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Migration, early axonogenesis, and Reelin-dependent layer-forming behavior of early/posterior-born Purkinje cells in the developing mouse lateral cerebellum
© Miyata et al; licensee BioMed Central Ltd. 2010
Received: 3 April 2010
Accepted: 1 September 2010
Published: 1 September 2010
Cerebellar corticogenesis begins with the assembly of Purkinje cells into the Purkinje plate (PP) by embryonic day 14.5 (E14.5) in mice. Although the dependence of PP formation on the secreted protein Reelin is well known and a prevailing model suggests that Purkinje cells migrate along the 'radial glial' fibers connecting the ventricular and pial surfaces, it is not clear how Purkinje cells behave in response to Reelin to initiate the PP. Furthermore, it is not known what nascent Purkinje cells look like in vivo. When and how Purkinje cells start axonogenesis must also be elucidated.
We show that Purkinje cells generated on E10.5 in the posterior periventricular region of the lateral cerebellum migrate tangentially, after only transiently migrating radially, towards the anterior, exhibiting an elongated morphology consistent with axonogenesis at E12.5. After their somata reach the outer/dorsal region by E13.5, they change 'posture' by E14.5 through remodeling of non-axon (dendrite-like) processes and a switchback-like mode of somal movement towards a superficial Reelin-rich zone, while their axon-like fibers remain relatively deep, which demarcates the somata-packed portion as a plate. In reeler cerebella, the early born posterior lateral Purkinje cells are initially normal during migration with anteriorly extended axon-like fibers until E13.5, but then fail to form the PP due to lack of the posture-change step.
Previously unknown behaviors are revealed for a subset of Purkinje cells born early in the posteior lateral cerebellum: tangential migration; early axonogenesis; and Reelin-dependent reorientation initiating PP formation. This study provides a solid basis for further elucidation of Reelin's function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives overall brain morphogenesis.
The cerebellum plays an essential role in the coordination of posture and locomotion, head and eye movements, and a wide range of motor activities. These functions depend on the structural organization of the cerebellar cortex, in which the Purkinje cells receive input from multiple sources in the central nervous system either directly or via parallel fibers of the granule cells [1–3]. Purkinje cells are generated during the early embryonic period from the ventricular zone (VZ) facing the fourth ventricle [4, 5] and migrate outward towards the pial side to subsequently form a monolayer (Purkinje cell layer) during the early postnatal days [6–10]. Just superficial to the perinatal Purkinje cell layer, there is a transient layer called the external granular layer (EGL) consisting of both differentiating granule neurons and their precursor cells. EGL precursors require Sonic hedgehog, which is supplied by the underlying Purkinje cells, to expand the granule neuron population postnatally [11]. Thus, the arrangement of the Purkinje cells during embryonic development is a key histogenetic event and consequently determines the overall cerebellar volume, the foliation pattern, and the intensity of the Purkinje-granule association, a lifeline of the cerebellar circuitry.
How this arrangement of Purkinje cells is established is only partly understood. In the cerebellum of reeler mice, Purkinje cells cannot form a normal layer under the pial surface and instead are clustered near the deep nuclear (DN) neurons [12]. Reelin, an extracellular glycoprotein encoded by the reelin gene mutated in reeler mice [13–15], begins to be expressed near the pial surface on embryonic day 13.5 (E13.5) by prospective DN neurons [16–18]. At E13.5, these DN neurons are still superficially migrating towards the anterior side from the posterior end of the cerebellum, the rhombic lip (RL) [18–20]. One day later (at E14.5), the arrangement of Purkinje cells into a structure several cells thick (called the Purkinje plate (PP)) is observed in normal cerebella; in reeler cerebella, however, abnormal distribution of Purkinje cells (lack of the PP) is clearly seen [7, 21]. The PP is formed just beneath a Reelin-rich zone to which RL-derived cells are continuously supplied; DN neurons, the first producers of Reelin, are followed by EGL cells [16, 17]. This in vivo spatial relationship between the Reelin-rich zone and Purkinje cells has been reproduced in experimental manipulations of Reelin-producing zones by explant culture and in utero transplantation [22], suggesting that Reelin may somehow promote or instruct nascent Purkinje cells to take a position close to the Reelin-rich zone. However, our understanding of the cellular scenarios involved in the initiation of PP formation at E14.5 is very limited. Furthermore, it is not known what nascent Purkinje cells look like in vivo.
To elucidate PP formation, there are three specific issues, each of which should be addressed with single-cell resolution studies. First, although a prevailing model suggests that Purkinje cells migrate along the 'radial glial' fibers connecting the ventricular and pial surfaces [6, 10, 23–26], whether or not this model fits with the actual morphology and behavior of Purkinje cells in the formation of the PP should be verified. Second, the key cellular behaviors that initiate or fail to initiate the PP in the presence or absence of Reelin need to be established. Third, whether the migratory and positioning behavior of Purkinje cells is linked with their polarization towards circuit formation should be determined. The time frame and manner in which Purkinje cells begin axonogenesis should also be examined.
By using an adenovirus-mediated pulse-chase method [27, 28] in combination with recently established Purkinje-cell-specific markers [26, 29, 30] and slice culture [31], the present study focused on the developing mouse lateral cerebellum until E14.5 and unveiled three important behaviors of a subset of Purkinje cells born at E10.5 in the posterior region: tangential migration (at approximately E13.5); early axonogenesis (at approximately E12.5); and Reelin-dependent 'posture' change initiating PP formation (between E13.5 and E14.5).
Purkinje cells that initiate PP formation at E14.5 are born on E10.5 in a posterior VZ
Following in utero injection of adenovirus vectors carrying a membrane-targeted green fluorescent protein (GFP) derivative (lyn-Venus) [32, 33] at E10.5, E11.5, or E12.5, the position of cells double immunostained with anti-GFP and anti-Lhx1/5 antibodies was analyzed in normal cerebella from E12.5 to E16.5 (Additional file 1). While 37% of the total GFP+Lhx1/5+ cells obtained by injection at E10.5 and analyzed at E14.5 were found in a region corresponding to the PP, incorporation of later-born cells into the E14.5 PP was very limited (3% for E11.5-born cells) or not observed (E12.5-born cells), indicating that the Purkinje cells that initiate the PP at E14.5 are generated mainly at E10.5. Histograms for birthdate- and track time-dependent changes of the distance of GFP+Lhx1/5+ cells from the ventricular surface show a general tendency that cells born earlier were seen further from the ventricular surface (Figure 1E). However, not all E10.5-born cells quickly moved towards the pial side to be integrated into the PP; some were still in a deep, periventricular region at E15.5 and E16.5. This result, together with the Lhx1/5 immunoreactivity results, is consistent with the possibility that Purkinje cells born in the VZ of the posterior cerebellar region may be more quickly incorporated into the PP than anterior-born cells. This interpretation was further supported by the observation of individual GFP+Lhx1/5+ cells in normal cerebella at E12.5 and E13.5, as described below.
Early/posterior-born Purkinje cells migrate tangentially in the lateral cerebellar primordium
Purkinje cells begin axonogenesis at E12.5
Collectively, these results suggest that nascent Purkinje cells (not only posterior-born cells, but also more anterior-born ones) may start axonogenesis from E12.5 through the expression of NF and then tau. This timeline, axonogenesis from E12.5, is consistent with a recent suggestion by Joyner's group, using visualization of nascent Purkinje cells by a novel transgenic system (Pcp2-CreER-IRES-hAP), that axonogenesis may begin between E11 and E14 [35]. This interpretation for early axonogenesis is further supported by our observations towards E14.5, as described below.
Migration and axonogenesis are normal in reeler cerebella until E13.5
Purkinje cells form a plate by changing their 'posture' between E13.5 and E14.5 in a Reelin-dependent manner through remodeling of dendrite-like processes
