of a few neurons in the facial nucleus, the cytoplasm of cell bodies of motoneurons of the adult cranial somato- and branchiomotor nuclei is γ-synuclein negative, whereas their axons and synaptic boutons in neuromuscular junctions are intensively stained with anti-γ-synuclein antibody. Previously published in situ hybridization data demonstrated high levels of γ-synuclein mRNA in cranial motor nuclei of adult brain (7). Therefore, in motoneurons during postnatal development, γ-synuclein undergoes a compartmentalization shift, which might reflect a functional shift. Our results contradict recently published data (32) which demonstrated the presence of γ-synuclein in cell bodies of motoneurons in brain stem motor nuclei of adult rats. This might reflect a difference between species; however, a more plausible explanation is the difference in the antibodies used in the two studies. In the present study, a highly specific antibody generated against mouse γ-synuclein C-terminal peptide was used, whereas Li et al. (32) used an antibody generated against human recombinant γ-synuclein whose specificity has been checked with recombinant human synucleins but not with samples from null mutant animals.
It is not clear why some neurons of the adult facial nucleus do not follow the common rule and continue to accumulate γ-synuclein in the cytoplasm of their cell bodies. In this aspect they resemble peripheral sensory neurons, in which no developmental changes of intracellular compartmentalization of γ-synuclein take place and which have equally high levels of γ-synuclein in their cell bodies and processes during embryogenesis and postnatally. We found that both neural crest-derived DRG and placode-derived trigeminal sensory neurons have this unchanging pattern of γ-synuclein intracellular compartmentalization. Taken together, our expression studies demonstrated that between neurons expressing the highest levels of γ-synuclein throughout development, two subpopulations could be specified. The first includes peripheral sensory neurons and some motoneurons of the facial nucleus, which localize γ-synuclein in their cell bodies and axons at all developmental stages. Most other motoneurons of the brain stem nuclei comprise the second group, which is characterized by the developmental shift of γ-synuclein compartmentalization.
The high levels of expression suggest that γ-synuclein should have an important role in the development and function of sensory and motoneurons. Consequently, the loss of this protein could affect the morphology and/or physiology of animal sensory and motor systems. To check this, we produced mutant mice with complete inactivation of the γ-synuclein gene. Similarly to previously reported α-synuclein null mutants (1, 8, 46, 48), these mice showed no obvious phenotypical changes. Detailed studies of sensory and motoneurons in vivo and in vitro failed to detect any difference between γ-synuclein null mutant and wild-type mice. The number of neurons is not changed in either of the subpopulations described above, suggesting that proliferation, migration, differentiation, or programmed cell death is not affected by the absence of γ-synuclein. Consistently, survival of γ-synuclein-deficient neurons in primary culture is not different from survival of wild-type neurons. Moreover, the absence of γ-synuclein does not render these neurons either more or less sensitive to any of the survival-affecting factors studied so far. Because mid-brain dopaminergic neurons seem to be most vulnerable to changes of synuclein metabolism (references 10, 28, and 33 and references therein), it is feasible that this neuronal population might be more sensitive to changes in the synuclein ratio than motoneurons and sensory neurons. We are currently testing whether the absence of γ-synuclein affects survival of dopaminergic neurons in the substantia nigra and ventral tegmental area of null mutant mice.
It has been suggested previously that γ-synuclein could be involved in axonal growth and stabilization of axon architecture (6). However, we did not find differences in the morphology and number of myelinated or unmyelinated fibers in the saphenous nerves of mutant and wild-type mice. Sensory reflex thresholds were also intact in γ-synuclein null mutant mice. Nerve injury led to similar changes in sensory function in wild-type and mutant mice. Normalization of sensory function after nerve injury is believed to be associated with neuronal plasticity in the spinal cord and axonal regeneration processes in the injured peripheral nerve (9, 14, 21, 58). These processes require remodeling of the axonal cytoskeleton, including the neurofilament network, and involvement of γ-synuclein in regulation of neurofilament network integrity has been suggested previously (6). However, the time course of sensory recovery after CCI in γ-synuclein null mutant mice is the same as that in wild-type mice, which suggested that nerve regeneration and plasticity of early somatosensory pathways in γ-synuclein mutant mice were unaffected.
The most straightforward explanation of our results is that despite high levels of expression, γ-synuclein is not essential for the development and function of motor and peripheral sensory neurons. Nevertheless an alternative explanation is also possible. This function(s) could be vital for vertebrate organisms, and therefore effective mechanisms of protection against its loss have been developed in evolution. The presence of three closely related synucleins in all vertebrates and substantial overlapping of their expression patterns readily suggest that they are able to compensate for each other's function(s). It is unlikely that compensation for the loss of one synuclein function in mice is achieved by a simple increase of expression of other synucleins. We found no difference in the levels of mRNAs encoding the two remaining synucleins in several neuronal populations of γ-synuclein null mutant mice, and the same has been demonstrated before for α-synuclein null mutant mice (1). However, it is possible that it is not necessary to boost an already high level of synuclein expression, because changes in compartmentalization, posttranslational modifications, or interaction with other macromolecules could be required and sufficient for functional compensation. Detailed studies of these processes in synuclein null mutant mice as well as studies of double and triple synuclein mutants should shed more light on this problem and finally reveal the normal functions of these proteins.
We are grateful to A. Smith and J. Ure for ES cells, help with the initial stages of generation of mutant mice, and valuable advice and to A. Oliver, S. Mitchell, and J. Wanless for excellent technical assistance.
This work was supported by grants from The Wellcome Trust.
- Received 9 June 2003.
- Returned for modification 18 July 2003.
- Accepted 14 August 2003.
- Copyright © 2003 American Society for Microbiology


