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Neural stem cells (NSCs) are present in the adult mammalian brain and sustain life-long adult neurogenesis in the dentate gyrus of the hippocampus. In culture, fibroblast growth factor-2 (FGF-2) is sufficient to maintain the self-renewal of adult NSCs derived from the adult rat hippocampus. The underlying signalling mechanism is not fully understood.
In the established adult rat NSC culture, FGF-2 promotes self-renewal by increasing proliferation and inhibiting spontaneous differentiation of adult NSCs, accompanied with activation of MAPK and PLC pathways. Using a molecular genetic approach, we demonstrate that activation of FGF receptor 1 (FGFR1), largely through two key cytoplasmic amino acid residues that are linked to MAPK and PLC activation, suffices to promote adult NSC self-renewal. The canonical MAPK, Erk1/2 activation, is both required and sufficient for the NSC expansion and anti-differentiation effects of FGF-2. In contrast, PLC activation is integral to the maintenance of adult NSC characteristics, including the full capacity for neuronal and oligodendroglial differentiation.
These studies reveal two amino acid residues in FGFR1 with linked downstream intracellular signal transduction pathways that are essential for maintaining adult NSC self-renewal. The findings provide novel insights into the molecular mechanism regulating adult NSC self-renewal, and pose implications for using these cells in potential therapeutic applications.
Neural stem cells (NSCs) represent a unique type of precursor cells that are capable of self-renewal and differentiation into multiple neural cell types, including neurons and glia [1-3]. During early brain development, NSCs in the germinal region generate numerous progeny in a highly organized manner to construct the nervous system. Adult mammalian brains also harbour a population of adult NSCs that are primarily located in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus to maintain regional ongoing neurogenesis [4-7]. Advances in NSC biology have highlighted the promise of NSCs in stem cell-based therapies for neurological disorders [8-11]. Understanding molecular mechanisms regulating the behaviour of NSCs, including their proper expansion in vitro with multipotentiality but not tumorigenicity, is a critical step towards these goals.
As the defining hallmark of stem cells, self-renewal refers to the process by which stem cells expand to generate at least one of the two daughter cells with the same range of developmental potentials as its parental cell [12,13]. Stem cell self-renewal is critical for both embryonic development and adult homeostatic tissue maintenance. In the mammalian brain, NSCs are subject to tight and complex regulation in different regions and at different stages of development. The earliest neuroepithilial NSCs, for example, self-renew and expand rapidly to produce a vast number of progeny in order to meet the need of brain histogenesis. Whereas most adult stem cells in vivo usually reside in a micro-environment (niche) and remain relatively quiescent [14], they engage in active self-renewal upon injury signals or under certain physiologic conditions that demand rapid production of new progeny. Due to the complex nature of self-renewal in vivo, stem cells in culture provide a better-defined system to investigate how self-renewal is controlled by intrinsic and extrinsic mechanisms.
Emerging evidence suggests that self-renewal is regulated by diverse mechanisms in different stem cells [13,15]. In the case of NSCs, it has long been noted that cell expansion is promoted by the growth factor FGF-2, although little is known about the underlying cytoplasmic signalling mechanism [16-20]. NSCs isolated from different regions of the brain or different stages of development, grown as either "neurosphere" or adherent monolayer culture, all undergo robust proliferation when supplemented with FGF-2 in serum-free defined medium [21-25]. Self-renewal entails not only proliferation but also maintenance of the stem cell state. Cellular sub-cloning experiments showed that the clonal progeny of NSCs still preserved multipotentiality after expansion by FGF-2 [23,26], and in vitro expanded adult NSCs retained multipotentiality in vivo even after serial transplantation [27]. Genetic ablation of FGF-2 locus in mice resulted in severe defects in the maintenance of a slow-dividing stem cell pool, providing in vivo evidence that FGF-2 is necessary for normal NSC self-renewal [28]. Interestingly, FGF-2 is present in normal adult NSC niches, can be induced by diverse types of pathological conditions, and is functionally capable of enhancing the inherently limited self-renewal of endogenous NSCs after ischemic stroke [29-35]. Under different biological contexts, FGF-2 may additionally act in coordination with many other types of extrinsic signalling molecules to exquisitely control adult NSC self-renewal in response to changes of cell physiological milieu, tissue homeostatic states and diverse environmental stimuli [5,10,36-41].
FGF-2 receptors (FGFRs) belong to the family of receptor tyrosine kinases [42,43]. The ligand binding, which is facilitated by heparin, leads to dimerization and autophosphorylation of FGFRs. Consequently, various phosphorylated tyrosine residues on the receptor serve as docking sites for adaptor or enzymatic proteins that link the receptor to downstream intracellular signalling pathways. Previous studies have implicated multiple pathways downstream of FGFRs, including the canonical MAPK (Extracellular signal-regulated kinase, Erk1/2) and phospholipase C (PLC) signalling [42,44]. However, it is unknown whether any of these pathways function in adult NSC self-renewal despite genetic evidence that has clearly implicated the role of FGFR1 in regulating adult NSC proliferation and neurogenesis [32,45,46]. Erk1/2 activation, for instance, has been shown to be important for myoblast proliferation, whereas its suppression promotes self-renewal of mouse embryonic stem cells [47,48]. These findings suggest that signalling pathways are largely conserved, yet their effects are context-dependent [42]. Thus, it is necessary to analyze the specific role of a given pathway in a particular cellular process.
In this study, we aim to gain molecular understanding on the role and mechanism of FGFR signalling in regulation of adult NSC self-renewal. Choosing the well-established rat hippocampal adult NSCs as our model system, we undertook multiple experimental strategies to assess whether specific FGFR signalling is sufficient to promote the self-renewal of adult NSCs, and further dissect out the functional requirement and cooperation of MAPK, PLC pathways in FGF-2-dependent self-renewal of adult NSCs.
Results and discussion
FGF-2 regulates the self-renewal of adult NSCs through promoting proliferation and inhibiting spontaneous differentiation
When grown as monolayer cultures, adult rat hippocampal NSCs remain multipotent and their self-renewal is strictly dependent on FGF-2 (Additional file 1). Initially isolated and purified from adult rat hippocampus, these adult NSCs can be maintained for long-term in serum-free F12/N2 medium supplemented with 20 ng/ml FGF-2 [22,24,25,49]. They give rise to neurons, astrocytes and oligodendrocytes both in culture and after transplanted into the dentate gyrus of adult rats in vivo [49,50]. Clonal-derived adult NSCs retain multi-lineage potentials, consistent with an FGF-2-dependent self-renewal of adult NSCs (Figure 1). To evaluate the effect of FGF-2 on adult NSC self-renewal, we assessed several integral aspects of stem cell self-renewal: proliferation, anti-differentiation, and maintenance of multipotentiality.
Additional file 1. FGF-2 promotes the self-renewal of both rodent and human NSCs. A. Sample images of mouse, rat and human NSCs cultured in the presence of FGF-2 and/or other growth factors. Scale bar: 20 μm. B. Sample images of rat NSCs in various conditions. Scale bar: 20 μm.
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Figure 1. FGF-2 regulates self-renewal of adult NSCs by promoting proliferation and inhibiting spontaneous differentiation. (A, B) Sample immunostaining images of adult NSC culture with or without treatment of exogenous FGF-2 (20 ng/ml). Nestin is a neural precursor cell marker; Ki67 is a cell proliferation marker; Tuj1 is a neuronal marker; RIP is an oligodendrocyte marker; GFAP is an astrocyte marker. Scale bar: 20 μm. (C) Quantification of the percentage of cells with characteristic markers in the presence or absence of FGF-2, or after treatment of RA (0.5 μM) and FBS (0.5%). Values represent mean ± SEM. (n = 6; *: P < 0.01, Student's t-test). (D) Multi-lineage differentiation potentials of adult NSCs after long-term culture in the presence of FGF-2. EGFP was used to label a single cell and allowed to expand in the presence of FGF-2 (20 ng/ml) and then induced to differentiate into Tuj1+ neurons (red) and GFAP+ glia (blue) with RA (0.5 μM) and FBS (0.5%) for 6 days. Scale bar: 20 μm.