Next, to examine how a Purkinje cell's orientation and polarization at the single cell level contribute to the overall structure of the PP, comparative immunostaining against NF, Corl2, and Lhx1/5 was carried out. In normal cerebella at E14.5, the PP was demarcated (that is, visible as a 'plate' near the pial surface) due to a bundle of NF+ fibers that ran below a group of Purkinje cell somata to form the PP (Figure 8F, G). This suggests that the change in orientation or 'posture' required for each Purkinje cell to make its axon relatively deeper may underlie the emergence of the PP at the population level.
In E14.5 reeler cerebella lacking the PP, a NF+ fiber bundle was observed above the Purkinje cells' somata (just beneath the pial surface; Figure 8H, I), raising the possibility that the lack of a PP is due to a failure to change the orientation or 'posture' of Purkinje cells between E13.5 and E14.5. Indeed, adenovirus-mediated pulse-chase examinations revealed that E10.5-born Purkinje cells in a dorsal region of E14.5 reeler cerebella remained parallel to the pial surface without showing a posture change (Figure 8J, K). While about 40% of the total E10.5-labeled Purkinje cells in normal cerebella (n = 71) were perpendicular to the pial surface (Figure 8E), none of the E10.5-born Purkinje cells adenovirally examined in E14.5 reeler cerebella (0/32 cells) showed the perpendicular orientation seen in normal cerebella.
To better describe this posture-change behavior as a distinct histogenetic step for migratory Purkinje cells, we compared the position of the Golgi apparatus between the most developmentally advanced Purkinje cells that had been incorporated into the PP at E14.5 and younger Purkinje cells exhibiting tangential or radial orientations of the soma in regions deeper than the PP or in E13.5 cerebella. Interestingly, nascent Purkinje cells before and after the formation of the PP distributed their Golgi completely differently (Figure 11E-J). In Purkinje cells displaying morphologies consistent with radial and tangential migration, the Golgi was located in a proximal part of the pially and anteriorly extending axon-like (tau+) fibers. In Purkinje cells from the PP, however, the Golgi was found in the dendrite-like process that was positive for microtubule-associated protein 2 (MAP2) and extended towards the pial side. It is likely, therefore, that the Golgi apparatus was excluded from the axon-like part of Purkinje cells and moved into the non-axon part during a transition from the tangentially oriented phase into the PP-forming step.
We carried out an additional three examinations to find out whether factors other than a Reelin-enriched superficial region and Purkinje cell polarization might also be primarily involved in the emergence of the PP. First, migration of RL-derived cells, which produce Reelin without expressing Reelin receptors and mDab1 (Figure 10A-E), was normal in reeler cerebella at least until E15.5 (as seen for DN cells positive for Tbr1 in Figure 8G, I and EGL cells positive for Pax6 in Additional file 7D, E, N, O). Second, we found that a portion of accumulating NF+ fibers that demarcate the PP in normal E14.5 cerebella did not contain afferent fibers that were labeled with DiI inserted into the cerebellar peduncular region (Additional file 8A). Third, Nestin+ fibers were radially oriented in reeler cerebella at E14.5 (Additional file 8B) in a pattern indistinguishable from that observed in normal E14.5 cerebella (Additional file 7A). These examinations suggest that migration of RL-derived cells, afferent fibers, and radial fiber morphology may not account for the differential Purkinje cell behaviors between normal and reeler cerebella during the E13.5 to 14.5 period.
The present study unveiled three important behaviors of nascent Purkinje cells derived from the posterior lateral cerebellum at E10.5: tangential migration, early axonogenesis, and Reelin-dependent 'posture' change to initiate PP formation. This early/posterior-born subset of Purkinje cells is the main contributor to the initiation of PP at E14.5 (Figure 1), and is a good model to investigate the mechanism through which Purkinje cells can be aligned under the regulation of Reelin.
The tangential mode of migration exhibited by these early/posterior-born Purkinje cells is very similar to that of RL-derived prospective DN neurons moving more superficially. We demonstrated immunohistochemically that the non-superficial, tangentially oriented cells were Purkinje cells. The existence of these cells was well recognized in previous Golgi studies, although they were regarded, together with the superficial cells, to be DN neurons [1]. The tangentially oriented Purkinje cells were negative for markers for RL derivatives (Math1/Atoh1, not shown; Pax6 (Additional file 7; Tbr1 (Figure 8)). Not only E10.5-born but also E11.5-born Purkinje cells exhibited tangential morphologies in a posterior part (Additional file 2E). While E10.5-born DN neurons (GFP+Lhx1/5-) migrated tangentially along the pial surface up to 600 μm from the RL in 2 days, the histogram for the overall E10.5-born Purkinje cells (GFP+Lhx1/5+) showed a peak at 100 μm from the ventricular surface, with the farthest cells around 160 μm at E12.5 (Figure 1E). This suggests a basic rule for the E10.5 to E12.5 period that neuronal migration velocity is much greater in the tangential mode than in the radial mode. Posterior-born Purkinje cells, which are inherently closer to the pial surface than anterior-born cells, seem to be the quickest to reach an outer zone for subsequent initiation of the PP mainly due to their greater dependence on the tangential mode.
According to the previous model [6, 9, 23–26], Purkinje cell behavior prior to initiating the PP could be likened to moving from one side across to another along radial fibers (Figure 12A). We propose here that migration of a population of Purkinje cells born early in the posterior part of the cerebellum is more akin to moving along a triangular perimeter (Figure 12A). The former model was coupled with a speculation that the front (outer extremity) of each migratory Purkinje cell may transform into a dendrite whereas an axon will emerge from the rear (inner extremity). Our results show that the origins of the axons and dendrite-like processes in the early/posterior-born Purkinje cells in the lateral cerebellar primordium are opposite of what is expected in the radial migration model. It is currently unclear whether the pially or anteriorly extended processes, which are ahead of outward migrating Purkinje cells, in cerebella at E12.5 can be immediately defined as axons. However, their traceability later during embryonic development along with the overall increase in number and length, as well as the sequential expression of NF from E12.5 (Figure 5) and tau from E13.5 (Figure 6), strongly suggest that they will directly become mature axons. The axonogenic behavior of Purkinje cells resembles that of neurons outside the neocortex; the extension of a prospective axon to the direction of migration (towards the pial/outer side) is suggested in motor neurons in the developing spinal cord [40], retinal ganglion cells [41, 42], retinal bipolar cells [43], tectal ganglion cells [44], acoustic nuclei neurons [45], and serotonergic neurons in the hindbrain [46]. Mechanisms regulating the elaboration of a new axon-like pial process from a nascent Purkinje cell (Figure 5C-E) are unknown. But the similarity between the course of axonogenesis by nascent Purkinje cells and that of afferent ingrowth reminds us of an intriguing possibility that afferent fibers might influence Purkinje cell behaviors [47]. Whether some of the axon-like fibers are formed through the inheritance of radial fibers from progenitor cells needs to be determined, as we have observed that progenitor cells in cerebella at E11.5 to E12.5 divide at the ventricular surface while keeping their pial processes (data not shown).
At E13.5, Purkinje cells reach an outer area (150 to 200 μm from the ventricular surface) that underlies the most superficial zone. Although this outer area normally becomes covered with Reelin from this point onward (Figure 10A), it cannot be distinguished between normal and reeler cerebella (Figure 7). The outermost Purkinje cells, which subsequently exhibit the earliest Reelin-dependent behavior, are horizontally oriented at this time-point (Figure 2). Then, differential posture-change behaviors between normal and reeler Purkinje cells are observed by E14.5 (that is, Purkinje cells in normal cerebella change their orientation as if bending upwards, while those in reeler cerebella remain laying down; Figures 8 and 12).
This difference is similar to that previously suggested in E14 to E15 cerebral walls [48, 49]. In the normal cerebral wall, early-born neurons that have migrated to an outer area are oriented vertically, with the dendrites arising from the outer pole of the soma and the axon from the inner pole. The axons extended from these early neurons demarcate these cells' somata above as a plate, forming the cortical plate [50]. In the corresponding outer zone in reeler cerebral walls, many neurons lie horizontally, which is similar to what we observed for Purkinje cells in E14.5 reeler cerebella (Figures 8 and 12). The normal bending up-like posture change of the early/posterior-born Purkinje cells enables the axons to become relatively deep and demarcate their somata above as a plate.