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With more than 10.4 million albums sold during his career, David Guetta is undoubtedly one of the most famous and globally recognized DJs. And the acclaimed French artist not only has an impressive sales record, but is considered the best in the world by DJ Magazine as well as EDM mega site, The DJ List.
The producer-songwriter is well-known for collaborating too, having worked with the likes of Madonna, Lady Gaga, Britney Spears and Rihanna. But Guetta hasn't always had it easy. Before his success, he began his career playing in local clubs in Paris before eventually becoming a producer and artist who has recorded No. 1 positions on lists such as Billboard Hot 100.
In fact, in 2010 he was awarded a Grammy for his album "One Love," which contains one of his biggest hits, "When Love Takes Over," alongside Kelly Rowland. He also won a Grammy in 2011 for his song "Revolver." His fame continues to rise like foam and he is now the most followed artist in the world on Spotify, the most searched on Shazam and has 56 million likes on Facebook.
What has been the key to his success?
"I do not think there has been only one key, but becoming a producer was definitely an important factor. There is a combination of having great recordings full of energy and excitement, and at the same time, what I create as a DJ and the kind of music I produce," says Guetta.
For him, the most important thing is to make his audience dance, while making them feel a balance of emotions and energy through electronic music. But no less important to him is to enjoy every second of what he does.
"That's what it is all about, because when I decided to become a DJ, there was no money or fame in this line of work," he explains. "I was ready to risk and sacrifice my life just because I wanted to do something I really enjoyed and I've been lucky because it became huge. To be willing to take those risks, you have to work with passion and give your 100 percent."
His 2014 album, "Listen," placed No. 1 on iTunes in 75 countries, and with his late 2015 follow-up, "Listen Again," he continues to make millions of fans and listeners around the world move.
"For me it is about making people dance but also about giving them a thrill," he says. "I want people dancing and having a wonderful time, but also to have an emotional impact and I think electronic music is probably the only music that does those things."

subset: commoncrawl


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How to install Red Hat OpenShift 3.11 on OpenStack 13
Red Hat OpenShift Container Platform is a platform-as-a-service (PaaS). It orchestrates and manages containerized applications through Kubernetes. Although OpenShift Container Platform supports cloud-native applications, it also supports custom-built applications. OpenShift Container Platform can run on a hybrid cloud configuration providing the flexibility to expand and grow.
Red Hat OpenStack Platform is an infrastructure-as-a-service (IaaS). This means it is a cloud-based platform that provides virtual servers and other resources. Users either manage it through a web-based dashboard, through command-line tools, or through RESTful web services.
If you are considering Red Hat OpenShift Container Platform on OpenStack Platform, there are several advantages, including easily increasing the number of compute nodes and using dynamic storage.
In this article, I will outline the main points required to successfully install Red Hat OpenShift Container Platform on OpenStack Platform. Because my OpenStack knowledge is limited, I reached out to my colleagues for help and will not address too many OpenStack technical details here.
Continue reading "How to install Red Hat OpenShift 3.11 on OpenStack 13"


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Where does lightening come from, clouds, ground or both?Asked by: Jason
AnswerFirst of all, to be vague, it comes from both but starts in the cloud.
A combination of air currents and moving ice and water particles in the cloud causes positive and negative charges to separate. Put simply, the positive charges gather near the top of the cloud and the negative charges gather near the base.
Once an appreciable negative charge has built up on the bottom of the cloud, a 'shadow' is created on the ground below where a positive charge is induced. The conditions are now right for a lightning strike.
Once the charge on the cloud is big enough, a small path of negative charge makes its way towards the ground. Once it gets close, a path of positive charge leaves the ground and heads up to meet it.
Answered by: Corey Taylor, Undergrad, Florida Institute of Technology