In the presence of FGF-2, the adult NSC culture comprised mostly Nestin+ (a neural progenitor maker) and Ki67+ (a proliferation marker) population (Figure 1A). Differentiation markers Tuj1 (neuronal), GFAP (astroglial), and RIP (oligodendroglial) were rarely detected (Figure 1B). By contrast, withdrawal of FGF-2 led to significant cell cycle arrest and spontaneous differentiation within 4 days, as shown by a significant decrease in the percentage of Ki67+ and Nestin+ cells and an increase of spontaneous neuronal and oligodendroglial differentiation (Figure 1B, C). Overall, the percentages of apoptotic cells were not significantly altered with or without FGF-2 under these culture conditions. When the multipotentiality of adult NSCs was examined at different passages (passage 15, 25, 35), the culture consistently generated both neurons and glia. Furthermore, EGFP-labelled clonal-derived adult NSCs gave rise to both Tuj1+ neurons and GFAP+ astrocytes (Figure 1D). These results suggest that FGF-2 promotes self-renewal of NSCs by stimulating proliferation, inhibiting spontaneous differentiation, and maintaining multipotentiality.
A chimeric receptor recapitulates effects of FGF-2 and implicates Erk1/2 and PLCγ1 signalling in adult NSC self-renewal
How does FGF-2 exert such wide-ranging effects on adult NSCs? Among the four members of FGFRs, FGFR1 was highly expressed in adult NSCs (Figure 2A). These adult NSCs exhibited little endogenous NGF receptor TrkA transcript during proliferation (Figure 2A). To test whether FGFR1 activation is sufficient to promote self-renewal, we derived an adult NSC line harbouring a chimeric receptor with the extracellular domain of NGF receptor TrkA and the intracellular domain of FGFR1 (TF1 line; Figure 2A). In the chimeric NSC line, NGF was sufficient to activate FGFR1 signalling and mimic effects of FGF-2 in promoting long-term proliferation and inhibiting differentiation of adult NSCs (Figure 2B, C). Importantly, the chimeric TF1 NSC line remained to be responsive to FGF-2, and multipotent after long-term culture in the present of NGF (Figure 2B, C, E), suggesting that FGFR1 signalling is sufficient to promote proliferation and maintain multipotentiality of adult NSCs.
Figure 2. A chimeric receptor recapitulates effects of FGF-2 and implicates Erk1/2 and PLCγ1 signalling in adult NSC self-renewal. (A) A schematic diagram illustrating the chimeric receptor and the amino acid residues within the FGFR1 intracellular domain that are linked to various downstream signalling pathways. Shown on the top is the RT-PCR analysis of the expression of endogenous TrkA and FGFRs under the proliferating condition in the presence of FGF-2. (B) A bright-field view (low and high density) of NSC lines with the TF1 chimeric receptor grown in the presence of FGF-2 (20 ng/ml) or the surrogate ligand NGF (50 ng/ml). Scale bar: 20 μm. (C) Growth curves of the NSC lines with TF1 chimeric receptors in the absence, and presence of FGF-2 (20 ng/ml) or the surrogate ligand NGF (50 ng/ml). Values represent mean ± SEM. (n = 3). (D) Quantification of NSC numbers of various chimeric receptor lines cultured in the medium alone, or with supplementation of FGF-2 (20 ng/ml) or NGF (50 ng/ml). For each line, the total NSC numbers were normalized to those cultures in the presence of FGF-2 (20 ng/ml). Values represent mean ± SEM. (n = 3; *: P < 0.01, Student's t-test). (E) Normal NSC characteristics and multipotentiality of the TF1 chimeric NSC line maintained by NGF. Shown on the top are sample images of immunostaining of proliferating NSCs with Ki67 (red) and Nestin (green). Shown on the bottom are sample images of immunostaining with Tuj1 (red) or GFAP (green) of cultures at 6 days after the treatment of RA (0.5 μM) and FBS (0.5%). Scale bar: 20 μm.
By expressing a chimeric TrkA-FGFR receptor, we used NGF as a surrogate ligand to activate FGFR1 and examined the influence of specific mutations from the intracellular domain of FGFR1 on adult NSC self-renewal. We established lines of adult NSCs with a series of chimeric receptor constructs, including TrkA-FGFR1 (TF1), TF1L422A, TF1Y463F, TF1Y653/4F, and TF1Y766F (Figure 2A). L422 is a critical leucine amino acid residue site for FRS2 binding, and its mutation leads to loss of downstream signalling through the FRS2-Ras-MAPK cascade [51]. Y653/4F (tyrosine to phenylalanine) is an FGFR1 kinase enzymatic inactive mutation, and Y463F and Y766F disrupt substrate actions of the tyrosine kinase Crk and a member of the PLC family, PLCγ1, respectively [44,52]. In NSC proliferation assay, NGF recapitulated the effect of FGF-2 for the TF1 line, whereas NGF failed to stimulate the proliferation of the TF1Y653/4F kinase dead mutant line (Figure 2D). In contrast, NGF-induced expansion of TF1L442A, TF1Y766F lines were significantly decreased compared to the TF1 line. Importantly, all these adult NSC lines retained normal self-renewal in response to FGF-2 (Figure 2D). Taken together, these results indicate that L442 and Y766 linked downstream Ras-MAPK and PLCγ1 activation are likely essential for maintaining adult NSCs, through direct regulation of NSC proliferation and/or maintenance of progenitor characteristics.
Using phospho-specific antibodies against Erk1/2 and PLCγ1, western blot analysis showed that FGF-2 induced prominent Erk1/2 and PLCγ1 activation (Figure 3A). While Erk1/2 phosphorylation persisted into 24 hours after the addition of FGF-2, PLCγ1 tyrosine phosphorylation appeared to be transient in nature. The dependence of Erk1/2 and PLCγ1 activation on L442 and Y766 residues was confirmed in chimeric NSC lines with respective signalling deficiencies (Figure 3C to 3E). Collectively, these results suggest that two key amino acid residues in the intracellular domain of FGFR1 are important for adult NSC self-renewal and mediate the effects of FGF-2 through ERK and PLCγ1 signal transduction pathways.
Figure 3. Specific activation of signal transduction pathways in chimeric receptor lines with various mutations of several key amino acid residues of FGFR1. (A-B) Western blot analysis of Erk1/2 and PLCγ1 phosphorylation in normal adult NSCs in response to FGF-2 (20 ng/ml). (C-D) Western blot analysis of NGF-induced Erk1/2 phosphorylation in the TF1 line and in the mutant TF1L422A or TF1Y766F chimeric lines. (E) Western blot analysis of NGF-induced PLCγ1 phosphorylation in the mutant TF1L422A or TF1Y766F chimeric lines.
Activation of Erk1/2 is both required and sufficient for the proliferation of adult NSCs
To directly examine the specific role of Erk1/2 activation in adult NSC self-renewal, we treated adult NSC cultures with U0126, a selective and potent inhibitor for the Erk1/2 kinase MEK1/2 [53]. As shown by western blot analysis (Figure 4A), FGF-2-stimulated Erk1/2 activation was inhibited by U0126 in a dose-dependent manner. In adult NSC culture treated with 2.5 μM U0126, the percentage of Ki67 or Nestin positive cells was significantly lower than the untreated culture (Figure 4B). In contrast, U0124, the inactive analogue of U0126, elicited no significant effects. When subjected to clonal analysis assay in measuring self-renewal expansion at the single cell level (Additional file 2), U0126 also suppressed FGF-2-induced clonal expansion of EGFP-labelled NSCs in a dose-dependent manner (Figure 4C, D).
Additional file 2. Clonal analysis assay. A. A schematic diagram of clonal analysis assay. B. Sample images and quantification of the effect of FGF-2 on clonal expansion. Scale bar: 20 μm.
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Figure 4. Erk1/2 Activation is both required and sufficient for adult NSC proliferation. (A) Western blot analysis of Erk1/2 inhibition in normal adult NSCs with the treatment of U0126 or U0124 at different concentrations. (B) Quantification of the cellular composition of adult NSC culture in the presence or absence of U0126, or its inactive analog U0124 (2.5 μM). Values represent mean ± SEM. (n = 6; *: P < 0.01, Student's t-test). (C) Sample images of clonal analysis of adult NSCs with MEK1 inhibition. Scale bar: 20 μm. (D) Summary of clonal analysis of adult NSCs in the presence of U0126 or U0124. (E) Western blot analysis of Erk1/2 activation in MEK1-WT, DN or CA lines. (F) Summary of clonal analysis assay for adult NSCs expressing MEK1-DN, WT or CA. Values represent mean ± SEM. (n = 3; *: P < 0.01, Student's t-test).
