text:
- Research article
- Open Access
Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina
- Jennifer J Stanke1,
- Bret Lehman2 and
- Andy J Fischer1Email author
© Stanke et al; licensee BioMed Central Ltd. 2008
- Received: 10 July 2007
- Accepted: 06 February 2008
- Published: 06 February 2008
Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT).
We found that as ChAT-immunoreactive cells differentiate they expressed the homeodomain transcription factors Pax6 and Islet1, and the cell-cycle inhibitor p27kip1. As differentiation proceeds, type-II cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells in the inner nuclear layer did not. Although there is a 1:1 ratio of type-I to type-II cells in vivo, in dissociated cell cultures the type-I cells (ChAT-positive and CRABP-negative) out-numbered the type-II cells (ChAT and CRABP-positive cells) by 2:1. The relative abundance of type-I to type-II cells was not influenced by Sonic Hedgehog (Shh), but was affected by compounds that act at muscarinic acetylcholine receptors. In addition, the abundance and mosaic patterning of type-II cholinergic amacrine cells is disrupted by interfering with muscarinic signaling.
We conclude that: (1) during development type-I and type-II cholinergic amacrine cells are not homotypic, (2) the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and (3) appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning.
- Retina
- Carbachol
- Amacrine Cell
- Cyclopamine
- Inner Plexiform Layer
Amacrine cells are a distinct class of retinal neuron that participate in the processing and refinement of visual information. The amacrine cells receive input from other amacrine cells and bipolar cells, release inhibitory neurotransmitters (GABA and/or glycine) at synapses that are formed with ganglion cells and other amacrine cells, and participate in retinal image processing. Amacrine cells are a highly diverse class of neuron; there may be as many as 30 distinct types [1, 2]. The classical findings of Cajál [3] and additional work [4–14] suggest that there may be as many as 30 different types of amacrine cells in the avian retina. Although many studies have identified mechanisms that promote or suppress amacrine cell fate, little is known about the mechanisms involved in the differentiation of specific types of amacrine cells. The factors that instruct cells from a pool of amacrine-fated neurons to form particular types of amacrine cells remain largely unknown. In this study we use the cholinergic cells in the embryonic chick retina as a model system to study the mechanisms involved in the differentiation of one particular type of amacrine cell.
Cholinergic neurons use acetylcholine (ACh) as a transmitter and are found in the retina of all vertebrate classes. Cholinergic amacrine cells have somata located at the proximal margin of the inner nuclear layer (INL; type-I) and displaced to the ganglion cell layer (GCL; type-II) with processes confined to two strata in the inner plexiform layer (IPL) [15–23]. Type-I and type-II cholinergic amacrine cells are arrayed in a mosaic pattern with near-mirror symmetry around a horizontal plane through the IPL [18, 19]. However, the patterned spacing of cells within one layer is independent of the spacing of the cells in the other layer [24]. The retinas of birds and reptiles contain a third type of cholinergic amacrine cell, with somata located near the middle of the INL and processes diffusely distributed in sub-laminae 1 through 4 of the IPL [21]. Furthermore, avian type-III cholinergic amacrine cells can be segregated into 2 subtypes: type-IIIa cells that contain enkephalin, neurotensin, and somatostatin immunoreactivities, and type-IIIb cells that do not [4].
During embryonic development, type-I and type-II cholinergic amacrine cells express ChAT (the biosynthetic enzyme that produces ACh) as early as embryonic day 6.5 (E6.5), whereas ChAT-immunoreactivity in type-III cells is not detected until 6 days prior to hatching at about E15 [23]. Cholinergic amacrine cells arise from a pool of undifferentiated post-mitotic neuronal cells and begin to differentiate in the middle of the presumptive IPL where they, coincidently, accumulate GABA [23, 25]. Although a great deal is known about the morphological and temporal development of these amacrine cells, little is known about the genes that they express during differentiation and the factors that influence their development. However, it has recently been shown that extra-cellular ATP and the P2X receptor coordinate, in part, the mosaic patterning and survival of the cholinergic cells in the rodent retina [26] and that visual activity is required for maintenance of the mosaic after eye opening in the mouse [27].
The purpose of this study was to better characterize the development of type-I and type-II cholinergic amacrine cells in the chick retina and identify factors that influence their development. We find that the type-I and type-II cholinergic amacrine cells express p27kip1, Pax6 and Islet1 during development. In addition, we show that these cells are not homotypic during embryonic development; type-II cells transiently express elevated levels of cellular retinoic acid binding protein (CRABP) and neurofilament, while type-I cells do not. Furthermore, we show that the relative abundance of type-I and type-II cholinergic cells is influenced by local microenvironment and signalling through muscarinic acetylcholine receptors. We propose that autologous signalling between cells acts to drive the differentiation and mosaic patterning of distinct types of retinal amacrine cells.
Cholinergic amacrine cells express Islet1 and Pax6 as they differentiate
In the developing rodent retina, cholinergic amacrine cells are known to express Islet1 [28]. Islet1 is a homeotic LIM-domain transcription factor that is known to be expressed by bipolar, horizontal, and ganglion cells in the retina [29]. In the developing avian retina at E6, Islet1-immunoreactivity was detected in the nuclei of presumptive ganglion and amacrine cells in the GCL and in differentiating cells in the distal retina (Figs. 1a and 1b). The Islet1-positive nuclei in the distal retina presumably were post-mitotic neurons migrating away from the ventricular surface of the retina to their final position within the retina. At E6, Islet1 was expressed by ChAT-immunoreactive cells prior to the formation of the presumptive IPL (Figs. 1c–e). These cells were intermingled with Islet1-expressing cells in the GCL, suggesting that the cholinergic cells begin to differentiate among a heterogeneous pool of post-mitotic Islet1+/ChAT- cells. At E7, Islet1-positive nuclei were found in the GCL, putative differentiating bipolar cells in distal layers (not shown) of the retina, and two rows of cells between the GCL and bipolar cells (Figs. 1f–h). The Islet1-positive nuclei at the vitread border of the INL and within the IPL were those of type-I and type-II cholinergic amacrine cells, respectively (Figs. 1f–h). At E9 and E11, the Islet1/ChAT-positive cells remained as distinctive rows of cells (Figs. 1i–n). By E16 the somata of the type-II cells had migrated into the GCL. In the retina of postnatal chickens, Islet1-immunoreactivity was present in horizontal cells, many bipolar cells, most if not all ganglion cells, and type-I and type-II cholinergic amacrine cells, but not type-III cells (Fig. 1o).
The cell cycle inhibitor p27kip1 has been shown to be important for the development of the retina and differentiation of Müller glia [34–36]. In E7 retina, immunoreactivity for p27kip1 was seen in numerous nuclei scattered throughout the retina, but was absent from the nuclei of proliferating progenitors in distal layers of the retina (Figs. 2g–i). The distribution of p27kip1-immunoreactive nuclei in E7 retina was consistent with the known location of differentiating cells. At this stage of development, immature ChAT-positive cells contained p27kip1-immunoreactive nuclei (Figs. 2g–i). At E11, numerous p27kip1-positive nuclei were observed in the INL, IPL and GCL (Fig. 2j–l). The p27kip1-positive nuclei in the IPL were immunoreactive for ChAT, and the labeling intensity of these nuclei was greater than that seen in the nuclei of non-cholinergic cells of the INL and GCL (Figs. 2j–l).
Cholinergic amacrine cells transiently express neurofilament and CRABP as they differentiate
Differentiation of cholinergic amacrine cells in dissociated cell cultures
Since the type-II cholinergic cells are in close proximity (<15 μm) to developing ganglion cells, whereas the type-I cells are further away (>30 μm), it is possible that signals provided by the differentiating ganglion cells influence the development and phenotype of the type-II cells. Ganglion cells are known to produce Sonic Hedgehog (Shh) as they differentiate [40, 41] and Shh is known to establish morphogenic gradients over short distances [42, 43]. Thus, it is possible that Shh stimulates type-II cholinergic amacrine cells to transiently express elevated levels of CRABP during embryonic development. However, the relative abundance of type-II cells was not significantly affected by the addition of Shh or the blockade of Shh-signaling with KAAD (3-keto, N-amino-ethyl aminocaproyl dihydrocinnamoyl) cyclopamine. Shh-treated samples contained 3.1% (± 10.8%; 151 cells) more type-II cells, and cultures treated with KAAD-cyclopamine contained 6.7% (± 5.7%; 140 cells) fewer type-II cells compared to control preparations. Statistical analyses indicated that these changes were not significant.
There is some evidence that type-II cholinergic amacrine cells express muscarinic receptors in the postnatal retina [44]. In addition, antibodies to the m2 and m4 isoforms of the muscarinic receptor in the embryonic chick retina appear to label type-II-like cells in the developing IPL [45, 46]. Thus, it is possible that the type-II cholinergic amacrine cells communicate to each other through paracrine muscarinic interactions. To test this hypothesis we applied a muscarinic antagonist (atropine) or agonist (carbachol) to cultures of E7 retinas. We found that activation and suppression of muscarinic signaling reduced the relative abundance of type-II cholinergic amacrine cells (Fig. 5f).
Since the relative abundance of type-II to type-I cholinergic amacrine cells is reduced by in vitro conditions and by influencing muscarinic signaling, we sought to assess whether similar mechanisms influence the phenotype of cholinergic cells in the intact retina. To test this, we made intraocular injections of 300 ng of atropine (a muscarininc antagonist) or 200 ng carbachol (a muscarinic agonist) into the vitreous chamber of E7 chick embryos. The E7 eye was estimated to have 75 mm3 of volume, which should have resulted in initial maximum vitreal concentrations of carbachol and atropine of about 25 μM. Atropine and carbachol have been used from 15 nM to 100 mM to elicit effects on retinal cells and chick muscarinic acetylcholine receptors (mAChR) [47–49].