This posture change is likely driven by dynamic extension of dendrite-like processes, coupled with repositioning of Golgi, towards the Reelin-rich zone (Figures 9, 11 and 12). Previous co-culture and transplantation experiments showed that Purkinje cells whose somata were aligned along an artificially provided Reelin-rich zone had extended dendrite-like processes into that zone [22]. Recently, involvement of Reelin in the growth of dendrites has been further demonstrated [36, 37, 51–56]. Taken together, we conclude that the posture change of early/posterior-born Purkinje cells, which drives the PP emergence in the lateral cerebellum, is explained primarily by the action of Reelin immediately above the Purkinje cells during the E13.5 to E14.5 period. Dendritogenesis may be tightly linked with layer formation.
Associated with the extension of the dendrite-like part, the soma moves towards the outer/pial side. The initial somal movement of the early/posterior-born Purkinje cells is oriented towards the anteriorly growing axon-like part (heading away from the posterior ventricular surfaces). The second step of somal movement towards the pial side can be viewed as a reverse or 'switchback'-like behavior (Figure 12), which has been suggested to occur during migration of tectal, acoustic, and facial nuclei neurons [44, 45, 56]. Although the initial migration step until E13.5 is tangential and considered to be independent of radial fibers, this second step between E13.5 and E14.5 is more radial and thus the radial fibers might be involved in this step. Further studies are required to determine whether the migration of Purkinje cells other than the early/posterior-born population is also biphasic (or multi-phasic). Comparison between Lhx1/5 and Nestin immunostaining patterns (Additional file 7) raises the possibility that Purkinje cell behavior after the emergence of the PP (Figure 1) might be explained by the guidance of their second (or final) phase of migration by radial fibers. This could correspond to what has been proposed previously [6, 9, 23–26]. As in the E13.5 to E14.5 period, the most intense extracellular expression of Reelin occurs subpially throughout late embryonic development [16]. Whether the short-range mechanism for Reelin's instruction that we propose for the early/posterior-born Purkinje cells (that is, targeting the action of cells near the Reelin-rich zone) in the lateral cerebellum might be applicable to later-arriving Purkinje cells should be examined further.
It is important to understand how the short-range action mechanism might coincide with other stage- or context-dependent mechanisms. These mechanisms include Reelin's 'encouragement' of migrating or premigratory neurons distant from their final arrangement position [57], Reelin's effects on radial fibers [58–62], and the Reelin-independent 'community effect' among Purkinje cells [63]. Transgenic mice artificially expressing reelin under the nestin promoter, which works within the VZ (the birthplace of Purkinje cells) until mid-embryonic development, showed an apparently normal arrangement of Purkinje cells by the postnatal period [64]. This may lead to an interpretation that Reelin is not necessarily required at the site of Purkinje cell assembly/alignment, and seemingly contradicts the present short-range action model. Nevertheless, the nestin enhancer/promoter is active not only in the VZ (until around E16.5) but also in glial-like cells radially migrating towards the pial side (from E17.5 onward; our unpublished observation using nestin-enhanced GFP mice [65]). Our current observations of the course of Purkinje cell migration inferred from Lhx1/5 immunostaining, the orientation of Nestin+ fibers (Additional file 7), and the movement of cells belonging to the glial cell lineage [24] during the E15.5 to E18.5 period suggests a potential role for glial cells. In reeler cerebella, glial cells can successfully migrate to the subpial region [66]. Reelin artificially provided by glial cells migrating from deep regions to subpial portions in the nestin-reelin transgenic reeler cerebella might have enabled Purkinje cells to approach the subpial position mainly during this late embryonic stage. It would be intriguing to check whether nestin-reelin transgenic reeler cerebella might not have a normal PP at E14.5.
By using an adenovirus-mediated pulse-chase method in combination with recently established Purkinje-cell-specific markers and slice culture, the present study focused on the developing mouse lateral cerebellum until E14.5 and unveiled three important behaviors of early/posterior-born Purkinje cells: tangential migration (at approximately E13.5), early axonogenesis (at approximately E12.5), and Reelin-dependent 'posture' change initiating their layer formation (between E13.5 and E14.5). Early axonogenesis by Purkinje cells contributes to their formation of a discrete layer. Our study provides a solid basis for further elucidation of Reelin's function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives overall brain morphogenesis.
B6C3Fe-a/a-rl mice (heterozygous for the reeler mutation) were obtained from the Jackson Laboratory. Heterozygous (+/rl) and homozygous (rl/rl) embryos were generated by pairing female heterozygotes and male homozygotes and genotyped as described previously [67]. For 'normal' cerebellar specimens, both the heterozygous mice and ICR mice were used. Only homozygous (rl/rl) mice are called reeler in this paper. The day of vaginal plug identification was defined as E0.5.
Adenoviral construction was carried out as previously described [27, 28]. The adenoviral vector Adex-CAG-Lyn-Venus expresses membrane-targeted Venus under control of the CAG promoter. Lyn-Venus was generated by fusing the sequence of the palmitoylation site of tyrosine kinase Lyn to the amino terminus of Venus [32, 33]. Adenoviral vector solution was injected into the fourth ventricle of embryos at each day from E10.5 to E12.5 using a trans-illumination system of in utero surgery [68]. For histological examination using frozen sections (16 μm), the titer of adenovirus vector solution was set to a level at which <5 labeled cells in each section could be seen. With this dilution, data used for making the histograms in Figure 1E were obtained from three to seven normal (ICR mouse) embryos at each stage. For live imaging on normal cerebellar slices (200 to 250 μm), viral solution was further diluted to ensure sporadic labeling. For comparisons between +/rl and rl/rl littermates at E13.5 (Figure 7C), adenovirus-mediated pulse-chase examination was carried out using a more concentrated viral solution.
Embryos at each day from E13.5 to E18.5 were perfused transcardially with periodate-lysine-paraformaldehyde (PLP) fixative [69]. Younger (E11.5 and E12.5) embryos were fixed by immersion in PLP at 4°C for 2 h. Isolated brains were postfixed in PLP at 4°C for 1 to 2 h, immersed in 20% sucrose, embedded in OCT compound (Miles, Elkhart, IN, USA), and then frozen and sectioned sagittally and in some cases coronally (16 μm). Frozen sections were treated with the following primary antibodies: anti-Lhx1/5 (Lim1/2; mouse, Hybridoma Bank [4F2]); anti-Corl2 (rabbit) [30]; anti-Nestin (rabbit, gift from Dr Yasuhiro Tomooka) [70]; RC2 (mouse IgM, gift from Dr Miyuki Mamamoto) [71]; anti-Neurofilament (mouse, Hybridoma Bank [2H3]; rabbit, BIOMOL, Plymouth Meeting, PA, USA); anti-GFP (rat, Nakarai Tesque, Inc., Kyoto, Japan; rabbit, MBL, Nagoya, Japan, anti-Tbr1 (rabbit, Abcam, Tokyo, Japan), anti-Reelin (mouse [CR50]) [14, 16]; Pax6 (rabbit, Covance, Berkeley, CA, USA); anti-Math1 (Hybridoma Bank); anti-tau (mouse, Chemicon, Billerica, MA, USA); anti-MAP2 (mouse, Sigma, St. Louis, MO, USA); and anti-giantin (rabbit, Covance). For immunostaining of live DiI-labeled cells in cerebellar slices, vibratome sections (50 μm) made after the slices were fixed with 4% paraformaldehyde at 4°C for 20 minutes were treated with antibodies at 4°C overnight.
Slice culture