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Memory foam mattresses are understood for their high density. They are also more support than an ordinary mattress. They are made from really thick strong viscoelastic memory foam and hence provide the bearer of a firm mattress and the comfort of a very soft mattress. Both the memory foam mattresses and mattress pads are made to entirely break down under the weight of the body. The foam mattress pads include cells of the holes to assist with the circulation of atmospheric pressure on the surrounding cells. This discusses their ability to be molded in the shape of your body as a response to the heat and the weight of your body. For that reason, they are highly efficient in distributing body weight evenly over the mattress or mattress pad. So, with memory foam mattress pads can be valuable, specifically in the case ofBack discomfort and arthritis. This question is a little controversial, as lots of have actually declared affirmed that they discovered no such favorable effects.
Measure the size of your bedroom before you put order for bed furniture and other add-ons. Ensure that after putting the bed and other things you will have some living space for relocating. In case you are having a huge bedroom then think about buying additional furniture like dressing table and cupboards as well as a reading table with light. Look for good quality pieces rather of low-cost ones since it is going to be one time financial investment which would be lasting and long lasting. You need to pay even more if you are picking more stylish furniture than comfortable ones. If you are trying to find high quality, stylish bedroom furniture at discount prices then our site is the right location. Below you will find great worths on magnificently designed, well-crafted bedroom sets.
What Every Person Ought To Learn About Sofas
This is not the case when you utilize a natural latex mattress due to the fact that its much more durable and long-term. Plus, its performance will not decrease for a minimum of three years from the date of purchase. If its raw product is pure natural rubber rather of synthetic ones, it will take longer than three years prior to you experience any wear and tear. The argument on which kind of mattress is better is a continuing one. Latex mattress individuals and visco-elastic supporters both have legitimate and sensible arguments. There are individuals who like all the advantages that latex mattresses provide whereas some prefer the sleeping condition that visco-elastic ones provide. In the end, its all a matter of individual inclination due to the fact that each specific differs in tastes and dispositions. If you're getting weary of your aged spring-coiled mattresses, then its time to provide the latex or visco-elastic mattresses a try.
A large well built option can be quite costly, while on the other hand a trendier space saving design could be less expensive and better matched to modern houses. These are leather recliners with a push back design when you lean back, while the bulkier designs will have a lever for the same operation. For those who love everything luxurious, the very best recliners to buy are those that come as speciality chairs and have a massager too in the backrest along with footrest. Though costly, these are a blessing.
You will certainly discover that microfiber reclining sofas can be found in lots of designs. You can get a standard one that is long or brief. You can get one that curves on the sides too so that you can get the most out of the corners in your space. Checking out the different looks provides you a chance to select the one you like the most. You can also compare rates to assist you get the one you desire at the least quantity of cost.
Compelling Types Of Rattan Sofa Sale
Foam bed mattress pads of natural latex are hypo-allergenic, and supply relief from dust mires. Foam bed mattress pads of Talalay latex are offered in differing degrees of firmness; all latex is biodegradable. The higher the density of a foam bed mattress pad, the longer it will certainly take to return to its routine shape. A quality foam bed mattress pad will certainly set you back from between $150 and $300, which, while is not as much as a foam bed mattress, can be a considerable financial investment. So unless your existing bed mattress still has some years of use ahead of it, you would be better off replacing it completely. You can do some Internet research to obtain an idea of exactly what foam bed mattress pads are offered where price varieties; however as with all things, you will certainly get exactly what you pay for.
Another innovative way of utilizing ottoman is by utilizing it to go shopping crucial documents, money, fashion jewelry, and valuables for sake keeping. So God forbid that your house is gotten into, intruders will unlikely appear into your ottoman for valuables and earnings. Your products are secure and sound in case you utilize ottomans as a storage area. Furniture of style is pleased to offer a massive range of top quality beds at incredibly low cost.
With the calming impacts that Asian inspired furniture designs can offer, increasingly more consumers are turning to Asian design contemporary beds in an effort to produce a level of calming Zen in their houses. Because the styles can differ from the minimalist to the complex, anyone can discover the perfect Asian inspired design to meet their requirements so they can experience its calming impacts for many years to come.
You'll likewise have to think of the convenience level you anticipate from the lounge. Sofa beds may be really strong and also exceptionally fluffy. The rear side could be firm and even set up with totally detachable cushions. Generally, folks choose the pillowed sofa beds since they are typically really relaxing and comfy. Other stuff to keep in mind would be the position at which the back of the lounge is kept and the depth belonging to the seat of the couch. Some individuals really like sofas that are fitted with seat designs weighing down on every sitting although some need a stiff lounge seat. Need to you still seriously feel baffled within the house furnishings retail outlet, under no conditions think twice to ask about aid from the salespersons.
Lazy Boy leather recliners are among the best liked furniture pieces. It is a financial investment to our convenience for that reason one should identified how to meticulously look after your Lazy Boy leather recliner to make it last for a long time. Having a Lazy Boy in the house can give you the benefit and the convenience at your own location. You don't need to go out simply to enjoy a seat on a Lazy Boy recliner made of leather. It will certainly be the favorite furnishings in the home. It one of those things that we purchase to reward ourselves after a long day of hard work and anxiety. Many people have feedbacks about the satisfaction they experienced in their Lazy Boy leather recliners. It is among the furnishings that we take a great deal of care. One should not take it for granted or else the next thing you identified, you recliner will certainly end up in the junkyard.
Wonderful Tips About Sofas From Unlikely Sources
Having a couch bed implies you do not have to devote that area as a bedroom and lose any dual functionality the space could have. Because of the ergonomic designs used by the makers of sofa beds, modern-day models can go with virtually any living area. Rather of having to tuck it away in a back space just to transport it out when needed, contemporary sofa beds are readily available in a range of designs and materials ranging from rustic amenities to the sheer luxury offered by a leather sofa.
Weave of the textile figures out the sheets level of softness, crispness, breathability, and toughness. Sateen weave produces a very soft but less durable product than percale or identify. Pinpoint weave is durable and not as soft as sateen, but will soften with frequent washings. Percale weave produces a sturdy and durable bed sheet. Percale sheets are breathable and have a crisp feel that will soften with repeated washings. Twill bed linen drape perfectly and are resistant to wrinkles. Jacquard is a weave with a pattern or tapestry design on the face of the textile.
Think Your Sofas Are Tacky? 5 Things To Change Right Now
Embellishing the kitchen, washroom, or living space is something, however when it concerns embellishing a child's space it can prove to be a lot more challenging. After all, it appears like every other day a child is into the following "in thing" so choosing a design that he/she will be pleased with for even more than just a couple of weeks can be a bit of a task! Rearranging rattan sofa sale is also a very simple method to alter the appearance of a room that can have a huge impact. If a child is bored of the method their space looks it is extremely simple to move the bed, closet, and cabinets around to offer it a fresh feel. If you thinking of utilizing rugs you can also think about removing the old carpet and leaving the wooden floorboards in location, as opposed to investing out on a brand-new carpet.
We have the tendency to be very hectic all day, and deserve to rest with our feet up. This is an excellent way to treat yourself. It likewise enables your body the opportunity to revitalize while you hang out. Many individuals are on their feet all day for work so this will assist them to be prepared for the next shift. Many individuals that struggle with a range of clinical troubles will likewise discover that this type of reclining sofa can assist them with flow.
Bedroom sets are offered in a great number of designs, colors, and patterns. For a striking and elegant result, you can pick soft bedroom sets and suit them with vibrant furnishings like attractively-colored bed linen and curtains. Bedroom arrives white or a pale color would stick out when put against an accent wall in deep purple or lime green or contrasted with table lamps in similar tones. For bigger spaces, visually extending the headboard with lively artwork or intriguing architectural details can work wonders for your bedroom space. Upholstered headboards assist produce an elegant and welcoming ambiance. Select brightly-colored or richly-embroidered material for your headboards. Bold shapes can likewise assist produce character in the room. Select your bedroom sets and headboards with care to keep the room open and large and make a traditional style statement.