In addition to a decrease in the percentage of type-II cholinergic amacrine cells, we found that the density of ChAT-positive cells was decreased by treatment with both a muscarinic AChR antagonist and agonist (ANOVA p = 0.0013, GCL; p = 0.0004, INL; Fig 6h). The number of ChAT+ cells per 10,000 μm2 in the INL (type-I cells) and in the GCL (type-II cells) was significantly decreased by intraocular injections of atropine (p = 0.024, INL; p = 0.035, GCL; n = 6). Similarly, treatment with the muscarinic agonist carbachol decreased the density of cholinergic amacrine cells in the INL and GCL (p = 0.0011, INL; p = 0.002, GCL; n = 4). Decreases in the density of cholinergic cells were not caused by increased apoptosis; we failed to find increased numbers of cleaved caspase3-positive cells in retinas treated with atropine or carbachol (Figs. 6i–k). Additionally, the muscarinic ligands did not have any obvious effect on non-cholinergic neurons; ganglion cells labeled for Brn3a and horizontal, bipolar, amacrine and ganglion cells labeled for calretinin displayed normal distributions when treated with atropine or carbachol (Figs. 6l and 6m).
The genes that are expressed by differentiating cholinergic amacrine cells are distinctly different from those that are expressed by other types of amacrine cells. Unlike other types of amacrine cells, the cholinergic cells express Islet1 with the onset of differentiation and into the mature retina, consistent with reports in the embryonic rat retina [28]. As the cholinergic amacrine cells differentiate they express proteins common to other types of amacrine cells such as Pax6, CRABP and p27kip1. However, the relative levels of immunolabeling for these proteins within the cholinergic cells are either decreased (Pax6) or increased (CRABP and p27kip1) compared to levels seen in other types of differentiating amacrine cells. It is likely that Islet1 and Pax6 play important roles in the development and maintenance of phenotype of the type-I and type-II cholinergic amacrine cells. The significance of elevated expression of p27kip1 and CRABP in the cholinergic cells remains unknown.
The type-I and type-II cholinergic amacrine cells are equally abundant, morphologically identical, and are arranged with near-mirror symmetry across a horizontal plane through the IPL [18, 19]. In addition to using acetylcholine, type-I and type-II cholinergic amacrine cells are known to utilize GABA as a neurotransmitter [50, 51] and begin to accumulate GABA soon after they begin to differentiate [25]. Furthermore, type-I and type-II cholinergic amacrine cells in the chick retina have been shown to originate from a common pool of migrating, post-mitotic undifferentiated neurons [25]. These findings have lead to the assumption that these cells are phenotypically identical and differ only by the position of their somata. However, we provide evidence that these two cell types differ by more than the location of their cell bodies. We found that during differentiation, type-II cells transiently expressed high levels of CRABP and neurofilament, whereas the type-I cells do not. Further, in the postnatal chick retina, type-II cells may express mAChR4, whereas type-I cells do not [44, 52], and these cells have different sensitivities to excitotoxins, suggesting that different types of glutamate receptors are expressed by these cells, and/or that they have different susceptibilities to large fluctuations in ion concentrations [4]. Embryonic expression of mAChR2, mAChR3, and mAChR4 appear in non-overlapping laminae of the developing IPL at E9 [52], suggesting that these receptors may be expressed by different types of amacrine cells. The mAChR1 may not exist in the chick and [53] the retinal expression of mAChR5 [54] remains uncertain, but has been detected in the sclera of Guinea pigs [55, 56] and human [57]. Differential expression of receptor isoforms is a possible mechanism to explain the differing effects of muscarinic ligands on the type-I and type-II cholinergic cells.
The cholinergic cells may be among the first types of amacrine cell to differentiate, with the onset of ChAT and Islet1 expression occurring while these cells are interspersed among the developing ganglion cells at E6. By comparison, amacrine cells that utilize different neurotransmitters such as dopamine, glucagon, enkephalin or vasoactive intestinal peptide differentiate 6–9 days after the cholinergic cells [58]. As development proceeds, the Islet1/ChAT-positive cells migrate away from the ganglion cells into the presumptive IPL where they begin to differentiate. When the type-I and type-II cholinergic cells become segregated into different laminae, the type-II cells begin to express elevated levels of CRABP and neurofilament. The close proximity of type-II cells to developing ganglion cells may expose these cells to environmental cues that influence the differentiation of ganglion cells. Accordingly, this microenvironment may promote the expression of genes common to both ganglion and type-II cells (including Pax6, Islet1 and neurofilament). Secreted signals produced by ganglion cells that influence the type-II cholinergic cells could include Shh [40, 41] and BMP2/7 [59]. However, we failed to find evidence that Shh influences the differentiation of type-II cholinergic cells.
We found that the 1:1 ratio of type-I to type-II cholinergic cells from the intact retina was disrupted when the cells were dissociated and grown in culture; type-I cells were twice as abundant as the type-II cells in vitro. In our in vitro paradigm, it is likely that CRABP expression is down-regulated by the type-II cells making them indistinguishable from the type-I cells. The disruption of the in vivo microenvironment did not favor the differentiation of type-II cholinergic cells – the co-expression of ChAT and CRABP. These findings suggest that the factors promoting the type-II phenotype are diluted under culture conditions and that the phenotype of type-II cells relies, in part, on the microenvironment provided by the intact retina. Alternatively, the survival of type-II cholinergic amacrine cells may be selectively compromised by acute dissociation and culture conditions (i.e. serum). It is unlikely that changes in the relative abundance of type-I to type-II cholinergic cells resulted from cell fate decisions made during the terminal mitosis. We did not find any cholinergic cells in our in vitro and in vivo paradigms that were labeled for BrdU, indicating that none of the cholinergic cells were generated after the cultures were established or after compounds were delivered to the developing eye.
Similar to the results of our in vitro studies, we found that the in vivo development of the cholinergic amacrine cells was influenced by muscarinic ligands. In saline-treated retinas, the vast majority of cholinergic cells in the GCL express CRABP. However, treatment with muscarinic ligands resulted in increased numbers of type-II cells that failed to express CRABP. These findings suggest that appropriate levels of muscarinic signaling are required for the maintenance of CRABP expression in the type-II cells; perturbation of optimal levels of muscarinic signaling between cholinergic cells by agonist or antagonist disrupted the phenotype of these cells. It remains uncertain whether the effects of the muscarinic ligands were mediated by direct or indirect actions on the type-II cells. The patterning of the cholinergic amacrine cells is likely coordinated by several distinct signaling pathways in addition to the muscarinic pathway. For example, extra-cellular ATP and the P2X receptor influence the patterning and survival of the cholinergic cells in the rodent retina [26]. In addition, Zhang and colleagues [60] have shown that maintenance of array of type-II amacrine cells in the mouse GCL is dependent upon visual activity; after 30 days of rearing pups in the dark, ChAT-positive cells are undetectable in the GCL, but remain present in the INL.
Inhibiting mAChRs with atropine or activating mAChRs with carbachol disrupted the regular spacing between the cholinergic amacrine cells. We propose that the proper patterning of the cholinergic cells requires optimal levels of muscarinic signaling similar to the Shh concentration-dependent differentiation of spinal cord interneurons [61]. In principle, release and diffusion of ACh from a cholinergic cell should result in a concentration gradient that decreases with increasing distance from the source. Regular spacing between cells can occur if like-cells are repelled from each other; and for the cholinergic amacrine cells this may involve migrating laterally away from neighboring, concentrated sources of ACh. Our data are consistent with the hypothesis that the regular spacing of the cholinergic cells requires an optimal level of muscarinic signaling, and if these cells encounter gains or losses in signaling the mosaic patterning is disrupted.
It remains uncertain what effect perturbations in muscarinic signaling have on the spontaneous calcium waves, spread through the cholinergic amacrine cells, that are necessary for the establishment of early retinal circuitry in the IPL [27, 62]. The mosaic pattern of the cholinergic cells may, in part, be required to establish normal waves of activity. However, this activity was shown to be regulated by nicotinic AChR in the embryonic retina [27, 63]. It is possible that the application of muscarinic compounds generally perturbed retinal development and thereby disrupted the patterning of the cholinergic cells. However, we found that muscarinic compounds did not affect non-cholinergic cells expressing calretinin, Islet1 or Brn3a-positive ganglion cells, and we failed to observe an increase in immunolabeling for cleaved-caspase 3. Taken together, these findings indicate muscarinic ligands do not overtly affect non-cholinergic inner retinal neurons.
The type-I and type-II cells are not likely to influence each other through cholinergic signaling across the developing IPL. For example, the mosaic pattern of type-II cholinergic amacrine cells in the GCL has no relation to the pattern of type-I cells in the INL [24]. Additionally, acetylcholine esterase (AChE) is first detected in the chick retina between E7 and E9 in the middle of the IPL when the differentiating cholinergic cells are segregating between the GCL and INL [48, 64]. AChE that is distributed into discrete laminae in the IPL should prevent any vertical diffusion of ACh through the retina. For example, ACh released from the type-II cells is not likely to act on type-I cells because AChE in the IPL is stratified between the dendrites of these cells and should degrade the ACh that diffuses distally through the IPL. However, given the dense intermingling of the dendrites of the cholinergic cells within narrow strata of the IPL, it seems likely that ACh released from the dendrites of one cholinergic cell could act on a neighboring cell with overlapping, co-stratified dendritic arbors.
It is possible that decreases in the density of cholinergic cells resulted from increases in cell death. However, we failed to detect increased numbers of cleaved-caspase 3 positive cells in retinas treated with carbachol or atropine. We cannot exclude the possibility that the death of cholinergic cells occurred in a caspase 3-independent manner resulting in reduced numbers of cholinergic cells and perhaps disruption of the mosaic patterning. Another possibility is that decreases in the abundance of ChAT/CRABP-positive cells in the IPL resulted from the down-regulation of ChAT and CRABP. Consistent with this hypothesis, ChAT may be transiently expressed by some cells in the GCL similar to the developing turtle retina, indicating that ChAT expression may be reversible in some types of retinal cells [65].