To further examine the role of Erk1/2 activation in adult NSC proliferation, we engineered retroviruses to over-express the dominant negative (DN), wild-type (WT) and constitutively active (CA) mutants of MEK1 in adult NSCs (Figure 4E, F). These mutants have been widely used to manipulate cellular Erk1/2 activity [54,55]. Bicistronic expression of EGFP was used to monitor transduced cells with infection efficiency over 95%. Western blot analysis using phosph-Erk1/2 antibodies confirmed that MEK1-DN NSCs effectively attenuated Erk1/2 activation, and MEK1-CA rendered Erk1/2 constitutively active in adult NSC culture (Figure 4E). In clonal analysis assay, MEK1-DN NSCs produced significantly reduced clone sizes in the FGF-2 treated condition, whereas MEK1-CA NSCs yielded significantly increased clone sizes even in the absence of FGF-2 (Figure 4F). Collectively, these data suggest that Erk1/2 activation is both required and sufficient for FGF-2-dependent proliferation of adult NSC.
Activation of Erk1/2 blocks both spontaneous and induced differentiation of adult NSCs
Spontaneous differentiation of some adult NSCs was also observed after U0126 treatment or MEK1-DN expression. Next, we examined the role of Erk1/2 pathway in the anti-neuronal differentiation effect of FGF-2 on adult NSCs in detail. Spontaneous NSC differentiation occurred at a very low basal level in the presence of FGF-2 (Figure 5A). Treatment of U0126 (2.5 μM), but not U0124, led to significant spontaneous differentiation of adult NSCs into RIP+ oligodendrocytes and Tuj1+ neurons (Figure 5A). Similarly, the NSC line expressing MEK1-DN exhibited a higher spontaneous differentiation rate than both MEK1-WT and MEK1-CA lines. In standard differentiation condition with the treatment of retinoic acid (RA; 0.5 μM) and fetal bovine serum (FBS; 0.5%) for 6 days [24,56], adult NSC lines expressing MEK1-CA generated a significantly lower percentage of RIP+oligodendrocytes and Tuj1+ neurons (Figure 5B). Meanwhile, most cells from MEK1-CA NSC lines remained in cell cycle as indicated by the significantly higher percentage of Ki67+ cells (Figure 5B). We further explored these changes in neuronal differentiation by performing western blot analysis. Tuj1 was up-regulated in the absence of FGF-2 and reached a higher level under differentiation conditions (Figure 5C). Such increase was accelerated in the MEK1-DN NSC lines and abrogated in the MEK1-CA NSC lines.
Figure 5. Erk1/2 Activation blocks both spontaneous and induced differentiation. (A) Cell number quantification for each differentiation marker (Tuj1, GFAP or RIP) in proliferating adult NSC culture in the presence of FGF-2 (20 ng/ml) and with U0126 or U0124 (2.5 μM) (n = 6; *: P < 0.01, Student's t-test). (B) Cell number quantification for each differentiation marker (Tuj1, GFAP or RIP) in adult NSCs expressing MEK1-WT, DN, or CA at 6 days after the induction of differentiation with RA (0.5 μM) and FBS (0.5%) (n = 3; *: P < 0.01, Student's t-test). (C) Western blot analysis of neuronal marker Tuj1 expression in adult NSCs expressing MEK1-WT, DN, or CA under the medium only, FGF-2 supplemented and normal differentiation with RA (0.5 μM) and FBS (0.5%) conditions. (D) Semi-quantitative RT-PCR analysis on the induction of NeuroD, CyclinD2 and Jag1 genes in adult NSCs expressing MEK1-WT, DN, or CA under the differentiation condition.
Considering the phenotype of MEK1 NSC lines, we also tested whether expression and regulation of key genes involved in proliferation and differentiation were affected in MEK1 NSC lines. Indeed, NeuroD1, an essential transcription factor for neuronal differentiation, was strongly down-regulated in the MEK1-CA NSC line compared to the MEK1-DN and WT NSC lines (Figure 5D). Interestingly, CyclinD2, one of the key genes for cell cycle progression showed a reverse expression pattern as NeuroD1. It has been shown that FGF and Shh control CyclinD2 and CyclinD1 expression to maintain the cycling and undifferentiated progenitor states at different brain regions with NSCs, respectively [57]. While it is likely that proliferation and anti-differentiation are coupled events, Erk1/2 may also promote cell cycle progression and inhibit precocious differentiation programs by independent mechanisms. One of the prominent targets is Notch signalling, which has been shown to inhibit neuronal differentiation from multiple types of NSCs and its constitutive activation led to astrocyte differentiation [58]. In adult NSCs, we detected abundant expression of a Notch ligand Jagged-1 in the MEK1-CA, but not in MEK1-DN or WT NSCs, suggesting that Jagged-1 and Notch signalling may mediate the anti-differentiation effects of MEK1 (Figure 5D). Taken together, these results indicate that the MAPK-ERK pathway of FGFR1 signalling prevents both spontaneous and induced neuronal and oligodendroglial differentiation, possibly through regulation of key genes including NeuroD1 and CyclinD2.
PLCγ1 maintains neuronal and oligodendroglial differentiation potentials of adult NSCs
In parallel with the MAPK pathway, we also examined how PLCγ1 might participate in regulating adult NSC self-renewal. To directly ascertain the function of PLCγ1, we designed and screened a number of small hairpin RNAs (shRNA) to knockdown endogenous PLCγ1 expression in adult NSCs (Figure 6A). Retroviruses carrying the shRNAs along with a visualizing marker ZsGreen were used to infect adult NSCs. Both western blot analysis and immunostaining confirmed the knockdown efficacy of one shRNA from the screen (Figure 6A–B). This shRNA targets the 3'UTR region to allow rescue experiments with the exogenous full-length cDNA of PLCγ1.
Figure 6. Impairment of neuronal and oligodendrocyte differentiation in PLCγ1-depleted adult NSCs. (A) A schematic diagram illustrating the retroviral shRNA construct and western blot analysis on the efficacy of knockdown by shRNAs against endogenous PLCγ1 in adult NSCs. (B) Immunostaining of endogenous PLCγ1 in NSCs infected with shRNA-i1. Scale bar: 20 μm. (C) Western blot analysis of neuronal differentiation marker Tuj1 and glial marker GFAP in PLCγ-depleted NSCs in the presence or absence of FGF-2, or after induction of differentiation with RA (0.5 μM) and FBS (0.5%) for 6 days. (D) Quantification of various cell types in PLCγ1-depleted NSCs after induction of differentiation with RA (0.5 μM) and FBS (0.5%) for 6 days. Values represent mean ± SEM. (n = 4; *: P < 0.01, Student's t-test). (E) Rescue of neuronal differentiation defects of endogenous PLCγ1-depleted adult NSCs by exogenous expression of PLCγ1 cDNA plasmid. (F) Western blot analysis of neuronal differentiation marker Tuj1 after differentiation of adult NSCs expressing WT and two different dominant negative mutants of PLCγ1 (LIM: lipase inactive mutant, SH3: SH3 domain deletion mutant).
Under normal proliferation conditions, PLCγ1-depleted cells exhibited decreased number of GFAP negative and Nestin positive cells, consistent with results from the mutant chimeric receptor NSC line TF1Y766F (Figure 2D). We next assessed the differentiation profile of adult NSCs infected with PLCγ1-shRNA and control shRNA viruses. Surprisingly, in the standard differentiation condition with 0.5 μM RA and 0.5% FBS for 6 days, the majority of the progeny of PLCγ1-shRNA cells consisted of GFAP+ astrocytes, instead of a mixed population of neurons and glia as seen in control (Figure 6D). Cell death rates were not significantly altered in these conditions. A large fraction of PLCγ1-depleted cells remained to be undifferentiated even with RA and FBS (Figure 6D), in sharp contrast to adult NSCs with Erk1/2 inhibition, which led to reduced proliferation and elevated neuronal differentiation of adult NSCs (Figure 5). Even in the normal proliferation condition, the number of GFAP positive and Nestin negative cells slightly increased among PLCγ1-depleted cells. Results from immunostaining-based quantitative cell counting were further supported by western blot analysis (Figure 6C and Additional file 3). The amounts of neuronal and oligodendroglial differentiation markers Tuj1 and CNPase, but not astroglial marker GFAP, were significantly less abundant in PLCγ1-shRNA cells than the normal, vector transduced, or control shRNA virus transduced adult NSCs after induction of differentiation. Importantly, introducing WT PLCγ1 cDNA back into the PLCγ1-shRNA cells rescued the impaired neuronal differentiation capacity of cells depleted with PLCγ1, excluding potential off-target effects of this particular shRNA (Figure 6E). Furthermore, adult NSCs expressing the lipase-inactive DN form of PLCγ1 [59] exhibited similar deficits of neuronal differentiation (Figure 6F). These experiments demonstrate that knockdown of PLCγ1 leads to largely exclusive commitment of adult NSC towards astroglial fates, suggesting an essential role of PLCγ1 to maintain the full developmental potential of adult NSCs for proper neuronal and oligodendroglial differentiation.