There could have never ever been a better time to buy brand-new furnishings and in certain brand-new leather sofas. Leather sofas are constantly a popular product but many times shoppers don't believe that they are within their price range therefore this has the tendency to frighten shoppers far from making this kind of purchase. With the current decrease in costs of luxury leather sofas now might be the ideal time to choose to progress and get that beautiful leather corner sofa. Despite whether your interior is much better fit for the black leather sofa or maybe the old trusty brown leather sofa now might be the very best time in years to purchase it.
The bedroom is the location which offers max convenience and leisure because we all require a great rest after a day's work. If you are newlywed couples setting your very own apartment or if you are a household who has just moved to brand-new location, you require to purchase bedroom furniture as well together with the huge list of information. There are great deals of factors to consider while picking bedroom sets for your personal. First learn how huge is your bedroom and whether it can accommodate a queen size bed with headboard, as well as have living area for walking around. Typically a bedroom consists of a big sized bed, one or two cabinets with drawers, and a dressing table. A lot of ranges of furniture are readily available in the store which is made of wood and metals. In the wood itself, you have different types of woods ranging from inexpensive wood to most expensive teakwood. Instead of buying all the furniture independently you can reserve a set at one purchase conserving money on deliverables.
Several Strategies To Master Sofas Without Breaking A Sweat
Little sofas are going to appear definitely tiny on the furnishings showroom floor but you do not stay in a furniture showroom so that furniture piece is going to appear larger in your house than it does in the showroom. If your brand-new corner sofa is planned for a huge room then there is no trouble with getting a huge corner sofa. This might even be the preferred selection if you wish to use the sofa in such a manner that it areas off an area of the room to provide it a more intimate setting. I recommend buying the best furniture piece that you can manage without breaking the bank.
Modern Sofas can be divided into 5 categories but there are lots of designs and patterns which you can choose from according to your needs. Mostly positioned in living room to entertain visitors, this can sit about 3 or more individuals comfortably. It is about 6 to eight feet long and consumes much area. If you are purchasing a regular sofa then make certain that you living room is roomy enough to accommodate this large furnishings.
The color scheme of your paint works must not be neglected, because your kids bedroom sets must blend well with it. Most likely not, which is why you desire to attempt to get it right, right from the start. Now, if you desire to ensure that you are getting the finest worth for your cash and that you are getting to pick from the greatest choice, you are going to desire to go shopping around online.
For children's spaces that are lacking in available floor space, a tall or vertical dresser is the functional choice. These dressers are generally 4 feet high at a minimum and contain each one of the storage space that a typical person would demand. Relying on the real drawer dimensions and width of these room dressers it is possible to find styles with anywhere from 6 to 8 drawers. A perfect discuss many tall room dressers is to obtain shiny brass hardware and manages. These added alternatives touches can make an otherwise plain room dressers really make a space have a touch of course.
Best Ways To Sell Rattan Sofa Sale
So, some things to consider when searching for a brand-new recliner. Whatever sort of back support does the recliner have? Does it have an excellent reputation by both users and physician? If so you should absolutely consider it as a possibility if it provides quality back support. Does it provide great neck support? Is it the type of chair that will leave you with a stiff neck or does it provide you head and neck great support? Leather or fabric is another choice you ll have to choose. Each have their advantages and is a selection just you can make. I hope these recliner shopping pointers will assist you find that perfect recliner that you ve constantly wanted.
Probably you awakened this morning with discomfort on your shoulders or back. Numerous understood futon producers originate from Japan. Nevertheless, since of the demand for futons, numerous other rattan sofa sale producers in different places have likewise turned to producing high quality futons. A futon sofa bed is actually a fantastic investment, as it has a dual function. It can be converted into a sofa and a bed. Thus, it is wonderful for use in the house and even in offices in case of late-night work. Because of this feature, futon sofa beds might likewise be the very best choice for houses with little spaces. This convenience and convenience is exactly what many people look for, and exactly what several futon business make every effort to give.
The 3 most popular and typically used types of recliners are the two-position recliner, the rocker or wall-saver, and the pushback or flex-back. The standard two position recliner is the most affordable and most basic of all types of recliners. Some of its areas are upholstered with authentic leather like the arm, seat base and leg. One can be purchased for less than a hundred dollars. Several parts such as the back are covered by vinyl, unlike others totally covered with leather. The normal warranty period for these recliners is simply one year. There are likewise couple of styles to choose from.
Slacker's Ebook To Real Sofas
A lot of Asian design contemporary beds are most generally made from rosewood, with the worlds most sought after manufacturers or professionals creating the most costly Asian design beds. These contemporary beds give off an air of richness that makes them stand out from basic bedroom designs. A lot of generally used to separate you from the floor, when they are used on the bed they are indicated to separate the mattress from the bed frame.
A leather couch is the very best furnishings for relaxation and resting at the end of the day. With just a little care and precision this remarkable piece of furniture will serve you for years of pleasure and fulfillment. A sofa is particularly made to support your lower back angled for full comfort and unwinding position while sitting whether it is at house or in the workplace. This sort of furnishings is readily available in lots of materials, but we will feature the advantages of leather furnishings. Leather is practically maintenance-free. A wet piece of cloth suffices to clean spots. Although, it's difficult to clean ink but is not impossible. Rubbing with a saddle soap for a minute suffices to clean difficult areas. Moreover, there are alternatives to repair pet scratches on your leather sofa. All in all, a leather couch is a perfect selection for your house.
The recliner chair is a cushioned chair that has a lumbar support. The recliner chair serves for individuals that have movement problem such as lame, and arthritis. Each recliner chair is equipped with distinct functions which accommodates the need of the clients. There are lots of types of recliner chair in the market, The recliner chair provides reclining positions for the user. The more reclining positions it can carry out, the more costly it is. Recliner chair than has 2 reclining position can be adjusted up to 45 degrees. They are the least expensive compare with other types of recliner chair. You can likewise customize the recliner chair into a various reclining angle.
There are likewise some great manual massage modes and the EP 1285 massage chair. You can pick from rolling, tapping, kneading as well as compression. Use these methods to massage your whole back or a part thereof. Another great function is the neck roller position. This massage chair enables you to set the height of the rollers. This is perfect for targeting precisely where you would like the shoulder and neck massage to concentrate.
While it prevails knowledge that the added deep fitted sheets are really comfortable, numerous individuals do not know why that is the case. Is it since they have such a high thread count? Is it since they are made from some elegant material? To be perfectly honest, the answer is a little of both. You require to make sure to select the right material in addition to a decent count of threads, though it doesn't have to be so high that it makes you spend more than you can afford to.
Can You Buy Used Sofas?
Search for something that blends well with the other pieces in your space. You may combine and match wood colors, but this isn't really the only aspect to take into consideration. The carving of the wood also makes a substantial distinction on its entire look. For example, there are dressers for sale which are simple and flat, but there are also pieces which have unique and elaborate carvings in them. Aim for a design, may it be sophisticated, modern, or contemporary and include it with the dresser that you wish to purchase. The dresser's height and mirror accessory are also elements of style. The taller pieces don't usually had a mirror, but can be more space-wise over the much shorter ones with a mirror. However if you wish to minimize your spending plan, dressers for sale with a mirror connected can be a great choice as you will not was required to was required to acquire a different mirror. The style of the drawer manages can also play a significant role in the dresser's entire style, so do not forget to have a great take a look at them when shopping.
There are lots of different types of cherry dressers on the market today. One can choose to have as few as four drawers or as lots of as ten. Relying on the type of storage wanted, possibly a closet style is required. The selection, naturally, depends upon the requirement in addition to the location included. The majority of bedroom suites include a bed, a mirrored dresser and a chest of drawers. Picture such a suite manufactured in a stunning cherry surface in a space especially embellished to complement its color. Finishing this off with the proper curtains will certainly bring the whole arrangement into stunning harmony. It may be possible that additional storage space is required. During that case it is possible to pick from other sizes available a selection that would complement the rest of the group.