The large diversity of amacrine cell types that are distributed in mosaic patterns with regular spacing implies that autologous signals are required to coordinate the patterning of unique cell types. The notion of homotypic interactions underlying mosaic patterning has been proposed for the cholinergic amacrine cells in the retina [65]. Furthermore, Rossi and colleagues [66] demonstrated that the establishment of regular spacing between horizontal cells in the rodent retina occurs independent of pre-synaptic input from photoreceptors, suggesting that patterning is mediated via homotypic interactions among the horizontal cells. It is possible that homotypic interactions are responsible for the regular spacing of the different types of horizontal, amacrine and ganglion cells. Consistent with this hypothesis, cones, horizontal cells, ganglion cells, and cholinergic amacrine cells migrate laterally away from their radial column of clonally derived cells, whereas bipolar cells and Müller glia do not [67]. To establish a highly ordered mosaic pattern, cells within the retina must have the ability to migrate laterally away from columns of clonally derived cells unless the order of the array is determined by the progenitors. A likely mechanism underlying the spatial patterning between like-cells is the use of peptide or amino acid-derived transmitters to provide homotypic signals between cells. For example, dopamine, glucagon, neuropeptide Y, substance P, vasoactive intestinal polypeptide or enkephalin, neurotransmitters that are used by distinct types of amacrine cells, are used as autologous signals to coordinate cell type-specific patterning. Consistent with this hypothesis, our data indicate that the disruption of muscarinic ACh signaling perturbs the numbers, spacing and phenotype of cholinergic amacrine cells in the developing retina.
We conclude that, during development, type-II cholinergic amacrine cells transiently express CRABP and neurofilament, while type-I cells do not. We propose that the transient expression of CRABP and neurofilament is regulated by local microenvironment. Additionally, we find that interfering with muscarinic signaling decreases the relative number of type-II cells and mosaic patterning of the cholinergic amacrine cells. We propose that the phenotypic differences between developing type-I and type-II cholinergic amacrine cells are elicited, in part, by paracrine cholinergic signaling; the local microenvironment of the in tact retina allows precise muscarinic signaling to establish proper cellular phenotypes and organization of the type-II cells. It remains uncertain whether muscarinic receptors are differentially expressed by the type-I and type-II cells.
The use of animals in these experiments was in accordance with the guidelines established by the National Institutes of Health and the Ohio State University. Eggs were obtained from the Department of Animal Sciences at the Ohio State University. Chick embryos were staged according to guidelines established by Hamburger and Hamilton [68]. Newly hatched leghorn chickens (Gallus gallus domesticus) were obtained from the Department of Animal Sciences at the Ohio State University and kept on a cycle of 12 hours light, 12 hours dark (lights on at 7:00 am). Chicks were housed in a stainless steel brooder at about 27°C and received water and Purina™ chick starter ad libitum.
Cell Culture
Embryonic chick retinas were dissected in sterile Hanks' buffered saline solution added with 3% D-glucose and 0.01 M HEPES buffer (HBSS+). Retinal cells were dissociated by mild trituration after a 10-minute incubation at 37°C in Ca2+/Mg2+-free HBSS plus 0.025% trypsin. Cell density was determined by using a hemocytometer. Between 50,000 and 200,000 cells were added to wells of a 24-well plate coated with poly-D-lysine (Sigma). Cell cultures were maintained at 37°C in 5% CO2 under culture medium (DMEM:F12 without glutamate or aspartate, plus 100 units/ml penicillin, 100 mg/ml streptomycin, 0.05 M HEPES; Sigma, and 1% fetal bovine serum; Invitrogen). Cultures were maintained for 1 to 4 days with 50% of the medium replaced every 24 hours. After one day in vitro, retinal cells were added with 20 ng/ml Sonic Hedgehog (rmShh-N; R & D Systems), or 40 ng/ml KAAD (3-Keto-N-amino ethyl amino caproyldihydrocinnamoyl Cyclopamine; Toronto Research Chemicals), 1, 10 or 50 μM of atropine (Sigma) or 1, 10 or 50 μM of carbachol (Sigma). Additional cultures were maintained for 3 DIV in the presence of 1 μg/ml Bromodeoxyuridine (BrdU). Cells were fixed and processed for immunocytochemistry at 1 or 3 days after the addition of Shh, cyclopamine, atropine, or carbachol.
In ovo injections
Time-staged eggs at E6 were windowed and access to the eye obtained by carefully tearing the chorioallantois and amniotic membranes around the embryo. The stage of the embryo was verified at this time according to the guidelines established by Hamburger and Hamilton (1951). A 40 gauge wire was used to pierce the dorsal side of the right eye. A pulled capillary pipette was then used to deliver 2 μl of a solution that contained 1 mM (200 ng) carbachol, 1 mM atropine (300 ng), or sterile saline solution (control) added with 0.4% fast green in glycerol water (1:1) and 3 μg BrdU. The chorionic membranes were placed back over the embryo, the window sealed with transparent tape, and the egg placed back into the incubator. Embryos were sacrificed 24 hours later and the injected eye enucleated. The anterior portion of the eye and vitreous were removed and discarded prior to fixation. After fixation, the ventral portion of the retina was removed and labeled for BrdU to verify the efficacy of the injection and to verify that the injection was made after terminal mitosis of the cells fated to differentiate as cholinergic amacrine cells. Retinas that labeled for BrdU in the GCL or presumptive IPL were discarded from the experiments. The remaining retina was processed for whole-mount immunolabeling.
Fixation and sectioning
Eyes were enucleated, the anterior portion of the eye dissected away, and the vitreous removed. Eyes or dissociated cells were fixed for 30 minutes at 20°C in 4% paraformaldehyde plus 3% sucrose in 0.1 M phosphate buffer, pH 7.4. Fixation was followed by 3 washes in PBS (phosphate-buffered saline; 0.05 M phosphate buffer, 195 mM NaCl, pH 7.4). Eye-cups were cryoprotected in PBS plus 30% sucrose, soaked in embedding medium (O.C.T.-compound; Tissue-Tek) for 30 minutes, and freeze-mounted onto aluminum sectioning blocks. Transverse sections nominally 12 μm thick were cut consistently from the posterior pole of the eye, near the dorsal portion of the ventral fissure (or pecten in postnatal eyes), and thaw-mounted onto SuperFrost Plus™ slides (Fisher Scientific). Sections were air-dried and stored at -20°C.
Antigen, species, immunogen, working dilution, and source of the antibodies used in the current study.
Working dilution
3B5/Developmental Studies Hybridoma Bank
Bromdeoxyuridine (BrdU)
BUI/75/Accurate Chemicals and Scientific Corporation
Choline acetyltransferase (ChAT)
1456/Dr. Miles Epstein, University of Wisconsin
Cellular Retinoic Acid Binding Protein (CRABP) (CRABP)
C1/Dr. Jack Saari, University of Washington
40.2D6/Developmental Studies Hybridoma Bank
Neurofilament (NF) (160 kDa)
RMO270/Dr. V. Lee, University of Pennsylvania
Clone 57/Transduction Laboratories
PAX6/Developmental Studies Hybridoma Bank
Dr. Stanislov Tomarev, NIH
Photography, measurements, cell counts, and statistical analyses
Photomicrographs were taken by using a Leica DM5000B microscope equipped with epifluorescence and a Leica DC500 digital camera. Confocal microscopy was done by using a Zeiss LSM 510 meta at the Campus Microscopy and Imaging Facility at Ohio State University. Confocal stacks of images were obtained for 1 μm-thick optical sections by using a 20× objective (0.75 NA) and multi-track, narrow-pass emission filter settings to exclude the possibility of fluorescence bleeding across channels. Images were optimized for color, brightness and contrast, and double-labeled images overlaid by using Adobe Photoshop™6.0. To avoid the possibility of region-specific differences within the retina, cell counts were consistently made from the same region of retina for each data set. Significance of difference among data sets was determined by using ANOVA. Data from treated and control eyes were compared statistically with the appropriate post-hoc Student's t-test.
Cell counts from cultured cells were performed on at least 100 cells from each coverslip per condition (n ≥ 5; untreated, Shh, KAAD, atropine and carbachol). Experiments were repeated three times. For whole mount images, one optical slice from a confocal z-series was selected for the INL and one for the GCL per individual. These slices were overlaid in Adobe Photoshop 6.0 and five 200 μm2 fields per retina were counted. At least four individual retinae and at least 600 cells per retina were counted per condition. As described elsewhere [70, 71], an index of regularity was calculated as the mean distance to the nearest neighbor divided by the standard deviation. The distance between cells was measured from confocal micrographs and was taken as the distance from the center of one cell to the center of the next nearest cell. Measurements were made from ten randomly selected cells per field of view for three individual retinas per condition (90 cells total). Data is presented as the mean and standard deviation.
The authors would like to thank Kanika Ghai for comments that contributed to the final version of this manuscript and technical assistance provided by Patrick Sherwood. J Spence and K Del-Rio Tsonis provided technical training essential to the success of this project. Confocal microscopy was done at the Campus Microscopy and Imaging Facility at The Ohio State University. The Islet-1 and Pax6 antibodies developed by Drs. T. Jessel and A. Kawakami, respectively, were obtained from the Developmental Studies Hybridoma Bank developed under auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by grants from the National Science Foundation (0413795), the National Institutes of Health (NEI, EY016043) and start-up funds from the Ohio State University to A.J.F.
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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Welcome to NEXT Village San Francisco!
NEXT Village SF is a neighborhood nonprofit providing services and support that empower members to live independently in their own homes as they age. Through regular get-togethers, local field trips, assistance with errands and more, our volunteers and staff work together to enrich, enhance, and nourish the lives of seniors in our community. As a vibrant, growing network for aging adults in northeast San Francisco, Next Village is connection you can count on. WATCH a quick video about NEXT Village SF.
"I feel lucky and so much more at peace with my aging process just by being a member and knowing that they are there." - John S.
Join NEXT SF
A membership in Next Village SF is a gateway to independence, connection, and fulfillment. If you'd like to get to know and trust your neighbors in a deeper, more meaningful way, we'd love to have you join us. You'll find a culture of mutual care that will comfort and sustain you in unexpected, delightful ways.