Cerebellar primordia were isolated from E12.5 or E13.5 embryos that had been infected in utero with GFP-adenovirus at E10.5 and sliced using fine microknives, as previously described for cerebral wall slices [31, 32]. Because the long axis of each cerebellar primordium is roughly perpendicular to the rostorocaudal (anteroposterior) axis of each embryo and bridges both sides of the metencephalon, the plane of each live slice (200 to 250 μm thick, 6 to 8 slices per embryo) was made by sectioning almost perpendicular to the long axis of the isolated primordium and was generally close to the in vivo sagittal plane. In a lateral region, where the cerebellar primordium is more oblique in vivo (less perpendicular to the embryo's long axis), the plane of the live slices was less sagittal. The slices were then transferred to a glass-bottom 35 mm plastic culture dish containing enriched culture medium, a 1:1 mixture of DMEM and Ham's F-12 (phenol red-free) supplemented with insulin (25 μg/ml), transferrin (100 μg/ml), progesterone (20 nM), sodium selenate (30 nM, Sigma), putrescine (60 μM), epidermal growth factor (10 ng/ml), basic fibroblast growth factor (10 ng/ml), horse serum (5%), and fetal calf serum (5%). Up to ten slices were then transferred with 100 to 150 μl of the enriched medium to the center of the dish, then mixed with 100 to 150 μl of 2× type I collagen gel solution (Cellmatrix IA, Nitta Gelatin, Tokyo, Japan; previously diluted to 0.5 to 0.6 mg/ml with distilled water, 5× phenol red-free DMEM, and a neutralizing buffer according to the manufacturer's protocol, and kept on ice until use). The gel (final concentration 0.25 to 0.3 mg/ml) was immediately spread onto the dish base using a pipette tip. After nearly allowing the gel to solidify (about 5 minutes) with the ventricular and pial surfaces of the slices perpendicular to the dish base (arranged with a tungsten needle), the dish was placed in an incubator (37°C, 40% O2 and 5% CO2 ) (Astec, Kasuya, Fukuoka, Japan) to allow further gel solidification (5 to 10 minutes). The enriched medium (0.8 ml) was dropped onto the gel and spread over the entire dish surface.
For on-stage culture, an inverted epifluorescence microscope (IX70, Olympus) equipped with a 10× objective lens (UPlanF1; numerical aperture = 0.30) was used. In this system, slices were continuously cultured in a chamber providing a humidified atmosphere of 5% CO2 and 40% O2 (Tokken, Kashiwa, Chiba, Japan). Imaging was performed at 5-minute intervals using a cooled charge-coupled device (CCD) camera (CoolsnapHQ, Roper Scientific, Tucson, AZ, USA), which was automatically controlled by METAMORPH 6.3r-1 software (Molecular Devices, Silicon Vally, CA, USA) as described previously [72]. Some live images of Venus-labeled cells were captured manually using a different system composed of an inverted epifluorescence microscope (IX71, Olympus) equipped with a 20× objective lens (UPlanApo; numerical aperture = 0.70) and a cooled CCD camera (ORCA-ER, Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan) and IPLab 3.6 software (Scanalytics, Inc., Rockville, MD, USA). The same manual capturing system was used for most of the presented immunofluorescence images. For some of the immunostained specimens, confocal microscopy was performed using a Yokogawa CSU10 (40×, 100×) (Tokyo, Japan) and an Olympus FV1000 (40×) (Tokyo, Japan).
DiI labeling
For single cell-level visualization within cerebellar primordia, fine crystals (< 30 μm) of DiI C18(3) (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; D-282, Molecular Probes, Eugene, OR, USA) were inserted into live cerebella from the pial surface using a small make-up brush, and slices were made and incubated at 37°C (40% O2 and 5% CO2) for 1 to 2 h. After live identification of DiI-labeled cells, slices were fixed and further processed for immunostaining. For labeling of afferent fibers from the vestibular ganglion and those of the cerebellar peduncle, crystals of DiI were inserted into fixed brains using forceps. The DiI-inserted brains were incubated at 37°C for several days, and then vibratome sectioned.
In situ hybridization
Probes to determine the expression of vldlr, apoER2, and mDab1 were designed according to Trommsdorff et al. [38]. Cerebella were fixed with 4% paraformaldehyde at 4°C overnight. Frozen sections (16 μm) were pretreated with 4% paraformaldehyde (5 minutes), 0.1 M triethanolamine/0.25% acetic anhydride (10 minutes), and hybridized at 60°C for 15 h in a solution containing 50% formamide, 0.1% SDS, 0.64 mg/ml tRNA, and 0.5 μg/ml riboprobe.
apolipoprotein E receptor 2
Dulbecco's modified Eagle's medium
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
deep nuclear
embryonic day
external granular layer
green fluorescent protein
Purkinje plate
rhombic lip
very low-density lipoprotein receptor
ventricular zone.
We are very grateful to T Shimogori for instructing on in utero surgery and critical reading of the manuscript, Y Tomooka, M Yamamoto, M Watanabe for antibodies, E Ito, K Ota, O Noritake, Y Noritake, T Miwa, and S Yamamoto for technical support, and the Developmental Studies Hybridoma Bank at the University of Iowa for monoclonal antibodies. This work was supported by grants from the Japanese Ministry of Education, Science, Sports and Culture (20021016, 21240027), Uehara Memorial Foundation, Brain Science Foundation, and Life Sciences Foundation.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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ALS has a broad scope of experience in providing analytical testing services to environmental emergency incidents.
ALS is a leader in providing analytical testing services to the environmental industry and has a broad scope of experience in providing analytical testing services to environmental emergency incidents such as plant explosions, ammonia and chemical fires, petroleum spills, train derailments, and accidental emissions or releases. These services are provided to consultants, government and industry in their efforts to make timely decisions in protection of the environment, mitigation of risk and maintaining business continuity.
ALS provides invaluable support for logistics and expedited testing services which can fall under the incident command structure during the initial stages of an environmental emergency. It is critical for stakeholders to be able to draw upon the technical expertise and support of the environmental laboratory to access not only sampling containers for the collection of water, soil, air and biota that may be potentially impacted, but to ensure that the analytical characterization results are processed on an urgent basis.
The initial data provided from environmental samples collected within the first 48 hours can provide critical insight into hazard identification, scope and environmental contamination zones, and indicators of efficacy of initial containment procedures. Ongoing expedited rush analysis services ensure timely cleanup of the site. Additional services such as mobile laboratory testing, specialty forensic analysis, and field screening tools are also available.
- 24/7 live operator emergency contact number
- Mobilization of ALS' team of experts and network laboratories throughout the United States
- Sample bottles and coolers
- Designated project lead
- Weekend and evening project coordination support
- Rapid deployment of sampling supplies to site
- Transport and logistics support between the site and the nearest ALS laboratory
- Convenient sample bottle supply centers and drop-off depots
- Around-the-clock expedited laboratory services
- Web-access to analytical results in real-time with screening tool to compare to regulatory limits
- Specialty forensic services
- On-site services
Analytical support in response to environmental emergencies can be challenging. Depending on the nature of environmental emergency and the surrounding environment, a variety of services are required of analytical laboratories. Usually, the company faces a number of questions from impacted or potentially impacted stakeholders. These include:
- Will the environmental emergency impact human health?
- Will the environmental emergency impact drinking water?
- Will the environmental emergency impact ground water?
- Will the environmental emergency impact wildlife and birds?
- Will the environmental emergency impact surrounding communities by exposing them to airborne toxic chemicals?
- What analyses are required to determine impact on humans, fish, wildlife, birds, vegetation, sediments, and water to determine impact or adverse risk?
- Following cleanup, is there confidence that the site has been fully remediated?
- Have sufficient samples been collected and analyzed to perform a legally defensible risk assessment?
There is no "one size fits all" solution to an environmental emergency, as every incident has unique characteristics and challenges. However, at a very high level, the following steps are essential:
- Early detection of the release
- Isolation of the release point to prevent further release
- Notification of incident to the appropriate regulatory agencies, municipalities, and other stakeholders
- Containment of the released product down gradient environmental receptors
- Recovery of the released product to mitigate further environmental impacts .
- Delineation and remediation of contaminated areas impacted by the release.*
- Reclamation of the remediated areas.*
- Monitoring of the impacted areas to ensure restoration efforts have been successful.*
* ALS provides support in these areas.
- Water analysis - Groundwater, surface water, and drinking water
- Soil analysis
- Air analysis
- Industrial Hygiene analysis
...and more!