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Enameling additions to The Silver Swann
Been a little while since I've blogged! I've been busy taking some classes with the Patuxent Lapidary Guild - wish I had more money and time to take everything I want to take NOW!
A more permanent expansion of The Silver Swann is the addition of ENAMEL! I've quickly fallen in loooove. For those who don't know what enamel is, it is glass that has been melted and fused to metal. It works perfectly with my designs and gives me a great opportunity to diversify my pieces and accomplish things I couldn't do with torch or patina finishes. PLUS enamel colors are predictable/consistent. As of right now I'm working with opaque colors on copper, but I am ordering transparent colors for my silver pieces.
Above I am working on earring sets with different copper cups.
The next two are colored pendants with several colors overlaid.
In-progress pictures of creating colored chevron necklaces.
A kiln I got to use at the guild that's around 200 years old!
I created pieces with PMC which will be for sale once I make the chains. They will be a limited line as I do not have resources to reasonably create pieces at this time consistently. Hopefully in the future I will be able to expand more permanently.


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This topic contains 1 reply, has 2 voices, and was last updated by Alexandr Smaga 6 years, 6 months ago.
I worked on the french translation in Crowdin of OroPlatform and OroCRM and I'd like to install it into OroCRM. How can I do it? By Akeneo, it's possible to install such packages into the folder app/Resources/, I did the same for OroCRM but it doesn't work.
I saw there is a process into OroCRM backend to install a translation package but this process bother me for two reasons 1) the downloaded package is not the up to date version from Crowdin 2) it is stored into app/cache, so once I want to deploy my code to a production server (it's not yet the case it will be done only when there is an official release).
Thank you for your answer.
The forum 'OroCRM – Installation/Technical Issues or Problems' is closed to new topics and replies.


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By Matt Kolberg, Application Specialist at SolidCAD
Have you ever saved and used Assembly Sets within the Intersection Wizard for saving your custom assemblies for future use? Have you ever had issues with them not appearing to work properly every time you use them? Have you ever wanted to share them with other team members?
This document will shed some light on why; and what you need to do to make them work every time.
Civil 3D intersections are complex corridors containing many assemblies, regions, and baselines. Creating and editing them without the Intersection Wizard takes skill, time, and patience. There are up to 8 assemblies required for a typical intersection and creating them is also time-consuming and prone to errors. The image below shows a corridor which contains just one intersection, complete with all of the baselines, regions, and assemblies applied.
Assembly Set Basics
If you're not familiar with them, Assembly Sets can be accessed and saved from within the Intersection Wizard when creating a new intersection or when Rebuilding Corridor Regions for an existing intersection. You simply create new assemblies or edit the ones that Civil 3D creates for you, and then export the set for use on future intersections, so you don't have to recreate the assemblies every time.
The Assembly Set is saved as an XML file initially stored in its default folder…
C:\ProgramData\Autodesk\C3D 2020\enu\Assemblies\Metric
The Problem
There are technical support cases where the user has indeed exported an Assembly Set, but the proper assemblies do not appear after the set has been selected. In the image below, the custom set was selected where all the assemblies were renamed to MPK… As you can see, it appears as if an out of the box set was selected; no MPK assemblies are listed.
The Solution
There are 4 different scenarios where Assembly Sets are used. One for each of the two corridor types, selected within the intersection wizard…
One for a T intersection…
And one for a cross intersection.
If you open the Assembly Set XML file with your internet browser, you will see the 4 scenarios listed and the assemblies applied to each scenario. In the second image below, note the AssemblyName is set to MPK Primary…
When you save the Assembly Set, only the current corridor type is exported, only 1 of the 4 scenarios. The next time you use the Assembly Set, if that corridor is of a different type, your custom assemblies will not be applied since that corridor type has not been exported.
To ensure your custom assemblies are used in all 4 scenarios, you must go through the intersection wizard 4 times, one for each scenario and export the Assembly Set for each of them.
- Create a cross intersection and specify All Crowns Maintained.
- Create a cross intersection and specify Primary Crown Maintained.
- Create a T intersection and specify All Crowns Maintained.
- Create a T intersection and specify Primary Crown Maintained.
Select the same XML file each time, the exported scenario will overwrite only the applicable section of the selected XML file.


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Liquid Glass Arts Hour Glass
Liquid Glass Arts did absolutely amazing work on this hourglass recycler the function on this piece is insanely effortless.
Liquid Glass Arts Biography
Liquid is originally from Boston, MA and is now living and working in Boulder, CO. He has been blowing glass since June of 2010. Liquid Glass Arts is always pushing the scientific glass game. Well known for their twisted scientific creations, you never know what kind of functional design Liquid Glass Arts is going to come up with next.
"In my spare time I like to blow glass, and try new projects with glass. Ha, only kidding, sort of. I'm a lot more introverted than I used to be so I do spend what seems to me to be an abnormal amount of time in the studio "working". My favorite thing to do in my off time is to backpack up in the Rockies with my girlfriend and our ridiculous canine companion named Alice." Liquid Glass Arts
What inspires you? "Nature, geometry, science, and fictional myths and legends inspire me the most. I value imagination slightly more than knowledge." – Liquid Glass Arts
Be sure to check out all of Liquid Glass Arts latest available work for purchase in our Online Store.
See Liquid Glass Arts history and progression here in our online gallery The Headies Collection.


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Caroline is determined to discover the truth at all cost.
Would you probe the secrets of an old house or let them rest in peace?
See how a woman finds something hidden from the world since World War II.
You will laugh, cry and feel her every emotion.
Author's note: this is my first novel inspired by research done on my own house.
Paperback ISBN: 9781784565084
Paperback: £5.99
* All services are subject to respective shipping companies terms of service. For more information see respective companies website.


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| Dean: how do we find him.
Castiel: With the boys powers, we can't. Not unless he wants to be found.
— Castiel and Dean Winchester in I Believe the Children Are Our Future
Supernatural Concealment is the power to hide oneself from anything, including angels, demons, etc. This can also be used to shield themselves (or their true forms) from creatures with Supernatural Perception (i.e., Edgar was able to mask his presence to take two angels by surprise).
Characters with this abilityEdit
- Primordial Entities
- God - God can only be found if he wants, he is in fact so powerful that even Amara or Death can't find him. While taking the form of Chuck, he was able to conceal himself from the Angels. Even the ones who saw him and would recognize his presence, such as Raphael. He was also able to conceal himself from the necklace that apparently burns hot in his presence.
- Amara - While God could hide himself from Amara, she also was able to hide herself from God. However, Donatello was able to sense her presence.[1]
- Gabriel - Gabriel lived among deities for centuries and concealed the fact that he was an archangel from them. After faking his death by Lucifer, Gabriel went onto hide himself from both God, Lucifer and Heaven for eight years.
- Lucifer - He was able to conceal himself from Amara.
- Angelic Beings
- Higher Beings
- Eve - Eve was able to conceal her presence from any angel, forcing the Winchesters, Castiel and Bobby to rely on a monster for her whereabouts.
- Leviathans - At least one high ranking Leviathan was able to conceal his true form from two angels which allowed him to take them by surprise to kill them. Normal angels, demons cannot distinguish Dick Roman and other Leviathans when they have a same appearance.
- Hybrids
- Nephilim
- Jack - When he decided he does not want to be found, Jack was subsconsciously able to block out the attempts of Lucifer, Asmodeus' and the Angel's to find him.
- Cambion
- Jesse Turner - According to Castiel, Jesse is hidden from angels, demons and humans unless he wants to be found.
- Nephilim
- Deities
- Baldur - He casted some sort of protection on Elysian Fields Hotel to make it untraceable to intruders.
- Demons
- Ruby - Using her knowledge of witchcraft, she produced hex bags capable of concealing herself and the Winchesters from angels and demons.
- Humans
- Witches
- Don Harding and Tracy Davis - Both were able to hide their presence from angels, as Castiel and Uriel couldn't find them on their own.
- Shamans
- Sergei - He implies that can conceal himself from anything. With his help, Gabriel went in his hiding during The Apocalypse.
- Hunters
- Sam Winchester - Ruby taught Sam her concealment spell.[2]
- Witches