subset: commoncrawl


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Jules Muck was spotted painting her latest installation by GTA on Abbot Kinney a few weeks ago. The new piece and her wonderfully grinchy green is viewable from inside the courtyard by GTA.
Jules Muck's Latest at GTA
June 16, 2011 By Leave a Comment
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Monday, 2 June 2014
Crasharama 2014!!
11 comments:
- Kizzy Von Doll said...
Ha, this looks super awesome!! Love it. Am glad you all enjoyed. I hope you have a gorgeous week doll xx
- 2 June 2014 at 11:48
- Castles Crowns and Cottages said...
WOW I've never seen the likes of such a race! My eight grade boys would love this! hahahahha
Hello my friend! I wish you a peaceful day. Anita
- 2 June 2014 at 11:52
- Willow said...
Oh i do love a good ole demolition derby ! My dad use to take us to them when we were kids . every now and again we will see one at the fair grounds.
- 2 June 2014 at 12:09
- Things and Thoughts said...
Never seen a show like this! I'm sure your boy has had an incredibly fascinating day!
- 2 June 2014 at 13:08
- Jen said...
Looks like a crazy fun day!
- 2 June 2014 at 15:25
- Vix said...
Proper boys' fun! xxx
- 2 June 2014 at 17:27
- Kelly Jackson said...
Definitely a boy thing. Is this what I'm in for? :)
Too bad we'll miss you in Annecy. It's such a wonderful place to visit; I'm sure we'll bring the little one back when he's older. Xo
- 2 June 2014 at 19:19
- Amy at love made my home said...
My goodness, what a contrast of two days!! Glad that you had such a good time on each of the days though. xx
- 2 June 2014 at 22:07
- greenthumb said...
Looks like lots of fun, I bet the boys loved it.
- 3 June 2014 at 10:20
- nilly said...
I think I'll give the smashing cars a miss - too exciting for my delicate constitution. But I hope we can get to the Bridport market soon. It's one of our favourite places.
- 4 June 2014 at 21:55
- Kristie Franklin said...
That looks like so much fun! Despite the smoke, screeching and banging around, I can just imagine that lil man really enjoyed this! Heck, I think I would have really liked this! LOL
- 14 June 2014 at 21:40