subset: commoncrawl


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Write an Android application
This project was awarded to businesstrip for £600 GBP.Get free quotes for a project like this
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We have an existing Epos software and need to get an android app developed for waiters to take orders on it - I would prefer Bangladeshi developers as our office is in Bangladesh and developer will need to work with our current developers of desktop software to develop the android app
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Category Archives: Health & Fitness
The Many Services of Dentists
Dentists perform a variety of dental procedures every day; many of which are done to prevent cavities, straighten misaligned teeth or perform cosmetic surgery.
Some of the dental services that are performed are: laser dentistry, dental sealants, dental bridges, dental crowns, fillings, dentures, tooth extractions and gum services. Dentists provide these services so that you and I can enjoy good dental health. This can be achieved with regular dental cleanings, x-rays and restorative dental services. Keep in mind; there are certain critical factors that are important for our dental health and our well-being such as teeth that function properly, taking care of our teeth and gums so that we can eat nutritious foods, maintaining our well-being and self-esteem and enjoying a nice smile and looking attractive. Carmichael dentists have the dental services you need!
Other dental services that dentists provide are dental braces and oral surgery. Examples of oral surgery are root canals and dental implants. In addition, besides the aforementioned services, other services are available such as mouth guards for TMJ and orthodontic procedures. All of these dental services keep a person's oral health in good shape. Dentists in Carmichael are happy to help with your dental needs.
To conclude, regular dental visits are important because they keep our teeth and gums healthy. Most dentists agree that regular dental visits should occur twice a year. During a dental visit your teeth and gums are examined and your dentist will check for cavities and see if there is tartar or plaque on your teeth. Your gums will be checked to see if they are healthy. Between visits you should brush and floss your teeth and use a mouth rinse to get rid of bacteria. Of course, your dentist will also ask if you have had any problems with your teeth such as pain or problems with your bite. It is important during the dental examination to be as honest as possible with your dentist.


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Changing Pesticide Use: Challenges for Water-Quality Monitoring and Ecological Implications WEBINAR
Date: May 3, 2017
Time: 10:00 AM - 11:00 AM
Contact: 415-473-4204
Sponsor: Grown in Marin
Location: online
Event Details
National Water Quality Monitoring Council Webinar Series
Pesticide use is constantly changing due to pest resistance, regulations, crops planted, and the introduction of new chemicals. In recent years, application techniques for agricultural use pesticides have also changed with an increase in seed coatings in addition to more traditional application methods such as foliar sprays. Most seed coatings consist of one insecticide and up to five fungicides; many of these pesticides have not been used extensively prior to their inclusion on seed coatings.
The webinar will be offered on Wednesday, May 3rd, 2017 at 10:00 am. Pre-registration is REQUIRED for this event -- register online now. Once your registration has been approved, you will receive a confirmation email with instructions for joining the webinar.
If you miss the webinar and would like to view it later, please visit the National Water Quality Monitoring Council's YouTube page to view their archive of webinars, or visit our website for more information.


text: video player loading . . . You Just Watched: Season 20, Episode 8 Tom Hanks Relives Famous 'Forrest Gump' Run replay Watch Next Harry Styles Reacts to Rumors Season 21, Episode 1 Why Peter Capaldi's Leaving 'Doctor Who' Season 21, Episode 1 Jennifer Hudson Wins a Bowling Alley Singing Contest Share close Facebook Twitter Tumblr Email Google+ Reddit Get embed code <iframe src="http://player.theplatform.com/p/M_UwQC/OGEJCrK0apNU/embed/select/media/TiRzgs1HISs_?form=html" width="480" height="270" frameBorder="0" seamless="seamless" allowFullScreen></iframe> Share on Facebook: Copy link below To share this on Facebook click on the link below Open Facebook back The Graham Norton Show Season 20 — Episode 8 Tom Hanks Relives Famous 'Forrest Gump' Run …for Mo Farah, naturally. Video Extra Copy the link below To share this on Facebook click on the link below. Open Facebook Advertisement Videos from this Episode Tom Hanks Jokes Clint Eastwood Treats His Actors Like Horses When Joseph Gordon-Levitt Met Edward Snowden's Parents Tom Hanks Shows His Bad Side Would Tom Hanks Run For President? Is it Tom Hanks or Bill Murray? View All Close


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The presentations on this page are about teaching testing to colleagues.
This talk was presented at the SAGWAS workshop about training and teaching testers. The slides are in German.
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2016-12-19 16:11
The Jubula team has released the latest official version of Jubula. Version 8.4.0 was released on 19th December 2016 and now allows users to write tests using conditions and loops as well as various other additions to simplify test creation, especially in more complex scenarios.
Read more … Jubula 8.4.0 has been released
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Entry requirements
Minimum 2:1 (or equivalent), with masters degree preferred
Months of entry
Course content
The Economics Department has built up, since the 1990s, an innovative specialism in industrial economics – notably in the leading-edge field of evolutionary industrial theory and its implications for industrial and regulatory policy. The department's expertise is acknowledged to have been one key element in the winning of the ESRC Innogen research centre, and the continuation of the Innogen Institute which includes several members of the department. The department took the lead in building up cross-faculty expertise in industrial development and innovation through the IKD research centre, which brings together innovation researchers in health, human capabilities and finance.
The current group of industrial economists in the department research the topics of industrial dynamics, industrial development, industrial finance and the implications of technical change, as well as the relationships and processes of finance and innovation.
Potential supervisors
Department specialisms
Topics could include: Industrial development and innovation Evolutionary economics and theory of development Financial markets and innovation Innovation theory and models of innovation Firm capabilities, growth and innovation systems (national, regional or sectoral) Health, innovation and equity Finance, innovation and development Financialisation and innovation Studies of pharmaceutical, biotechnology, environment and renewables firms and industries.
Fees and funding
Please see The Open University website http://www.open.ac.uk/postgraduate/research-degrees/fees-and-funding
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- email@example.com
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- +44 (0)1908 654456


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Young people who come through Restorative Justice are checked for later violations of the law. This is called the recidivism rate, the percentage who commit another offense.
The following recidivism rates are based on young people who entered the RJS programs from February 2008 - April 2011 and completed the program. Checks at 12 months past program completion were done for all these young participants. The recidivism checks were done on the Fort Collins Police Services Tiberon system, which covers Larimer County.
Recidivism Rate at 12 months past program completion: 8.7%
This check includes all offenses, including drug and alcohol, but not smoking violations.
Recidivism Rate at 12 months past program completion for a same or similar crime: 2%
This check includes offenses that are the same type of offense, or a similar offense to the offense for which they came to RJS. For example a shoplifting theft would be same or similar to any other kind of theft, including fraud or larceny but a theft would not be the same or similar to a drug or alcohol violation.


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Bus Mikolow Mikołów • Silesian Voivodeship » Carlisle
Coach and bus timetable from Mikolow to Carlisle
Mikolow to Carlisle timetables
You can go with Super Busy from Mikolow to Carlisle.
At Mikolow - United Kingdom page you will find more online tickets from Mikolow to cities in United Kingdom. Check rebrutto.com offer for return tickets at Carlisle to Mikolow or Carlisle to other cities in Poland bus services.
If you have any questions on your journey or you experience any problems with booking your ticket, please visit our FAQ or contact us.
Choose your best option from rebrutto.com offer.