Additional file 3. Defective neuronal differentiation in PLCγ1-depleted cells. Immunostaining of differentiated culture from adult NSCs with PLCγ1 depleted. Shown are Tuj1 (A), DAPI (B), ZsGreen (C) and Merged image (D). Scale bar: 20 μm.
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Intriguingly, the phenotype of PLCγ1-depleted cells resembles glioblastoma, a type of brain tumor cells that also exhibit impaired capacity for neuronal and oligodendroglial differentiation [60,61]. Accumulating evidence also suggests that adult NSCs in vivo express GFAP, an astrocytic marker, and glioblastoma may originate from adult NSCs [60,61]. In addition to the impaired neuronal and oligodendroglial differentiation, PLCγ1-shRNA cells exhibited enhanced GFAP expression in the presence of FGF-2 (Figure 6C). It is thus plausible that PLCγ1 normally regulates the transition of multipotent NSCs into astrocyte versus other fates, and its depletion may predispose NSCs to glial differentiation thus compromising multipotentiality. Consistent with this notion, PLCγ1 is abundantly expressed by embryonic radial glia during fetal brain development, and its overall expression is inversely correlated with GFAP expression from the embryonic stage E14 to adulthood [62].
Our results support a model that FGF-2 induced Erk1/2 activation promotes the proliferation and blocks the spontaneous neuronal and oligodendrocyte differentiation of adult NSCs, while in parallel FGF-2-induced activation of PLCγ1 pathway maintains the full differentiation capacity of NSCs into neuronal and oligodendroglial lineages by preventing excess astroglial commitment of adult NSCs (Additional file 4). Other pathways downstream of FGF-2 or alternative signal transduction machineries, such as EGF, BMP, WNT, SHH, and cytokine signalling molecules, may also interact with the pathways studied in our work, and converge on the regulation of adult NSC self-renewal in a context-specific and coordinated manner [36,38,40,46,63-70]. Given that FGF-2 is normally expressed in adult NSC niches, induced by diverse injuries such as ischemic stroke, and promotes the mobilization and self-renewal of adult NSCs in certain physiological and pathologic conditions [29-35], it will be of interest in the future to investigate the involvement and functionality of these FGF-2 dependent intracellular signalling pathways in regulating adult NSC self-renewal in vivo in normal and disease contexts.
Additional file 4. Model. A model of the intracellular signalling mechanisms by which FGF-2 promotes the self-renewal of adult NSCs.
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Understanding molecular mechanisms of stem cell behaviour regulated by extrinsic factors is an important step towards therapeutic application of NSCs for neurodegenerative diseases. Here we identified two key intracellular signalling pathways that dictate distinct aspects of adult NSC self-renewal in response to FGF-2. Erk1/2 pathway mediates both the proliferation and anti-neuronal differentiation effects of FGF-2, whereas PLCγ1 maintains adult NSC characteristics and developmental potentials of adult NSCs for neuronal and oligodendroglial differentiation. Coordination of these two pathways ensures that adult NSC self-renewal is under the stringent control of growth factor signalling, and to potentially prevent adult NSCs from transforming into cancerous stem cells such as glioblastoma, and losing precocious multipotentiality.
FGF-2 signaling is essential for self-renewal of adult neural stem cells from multiple mammalian species, including humans (Additional file 1). Our findings provide mechanistic insights into the molecular and cellular machinery regulating adult NSC self-renewal. Molecular genetic dissection of the FGFR1 pathway in this study also suggests novel biomarkers and interventions for monitoring and preserving desired NSC states, and thus have clear implications for potential uses of adult NSCs expanded in vitro in therapeutic applications.
Isolation, Culturing and Differentiation of Adult NSCs
The adult NSC line was initially established from primary adult rat NSCs [22,24]. These adult NSCs were isolated from hippocampi of adult (3-month-old) male Fischer 344 rats. Briefly, hippocampi were dissected and transferred to PBS medium containing penicillin and streptomycin. Tissue was washed, minced, and enzymatically digested for about 30 min in a mixture of 0.1% neural protease, 0.01% papain and 0.01% DNAse I. Tissue was then mechanically dissociated and cells were washed, centrifuged, and resuspended in DMEM containing 10% FBS. Equal volume of Percoll was added, and cells were centrifuged at 12,700 RPM for 30 min. The middle layer of the gradient were removed and washed 3 times with PBS. Cells were then resuspended and counted before plated on laminin-coated flasks in DEME/F12 medium containing N2 supplement, L-glutamine (2 mM) and FGF-2 (20 ng/ml) as described [22,24]. Cells were passaged for expansion when reaching 70% confluence or seeded at clonal density for experiments. For differentiation studies, fresh RA (0.5 μM) and FBS (0.5%) were added to FGF-2 free culture for six days and the medium was changed every 3 days with fresh RA and FBS.
Constructs and molecular biology
The original chimeric TF1 constructs [71] were sub-cloned into the retroviral vector pBMN-IRES-EGFP upstream of IRES-EGFP. Mutagenesis was performed by QuickChange (Stratagene) and confirmed by sequencing. The vector pSilencer-RetroQ (Clontech) was used to amplify the fragment containing the U6 promoter by a universal sense primer and an shRNA-containing antisense primer. PCR products were cloned into pSilencer-RetroQ to generate retroviral vectors. The primer sequence for PLCγ1 shRNA was as follows: PLCγ1: 5'-CTAGAATTCACGCGTAAAAAAG
Virus Production and Transduction
Phoenix Eco-packaging cell line or 293-gp cells (Clontech) were transfected with retroviral vectors pseudotyped with VSVG by calcium phosphate methods as previously described [72,73]. Supernatant was collected and subject to ultra-centrifugation (25 k rpm, 90 min). Titer of virus was determined in NIH3T3 cells and aliquots were frozen at -80°C. Transduction was performed overnight with 2 μg/ml polybrene in a minimum volume of medium as previously described [74].
Immunocytochemistry and in Vitro Quantification
Cells were fixed with 4% paraformaldehyde, followed by immunocytochemical staining as described [22,24,56]. The following primary antibodies were used: rabbit anti-Tuj1 (1:7500; Covance), mouse anti-RIP (1:50; Hybridoma Bank); guinea pig anti-glial fibrillary acidic protein (GFAP; 1:2,500; Advanced Immunochemical), mouse anti-PLCγ1 (1:1000; Upstate). After incubation with secondary antibodies (1:250; Jackson Immunoresearch) for 90 min at room temperature, cultures were rinsed, stained with 4',6-diamidino-2-phenylindole (DAPI), rinsed, mounted and stored at 4°C. Images were taken with fluorescence confocal microscopy system (Zeiss LSM510). All experiments were independently replicated at least three times.
Clonal analysis assay
Retrovirus transduced NSCs were mixed with WT NSCs at a clonal ratio (generally 1:40) and assayed for clone size and clonal composition in various conditions. For each chimeric receptor, infected culture was grown in the presence of FGF-2 (20 ng/ml), NGF (50 ng/ml), or medium only. After 4 days, the size of EGFP clones was counted and quantified; cell death events in each clone was assessed alive by propium iodide and Hoescht staining; differentiation states was examined by immunostaining with Nestin, Tuj1, RIP and GFAP. Another duplicate set of cultures was allowed to differentiate in the presence of RA (0.5 μM) and FBS (0.5%) for six days and the clonal composition (Tuj1+/RIP+/GFAP+) was examined for multipotentiality.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DKM and KP: conception and design, collection of data, data analysis, manuscript writing; MS: original chimeric receptor constructs; GM and HS: conception and design, financial support, data analysis, manuscript writing.