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Sexual Orientation Discrimination Under Title VII: Exploring the Developing Circuit Split
Is sexual orientation discrimination the same as gender discrimination? This is currently a very important topic, and we continue our explanation of what the courts are doing with it.
Last month, we reported on how the United States Court of Appeals for the Seventh Circuit became the first federal appellate court to hold that discrimination on the basis of sexual orientation is covered by Title VII of the Civil Rights Act of 1964. But not only did the Seventh Circuit become the first, it also parted ways with every other circuit to have decided the issue. Recently, the Second and the Eleventh Circuits were also urged to reconsider prior precedent. So far, those circuits have adhered to their prior decisions. Ultimately, the Supreme Court is likely to weigh in to settle this score, but for now, the state of the law remains a patchwork quilt across the nation. This post explores the Second and Eleventh Circuit decisions and discusses what may happen in the future given the split among the appellate courts.
The Second Circuit Holds Firm: Christiansen v. Omnicom Group, Inc.1
Like the Seventh Circuit, the Second Circuit (which covers Connecticut, New York, and Vermont) was recently asked to reconsider its prior holding that sexual orientation discrimination is not covered under Title VII. Unlike the Seventh Circuit, however, the Second Circuit (at least for now) has stuck with the previous line of cases.
The case at hand involved the claim of an openly gay man who is HIV-positive. During his time with the company, he was subjected to humiliating harassment. Eventually, he filed a lawsuit in federal district court arguing that he was subjected to discrimination. The district court dismissed the lawsuit, holding that sexual orientation discrimination was not covered under Title VII under the Second Circuit's established precedent.
The Second Circuit reversed, but not because it held that sexual orientation discrimination was covered. On the contrary, the panel recognized that the law was settled in its jurisdiction, and that it could not be overruled except by the entire appellate court sitting en banc, or the U.S. Supreme Court. Nevertheless, the panel concluded that the employee's claim should proceed as a gender stereotyping claim. Because the employee alleged that he was perceived by his supervisor as effeminate and submissive and that he was harassed for these reasons, gender stereotyping as discrimination on the basis of sex was possible.
It is likely that this case turned out the way it did was because one panel cannot overrule another. The opinion was per curiam, which means no one judge is attributed as the author. But the majority of the panel wrote separately to express the view that the court as a whole should revisit its precedent and hold that sexual orientation discrimination claims are covered under Title VII.
Late in April, this plaintiff petitioned for the entire court to rehear the matter en banc. Whether the entire court will take this issue up remains to be seen. But if the court takes up this request and follows the Seventh Circuit's lead, it is possible that the circuit split could continue to grow, making it even more likely that the Supreme Court will step in.
The Eleventh Circuit Declines the Invitation: Evans v. Georgia Regional Hospital2
The Eleventh Circuit, which covers Alabama, Florida, and Georgia, also recently had to decide whether sexual orientation discrimination is covered by Title VII. Unlike the Second and Seventh Circuits, however, the Eleventh Circuit had not yet itself decided this issue.
In this case, the employee argued that she was discriminated against at work on the basis of her sex and targeted for termination for failing to conform to gender stereotypes. In essence, the case asked the court to decide for the first time that sexual orientation discrimination was actionable under Title VII. On appeal, the panel explained that its hands were tied. The court ruled that binding precedent from the Fifth Circuit required it to rule that sexual orientation discrimination was not actionable under Title VII. Here is the reason for this seemingly strange result. In the early 1980s, the Fifth Circuit (which had become too large) split up into two, creating the Eleventh Circuit in the process. As part of this transition, the Eleventh Circuit has decided to adhere to those opinions of the Fifth Circuit decided prior to 1981. In this case, such a decision had been issued holding that sexual orientation discrimination was not a part of Title VII. Accordingly, the Court was compelled to abide by that precedent.
Again, however, judges wrote separately to express their own views about whether sexual orientation discrimination is a part of Title VII. The dissent put forth similar reasons to those of other judges around the country advocating for a change in the law.
Exploring the Circuit Split: What Comes Next?
Now that the Seventh Circuit has ruled that sexual orientation discrimination is actionable as a separate claim of sex discrimination under Title VII, it is at odds with these and many other circuits to consider the issue. This creates a split among the federal circuit courts, which is something the Supreme Court looks to when deciding whether to take a case. Because the Supreme Court ultimately decides federal law, if the split continues to widen, it is likely that it will need to step in and settle the issue once and for all.
For now, only those employers covered by the Seventh Circuit must comply with its ruling. However, state and local laws may apply in other jurisdictions and must be followed. And the dissenting and concurring opinions from the recent cases out of the Second and Eleventh Circuits may signal the changing tide of the law. Telios Law will keep tabs on this timely issue as it continues to be litigated across the country.
1Christiansen v. Omnicom Group, Inc., No. 16-748 (2d. Cir. Mar. 27, 2017).
2Evans v. Georgia Regional Hospital, No. 15-15234 (11th Cir. Mar. 10, 2017).
Because of the generality of the information on this site, it may not apply to a given place, time, or set of facts. It is not intended to be legal advice, and should not be acted upon without specific legal advice based on particular situations


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Trave Money started out entertaining crowds at the young age of twelve, drawing his inspiration from local Cashville artist Kool Daddy Fresh. During the time span of his mother working under Carylon Miller for Great Vizions, Miller had relocated to Atlanta to work with The Funkaholics, which well known artist Jazzy Fae was also a member of.
Trave Money at the age of fifteen continued to find and develop his own style while forming a group called Soljanize, with his cousin Brandon Harris and friend Reggie McClain. "We rocked a couple of shows at Nashville Street Fest," states Trave Money. They later split up and he continued with the name Singalo.
"I started taking my career seriously when partnered with Nathaniel Simpson A.K.A Nate Tha Great when we formed our label Letz Go Get It Ent. The label consisted of us and an artist by the name of Unkle Mike, another childhood friend of mine. Things continued to get better and I moved to ATL the year of 2004 at the age of 18. At this time I started working with The In-Control Clique which included artist from all over Atlanta, New Orleans, New York, and Chicago. With my down south flow, crazy show swag and energy things manage to to keep peeking for me and Nate Tha Great."
As the partnership continued to climb the ladder of success they were discovered at a Showcase by Columbia Records where things took a turn for the worse and a Deal was turned down by Manger Carylon Miller. Trave Money continued, "Well we looked for things to get better for us before worse. When funds got low and The Clique started to divide over who was the best and the worst everyone began going home."
Trave Money and Manger Miller moved outside of Peachtree City to keep focused. Returning home in 2005 drained and down because of the out come of events Trave Money began to hit a low. Manger Carylon Miller continued to push, adding an artists by the name of Young Joc and Miss Behavin (Miss B. from the hit song "I Know You See It). Trave Money was at a stand still while they where on the road promoting "Bottle Action" by Miss Behavin. He performed live with her at The Tennessee State University Gentry Center. "It was not long before I saw Young Joc and her take off after later getting signed by Sean Puffy Combs AKA P.Diddy in 2006. By me becoming a father and dealing with legal battles I manged to get things back on the road and in 2008." Trave Money and Nate Tha Great decided to take things on the road in his Hometown Grenada Mississippi where they got a lot of work done. By the year of 2010 the partnership had ended and Trave Money formed Heavy Ent. Trave Money stating,"Working on Self and getting my younger Artist by the name of Ealton Davis A.K.A G-Eazy on, starting back on my career hitting the ground running at a 100mph putting everything I learned from the game, Manger Carylon Miller and Boss John Smith for Marketing and Productions."
Working a few Cashville Night Clubs; Roxy's. Boots, Reverb, Love Noise etc..and learning too play too win later on of 2011 Trave Money and NateThaGreat rejoined with a label called The Premier Seat (TPS) after a huge local artist showcase . Heavy Ent,/The Premier Seat Ent. released their first mixtape, Trave Money Im Winning Vol 1..


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Drug Rehab:
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When deciding that you require aid with a chemical addiction problem you have made the first and hardest step on your path to sobriety, however the next step of finding that help might be a difficult task if you do not know where to look. This page provides you with a list of public, private, non-profit, gay, and Christian drug rehab centers and addiction specialists near Stormville, New York.
Stormville is located in Dutchess County and contains the following zip codes:
Drug Rehab Centers for Stormville, New York
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Centuries ago, in the time before electricity, the internet and siren's song of American Idol, I was a Boy Scout. Or rather, I am an Eagle Scout. During these formative years that I, like many, were inoculated with the Scout oath, laws and the mental tattoo, "Be Prepared." I went on to work with scouts during the summer, teaching Emergency Preparedness and survival skills.
And so, with what sometimes feels like a fanatical zeal, I plan contingencies. For everything. "What if there is traffic on this route? What if the client asks us to deliver early? What if my top performer decides to leave? Then what?"
In business the impact of the unknown "what if" question could be small, with no effect, or there could be significant consequences. Losses can be quantified financially, in terms of productivity, or perhaps damaged personal prestige.
Here's the idea, if someone walked in from the gas company announcing a gas leak with the threat of explosion, what would you do? What if the problem can't be resolved for three days? Could you work somewhere else? Are the tools, files, or resources you need easily relocated, or will you be up a creek? How would staff be notified? Could you still make money during this time?
You can't plan for everything, but if you follow the guide below, you can prepare for many of the curve balls thrown your way.
1. Define critical systems. What are the essential tools for your business? Computers? A kitchen? Telephones? Transportation? Make a bare-bones list of the minimum number of things needed to successfully operate. Notice I didn't say easily or comfortably – because we want to be ready for the worst of it. Start your planning there and anything above this line will seem easy.
2. Prioritize. Now that you have the list of essentials, list the in order of importance. At a large financial firm I worked with recently, it was critical to have customer data online all the time – even in the event of an emergency. Marketing data, such as their website or files for printed materials, could wait as long as 72 hours.
3. Document. Put your plan in writing including contact names and numbers, addresses of offsite resources or suppliers that will be able to help when things go wrong. If your plan relies on moving supplies or backing up data, make sure your plan includes who will be accountable for this task and how often they will do it. There's nothing worse than executing a contingency plan only to find that when you need it most, it's incomplete or missing altogether!
4. Keep a copy offsite. Copies of the emergency plan should be in the hands of key personnel both at work and at their homes. If anything happens in the middle of the night you could be shut out of your location and unable to reach the plan. Having offsite copies ensure that key people can get in contact and begin to activate the plan to get the business back up and running.
5. Test the plan. Don't wait for something to happen! In conjunction with senior management test your plan at least once a year. Dream up different scenarios that designed to test critical parts of your plan to ensure that, should something happen, all of the resources needed to keep your business operating are available.
I wish you luck developing an Emergency Response Plan. If you have questions, or thoughts you'd like to share – not only do I appreciate the feedback, but it will help me with my merit badge!
Monday, October 5, 2009
On Being Prepared
Posted by John Tigh at 8:15 AM
1 comment:
- AnonymousOctober 8, 2009 at 9:33 AM
Wish I had this info a week ago! Our remote email server is down, and the manager happens to be in another country for the next 5 days. Ouch.ReplyDelete
Thank you - will start implementing an ERP pronto!
Subscribe to: Post Comments (Atom)