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Product Description
If You Are Looking For A Wooden Tray That Will Be At Your Service In Multiple Ways, Then The Wood Metal Tray Is What You Are Searching For. Over Two Feet In Diameter, This Tray Is Not Just Pleasant To Look At, But It Also Has Multiple Uses. For Example, You Can Use It To Carry Pizzas, Or Dishes Filled Of Food, Or Even Cups Brimming With Beer. The Tray's Looks Are Awesome And It Has Handles On Two Ends Which Make Carrying It A Simple Task. The Color Of This Tray Is Brown, And It Has A Light Black Wash On It: This Creates The Effect Of Royalty And Imparts It With Certain Agedness. Made From Quality Wood, This Tray Is Sure To Last For Years To Come. Suitable For Modern As Well As Traditionaldecors, This Tray Will Garner A Lot Of Plaudits For You From Your Guests. So Get One Of These Beauties Today!
- Manufacturer: Woodland
- Brand: Benzara
- Category: Kitchenware
- Assemble Required: No
- Ship Type: Small Package
- Country Of Origin: China
- UPC Code: 758647939558
- Dimensions: 29" W x 19" L x 4" H
- Carton Dimensions: 30" W x 19" L x 4" H, Weight: 11 Lbs
Additional Information
Additional Information
|Free Shipping
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|Fedex/UPS Drop off Delivery (If bought alone ) without Appointment / For large quantity, LTL Curbside Delivery with appointment
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PortCityDaily.com is your source for free news and information in the Wilmington area.
1 Flytrap Brewing | Directions
Why you should visit: Named after the Venus flytrap whose native habitat is within a 60-mile radius of Wilmington. Flytrap Brewing, located in the Brooklyn Arts District of Historic Downtown Wilmington, is a neighborhood brewery that offers a large selection of beer to choose from, serving signature brews as well as five rotating guest taps. One of the Flytrap's most popular signature ales is the Rehder's Red Ale, named after Stanley Rehder, a local horticulturist with a special interest in the Venus flytrap. WilmingtonAndBeaches.com
2 Front Street Brewery | Directions
Why you should visit: Front Street Brewery, located in historic downtown, has been a favorite restaurant to locals since it opened in 1995. It is home to Wilmington's only restaurant with a micro-brewery on site. History is on their side as the building where they serve the suds dates back to 1865, the same year that the Second Battle of Fort Fisher began during the Civil War. The brewery offers daily tours that teach the brewing process and offers samples of seasonal brews and flagship brews including Coastal Kolsch and Riptide Raspberry Wheat. WilmingtonParent.com
3 Waterline Brewing Company | Directions
Why you should visit: This 1940's preserved original structure, located just under the Cape Fear Memorial Bridge, gives the atmosphere of a rustic and historic feel with plenty of locally-crafted beer. They distribute kegs locally and Waterline even offers tours where the staff outlines not only the brewing process but the historical aspects of the renovated building. Original concrete floors and rough cut heart pine beams throughout their space makes for a wonderful setting to brew and taste craft beers. WaterlineBrewing.com
There is no doubt that Wilmington and the surrounding areas have an emerging craft brew scene and everyone likes to drink locally, but for those who are thinking globally, here is a breakdown from PortCityDaily.com of the three best breweries outside of America that we recommend visiting. Please drink responsibly.
1 Meantime Brewery in London, England
Why you should visit: The Greenwich site is the largest craft brewery in the British capital, and visitors can hear about the history of craft brewing, take in the sights and sounds, and taste a range of the different products during their visit. They offer a brewery tour that is billed as "one part stand-up comedy, one part history lesson, one part science lesson and one part going down the pub". MSN.com
2 Evil Twin Brewing in Copenhagen, Denmark
Why you should visit: Evil Twin is a nomadic (or the ethnically and politically incorrect, but infinitely more popularly described gypsy) brewery that traces its origins to Denmark. Evil Twin literally brews all over the world utilizing excess capacity from other breweries to create their beers. Due to the nature of being a nomadic brewery, many of these beers are one-off production runs. Its12oclockSomewhere.com
3 Amburon Belgian Craft Brewery in Tongeren, Belgium
Why you should visit: The love of beer was instilled in Davy Daniëls from a very young age. His grandfather opened a drinks store in 1961. Davy's father worked in the store throughout his life and Davy followed in his footsteps. He was a first-hand witness to the growing success of Belgian specialty beers and a new wave of microbreweries and beer firms. Today the brewery features some of the world's best craft beer, located in Belgium's oldest city. BeerTourism.com
For more information or to nominate a Wilmington establishment to be featured in future articles, please email travel editor Aaron Gray at email@example.com.


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Based on 548 Sales and 3 Service Reviews
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Miranda Hart or Jodie Whittaker? I think it's safe to say that Jimmy McGovern wasn't troubled by this casting dilemma for last week's episode of Accused.
I only mention it because who should turn up, playing a sexy barman, but Tom Ellis. Gary! I knoooow! Such fun!
The focus of McGovern and co-writer Danny Brocklehurst's sweaty little morality tale was Liam (Andy Serkis), a cab driver with a life as clapped out as his old Volvo: a barely functional relationship with a wife in the advanced stages of multiple sclerosis and a gambling habit so bad that even his poker opponents are telling him to ease off. Into his cab steps Emma (Whittaker), a young woman with a shampoo commercial for a life. Her business trip gives Liam the chance to burgle her flat and pinch a piece of jewellery that he can give to his bright daughter for passing an entrance exam to a posh school. Would that Liam had left it there – but no.
He takes the memory stick from the side of her laptop, and downloads the personal details therein to engineer the break-up of her relationship with the sexy barman and offer his convenient, if lightly dandruffed, shoulder to cry on.
An amour fou between a beautiful estate agent and a needy minicab driver twice her age? It gives me pause for thought too, now I think about it. But so neatly did the production flesh out Liam's desperate sense of domestic imprisonment that the tale was well into its tragic dénouement before you started wondering exactly what Emma saw in Andy Serkis's rheumy, guilt-ridden eyes.
Much of Accused played cleverly on the intimate frisson of a cab ride and the anonymity of the man at the wheel. That curious tension was also to be found in the documentary I Was Bin Laden's Bodyguard. Large parts of this rambling film were set in the cab of Abu Jandal as he pootled his way round Sana'a in Yemen, hustling for fares and recalling his days as Osama bin Laden's constant chaperone in the late Nineties. The tragic irony, he reflected, was that it wasn't he who ended up in solitary confinement in Guantanamo for seven years, but his brother-in-law, Salim, a driver to the senior figures of al-Qa'ida in Afghanistan and, according to Jandal, nothing but a stooge. Worse for poor Salim, he became the first of Gitmo's inmates to face trial by military commission.
Abu Jandal was, it turned out, a nom de guerre, meaning "the killer". And though it emerged that plain old Nasser al-Bahri had undergone a Yemen government rehabilitation programme called The Dialogue and been given the money to buy his taxi, it was clear that he still yearned for those crazy days with the guys in Tora Bora. He had an eye for the main chance too, tailoring his self-justification to fit his audience. One minute he was filmed telling young Yemeni lads about why the 9/11 attacks could be justified; the next he was testifying, at length, on Al-Arabiya, that he had simply changed his mind about al-Qa'ida (not an argument, I imagine, that would cut much ice with brother Bin Laden).
This odd mixture of courage and opportunism was off-putting, at first. But as he careened from a New York Times interview to haggling with fares over a hundred riyals, broke and exposed to god-knows-what retribution on the streets of Sana'a, it was hard not to warm to Nasser, an all-too human ex-jihadi. As for Salim, he eventually returned to the Yemen, refusing to be filmed, a changed man and – the implication was left hanging – ripe for jihad.
Finally, a fond farewell to The Trip. As Steve Coogan and Rob Brydon toured the North's finest rural hostels, indulging their mid-life crises, it became clear, I think, what this amusing, infuriating series was: a journey in which, quite winningly, our travellers learned almost nothing at all.
- More about:
- Al-Qaeda
- Andy Serkis
- Osama Bin Laden
- Yemen


text: Next Story > MoneyBeat BitBeat: Mt. Gox Halts Withdrawals, Bitcoin Price Drops Feb 7, 2014 5:16 pm ET Oh, Mt. Gox. When one of the larger crypto-currency exchanges has a problem, the entire bitcoin trading universe has a problem. So it was with BTC China back in December, and so it was with Japan-based Mt. Gox today. To Read the Full Story, Subscribe or Sign In Previous Can Apple's Buybacks Bring the Stock's Shine Back? Next Podcast: Will the Fed Pause the Taper?


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We are a young, dynamic, international team of 30+ professionals dedicated to improving patients' lives through innovation.
Innovate to improve patients' lives
Listen and challenge with respect
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Act like owners for a common goal
Work with joy!
Xeltis is dedicated to transforming the standards of care in cardiovascular treatment by developing the first-ever bioabsorbable cardiovascular valves and vessels designed to allow Endogenous Tissue Restoration (ETR). Xeltis is striving to enable ETR to potentially help reduce the risk of complications and repeated interventions in patients requiring cardiovascular valves and vessels replacement, for them to live better, longer lives at lower cost for the healthcare systems.