We would like to thank J.O. Liu, D.D Ginty, S.H. Snyder and M. Bonaguidi for comments and suggestions, L-h. Liu for technical support, Drs. X. Yu and L. Cheng for help on flow cytometry, and A. Ghosh, S.H. Snyder, D.D. Ginty, and V. Dawson for reagents and assistance. This work was supported by the National Institute of Health (NS047344, AG024984, MH084018), McKnight Foundation, NARSAD, The Robert Packard Center for ALS Research at Johns Hopkins and MDA, Maryland Stem Cell Research Fund and IMHRO to H.S. and by Maryland Stem Cell Research Fund to G.L.M. D.K. Ma was a pre-doctoral fellow of the American Heart Association.
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text:
National Boxing Association
From Barry Hugman's History of Championship Boxing
Formed on 10 January 1921 in Rhode Island, USA, the National Boxing Association (NBA) became the World Boxing Association (WBA) in 1962. It is not to be confused with the current National Boxing Association, a completely new and different body.
On 13 January 1921 it was reported in the Tacoma News Tribune that the newly-founded NBA is composed of 17 American states.
By late 1948, New York and Massachusetts were the only states where boxing was conducted which were not members of the NBA.

subset: commoncrawl


text:
School is just around the corner and we are accepting teacher book pack requests at the Info Desk in the Children's Room. If you are teaching children 5th grade and under, we can schedule a book pack for you! Stop in or call the Children's Room at (920) 967-3670.
We can create automatic packs which rotate every six weeks or a one time use theme pack. If you are looking for a theme pack, let us know what type of books you will be using. Theme packs can differ in number of books depending on theme demand at the time of your request. You can also request DVDs, music CDs, books on CD, playaways, puppets, and more! We would love to pull a teacher pack for you!
To order your teacher pack for the 2012-2013 school year, stop by the Info Desk in the Children's Room or give us a call at (920) 967-3670. We can't wait to hear from you!
Read the Library Newsletter


text:
So much mystery surrounding this "Prometheus" flick... how much "Alien" DNA will it really have? Which characters will live and which will die? Who will make an awesome cameo? So many questions and so many months to mull them over while we wait for the film's release on June 8.
In addition to the newly-released still of star Noomi Rapace all space-suited up and looking on in wonder, we had a little chat with actor Guy Pearce at the Golden Globes during which he confirmed that he is indeed in the film, if only for a hot second or two.
When asked about his experience on set, Pearce said it was everything we'd assume it would be, amazing and fantastic.
"I'm only [in the film] for a minute, but it was a real honor to work with Ridley [Scott], to see what was going on," he said. "I think Michael Fassbender, Noomi Rapace and Charlize Theron and everybody will do a really spectacular job."


text:
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Transformation tells the story of the reconstruction of the Royal Shakespeare Theatre and the building of the temporary Courtyard Theatre.
Through interviews with the key figures involved in the Transformation Project including the architects, project team, RSC staff and actors, the book reveals how the Company's plans developed, from the controversial proposal to demolish the building through to the reopening in November 2010 of the transformed RST theatre space.It describes how the original plans foundered, how new ideas and solutions developed, how the Courtyard Theatre was conceived and built in record time as a temporary home for the Company and how the Swan Theatre was improved and updated. The 144-page book has been written for the RSC by David Ward. David is a freelance journalist who worked for The Guardian for many years and who has followed the Transformation Project from the start. The soft cover book is printed on high-quality paper with a sturdy, attractively foil-blocked cover. Printed in colour throughout, it is fully illustrated with photographs and architectural plans. The book is a must for those wanting to know the full story of the Transformation project. View the opening chapter (PDF, 180KB)
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text:
Scolari confident of World Cup glory
Brazil coach Luiz Felipe Scolari says his team have one hand on the World Cup as he rounded angrily on his critics ahead of Friday's quarter-final against Colombia in Fortaleza.
Scolari insisted during a tense news conference that the 2002 World Cup winners are on course to reclaim the trophy on home soil.
"Does Brazil continue to have one hand on the trophy? Yes," he said.
"We are going on to the fifth step and there are seven steps."
Scolari has been criticised by former players, including 1970 World Cup winning captain Carlos Alberto, for his use of sports psychologist Regina Brandao.
Brandao, who has worked regularly with Scolari, visited the Brazil camp after several of the players broke down in tears during the national anthem, and also after the penalty shoot-out win against Chile in the last 16.
He said: "It was scheduled and organised. She will come again on Sunday and Monday. She has participated in a very nice way and I will tell you I don't think she makes us win.
"She isn't paid anything to do this. I admire psychologists. You can try to seek more information."
The 65-year-old, who led Brazil to 2002 World Cup success in Japan and South Korea, also took issue with members of the media who were excluded from a briefing with him earlier this week, telling them: "If you don't like it, go to hell."
1 Comment
July 04, 2014
Brazil needs to up their tempo and win this fifth step and continue
on with the sixth and seventh steps and make nearly a quarter
of a billion Brazilians happy and contented by lifting the sixth WC.


text:
Winning Fantasy 5 ticket from Thursday night's drawing sold in Port St. Lucie
PORT ST. LUCIE -- A winning Fantasy 5 ticket from Thursday night's drawing worth $60,015.22 was sold at a local convenience store.
The ticket, one of four winners in the drawing, was sold at Milk N Things, 1288 S.W. California Blvd.
The winning numbers on the ticket are 3-10-11-12-16.
The other three winning tickets were sold in Citra, Miami and Fort Lauderdale.
According to the Florida Lottery website, 465 tickets in the drawing matched four numbers, 12,764 matched three numbers and 114,516 matched two numbers.
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text:
Calliope Games' First Kickstarter: Tsuro of the Seas
- 05.26.12
Calliope Games has just announced this Kickstarter for a beautiful new version of the game Tsuro which was introduced back in 2004.
In this new version, you're captain of an Imperial Red Seal ship. Your mission is to spread the word of the Emperor, avoiding monsters and staying on the board. This tile-based game is simple to play, yet requires strategy and critical thinking to make sure that your ship stays on the board while forcing other players off the edge. Depending on your donation, you've got the chance to earn some serious swag including artwork and even a chance at tickets to GenCon this August to attend the game's launch party.
You can see all the details, including a video with Calliope Games owner Ray Wehrs over at their kickstarter page.
Nicole has her own blog, Total Fan Girl and a segment on The D6 Generation gaming podcast. She contributes to Nerd Approved, Fashionably Geek, That's Nerdalicious and The Fast Lane Car.
Follow @nicolewakelin on Twitter.


text:
Kolkata celebrates Kali Puja today
Amid bursting of crackers, fireworks and chains of dazzling lights adorning the City of Joy, Kolkatans along with the rest of West Bengal is all set to celebrate Kali Puja on Saturday and Diwali on Sunday.
In Kolkata, the celebration of Kali Puja is considered to be the second most important festival after Durga Puja.
Dedicated to the Hindu goddess Kali, it is celebrated every year on the new day of Hindu month Ashwin in Bengal.
Interestingly on the occasion of Diwali, the entire India worships goddess Lakshmi- the Hindu goddess of wealth while the Bengalis, Assamese and Oriyas adore Goddess Kali.
In Kolkata, Kali puja, the festival of lights is celebrated with immense fun and mirth.
Like Durga Puja, the goddess is worshipped by the people in this city in both their homes as well by the local organizers in pandals (marquee).
A lot of rituals is associated with the worship of goddess Kali, who is worshiped at night with Tantric rites and mantras.
She is offered red hibiscus flowers, animal blood in a skull, sweets, rice and lentils, fish and meat.
It is prescribed that a worshipper should meditate throughout the night until dawn.
Homes may also practise rites in the Brahmanical (mainstream Hindu-style, non-Tantric) tradition with ritual dressing of Kali in her form as Adya Shakti Kali.
Animals are ritually sacrificed on Kali Puja day and offered to the goddess though the practice is now less in vogue. Animal rights groups have protested too this ritual of sacrifice.