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Events for 7th December 2018
Events Search and Views Navigation
All Day
COP 24 – 24th Conference of the Parties to the United Nations Framework Convention on Climate Change
COP24 is the informal name for the 24th Conference of the Parties to the United Nations Framework Convention on Climate Change (UNFCCC). The UNFCCC is a "Rio Convention", one of three adopted at the "Rio Earth Summit" in 1992. The UNFCCC entered into force on 21 March 1994. Today, it has near-universal membership. The […]Find out more »
Water Security and Climate Change Conference
The WSCC 2018 at Kenyatta University takes up the "bridge-building" aspect of WCSS 2017 and will provide continuity in serving as a platform for discussions towards water secure societies. The conference will be a stimulus for discussing new and innovative ideas with special attention to SDGs dealing with "Zero Hunger and Food Security", "Ensuring availability […]Find out more »


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Stats: - 3 generous servings, 1 propoint per serving
Ingredients: -
One whole cauliflower, separated into small florets
One tablespoon olive oil
Three fat garlic cloves, bashed a bit to release juices
Salt and pepper
Preheat oven to gas mark 6. Toss the cauli in the oil and garlic cloves and sprinkle with a generous amount of salt and pepper. Roast for half an hour, turning the florets over half way through.
I'm thinking that the boiled version will be no more than a distant memory.
What unusual sides do you make with your vegetables? What unusual vegetable do you like to roast? Please comment and let me know!


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Liverpool's legendary European Cup winning captain and Sky Sports pundit Phil Thompson will be guest speaker at Nantwich Town's Sports Dinner.
Regulars who watch Sky's Soccer Saturday with Jeff Sterling will know "Thommo" as a popular pundit who is also highly regarded on the dinner speaking circuit.
This year he was a finalist in the "Soccer Speaker of the Year" held at the Hilton in Manchester, thanks to his stories about his time as Liverpool captain under both Bill Shankly and Bob Paisley and as England captain.
Stories will also include his close relationship with fellow pundits such as Paul Merson and Matt Le Tissier.
Phil is supported at the Nantwich dinner by stand up comedian Jimmy Carrol, with a three-course meal, including cheese and biscuits.
There is also a charity auction and raffle prizes.
The Sports Dinner is on Thursday May 3.
Tickets are £39.95 with just a gew tables available for corporate hospitality.
Contact Vicky Williams on 01270 621771 or email [email protected] to reserve your tickets.


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Our Tallahassee funeral home values
Commitment, caring, understanding and compassion defines Culley's MeadowWood Funeral Home. These are the qualities that have been a tradition with us since 1903. For more than a century, Culley's MeadowWood has earned an exceptional reputation by providing funeral, cemetery and cremation services in Tallahassee, Florida, and surrounding Leon County.
Custom funeral and memorial services
We take a careful approach to funeral and memorial planning, helping our families create a tribute that best represents their loved one. Our Tallahassee funeral home team believes that services should reflect individuality and celebrate life. We'll help you design a service that showcases your loved one's personality, accomplishments and what they loved most.
In addition, our services are designed to help families through this challenging stage of life. We understand that losing a loved one is an emotional and difficult experience and will help ease the funeral planning process. Our staff will support you every step of the way.
We honor veterans
At Culley's MeadowWood Funeral Home, we understand the unique needs of America's veterans and their families. As a Level One Founding Community Partner with We Honor Veterans, our staff is knowledgeable in the benefits available to veterans and their families. With respect, integrity and dignity, we will walk you through the steps of creating a veteran's memorial service that truly honors the life and sacrifice of your loved one.
Contact our team
Culley's MeadowWood Funeral Home strives to build long-standing relationships with every family we serve. Please contact us if need immediate assistance or would like to know more about funeral and cremation options in Tallahassee.
Everyone at Culley's MeadowWood is proud of our heritage and the everlasting relationships we have established within the community for more than 100 years.
Our Tallahassee funeral home legacy
James Victor Culley founded Culley's MeadowWood Memorial Park and Funeral Homes in 1903. It was shepherded through the previous century by many generations of the Culley family. From the firms beginning with a single location in downtown Tallahassee, Florida, to today, Culley's MeadowWood has expanded the business to offer funeral, cemetery and crematory locations throughout Tallahassee and surrounding Leon County.
James Victor Culley began with a commitment to a high standard of service. Throughout our history, we have continued to provide funeral, cremation and cemetery options in Tallahassee while delivering personal attention to the families we are privileged to serve.
Recent Obituaries
Here for you when you need us
Meet our associates
"Felt somewhat pressured to purchase memory items; otherwise everything went extremely well. I also appreciated the after service meeting."
Verified Burial & Memorial Service Customer
February 2019
"The staff was very professional and sympathetic to myself and the situation."
Verified Cremation & Memorial Service Customer
December 2018
"My family is grateful to all of the people at culleys for making the celebration of moms life a wonderful, meaningful experience."
Verified Cremation & Memorial Service Customer
December 2018
"Everything was perfect."
Verified Burial Customer
October 2018
"Everything was great. Staff were very helpful."
Verified Cremation Customer
August 2018


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Similar to any business model, health and medical organizations are faced with a number of operational challenges. One of the most common involves improving the internal billing processes to ensure a sufficient revenue stream to keep the organization financially healthy.
Since sending-out patient billings in a timely manner is directly tied to the amount of income received, improving this area of the medical practice is not a luxury that must be addressed, but rather a necessity.
For this reason, it is important for these organizations to identify and implement initiatives and programs that will help to resolve any medical billing problems that exist. Many of which may include utilizing the following strategies, tools, and resources.
As technology progresses, its not uncommon for both small and large health and medical organizations to review their billing systems for inefficiencies. Specifically, those that cause the bills to their patients and clients to be sent out on an untimely basis. When this occurs, the organization can begin to lose money in a wide variety of ways including in the interest that may be loss be depositing the funds late instead of late.
Because each day can make a major difference in many large medical practices, it is important to have the best billing services at all times. With this being the case, one of the best ways to solve some of these issues is to benchmark with similar organizations to see how they are handling the same. The health and medical organizations selected should be those who are known for having stellar technology and billing practices.
Investing in Training
Investing in training is also essential to the success of a medical billing organization as well. For instance, once a new system has been selected, the organization should make sure that they have set aside time and money for their staff to be trained properly. This type of training is designed to help the entire organization to be up and running efficiently when new systems are being implemented.
Attending Medical Billing Conferences & Seminars
Many times businesses pay for their employees to attend medical billing conferences and seminars that apply to their job responsibilities. These conferences and seminars are usually very beneficial in numerous ways, especially since they are designed for motivating people to be better at their craft. These medical billing seminars and conferences are also ideal for teaching employees when legislations that apply to their jobs change, and when they must be acted upon to remain in compliance. Some seminars also vital for those who will be selecting new billing systems that the organization uses in future times.
Investing in Medical Billing Software
Sometimes a medical organization may be hesitant about investing in new medical billing software. Specifically, when the cost of buying it makes a significant dent in the practice's budget. However, it is important for the medical admin staff to do their homework before turning these new systems down.
One of the most important reasons is, some of the newer systems can easily pay for themselves in effectiveness and efficiency within a short period of time. Thereby, saving the organization time and money in the long run instead of reducing the budget. Check out some of the top medical billing software platforms by clicking here.
Hiring a Medical Billing Consultant
Its not uncommon for any organization to hire a third party medical billing agency to assist them with improving their billing systems. In fact, an outside party is normally skilled in their craft and they can identify inefficiencies in medical billing systems quickly without having to spend a lot of time. For instance, some agencies specialize in certain aspects of medical billing, such as chiropractic billing, cardiology billing, and even Federally Qualified Health Center (FQHC) billing services
Also, by hiring a medical billing consultant, they can share the information that they have already learned about industry standards and the best ways to improve an organization's with minor to major recommendations.