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People need to feel safe and secure in their homes, protected from both intruders and the elements. Our composite doors are made of weatherproof, low maintenance materials and designed to be attractive as well as provide you with peace of mind.
Beautifully moulded with a richly grained grained exterior surface, you get the look and feel of traditional timber but without the environmental and maintenance disadvantages. A wide range of colours with wood grain our white frames, there's also a selection of beautifully crafted glass patterns to suit your own style and taste.
Composite doors have a solid core which provide a higher level of thermal and sound insuation compared to most doors.
A combination of a solid core, multipoint locking system, centre deadbolt and a full 72mm PVCu steel reinforced frame will foil even the most determined intruder.
The vacuum formed outer skin, bonded to its solid core, with fully sealed edges ensure that yur door will not flake, twist or warp. It will also never need painting!


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Heat Treatment And High Tensile (Strength) Fasteners Two Industry Standard Conditions
In order to promote structural adjustment and transformation and upgrading of the heat treatment industry and high-strength fasteners industry, the Ministry of Machinery has formulated the "Heat Treatment Industry Standard Conditions" and the "High-strength Fastener Industry Standard Conditions", which are now publicized. If opinions and suggestions are made, Please write comments during the public feedback to the Ministry of Industry and Information Technology Equipment Industry Division.
Ministry of Industry May 6 news, in order to promote heat treatment industry and high-strength fasteners industry restructuring and transformation and upgrading, the Ministry of Industry formulated the "heat treatment industry standard conditions" and "high-strength fasteners industry standard conditions", is to be publicized.
According to the conditions of publicity, heat treatment industry standard conditions, the new or expansion of heat treatment processing enterprises should have a production capacity greater than (inclusive) 10 million yuan / year output value of production capacity.
All heat treatment specialized processing plant set up must be resolutely eliminate backward production capacity, to speed up the "development of advanced technology, limiting obsolete technology, eliminating backward technology" as a guide. Promote the transformation and upgrading of enterprises to ensure safe production and strengthen energy conservation. Promote the development of low-carbon technology projects, the development of high-tech value-added heat treatment enterprises.
High-strength fasteners industry standard conditions, the layout of the fastener business and site should be determined in line with national industrial policies and relevant laws and regulations, in line with the provinces, autonomous regions and municipalities fastener industry and equipment manufacturing industry development planning.
New (expansion) construction of fasteners enterprises should be "approved by the construction project environmental impact assessment" and "occupational health and safety pre-assessment" and through the project environmental protection and occupational health and safety protection facilities "three simultaneous" acceptance.
High-strength fasteners industry standard conditions
Management system and product quality
(A) fastener enterprises should be in accordance with GB / T19001 a 2008 standard (for automotive products in accordance with ISO / TS16949, QS9000 or VDA6.1 standards) to establish and implement a quality management system certification, the system should also meet the GB / T 90.3 Request. Enterprises should be equipped with independent quality management and testing departments, with full-time quality testing staff, a sound quality management system.
(2) Fastener products mass-produced and supplied by enterprises must comply with the current valid national standards, industrial standards, international standards, foreign advanced standards or enterprise standards that have passed the filing. Built product inspection files and quality traceability system. Quality inspection standards and catalogs see Annexes I, II and III.
(C) product quality should be approved by the "Industrial Products Quality Control and Technology Evaluation Laboratory" by the accreditation (accreditation) and China Accreditation Board (CNAS) accredited third-party testing agencies issued inspection reports or within three years The above major customers to provide the use of the report. "" "" Unless "" Being "" "" GE GE Organism "" "" "" "" "" "" "" "" "" "" "" "" "" "" "" "" "" "" "" "" KM " Customer use report should have a detailed discussion and evaluation of product quality status, accompanied by the above-mentioned third-party testing agency issued inspection reports or data over the same period. The applicable testing items and standards shall be submitted by the accepting agency, be separately announced upon approval by the Ministry of Industry and Information Technology, and be changed as needed.
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Are you a book blogger? I'd love to interview you for my Behind the Blog feature. Make new friends and gain new followers!
I'd love to feature more new-to-me bloggers!
Acquired Books*
The Secret Diary of Lizzie Bennet by Bernie Su and Kate Rorick [library]
Reading Plans
I'm reading Little Women by Louisa May Alcott for the first time! I'm enjoying it but my copy is massive. It's almost 800 pages long. I'm moving through the book quickly enough but it doesn't feel like it because of how big the book is. Oh well, I'm enjoying my time with Meg, Jo, Beth, Amy and Laurie. I know I'll be very sad to leave them when the book is over.
After that, I'm going to read the Secret Diary of Lizzie Bennet next! Should be fun.
What did you get this past week? What are you planning on reading this week?
*I'm linking up to Stacking the Shelves hosted by Tynga's Reviews. I miss seeing all the good books people get! I'm sure my TBR list doesn't miss it though.