A celebration of Kali Puja in Kolkata is also held in large cremation grounds where she is believed to dwell.
Kalighat temple and Dakshineswar Kali Temple are two of the famous Kali temples in the city and on this day the priests worship goddess Kali in different ways in this two temples.
In Kalighat temple, Kali is worshipped as Lakshmi on the day of Kali Puja, reflecting an essence of Vaishnava Haldars on Kali worship.
Thousands of devotees flock to the temple on this day.
On the other hand in Dakshineswar Kali Temple, where famous Kali devotee Sri Sri Ramakrishna was a priest, the celebrations have changed little from his times.
Chetla area in South Kolkata hosts few of the popular pujas in the city.
Revellers goes pandal hopping even on Kali Pujas with theme based decorations setting in this festival as well.
In households, people lights diyas and decorates homes with chains of lights to mark the occasion.
Many Bengali homes also hosts Lakshmi Puja on this day.
(Posted on 02-11-2013)


text:
Congratulations for all of you who work on the article.
I hope you'll keep in mind that, just because the article is on Shia Islam, it doesn't mean it has to present only the Shia point of view ; that would violate Wikipedia's NPOV rule.
Yes, we will abide by net-neutrality as much as is within our power, we will try to be objective, the whole point of this project was not an issue with neutrality it was with gross inaccuracies regarding our belief system.
LOL oh darn...and i was SO looking forward to putting a whole row of after ammi jaan and umars names! oh well ill try not to sink under the weight of my dissapointment.
thanks for the heads up bro; ill focus on finishing the first paragraph today inshallah so hopefully we will be in a position to finalise and upload the article at some point before tomorrow. i just need to improve those flipping references, they suck
Wow, bro, you've really been working hard on it, I'll let you know later tonight, I just bought a laptop today so I can help with
stuff till late, while lying in bed in the evenings.
I have some plans I want for the team, if you have anything else you can finish posting it BUT otherwise, I need to call a TIMEOUT!
Till tonight. Please wait for my next post where I will explain in better details some important points about how we need
to work on this, some people are confused and falling behind....
...So till later:
I am busy just working out some order to this whole situation, so far two-three members are working hard and the others don't know what's going on...


text:
January 24, 2013
Star Features
Spring Garden - St Thomas
Crystal Harrison, Staff Reporter
Apart from an obvious cry for water in Spring Garden, St Thomas, the residents say they are more than pleased to have the main road to their community, Morant Road, fixed.
When THE STAR journeyed to the tranquil community yesterday, several residents were out in their numbers at a main standpipe filling their buckets and jugs.
The residents say that for over two weeks they have not seen running water in their homes.
"Sometimes, when you come to the standpipe there is a long line. We have to go to the river or go to the spring because the water problem is very bad, and I don't know what the problem is. They say it is because of a broken pump or something like that. Sometimes, I have to make like two trips for water a day," Sandra Turner told THE STAR.
Justice of the peace for about 10 years in the community Roger Smith told us that road repairs in the area come as a relief to residents, as it put the community in a better light.
"The roads were in a deplorable condition, so it is now a relieve. The main problem right now is that the water supply is poor, as the water pumps break down frequently. Residents have to be using spring which is unsafe and untreated. Here unemployment is also a problem and we need a functioning community centre," Smith said.
Several youths who entertained us in the community said that they have been keeping their area active by keeping regular events such as Chicken Back Mondays and Tuesdays, Bingo Wednesdays and so on.
"We have we little entertainment because di people dem have to juggle because di place is jobless, but the police dem nuh stop lock-off we music early. I think that the cops should allow us to play our music on weekends until 6 a.m., the following day and use the regular rule that they have now for weekdays," a man who identified himself as only Rasta told THE STAR.
Meanwhile, a senior teacher at the Spring Garden Primary School says the small institution is doing very well in literacy.
"The children here are passing for the traditional schools and we enter the Spelling Bee competition, so we are doing well. What we would love to see is more parents sending their children here because we find that a lot of parents are sending their kids to schools outside the community," Beverley Richards told THE STAR.
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Monday was a busy day out at University Ridge Golf Course with more than 30 former Badger athletes reuniting for the 2009 Legends of Wisconsin Golf Classic. This annual event pairs Badger Fund donors with some of the biggest names in Wisconsin athletics for a round of golf, lunch and great conversation about UW athletics. View the mid=3455%20"> --> video .
There was a lengthy list of UW legends at the event, including former basketball, hockey and football players along with a former Badger Olympic track and field athlete. Joe Thomas, Adam Burish, Brian Butch, Suzy Favor-Hamilton, Owen Daniels and Jim Leonhard were just a few of the legends at this years event. A number of Badger head coaches including Lisa Stone, Mike Eaves, Todd Oehrlein and Mark Johnson also golfed at the classic.
Before the legends and donors took to the course, they had the unique opportunity to receive a free clinic from PGA veterans Andy North and Jerry Kelly. The clinic lasted for more than 30 minutes and a question and answer session with the golf pros followed.
Lunch followed the clinic and despite the cloudy skies, the legends and donors were on the course promptly at 1 p.m. for the shotgun start.
Be sure to check out the galleryID=430"> --> photo gallery from the 2009 Legends of Wisconsin Golf Classic and check back to UWBadgers.com tomorrow to see official results along with the complete list of legends from this years event.
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Well how about that hairdo? It's like a comb forward instead of a comb over. And I have to tell you the wet, leathery beak is a bit gross to me. Of course my daughter thinks this is adorable.
Let me know what you think it might be. I'll let you know in a couple of days who our smartest reader is.


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Your satisfaction with our products and services is our utmost concern and Victorinox Swiss Army strives to provide a high level of service. By selecting one of our authorized dealers you can be confident of receiving an authentic quality product with the full Victorinox Swiss Army 3 year's limited warranty and the service you expect.
This requires Victorinox Swiss Army to preserve your interests by warning you against unauthorized dealers and internet sales offering timepieces of dubious origin, second hand product and any risk of fraud, particularly relating to counterfeits.
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Item #: 46058
Your Price: $232.00
6-Piece Round-Tip Steak Set
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The 6-piece Round- Tip Steak Set by Victorinox Swiss Army comes with 6 stainless steel knives perfect for weekend dinner parties or weeknight family meals.
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Our 6-Piece Steak Knife Set helps you set a perfect table whether you're entertaining friends or making dinner for the family. The wavy-edge high-carbon stainless steel blades carry a lifetime warranty against defects in materials and workmanship. And the natural beauty of our Rosewood design is ergonomic, lightweight, balanced for extended use, and will not discolor or corrode.
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Customer Service: 1-800-331-7993
July 29, 2014
There are a significant number of handpiece brands on the market and various terms are thrown out to refer to the different slow-speed handpieces and components available. Names like "attachment" or "contra angle" or "handpiece" can all be used to refer to the same thing. No wonder so many practitioners are often confused.
Most slow-speed handpiece systems consist of a separate motor and various attachments. The motor provides the force to drive whatever type of rotary instrument will be used for a given procedure. The motor will not directly accept any type of rotary instrument (e.g. burs). Most motors operate at a given maximum rpm -- 20k and 5k being the most common speeds available.
E-TYPE This is the most common type of slow-speed motor and is the closest to a "universal" type on the market. Most house brand slow speeds use an E-type motor. NSK is one of the best known name brands to use an E-type motor. Most electric motors are also E-type motors and will accept any E-type attachments. E-type motors primarily rely on friction to hold any attachments on using o-rings and a "split ring". Some E-type motors (long shaft- #15-105 & #15-113) also incorporate an attachment lock to further secure the attachment.
Some common brand names associated with E-type motors and attachments are: NSK, Lynx, Micro Mega, Medidenta, American and Champion, although there are many others.
STAR TITAN or "T" TYPE Star Dental has a proprietary system they use for their motors and attachments. As it is a proprietary system it will only work with components designed to work within it (ie: You cannot use components designed for a Star Titan with an E-type or Midwest Shorty (see below) component or vice versa). The Star systems use a short shaft on the motor with a groove and attachments with a spring-loaded collar covering ball bearings that seat into the groove on the motor shaft.
MIDWEST SHORTY & RHINO Midwest motors use their own proprietary attachment system. The end of the motor is recessed allowing the attachment to seat into the motor housing. The motor also incorporates a locking lever that clips onto a lip at the end of the attachment to hold it on the motor. As with the Star type, Midwest compatible components will only work with other components specifically designed for this system.