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To say this album is long overdue is an overwhelming understatement. Witness Tha Realest was originally said to be dropping a decade ago, held back by loyalty and an infamous label's demise. Patiently bubbling, Tha Realest [click to read], a Dallas native and Los Angeles transplant, reportedly took his rhymes away from Death Row after many years of bench work. With this album and a new label deal, Tha Realest's finally able to get in the game. However, the crowd has already given him a fair amount of criticism before his official initial run.
To discuss the similarities between Tha Realest and the late, great Tupac Shakur would be to engage in overly drawn out discourse. That's been going on since Realest's early work with Death Row was released. Still, ignoring this facet of the album would be irresponsible because the similarities are not only striking, but also unavoidable. As with knocks on flow-ripping, jabs at simplified rhyme patterns are legitimate. Still, commendable is the emcee's topical diversity from love ("N Luv Wit Ah Ghetto Gurl") and lust ("Number 1 Hoe") to disparity ("Mind of Ah Madman") and paranoia ("Y I Keep My Burna On Me"). With 17 cuts, he navigates through length by taking us back ("Memory Lane"), showing pride ("Tha Good Lyfe") and supplying street-generated dedications like "All or Nuthin," "When Ya Time Iz Up," and "Kuz It Just Ain't N U." Guests bring healthy breaks including notable appearances from Devin The Dude's [click to view] smooth crooning and Crooked I's [click to view] scene-stealing 16. WC [click to read], Fat Joe [click to read], Sean P. of The Youngbloodz [click to read], Yukmouth [click to read], Ray J [click to read] and E.D.I. Mean from The Outlawz [click to read] all contribute throughout.
Shove the vocal comparisons to the side when acknowledging beats. "Get It N," manned by Detail of Konvict Muzik, is a Pop-friendly beat that doesn't lose its edge, which is the perfect background for a first single. The electric guitar riff on "That's Juss Lyfe," incorporated with the soulful backing from Val Young's voice provides a peak on the disc in beats and rhymes. "Memory Lane" serves as a throwback worth the trip and the piano's gothic keys enhance the dark texture on "Y I Keep My Burna On Me." Still, many of the instrumental selections come from a few pages back in the calendar as "Ice Kold," "Number 1 Hoe" and "Grown Ass Man" all sound dated, making the seventeen cuts have many valleys to walk through.
Haunted by criticism, it's difficult for anyone to truly thrive within shadows. Living up to criticism and having less than stellar production doesn't help the cause. Despite all of this, Tha Realest does bring forth a platter full of different topics, which his fans may appreciate. After 10 years of waiting for this album to hit store shelves, it's hard not to commend the grind but it's almost as difficult to compliment the result.


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If you ask me how many elevators I have ever been in with how many different people since nearly four years ago (when I arrived in Singapore), I must say that I would like to divert the question into something more interesting than that.
Something surprising happened to us this morning. Noe walked by himself to the bathroom/toilet and pee on its floor.
(For background info, these past two weeks we let Noe diaperless and barebottomed where possible, in order to study his elimination needs. We got 50% success rate so far of carrying him to the bathroom to encourage him to do it. The other 50% were accidents on the living room floor)
A last minute decision, I went to Esplanade to watch Paquito D'Rivera with New York Voices as part of the Mosaic Festival. This decision only arrived 45 minutes before the show started, and I was lucky to get a taxi that rushed there.
This recipe is adapted from Gumbo Pages
This recipe is adapted to suit the availability of ingredients here, and to suit my unwillingness to spend five hours making gumbo broth.
It's Noe's first birthday. We made a small celebration with neighbours and relatives.
This is Noe's birthday cake. Rani had to decorate it because we ordered the cake without allowing time to create custom decoration. So Rani rushed to National Library and borrowed book about cake decoration, and then dashed to Cold Storage to buy materials for royal icing. She made the blue royal icing and lay it on top of the cake, being inexperienced with cake decoration and all. And to complicate matters, the cake that Indi bought was a mango cake with jelly topping, which caused the royal icing to melt. So we had to blow the candle rapidly. Here's the picture (Photos courtesy of Judhi Prasetyo):
Ever since I went to France to learn french ten years ago, I changed from a cheese-hater into a cheese-lover. How could I not! Everyday for three weeks my host parents (Les Delforges de Reims) indulged me with french food, and closed the lengthy dinner session (which can last up to two hours) with a plate of various cheese, consisted of different kinds of cheese, from Camembert, Brie, swiss cheese which has holes in it, blue cheese, smelly cheese, etc. It was quite challenging, but if I want to learn about the french, I gotta eat what they eat. Until now, my favorite is Camembert Cheese which should be slightly aged.
What do you see in some of the most recent Indonesian movies? I see a mass masturbation of visual elements, to compensate for a lack of storytelling abilities. Or some may say Indonesian movies have too much icing without much chewable cake in it.
I wanted to cook pasta today, but then I realized that I didn't have any pasta. Instead, I found udon. So I decided to experiment a bit, and discover this nice easy recipe, a fusion between japanese and italian cooking. It is very easy and quick to cook. Try it for yourself!
I got tagged by Yanti to reveal my five dreams. Well, since this website is owned by my whole family, I guess the tag is also applicable to the other members of the family.
Late afternoon today, I took MRT to Jurong East while carrying Noe in the babybjorn carrier. Noe was really getting heavy for a carrier! It was unusually crowded for that time of the day. I walked towards the seat intended for "people who need it more than you do". There are two students there, from one of the prestigious junior college. I stood right in front of them. Apparently those two students were a bit annoyed by my presence, hence they were trying hard to avoid any eye contact with me. Hmm.
A while later, I took out my phone, which happened to be a camera phone, to check out my schedules in the calendar. Moments later, the two students seemed panicked, and once the train stopped at the next station they quickly got out of the train.
I overheard them saying:


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Aspirin was the brand name given to acetylsalicylic acid (ASA) and its evolution stretches back to ancient times when medicines were made from willow bark and other plants. Hippocrates wrote around 400 BCE of salicylic tea being used to reduce fevers.
By the nineteenth century pharmacists and chemists were experimenting with various chemicals related to it and a form of it developed by Bayer was named Aspirin. Its popularity grew over the first half of the twentieth century.
Read more about its history and its pharmacological profile at Wikipedia.
Aspirin relieves headaches because of its effect on a chemical called cyclooxygenase. Cyclooxygenase is an enzyme the body uses to produce a chemical called prostaglandin. Prostaglandin is activated when the body is hurt. It tells the brain there is an injury somewhere, and the brain in turn activates the sensory response we know as pain.
So, if you have an infection and your body temperature rises, causing a fever, prostaglandin tells your brain there's a problem, and your brain triggers a headache.
When you feel a headache and take an aspirin, the result is that cyclooxygenase production is inhibited, leading to a decrease in prostaglandin production and therefore a decrease in pain response.
People have used aspirin for things like headaches for more than a hundred years. In the 1980s, a group of 22,000 men, all doctors, did a little experiment. Half the subjects took an aspirin every other day, and half took a placebo. At the end of the study, twice as many members of the group taking the placebo had experienced heart attackscompared with the aspirin group [source: YHF].
People who take the recommended dose of between 80 mg and 325 mg a day have a better chance of avoiding heart attacks and, if they do have one, of surviving it [source: MedicineNet]. Why is aspirin so good for people with heart disease?
It's because it blocks cyclooxygenase and, in turn, certain prostaglandins. As it turns out, prostaglandins don't just trigger the feeling of pain. Some of them also help blood platelets clump together to form clots. Aspirin is an antiplatelet agent. It inhibits that prostaglandin that makes platelets stick together. If clots can't form in arteries, the chances of a heart attack decrease dramatically.
Source: How Stuff Works – Read More
Some people are allergic to aspirin, and they should always avoid it. Also, since aspirin inhibits blood clotting, people with bleeding disorders and things like ulcers can't use it, because it can lead to uncontrolled internal bleeding. It can interfere with certain medications and it can cause stomachaches and other gastrointestinal problems. In high doses, it can even cause a ringing in your ears.
Because it does have these side effects, doctors usually don't recommend an aspirin regimen for healthy people who do not have disease. For people at low risk, the benefits just don't outweigh the dangers.
Source: How Stuff Works
Daily Aspirin Significantly Reduces Cancer Risk
A new study from Queen Mary University in London concludes that taking 75 to 80mg of aspirin a day significantly lowers the risk of cancer.
The University says in a news release that to reap the benefits of aspirin, the evidence shows people need to start taking a daily dose of 75-100 mg for at least five years and probably 10 years between the ages of 50 and 65. No benefit was seen while taking aspirin for the first three years, and death rates were only reduced after five years.
If taken for 10 years, bowel cancer cases decreased by 35 percent and deaths from the disease by 40 percent, according to the research. The risk of esophageal and stomach cancers are reduced by 30 percent.
Dr. David Agus told "CBS This Morning" that the pill is most effective if taken long-term.
"At year five, the benefits dramatically outweigh the risk, and it continues on where the benefits grow and grow," Agus said. "So if you take aspirin for 20 years, there's a dramatic reduction in the overall death rate of people compared to the people who didn't take aspirin."
Agus says aspirin is effective against cancer because it is an anti-inflammatory.
"We think the daily dose is something that lowers or tempers the inflammation, reducing the risk of cancer and heart disease over time," he said.
For 60-year-olds, a daily aspirin regimen for 10 years increases the risk of stomach bleeding from 2.2 percent to 3.6 percent. Agus advises patients get the okay from their doctor before adding the pill to your daily routine.
"Doctors really need to talk to their patients," Agus said. "It's not for everybody. If you have upset stomach or bleeding, it's probably not for you."
Overall, rates of serious or fatal gastrointestinal bleeding are very low under the age of 70, but increased sharply after that age. Another side effect of aspirin use is peptic ulcer, the risk of which is increased by 30-60%.
The study also uncovers uncertainty over the most appropriate dose of aspirin required to maximize the benefit / harm ratio, with doses varying between 75 mg to 325mg a day in different clinical trials and studies. It is also not clear whether taking aspirin for longer than 10 years will result in greater benefits.
Professor Jack Cuzick, Queen Mary University of London, comments: "It has long been known that aspirin – one of the cheapest and most common drugs on the market – can protect against certain types of cancer. But until our study, where we analysed all the available evidence, it was unclear whether the pros of taking aspirin outweighed the cons.
"Whilst there are some serious side effects that can't be ignored, taking aspirin daily looks to be the most important thing we can do to reduce cancer after stopping smoking and reducing obesity, and will probably be much easier to implement."
"Our study shows that if everyone aged between 50-65 started taking aspirin daily for at least 10 years, there would be a 9% reduction in the number of cancers, strokes and heart attacks overall in men and around 7% in women. The total number of deaths from any cause would also be lower, by about 4% over a 20-year period. The benefits of aspirin use would be most visible in the reduction in deaths due to cancer.
"The risk of bleeding depends on a number of known factors which people need to be aware of before starting regular aspirin and it would be advisable to consult with a doctor before embarking on daily medication."
Prof Cuzick said the recommended dose was 75mg per day and that the risk of internal bleeding associated with aspirin could be cut by around one third by testing and treating anyone found to be carrying the infection, H.pylori in their stomachs.
Agus says that while the preventative benefits are the same for men and women aged 50-65, women should start taking aspirin later in life than men.
"Women get heart disease a little later and cancer a little later," he said. "So while it's beneficial in both, the recommendations are starting it a little later in women than in men."
Sources: Queen Mary University London | CBS News
Vintage Aspirin Advertising & Packaging


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Scrap Car Natland, LA9, Scrap your car for free
Here at Scrap Car Natland we collect vehicles free of charge throughout Natland (LA9), Cumbria. Your old scrap vehicle can cause you serious problems if you do not get them scrapped, for instance to keep your car on the road it will need to be taxed. Do not pay to have your old scrap car collected. We can take away your scrap car for free!
Recent News in Natland
As well as offering a completely free collection, we are also currently able to offer cash for scrap cars in Natland depending upon the weight of the vehicle!
If you'd like us to make you an offer just ask when calling or add 'CASH QUOTE PLEASE' in the 'Additional Info' box when submitting our online form.
- DVLA notification
- Convenient collection times
- Nationwide coverage
- Environment agency approved recycling facilities
- Completely free collection!
Book today with our team on 0800 043 5100 or fill out the online booking form to the right of this page!
Choose Scrap Car Natland
Scrap Car Natland have built up a service to be proud of with many years experience in the trade and a lot of happy customers. Covering Natland we know the area well and will be with you at the agreed time and we will not be late. We are proud to be able to offer an unrivalled service and will arrive to collect your car at the agreed time slot. Our collection service is there for you so we will dedicate a specific time slot to work around any commitments you may have.
We always ensure that you car is disposed of the correct way. Unreliable scrap car collection companies might sell on your car and try to make more money than if they recycled it, sometimes they will not even inform the DVLA of any change of ownership. This can cause you to receive fines, legally the car is still registered to you. We will let the DVLA know that your vehicle has been declared of road, giving you complete peace of mind. Any car that we scrap we ensure that it is scrapped in the most environmentally friendly way possible, we like to do our bit.
Make sure your old scrap car is disposed of correctly as we want to keep Cumbria clean and tidy. When your street is filled with old abandoned cars it can seriously damage the image of an area, scrap your car and clean up your street. Get rid of that old car, book today!
Reasons to scrap your car
There will be many reasons for you to scrap your car. In our experience the main reason is that the vehicle has failed its MOT and the required work to repair it is too expensive. Your car may not work but if it is on the road it still needs to be taxed, costing you a lot of money
The car scrappage scheme
The scrappage scheme may seem like a superb idea. However, you will be limited to what you can buy. You can't just go out and purchase any vehicle you want This can prove to be very frustrating as you have to choose a car that is currently in the scrappage scheme.
How to scrap your car
It really is easy to scrap your car in Natland. Just by picking up the phone you can learn the process from our helpful phone operators. Rather than calling if you would like to find out about our services please feel free to look at the guide on scrapping your car.
Just call us on 0800 043 5100 or fill out the following form:


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Effective visualisation. September 2012. Applied Statistics 2012, Rigno, Slovenia.
Data science in R. July 2012. OSCON, Portland, OR.
R development master class. June 2012. Bay Area/New York City.
Big data summer institute @ Rice University, Houston, TX. June 2012.
Package development @ CSIRO, Canberra. October 2011.
Data visualisation in R with ggplot2 and plyr @ Rice University. September 2011.
Visualisation and data manipulation in R @ ebay. August 2011.
R development master class. June 2011.
ggplot2 @ The Institute of Statistical Mathematics. November 2010.
Visualisation and data manipulation in R @ The World Health Organisation. July 2010.
Data manipulation and visualisation with R @ Dept of Biology, University of Missouri. October 2009.
Data visualisation with ggplot2 @ ISMI manufacturing week. October 2009.
Introduction to plyr @ useR! 2009. June 2009.
Are you interested in a custom short course at your organisation? Please email me with what you're interested in.


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Today I attended a really interesting, but in some ways frustrating University of Leicester Distance Learning (DL) Forum (#dlforum). The subject of today's forum was Assessment and Feedback, during which several of our DL departments described their methods of assessment, feedback and administration.
There were several interesting discussions around:
- Anonymous marking and is it really necessary
- Second marking and whether that is always necessary
- Expected turn around times for marking
- The amount of feedback required, we saw examples of 600-800 word feedback statements on assignments of 2500 words, is that necessary
- Phsyical vs. Digital submission. How are physical assignments tested for plagiarism. Should all digital submissions be submitted via Turnitin. When should Turnitin reports be shared with students
However the underline theme that was coming out of many of the examples was the fact that almost all departments still print all assignments to mark them, because of the classic reasons of "I can't read on a screen", "need to write comments in the margin" and "markers like to mark during commutes". However I feel the these issues and also the huge printing costs could be over come with the use of iPads. OK the initial outlay would be costly, but I'm sure this cost would be gained back in no time.
So I put it to my trusty twitter network, asking if anyone knew of any examples of institutions using iPads for making? After a retweet from Alan Cann some examples came in:
So it would seem that there are some small scale pilots going on, but nothing substantive as yet, this is definitely sounds like an interesting research project in the future. It's not like the tools aren't already there, as Nick Morris said in his blog post Goodreader allows comments on documents, as does iAnnotate, both apps I have been using for sometime.
During the forum we saw some examples of administration of assessment marking, this included a number of Heath Robinson style spreadsheets and databases. At the Univeristy of Leicester this will be overcome by the Sunrise Project. The project will develop a central administration for DL linked to the Student Record System, which will enable the recording of marks and feedback comments onto the system. One great outcome of the project would be an iPad app, this would require a login, that would give assessors access to download assignments and make comments. When complete the marks and comments can be uploaded back onto the administration system.
It will see interesting how developments in this area progress.