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Hamilton: Floor damage costing me a "good couple of tenths"
Lewis Hamilton says the extensive floor damage he suffered in the Mexican Grand Prix left him unable to attack as much as he wanted to and cost him several tenths per lap.
The Mercedes driver suffered a broken floor in a chaotic first lap at Mexico City, as the right side of his car was struck by Max Verstappen's left rear wheel as they battled for position through Turn 1.
Hamilton could not see the damage when driving, but having felt a big difference in the car's handling he made sure to check it out straight after the race.
Mercedes AMG F1 W10 floor comparison
Photo by: Motorsport Images
As can be seen in the above photograph, the damage is to the vertical slots alongside the edge of the floor, along with the diagonal ones.
These let the airflow moving over the top of the floor spill outwards to build a seal to the floor, stopping any dirty air from leaking in and disrupting diffuser performance. With that damage, one half of the diffuser would have been working harder than the other, meaning the aero balance would have shifted towards one side of the car.
Read Also:
Hamilton said that he had to adapt his car's settings to try to overcome the handling imbalance, but was never totally comfortable.
"I didn't actually know how much was missing or what was damaged, but the balance was quite a lot different," he said.
"The rear end was quite weak so I was particularly sliding around a lot in the high-speed, so I had to change my settings quite a lot and had to drive it a little bit differently because I couldn't attack the same way on the entry of corners because the rear stability wasn't the same.
"I'll probably find out later on just how much we were losing but I would imagine a good couple of tenths probably, just from rear floor damage. It's quite a sensitive area around where the tyres is."
The scale of the damage to Hamilton's floor in Mexico is much bigger than in Australia, when a missing element of the floor cost him performance that contributed to him failing to get on terms with teammate Valtteri Bottas.
Additional reporting by Scott Mitchell and Jake Boxall-Legge
Previous article
Vettel no fan of "shitty" F1 trophies or Mexico's "selfie guy"
Next article
How Hamilton went from "no hope" to victory in Mexico
About this article
|Formula 1
|Mexican GP
|Lewis Hamilton
|Jonathan Noble


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I saw this news story the other day and I knew immediately that it fell right into the wheelhouse of what I have been discussing on this site since I began: Government Accountability. It seems that an editor of the St. Petersburg Times decided he would research, document, and track every one of the 510 campaign promises that Barrack Obama made during his campaign for the Presidency. I initially thought, "yeah, right. Some newspaper will realistically track this…"
Some Thoughts and a Good Resource for All of You
Holding Barrack Obama Accountable
I saw this news story the other day and I knew immediately that it fell right into the wheelhouse of what I have been discussing on this site since I began: Government Accountability. It seems that an editor of the St. Petersburg Times decided he would research, document, and track every one of the 510 campaign promises that Barrack Obama made during his campaign for the Presidency. I initially thought, "yeah, right. Some newspaper will realistically track this…"
Where Do Republicans Go From Here?
I had some good comments on yesterday's post, but one commenter really struck me. They were angry, but wanted to have hope, and wondered "where do I go from here?" This struck me because so many people have asked me lately what the next step for the Republican Party is. They figure I am more politically active than most so I will have an answer. I don't have an answer offered by the Republican Party. But I do have an answer for where I intend to go from here. So I figured I would share it and hopefully help others sort their own feelings out as well.
Why I am Angry and How I Will Move Forward
Reflections on the Inauguration
So here we sit on Inauguration day. In a couple of hours we will swear in a new President and bid goodbye to George W. Bush as Commander in Chief. I do so with mixed feelings on the matter. I am sad to see George W. Bush go away. I liked who he was despite his flaws. But I am also looking forward to President Obama and learning about how he is going to change our country. So I thought I would share my thoughts as we move on to the next chapter in American history.
A Sad Goodbye and a Hopeful Hello
Is Obama's Economic Stimulus Plan a Good Idea?
The Office of the President Elect (a truly made up office for the record) has issued several statements over the last several weeks that clue us in to the intentions that the messiah has for fixing the economy. One of the most covered and almost universally accepted in the media is the trillion dollar plus economic stimulus plan that he intends to get through Congress within the first couple of weeks in office. The question is: Is this a good idea?
See USWeapon's Alternative Economic Recovery Plan
Should Burris be seated?
We are watching the petty bickering in the United States Senate over the embattled Illinois governor's selection to replace Barack Obama's Senate seat. Today they refused to allow him admission to the United States Senate. Harry Reid led the charge. So the question is, should he be seated or should he be barred from being the next Senator from Illinois?
Let's Look at Some More Childish Behavior by Our Democratic Senators
I Shouldn't be Subjected to Yankee's Fans, Either…
Today we see in the news that the Freedom From Religion Foundation is suing to keep Barack Obama from using the phrase "So Help Me God" during his official taking of the oath of office on January 20. Additionally the group is suing to keep Pastor Rick Warren from giving the invocation. According to the group, failure to eliminate these things is a violation of the separation of Church and State. Nut Jobs in America pushing themselves on the rest of us is what I say…
Why Aren't Christians Protected like Everyone Else?
Chicago Stinks… and We aren't talkin about the Cubs
I initially told myself that I wasn't going to write about this subject. "Too much of a layup" I said. "Too easy to take a shot at this" I told my friends. But see that is the beauty for a guy like me, who spent most of his youth getting rejected by basketball players far better than I. A layup is an awfully inviting thing. I mean it is a slam dunk in the box score, right? So I am taking the shot, and letting myself vent on not only sleazy Illinois Governor Blagojevich, but the entire Chicago machine, which I think once or twice has given us a national figure…..
The Chicago Machine is Back…and in Power?
Cabinet Positions are Too Tough to Keep up With!
I had a request tonight for a running update on the selections for the President Elect's Cabinet. Initially this looked like a good post topic, as I have had a lot of people calling me to ask about the people selected. While others have no idea who I am talking about when I mention one. However after some contemplation, I have decided to not do separate posts for these picks. They would simply be too long and update too often. I have instead decided to have a page up there at the top of the blog where the picks will be updated as they are announced along with my thoughts and analysis on each one. The page will be titled "Obama's Cabinet Picks". We can have discussions there around the picks and it will keep that page right up here near the top where it needs to be. So Check it out as it gets updated over the next couple of days and learn who these folks are that are being chosen to be in Obama's new Cabinet.
I Always Felt Farrakhan Would Bring Whites and Blacks Together Too
We all know "Calypso Louis" (yes he was a successful calypso singer until the Nation of Islam banned members from being musical performers). "Minister" Farrakhan made our hearts warm and fuzzy with his great hits "Kill Whitey", "Raise up Against the White Devil", and "Dirty Jews injected blacks with the AIDS virus". Such classic hits from a man who has lived a life hating anyone who isn't black and muslim. So he is the perfect choice for a new cabinet position for repairing race relations. OK, I jest. But I don't jest when I say one of the most partisan members of Congress is our next Chief of Staff. Rahm Emanuel is as partisan a Democrat as any I have seen. Let's meet the man, shall we?
[Read more…]