All of these motors are available in different speeds or speed combinations (some Midwest motors are available in a 2 speed configuration and the user can select the speed of the motor before operating it). The speed of the motor will dictate the speed of the rotary instrument but can be affected by the attachment (q.v.).
The first step in procuring a slow-speed system or component should always be determining what type of motor you have so that you can get components that will be compatible with it.
An attachment is the next component in the series and will attach directly to the motor. Attachments come in two primary types: nosecones and contra angles. We'll discuss attachments in next month's episode.
June 29, 2014
The turbine is the heart of the high-speed handpiece and bearings are the heart of the turbine.
Bearings are the most common failure point of a turbine and are often the primary differentiator between one turbine and another.
Way back in Practice Tips #22, "High-speed Handpiece Design," we covered all of the components that make up a complete bearing assembly. For ease of reference, we've included the diagram of a bearing assembly below. For further explanation of the components, check out Practice Tips #22.
Today, we're going to look at some of the different bearing materials and designs on the market.
Many turbines use stainless steel bearings that require lubrication. The balls, inner ring, outer ring, and shield are all made of stainless steel. The ball cage will be made of a polymer (there are a few more variations within this broad category and different types of polymers for the cage, but all these permutations require lubrication).
Stainless steel bearings have been in use for decades and are a proven design with good performance and good reliability. They are manufactured in large quantities for dental turbines and many other industries so cost is comparatively low. As these turbines incorporate metal bearings in metal housings, they require lubrication. Most handpiece lubricants on the market are designed to withstand the rigors of sterilization, but these turbines should still be lubricated every time they are used (see our handpiece maintenance products here). Lubrication after using and before sterilization is generally adequate, but consult the manufacturer of your turbine and lubricant to determine if post sterilization lubrication is required as well.
Many turbines currently on the market are advertised as "lube free." There are 2 primary methods of manufacturing lube free bearings:
LTL bearings are still the same basic stainless steel design so they share many of the features of standard stainless bearings. The greasing and sealing process adds to the cost (and they aren't manufactured in quantities like the standard bearings), so they will add to the cost of the turbine or handpiece that uses them. The sealing process, also, prevents debris from getting into the center of the bearing assembly and on the actual steel balls, so maintenance is a little easier. Nonetheless, these bearings (or, more accurately, turbines that incorporate these bearings) still need to be cleaned after every use and before sterilization.
Last of all, there are ceramic bearings. Ceramic bearings are actually made of a ceramic silicon nitride, so they have a very smooth low-friction surface. The low-friction surface removes the need for lubrication and also minimized heat build-up during use. They, also, withstand high temperatures very well, so they withstand repeated sterilization better than stainless steel bearings.
Both LTL and ceramic bearings will run at higher rpm than stainless bearings and are better able to handle higher air pressure (they typically require in excess of 40 psi drive air pressure). The higher rpm can help these turbines cut faster so some practitioners feel they perform better (naturally, this is subjective).
In summary, the materials and techniques used to manufacture lube free bearings are more costly than standard stainless bearings, so these bearings (or the turbines that incorporate them) often cost twice as much as turbines that need to be lubricated. It's up to the practitioner if the advantages are worth the expense.
January 29, 2014
A dentist's handpiece is one of the ultimate tools in his dental office. Making sure it runs smoothly is very important.
The primary function of a handpiece is to provide power for rotary instruments, i.e. burs. If your handpiece won't hold the bur, or won't release the bur (so you can't change burs) it effectively ceases to function.
Regular readers of Practice Tips will know that the chuck is the part of the turbine responsible for bur retention. Consult our issues on handpiece design and chucks for more information on this component.
There are basically two reasons for a chuck to cease functioning. One is simple break-down of the chuck itself: wear, breakage, or other alteration from the original specification preventing it from functioning as designed. The other is simple debris. If the chuck is clogged with debris, this will impede proper function.
This month we'll show you how to properly maintenance the chuck in an auto-chuck handpiece to minimize debris build-up and eliminate it when present.
The steps illustrated below should be taken at least once a week on all auto-chuck handpieces to keep the chuck working as well as possible for as long as possible.
Materials required (see image below):
Step 1. Place a drop or two of lubricant into the chuck. If using a spray lubricant (such as Once-A-Day), a quick shot of lubricant should be sprayed into the chuck.
Step 2. Activate the chuck and insert a bur.
Step 3. While activating the chuck, (hold down the button if a push button handpiece) work the bur in and out to loosen any debris.
Step 4. Remove the bur and clean any debris off of it with the swab. Some debris may be on the exterior of the handpiece, swab this off as well.
Step 5. Re-insert bur and verify proper function. Bur should be held securely and remain in place if tugged.
Step 6. Repeat the above steps as needed. It is not uncommon to have a significant debris build-up so you may need to flush the chuck several times.
NOTE: We've demonstrated the technique with a Star 430 push button handpiece. We encounter clogged chucks on this make and model of handpiece frequently, but chuck cleaning should be a part of your regular weekly routine with all models of auto-chuck handpieces, Lares, Kavo, Midwest (push button or Power Lever™) or any others in your inventory. Add chuck cleaning once a week to your handpiece maintenance routine to help keep your handpieces functioning well.


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black quilted coyote fur trim hooded down coat
designer's notes
Looking good and staying warm shouldn't be a test! This quilted down jacket from Marc New York will pass with flying colors!
color: Black Quilted, water resistant poly sateen exterior Zip front with snap close placket and standing collar Zip removable snap close hood with natural coyote fur trim; Fur origin - Canada Snap close flap front pockets with welt pockets underneath and zip close angled chest pockets Inset fleece cuffs Sateen lined with dual interior zip pockets Mid-weight down fill Size S measures approximately 40'' from base of back collar to hem, 24'' sleeve length from top shoulder seam; may vary by size This item cannot be shipped outside of the US Shell / Lining: 100% Polyester; Fill: 60% Down / 40% Waterfowl Feather; Fur: Real Natural Coyote Fur; Machine Wash; Imported; style# 312149501


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No, Apple's Tim Cook won't be HP's next CEO 5
The rumor going around the internets this morning (we first alluded to it in passing here) is that Apple's former interim-CEO and current COO Tim Cook is on the short list to become HP's next CEO.
Not so, said Cook himself to an analyst over coffee, reports Barron's:
Not true, says Gleacher & Co.'s Brian Marshall, who follows Apple. He called me a short while ago to say he'd just run into Tim Cook in Palo Alto, had coffee, and asked him about the rumor.
"Tim Cook will not be going to HP, he loves Apple," Marshall tells me Cook told him.
Even though the above is a fairly long chain of reporting, we can't say we're surprised to hear Cook isn't interested. After running Apple in Steve Jobs' absence for some time (and getting a cool $22 million in cash and stock for his trouble), we suspect he's on the short list to take over after Jobs
ascends retires.
Back to more likely candidates, we also are going to give HP's own Todd Bradley the benefit of the doubt after his interview with TechCrunch yesterday when he said "you can ask me next year, if I take the position." We're not saying he's not on the short list, we're just saying interviews are hard and, well, we know from hard experience that HP execs need to be given some leeway when it comes to off-the-cuff statements.
Sources: Business insider (rumor), Barrons (debunk)
5 Comments
LOL after he ascends. That's priceless!
+1 cracked me up.
*cricket sounds*
wow this is a slow news day. hopefully that will improve once you start reporting on the analyst meeting at HP.
Until then, if everyone could stop by and nominate webOS:


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PROVIDENCE — Deep down, Ed Cooley has to know that the basketball gods have not completely forsaken his Providence College Friars.
While injuries continue to decimate Cooley's already razor-thin core, PC somehow continues to avoid getting buried under an avalanche of excuses and naysayers proclaiming it's impossible to play major college basketball with a depleted lineup. The fact that the Friars sport a 6-2 record following Saturday's too-close-for-comfort 73-63 win over SEC foe Mississippi State reiterates the adage "what doesn't kill you makes you stronger."
"I couldn't be more proud of the group. When you look at the adversity that we have dealt with, it's a credit to our players, coaching staff and medical staff," stated Cooley. "I think we need to give (the team doctors and trainers) a raise based on what we've been through."