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MediaDo Reader is a browser-based eBook reader that works on most tablets, phones, and computers that run up-to-date web browsers.
If you're not sure whether or not your device is supported, you can test it using the sample below. Just click or tap the jacket cover.
For the best reading experience, we recommend using the most up-to-date version of one of these web browsers:
MediaDo Reader is also compatible with the default browser on Android and iOS (iPhone/iPad/iPod touch) devices.
MediaDo Reader compatible devices
Note: This is not a complete list of compatible devices. If you do not see your device here, you can use this sample to test your browser.
- Android v2.3+ (we recommend 4.0+)
Example: Samsung Galaxy and Google Nexus devices
- iOS v6.0+
Example: iPhone/iPad/iPod touch
- Mac OSX (10.6 or later)
- Windows RT
Example: Microsoft Surface RT and Surface 2
- Windows XP, Vista, 7, 8, or 10


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Xian Lim gets hospitalized?
What happened to XIan Lim? Was it just a regular check-up or something should fans be alarmed?Tuesday, March 15, 2016
Actor Xian Lim surprised fans and followers this afternoon on Instagram after he posted a photo of himself laid down before a CT-scanner in The Medical City.
Xian wrote on the caption, "Thank you po sa lahat ng nag accommodate kanina sa The Medical City and kay ms. Cheryll Gonzaga for helping me out ❤"
Fans expressed worries about what happened but The Story of Us actor still have not answered to fans' queries.
One fan noted that Xian should stop going on an excessive diet.


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By Joan Hallmark - doctruyen cogiaothao video | pomeranian dog picture
Posted by Dana Hughey naruto shippuden mugen characters | el rey de oros nightclub com
The term I've come across for this is Scratch-N-Dent. I know someone who saved over $1000 on a large refrigerator because of a slight scratch on one side. Here's my problem... how do you find out where these scratch-n-dent appliances are sold? Do the manufacturers have outlet stores? Thanks, Tamara C. We always buy scratch and dent appliances from Sears outlet stores. The outlets carry the Sears brand name and also many of the other major appliance makers. Our most recent purchase was a Maytag dishwasher priced $150 off of the regular price due to a small ding on the front panel.
Before that, we purchased a $600 GE smooth top stove for $400 with a slight scratch on stove top. A few years ago, we purchased a $1499 Amana side-by-side refrigerator for $830; it had a small ding on the side which didn't show. We have been 100% happy with these products. Here's a list of Sears Outlet Store - Southwest - Denver, CO to help. Marie H. I found most scratch and dent places in the outlet books at the library or purchased in book stores. There seems to be a book out for every area.
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Computer Surfing for 50+
November 15, 2012
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Hands on using wireless laptops. Explore websites of special interest to active older adults. Online registration begins Thursday, November 8 at 1:00pm. This class takes place on the 4th floor from 6:30pm-8:30pm.
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Anonymous, after a relatively large period of doing nothing, are back with a vengeance. Even without their (arrested) de facto leader Topiary, they've punched Texan law enforcement squarely in their gut: a giant email leak, internal documents, addresses. Anon's back.
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Kinja is in read-only mode. We are working to restore service.


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Botanical name: Cananga Odorata
Botanical family: Anonaceae
Note: Middle
Part of plant used: Flowers
Origin: Native trees to the Philippines as well as islands like Tahiti, Seychelles and Madagascar. A tree takes 4 years to produce 5 kilos of oil. The yellow flowers have to be picked at monsoon time.
Description: The smell of Ylang-Ylang is compared to hyacinth and narcissus.
History: A tribe called the Bori Bori from the Tahiti region (Maluc) used the flowers to put in their hair as decoration and used Ylang-Ylang as a hair conditioner as well as rubbing over their bodies to keep in good health. They used it in their tea for malaria, typhoid and to calm down diarrhea.
Properties and Indications:
Back to Aromatherapy Essential Oils


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Considering the fact that America is built on white supremacist ideologies, it is very difficult for those who are fully aware of the history of the exploitation of Black labour in the Americas to simply take this new meme with a smile. While Ellen herself might not be racist, her response demonstrates a lack of understanding and sensitivity for the pain and anger her meme holds.
Going to the movies with little kids can be a stressful experience. There's always the fear a tantrum will break out and that you'll interrupt other viewers, but with these tips you can ensure a stress-free and controlled experience. From stocking up on snacks to strategies on keeping them as attentive as possible, these tips will save the day.
Many Muslim communities around the world are calling the killing of three young Muslims at Chapel Hill a hate crime. Others are calling it a parking dispute, a sort of road rage over space. According to his Facebook page, Craig Stephen Hicks, the alleged murderer was a supporter of gay rights, but didn't seem to extend the same warm feelings towards Muslims and Islam. Why tolerance is higher for one minority group and not another has complex roots.
Commercial sealing advocates find it exceptionally difficult to win hearts and minds with the truth. Because the truth is an industrial scale, non-aboriginal slaughter in which defenseless seal pups less than three months of age are horribly beaten and shot to death for their fur. It is a wasteful kill, in which the carcasses are normally dumped at sea.


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The CGV class simplifies the work with global variables of MetaTrader 5 client terminal.
Class methods:
Translated from Russian by MetaQuotes Software Corp.
Original code: https://www.mql5.com/ru/code/60
The Stochastic of all oscillator-like indicators, included in MetaTrader 5 client terminal.iUniMA
The Universal Moving Average, it allows to select any type of moving average, included in the MetaTrader 5 client terminal.
Color Parabolic 2. The color depends on the acceleration factor.TimerClosingPeriod v.2
The updated version of TimeClosingPeriod indicator, it plots the time, remained to the current bar complete. At the last second it plays the sound file, specified in the input parameters.