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|Review by a1Jim
|posted 12-18-2009 02:52 AM
|5077 views
|0 times favorited
|23 comments
- Charles Neil CASES AND BASES DVD SET
- Brand: Charles Neil | Category: DVDs
I received my long awaited DVD set "Cases And Bases" by Charles Neil. Charles is a one of a kind he's as real as it gets and as knowledgeable as anyone I've ever watched there Videos or read there books . I own most of Charles DVDs and have been more than pleased with every one I've ever purchased or seen on you tube.
This DVD set has a total of 8 disc. covering every thing you could imagine and more about many difference
styles of base and case work and techniques dealing with everything from kitchen cabinets to Bombe chest.
As a 20 year professional furniture maker and cabinet maker every time Charles would start a particular subject I would say to my self OH I know how to do that, WRONG! I may have done the that particular operation before but Charles always as a unique ,innovative and simplified way to do some of the most complicated and even the more simple pieces. As he would say"it just makes sense". If you want to learn more about building furniture This DVD set can't be beat . The only thing I didn't like about it is that it ended ,even though it's 600 minutes long( 9 hours) it seemed to just fly by.
-- https://www.artisticwoodstudio.com/videos
23 comments so far
#1 posted 12-18-2009 03:05 AM
Hey thank Jim I did not know he put out that set I have the finishing set and it looks and sound like this is a must.
#2 posted 12-18-2009 03:14 AM
Thanks for the review Jim. I have been mulling over buying this video set for awhile. 535 minutes is alot of air time. Cost?
#3 posted 12-18-2009 03:49 AM
Waiting on mine… looking forward to seeing what he has to say… a lot I imagine..
-- Don't drink and use power tools @ lasercreationsbylarry.com.au
#4 posted 12-18-2009 04:19 AM
Mine arrived last week. I'm just trying to find the time to view it; tough time of year to find free time for anything.
-- Failure is the road to success if you learn to learn from your mistakes - Vince, Greenville, SC
#5 posted 12-18-2009 04:30 AM
hey jim…i went to his site and looked at the dvd's available and its not listed….am i missing something..i would like to maybe purchase it…can ya let me know
#6 posted 12-18-2009 04:44 AM
Hey Grizz I don't think they have it posted yet just shoot them and e mail
#7 posted 12-18-2009 05:52 AM
Hey gang
#8 posted 12-18-2009 08:41 AM
-- Bob Kenosha Wi.
#9 posted 12-18-2009 02:05 PM
Thanks for the review Jim. I did not watch any charles Neil videos until i joined LJs, but you are right his methods seem very simple with excellent results. I am building a dining set right now and i am going to try and scoop the chairs like on his video i seen on you tube. Do you have his finishing dvd set and if so is it good?
-- Mert,Oshkosh WI,
#10 posted 12-18-2009 03:49 PM
Hijacked Charles Username – Sherri here.
When we release a new set to our subscribers at a pre-sale price, we wait for a bit before releasing retail giving us enough time to recover! LOL! Our plan is to release on our website the first week of 2010 and then to retailers about 6 weeks after that. But…...since members of LJ's are special too, Charles wanted me to pass along the pre-sale purchase price to you guys. The set is 8 discs and the retail is $89.95, special price is $69.95. Here is the link where you can purchase at the special price and I'll leave it up for you until we go live on our site at $89.95.
Click Here to Order 'Cases and Bases' DVD set for $69.95
Note – Just ignore the expiration date, the link to the cart is still active. Also, we do have several in stock but we are in the process of re-stocking but these sets will ship within 2-3 days of receipt of order,
On another note, in the next few days our DVD store at our web site will be changing formats making it easier to find all of our
#11 posted 12-19-2009 12:58 AM
Just want to say that, as usual, a1Jim has it right. My set arrived a week ago, and I have watched all but disk #8. I have a bunch of Charles Neil's DVD sets, have thoroughly enjoyed them all, and this one could be his best yet.
-- Gerry -- "I don't plan to ever really grow up ... I'm just going to learn how to act in public!"
#12 posted 12-19-2009 12:59 AM
Uh …. Jim?
Thank you for an excellent review, but …. this morning …. I was finally telling myself that I didn't really need anything else, and was done spending money for a while :-)
Now … I have Sherri's link open in my browser!! Uh-oh!!!
Particularly since reading is tough for me, I'm sure I'd get a TON of good info out of these vids. Thanks much!
-- -- Neil
#13 posted 12-19-2009 01:03 AM
Come on Neil go for it. Maybe Charles will come out with a DVD on how to control electrictions LOL
#14 posted 12-19-2009 01:04 AM
$ 89.95 is already a great deal for a wonderful brain dump – which I am SURE it is after seeing only one of his DVDs so far. $69.95 is a steal.
Jim, thanks for this great alert! Oh oh, I also have Sherri's link opened….
#15 posted 12-19-2009 01:09 AM
Hey mtkate
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New York City Children's Theater is currently running a Kickstarter campaign to help fund our Literature at Play workshops in homeless shelters as part of the After School Reading Club (ARC). But what is ARC?
This year, the Department of Education (DOE) is piloting Afterschool Reading Club (ARC). ARC is a new literacy enrichment program for elementary school students living in shelters. The program was piloted in 18 shelters in the Bronx, Brooklyn, Manhattan and Queens throughout the school year.
Afterschool Reading Club meets three days a week for 3 hours a day, and is taught by DOE teachers.
For 20 years, New York City Children's Theater has been dedicated to reaching all children in NYC with innovative theater programs that enhance their social, emotional, and educational gains. We work in over 70 public schools each year, serving approximately 15,000 students. We are honored to be chosen as a partner in this important program
Brooke Boertzel, Director of Education
The pilot program began in October and reached hundreds of students and their families within the first six weeks. Overall there are 1,400 students in grades K-5 living in these 18 shelters.
In addition to New York City Children's Theater, the DOE partnered with Studio in a School, The New York Public Library, Brooklyn Public Library and Queens Library for this program.
"As a longtime elementary school teacher and principal, I know how critical building an early foundation in literacy is – it's truly the foundation of equity and excellence in education. The Afterschool Reading Club pilot demonstrates our commitment to reaching every student and family and meeting their needs."
"We know that homeless families need more than a roof over their heads. That's why we are proud to partner with the Department of Education and others to promote literacy and provide other critical educational support for children living in shelter."
"The Afterschool Reading Club is welcome news for elementary school students living in temporary housing. It is crucial that we provide our city's most vulnerable children with sound early literary instruction. This important pilot demonstrates New York City's commitment to equipping all students with the knowledge they need to achieve academic excellence and promising career opportunities. I am pleased to support this exciting project."
"There are far reaching educational consequences when children are being raised in a shelter system that often falls short of providing the nurturing and encouraging environment they need to succeed. Genuine initiatives like– Afterschool Reading Club – are not only an essential part of effectively responding to the City's ongoing housing crisis, but reflects commitment to curtailing the crisis level chronic absenteeism of Bronx school children living in homeless shelters"
Support powerful arts programming for children in need.
Homeless Shelters
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