Bryce Cotton became the latest casualty of PC's walking wounded, the team's leading scorer exiting with 3:54 left in the first half and nine points under his belt. Originally the injury was described as an ankle injury first suffered a couple weeks ago in Puerto Rico, but afterwards Cooley confirmed that Cotton injured his knee.
Cotton officially left the Dunkin' Donuts Center early in the second half, heading to Fatima Hospital to undergo a MRI that revealed no structural damage. Wishing to keep his players focused on the task at hand, Cooley didn't have Cotton sit in the locker room during halftime.
Without Cotton, the MASH unit that is Providence took the floor in the second half with two guards among the six healthy and available bodies. The good news was that up until that point, Mississippi State proved to be the gang that couldn't shoot straight. The Bulldogs limped off the floor trailing 39-21 after hitting 5-of-26 field-goal attempts for 19 percent. The woeful mark includes a cover-your-eyes 0-of-13 showing from the 3-point stripe.
Over the final 20 minutes, the Friars nearly relinquished the sizable advantage they had built. PC led 61-35 with 10:54 to play but hobbled to the final buzzer. Trailing 66-47 with 5:13 remaining, Mississippi State ripped off 13 straight points to close to within five (68-63) with 46.9 ticks remaining. The Bulldogs' sharp turnaround stemmed from a full-court trapping scheme that wasn't unleashed until very late, which seemed sort of puzzling considering the Friars were down to only one adequate ball hander – freshman Josh Fortune, who logged 40 minutes.
"Thankfully Josh gained experience at running the point in Puerto Rico when Cotton rolled his ankle," said PC junior Ted Bancroft about Fortune, who distributed seven assists but turned the ball over five times. "Coach did a great job getting Josh into a position where he can still play off the ball."
Mississippi State looked to move closer when Roquez Johnson stepped to the free-throw line with 35.5 seconds showing on the clock, but the sophomore missed both. The pressure then shifted over to PC with Kadeem Batts stepped to the charity stripe for a 1-and-1 opportunity with 29.3 seconds left. The junior calmly swished both to cap off a day that saw him net a career-best 32 points.
"Kadeem was a lottery pick today. He had an incredible presence on the floor, a demanding presence," Cooley proudly remarked. "He was great in the huddle, great in the locker room."
A turnover by Mississippi State sent PC's LaDontae Henton to the line with a chance to make Cooley and the 6,156 fans breathe a little easier. Just like Batts, Henton was on target on both tries on his way to an effective 18-point outing.
"We were definitely in survival mode, but we did a great job staying together," said Batts.
Fortune and Bancroft, who with each passing game is being viewed less and less as strictly a walk-on, did not come off the floor once during the second half. While the pair combined to shoot 4-of-18, they worked in unison as the first line of defense in PC's 2-3 zone.
"We're both getting deflections and the team builds off that energy," said Bancroft.
Added Cooley, "I don't know if anybody in the country can do what we've done over the past eight weeks, to persevere through everything we've been through. It's a great win under the circumstances and I'm very proud of the group."
RIM RATTLERS: When asked if freshman Kris Dunn (shoulder) could have been called upon to play his first PC game Saturday, Cooley responded, "He's not ready to go yet. I'm not going to jeopardize somebody's career for one game. That young man has a bright future ahead of him; if he's ready to play the next game, great. If not, we'll play the guys available." … Saturday's game was part of the SEC-Big East Challenge. The Big East wound up winning the 12-game series, 9-3. … Saturday was just the second time that PC lost the rebounding battle. Mississippi State ended up prevailing by a 38-36 count. … Next up for the Friars is the annual intrastate showdown with URI, which will take place this Thursday night in Providence. Said Batts: "It's going to be a high energy game against our rival."
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Democratic Convention
By NBC26 Staff. CREATED Jun 8, 2012
APPLETON--State democrats descend on Appleton for their annual Democratic Convention. And they're more fired up than ever. Even though they may have lost in Wisconsin's recall election, they hope to get a win in Washington.
In the spotlight this year, President Obama's bid for re-election, and Tammy Baldwin's run for U.S. Senate.
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May 19, 2010
MADISON, Wis. – The University of Wisconsin's Academic Advancement Program Student Organization (AAPSO) will be holding a fundraiser at bd's Mongolian Grill of Madison on Thursday. All UW Badger fans are invited to attend the event to benefit underrepresented students of color.
Former Badgers Ron Dayne, Anthony Davis, Jason Jefferson and Jonathan Clinksale will show their support for the AAPSO as special guests in attendance at the fundraiser.
The AAPSO fundraiser looks to raise money to help support study abroad opportunities for underrepresented students of color from the Center for Academic Excellence in the College of Letters and Science.
The fundraiser is scheduled to take place from 11 a.m. to 10 p.m. at bd's Mongolian Grill, located at 4722 East Towne Blvd in Madison. Please visit the bd's Mongolian Grill website for location and directions.
The AAPSO has partnered with the LEAP Initiative, which stands for Liberal Education and America's Promise, to help students participate in valuable international experiences.
"A core goal of the LEAP Initiative, which the UW system and specifically UW-Madison is a national leader in, is to prepare our students to live in a global and diverse community," Academic Advancement Program Director and Assistant Dean DeVon Wilson said. "One of the important ways that this happens is through high-impact educational activities to study abroad.
"The event at the Mongolian Grill will be critical to sending 14 students to actually witness the goals articulated in the LEAP Initiative."
The AAPSO is committed to creating an inclusive atmosphere, thereby developing well-rounded scholars and focusing upon community building not only on campus, but greater Madison, and nationwide volunteerism. It is a registered student organization at the UW, affiliated with the Academic Advancement Program (AAP).
The AAP, a four-year program, focuses on academic advising, academic instructional support, academic engagement and enrichment and community building. For more information, please visit the AAP homepage.
2015 Board of Regents of the University of Wisconsin System. All rights reserved worldwide.
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- Home
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Doug Burlison
Springfield City Council, Seat C, Missouri
Election Date: April 1, 2011
Term: 4 years
Non-Partisan Office
Term: 4 years
Non-Partisan Office
About the Candidate:
Doug Burlison was elected as an at-large Councilman in April 2007 and re-elected in April 2011. He currently serves as Chair of the Plans and Policies Committee and is a member of the Finance and Administration Committee.
Doug studied Public Administration, Economics, and Environmental Sciences at SMSU from 1989-1992. Doug has served in the U.S. Army's 82nd Airborne Division. He was honorably discharged as a Sergeant. Doug also volunteered as a Firefighter/EMT for the Battlefield Fire Protection District. Additionally, he attends many community events and meetings as a member of the City Council. Doug was the original Chief Petitioner seeking the first State Audit for the City of Springfield.
Doug, an Assistant Manager at Ozark Tape and Label Co., is a life-long resident of the City of Springfield. He married Mellony in 2004 and is the proud father of eight children.


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Republican Presidential contender announced on Tuesday that he would be dropping his bid for President of the United States.
"We made the decision over the weekend [that] this presidential race is over for me, and we will suspend our campaign today," Santorum said at a news conference in Gettysburg, Pa.
Santorum had been on a break from the campaign for the past few days because of the Easter holiday and the hospitalization of his 3-year-old daughter.
The former Pennsylvania Senator, who had been called Mitt Romney's top opponent, suffered a trio of defeats last week in Wisconsin, Maryland and Washington, D.C. Over the weekend, his daughter, Bella, was hospitalized for the second time during his Presidential campaign due to complications from a rare genetic disorder.
Santorum has yet to offer an endorsement and has not yet urged his delegates to support another candidate. Some pundits speculate that he is hoping Romney will fall short of the required delegate threshold, allowing him to re-involve himself in the race during the Republican National Convention in August.
Romney remains the media-declared winner of the GOP, though Ron Paul and Newt Gingrich remain in the race. Gingrich's campaign, however, has been embattled of late and revealed last weekend that it is $4.5 million in debt.


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Another great product from Crafty Games arrives on your doorstep! This time it's the Modern Arms Guide for Classic Spycraft!
The Spycraft RPG (Roleplaying Game) is a ground-up retooling of the classic d20 fantasy rules, updated for all styles of modern day gaming. It contains original classes, skills, feats, combat options, rules of adapting to nearly any genre, a unique system for resolving dramatic conflicts, and more!
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