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Automechanika 2016
13.09.2016 - 17.09.2016 — Hall: 5.1 Stand: A 82
GETCO, General Trading Company is a French company specialized in the import/export and supplying of auto parts.
GETCO was founded in 1992 by Kamel Hagot, a veteran businessman with 40 years experience in the trade of auto parts. The company has since been successful in the international market. It has been constantly growing and exporting certified Original quality auto parts to more than 15 countries in Africa, the Middle East, Latin America and the French Overseas Departments and Territories (DOM-TOM).
GETCO has developed partnerships with various international companies such as Continental Contitech group, ATE, VDO, MECAFILTER, FTE,or NRF,
... . Since 2009, GETCO has been also selling its own range of products, since 2013 we started business with Comline and Borg and Beck, and since 2015, we are developing our own Timing belts Kits Syncronix and shock absorbers, all the while expanding its suppliers to be able to respond effectively to customer demand.
Thanks to the quality of its automotive parts and service policy, logistics and high performance rates, GETCO has been constantly gaining ground in new markets.
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New Hampshire Laws Regarding Safety
AN ACT relative to Occupational Safety and Health Administration certification requirements for state contracts and establishing a commission to recommend a comprehensive program for increasing the use of passenger restraints in New Hampshire.
Be it Enacted by the Senate and House of Representatives in General Court convened:
326:1 New Section; Occupational Safety and Health Administration Certification. Amend RSA 277 by inserting after section 5 the following new section:
277:5-a Occupational Safety and Health Administration Certification.
I. Any person signing a contract to work on a construction, reconstruction, alteration, remodeling, installation, demolition, maintenance, or repair of any public work or building by a state agency, municipality, or instrumentality thereof, and with a total project cost of $100,000 or more, shall have an Occupational Safety and Health Administration (OSHA) 10-hour construction safety program (OSHA 10 Hour Training) for their on-site employees. All employees are required to complete the program prior to beginning work. The training program shall utilize an OSHA-approved curriculum. Graduates shall receive a card from OSHA certifying the successful completion of the training program. This section shall apply to the construction, reconstruction, alteration, remodeling, installation, demolition, maintenance, or repair of any public work or building paid for in whole or in part with state funds.
II. Any employee required to complete the OSHA 10-hour construction safety program under this section who has not completed the program shall be subject to removal from the worksite if the employee does not provide documentation of having completed such program by the fifteenth day after the date the employee is found to be in noncompliance. Proof of such documentation provided by the employer shall constitute compliance with this section. The labor commissioner or commissioner's designee shall enforce this section.
III. The labor commissioner shall adopt rules under RSA 541-A relative to implementation and enforcement of this section. The commissioner may also assess a civil penalty of up to $2,500; in addition, such an employer shall be assessed a civil penalty of $100 per employee for each day of noncompliance. All funds collected under this section shall be deposited into the workers' compensation safety inspection fund established in RSA 281-A:64, IX. The labor commissioner shall appoint as many individuals as necessary to carry out the department's responsibilities under this section.
IV. The following individuals are exempt from the requirements of the OSHA 10-hour construction safety program:
(a) Law enforcement officers involved with traffic control or jobsite security.
(b) Flagging personnel who have completed the training required by the department of
(c) All relevant federal, state, and municipal government employees and inspectors.
(d) All individuals who are not considered to be on the site of work under the federal Davis-Bacon Act, including, but not limited to, construction and non-construction delivery personnel and non-trade personnel.
326:2 Safety Enhancement Program. Amend RSA 281-A:67, I to read:
I. There is hereby established a safety enhancement program to be administered by the commissioner. Such program shall include regional training and safety seminars open to all interested employees and employers.
326:3 Applicability. Sections 1 and 2 of this act shall apply to all contracts entered into on or after July 1, 2008.
326:4 Commission Established. There is established a commission to recommend a comprehensive program for increasing the use of passenger restraints in New Hampshire in order to reduce motor vehicle fatalities and serious injuries and the resulting costs.
326:5 Membership and Compensation.
I. The members of the commission shall be as follows:
(a) Two members of the senate, appointed by the president of the senate.
(b) Three members of the house of representatives, appointed by the speaker of the house of representatives.
(c) The commissioner of the department of safety, or designee.
(d) The commissioner of transportation, or designee.
(e) One member of the automobile insurance industry, appointed by the insurance commissioner.
(f) One member from the New Hampshire Medical Society, appointed by the society.
(g) One member from the New Hampshire Emergency Nurses Association, appointed by such association.
(h) One member from the New Hampshire Association of Emergency Medical Technicians, appointed by such association.
(i) One representative of the New Hampshire Association of Fire Chiefs, appointed by such association.
(j) One representative of the New Hampshire Association of Chiefs of Police, appointed by such association.
(k) One member of the Brain Injury Association of New Hampshire, appointed by such association.
(l) One member of the New Hampshire Civil Liberties Union, appointed by such organization.
(m) One member of Safe Kids New Hampshire, appointed by such organization.
(n) One member from the American Automobile Association, appointed by such association.
(o) Two members of the public, one of whom shall have expertise in the field of public relations, appointed by the president of the senate.
(p) Three members of the public, one of whom shall be a member of an organization that opposes mandatory seatbelts, appointed by the speaker of the house of representatives.
(q) One member of the public, appointed by the governor.
II. Legislative members of the commission shall receive mileage at the legislative rate when attending to the duties of the commission.
326:6 Duties.
I. The commission shall:
(a) Study the most effective strategies to increase the use of passenger restraints.
(b) Analyze the insurance costs and economic benefits of increasing the use of passenger restraints.
(c) Review the fatality and injury data regarding the health benefits of increased use of passenger restraints.
(d) Identify public education strategies that would encourage voluntary use of passenger restraints.
(e) Make recommendations for legislation to advance the goal of increasing the use of passenger restraints.
II. The commission may accept grants, donations, and other moneys from any source and may expend such moneys for the purposes of the commission.
326:7 Chairperson; Quorum. The members of the commission shall elect a chairperson from among the members. The first meeting of the commission shall be called by the first-named senate member. The first meeting of the commission shall be held within 45 days of the effective date of this section. Eight members of the commission shall constitute a quorum.
326:8 Report. The commission shall report its findings and any recommendations for proposed legislation to the president of the senate, the speaker of the house of representatives, the senate clerk, the house clerk, the governor, and the state library on or before July 31, 2008.
326:9 Effective Date.
I. Sections 1-3 of this act shall take effect 60 days after its passage.
II. The remainder of this act shall take effect upon its passage.
Approved: July 16, 2007
Effective: I. Sections 1-3 shall take effect September 14, 2007.
II. Remainder shall take effect July 16, 2007.
For on-site training in New Hampshire please visit OSHA Training in New Hampshire
Set Up A Corporate Billing Account
Corporate billing accounts enable your organization and your clients to utilize a special code for multiple employees and students that may need training. When your students or the client's students register for online safety training courses they will enter their corporate billing code and it will not be necessary for them to enter any of their credit card information.
By utilizing a corporate billing code, an invoice will be sent in the middle of each month and can be charged to a Purchase Order # or to a credit card of choice.
Corporate billing accounts can also be tracked.
Set up A Corporate Account by FAX
Follow these 3 simple steps:
- 1. Download our corporate billing document .
- 2. Fill out the form.
- 3. FAX it back to us at (951) 698-3552.
We will call you to confirm that your account has been setup successfully. You will reveive a "Special Code" which will allow your employees or clients to log in and begin their training immediately. You will be billed according to the agreement.


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Outdoor Lighting Plans When Building or Remodeling
by Lani Furbank
When you're building a new house or having remodeling work done on your existing home, it seems like there are a million and one things to consider. From tile choices and paint colors to ceiling heights and window placement, it's easy to get caught up in the seemingly endless list of decisions. But, there's one important detail you could be forgetting…outdoor lighting.
Even if you're unsure whether you want to install outdoor lighting right away, you should think ahead and plan for the lighting work while you're in the construction phases. Having a contingency plan will save you time and money in the long run. Trust us, you'll be grateful if and when you decide to add lighting!
Here are five tasks that homeowners should do when they're building or remodeling.
What homeowners need to do during the construction phase, to save money on outdoor lighting.
Plan Ahead For Outdoor Lighting: 5 Things You Need To Do When Building or Remodeling Your Home
1. Plan for electricity outside.
This one seems rather basic, but you'd be surprised how many people don't consider this crucial step. In many new homes, there is only one outlet in the garage. Building code requires just one, and most people don't think they'll need more than one. After all, one outlet is typically enough for a fridge, freezer, or work station.
However, for outdoor lighting, you'll need a dedicated outlet. You should ideally run an additional outlet in an unobtrusive space, such as on an outside wall. This will also be useful for any irrigation needs.
2. Have a controlled outlet.
After planning a dedicated outlet, you also need to think about whether or not you want your landscape lights on a switch. If you do, you'll need a controlled outlet. It's much easier and less expensive to put this in while you're building or remodeling, rather than after the construction is finished. Place the outlet in a hidden location, because you don't want a transformer exposed on the front of the house or on the deck where you'll be spending a lot of time.
3. Plan ahead for wiring under pavement.
Digging under pavement to run wire is challenging and time-consuming. Anytime you're laying or repairing a walkway or driveway, have a couple of PVC pipes put in to act as sleeves for future wiring. Place them in an easy to find spot – line them up with memorable landmark or stationary feature of the house, such as the corner of the house or by the front walkway – and then take pictures documenting where they are located for future use. These sleeves will save you money on the cost of installing landscape lights by eliminating the costly step of having your lighting contractor rent a boring machine in order to dig under the pavement.
4. Think about how to integrate lighting into your existing automation system.
It's a growing trend that more homes have automation systems these days, whether they are control4, DIY, or something set up through your security company. If you have an automation system, make a plan to integrate your new lighting. This means you'll want to place the transformer in a location where the signal is strong. Otherwise, you could have to deal with glitches in your lighting automation.
5. Consider how to incorporate lighting into masonry work.
Adding lighting into masonry work after it is built is not an easy feat. To save you and your lighting contractor a whole lot of trouble, plan your lighting while the stone work and building is being done. When installing lighting into stone or concrete, make sure you choose high-quality, long-lasting lighting fixtures, like our solid brass Sterling Lighting line. Cheap aluminum fixtures will quickly degrade in concrete. Your stonework is built to last, so your lighting should be, too.
If you're unsure of how to plan for outdoor lighting on your property, give Enlightened Lighting a call and we can help answer your questions! We want to help make the process of installing lighting as easy and affordable as possible.
Related Articles
Why More Homebuilders Are Offering Outdoor Lighting
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How To Get Your Lighting Project Approved by Your HOA
Organized under Home Improvement, landscape lighting, landscape lighting, pool lighting, patio lighting, lighting design, Outdoor Lighting Contractors. Labeled as automation, building, construction, electricity, home improvement, homeowners, landscape lighting, Lani Furbank, lighting, masonry, Outdoor lighting, outlets, planning ahead, remodeling, Sterling Lighting.