text:
Mouse incisors grow continuously throughout life. This growth is supported by the division of dental epithelial stem cells that reside in the cervical loop region. Little is known about the maintenance and regulatory mechanisms of dental epithelial stem cells. In the present study, we investigated how transforming growth factor β (TGF-β) signaling-mediated mesenchymal-epithelial cell interactions control dental epithelial stem cells. We designed two approaches using incisor organ culture and bromodeoxyuridine (BrdU) pulse-chase experiments to identify and evaluate stem cell functions. We show that the loss of the TGF-β type I receptor (Alk5) in the cranial neural crest-derived dental mesenchyme severely affects the proliferation of TA (transit-amplifying) cells and the maintenance of dental epithelial stem cells. Incisors of Wnt1-Cre; Alk5fl/fl mice lost their ability to continue to grow in vitro. The number of BrdU label-retaining cells (LRCs) was dramatically reduced in Alk5 mutant mice. Fgf10, Fgf3, and Fgf9 signals in the dental mesenchyme were downregulated in Wnt1-Cre; Alk5fl/fl incisors. Strikingly, the addition of exogenous fibroblast growth factor 10 (FGF10) into cultured incisors rescued dental epithelial stem cells in Wnt1-Cre; Alk5fl/fl mice. Therefore, we propose that Alk5 functions upstream of Fgf10 to regulate TA cell proliferation and stem cell maintenance and that this signaling mechanism is crucial for stem cell-mediated tooth regeneration.
The growth of mouse incisors continues throughout life and is supported by dental epithelial stem cells localized within the cervical loop of the incisor tooth organ. The dental epithelium gives rise to the inner enamel epithelium (IEE), stratum intermedium, stellate reticulum (SR), and outer enamel epithelium (OEE). The IEE was previously proposed to represent transit-amplifying (TA) cells and will differentiate into ameloblasts (9). Investigations of adult stem cells have been difficult because of their low abundance and the lack of specific markers to differentiate them from surrounding cells. A bromodeoxyuridine (BrdU) pulse-chase experiment, in which stem cells are identified by taking advantage of their relative quiescence within the surrounding population of cells, is a widely accepted approach for identifying stem cells (8). This method allows the characterization of stem cells with few or no identified stem cell markers. Label-retaining cells (LRCs) have been found in various epithelial tissues, including the cornea (3), hair follicle (4), intestinal crypts (20), mammary gland (31), and skin epithelium (27). Pulse-chase experiments conducted on mouse incisors indicated that most LRCs are in the cervical loop region, localized among the stellate reticulum cells (24).
Adult stem cells typically reside in specialized niches where they receive microenvironmental cues to maintain homeostasis and function. Previous studies demonstrated that the inductive signals for dental epithelial stem cells originate from the dental mesenchyme (10). Different pathways have been proposed to regulate the dental epithelial stem cell population, including the Fgf, Spry, Follistatin, and Bmp pathways (10, 13, 23, 25, 28). Fgf10 appears to play a crucial role, because the knockout of Fgfr2b or Fgf10 in mice results in the absence of the cervical loop structure (10, 19). Nevertheless, the molecular mechanisms involved in the regulation of fibroblast growth factor (FGF) signaling and the maintenance of dental epithelial stem cells through mesenchymal-epithelial cell interactions remain largely unknown.
Transforming growth factor β (TGF-β) family members signal through a heteromeric complex composed of type I and type II receptors. Upon ligand binding, type II receptors recruit and phosphorylate type I receptors, which then propagate the signal via phosphorylated Smad proteins. Both type I and type II receptors play important roles in tooth development (18, 32). In our previous studies, we have shown that interactions between TGF-β and FGF signaling pathways in cranial neural crest (CNC)-derived mesenchymal tissues play crucial roles in the development of the tongue and calvaria (11, 22). In this study, we designed experiments to test the hypothesis that TGF-β-mediated FGF signaling is well conserved in other areas of embryonic development and may function to regulate dental epithelial stem cells. We used Wnt1-Cre; Alk5fl/fl mutant mice as a model to study how the TGF-β family may regulate dental epithelial stem cells through mesenchymal-epithelial cell interactions. We found that the loss of TGF-β type I receptor (Alk5) expression in the dental mesenchyme severely affected the maintenance of dental epithelial stem cells. Wnt1-Cre; Alk5fl/fl incisors no longer had the ability to support continuous growth in vitro, and the number of LRCs was significantly reduced. We also detected a reduced expression level of Fgf10 in Wnt1-Cre; Alk5fl/fl incisors. Strikingly, we found that the addition of exogenous FGF10 into organ cultures was able to rescue the dental epithelial cell defects of Wnt1-Cre; Alk5fl/fl incisors. Therefore, we propose that Alk5-mediated TGF-β signaling functions upstream of Fgf10 to regulate dental epithelial stem cells during tooth development.
Mouse maintenance and genotyping.The Wnt1-Cre transgenic line and Alk5 floxed alleles were described previously (5, 6, 14). Wnt1-Cre; Alk5fl/fl embryos were generated by mating Wnt1-Cre; Alk5fl/+ male mice with Alk5fl/fl female mice. Genotyping was carried out by using PCR on tail tip or yolk sac DNA. Mutant embryos were identified by PCR genotyping for the presence of the Cre transgene (Cre1 [TGATGAGGTTCGCAAGAACC] and Cre2 [CCATGAGTGAACGAACCTGG]) and the Alk5fl/fl allele (lnl5′ [ATGAGTTATTAGAAGTTGTTT], lnl3′ [ACCCTCTCACTCTTCCTGAGT], and llox3′ [GGAACTGGGAAAGGAGATAAC]). Noon on the plugging day was designated embryonic day 0.5 (E0.5). All mouse embryos used in this study were maintained in a C57BL/6J background.
Histology and in situ hybridizations.For routine histological analyses, embryos were fixed in 4% paraformaldehyde, embedded, and sectioned at 7 μm. Paraffin sections of embryos were stained with hematoxylin and eosin (H&E) according to standard procedures.
For in situ hybridization, embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 8 μm. RNA probes were labeled with digoxigenin (Roche). In situ hybridization was performed as previously described (30).
BrdU incorporation analysis.Cell proliferation was assayed with BrdU incorporation experiments. Pregnant mice were injected with 1 ml 10 mM BrdU in water per 100 g of body weight, and embryos were collected 2 h later. For samples in organ cultures, the explants were incubated with BrdU (10 μM) for 2 h. Samples or embryos were then fixed overnight in 4% paraformaldehyde and embedded in paraffin. Histology sections (7 μm) were prepared according to standard techniques. BrdU was detected by using a BrdU staining kit (Invitrogen) according to the instructions provided by the manufacturer.
For BrdU pulse-chase experiments, samples were incubated with BrdU (10 μM) for 24 h. BrdU-containing medium was then replaced with regular medium and refreshed every day. Samples were continuously cultured for 14 days and then fixed with 4% paraformaldehyde and embedded in paraffin. Histology sections (7 μm) were prepared according to standard techniques. BrdU was detected by using a BrdU staining kit (Invitrogen).
Apoptosis assay.Apoptosis was detected by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay using the In Situ Cell Death Detection kit (fluorescein) (Roche) according to the manufacturer's instructions. The paraffin sections were rehydrated and treated with proteinase K according to the manufacturer's instructions. After the substrate reaction, slides were mounted with Vectashield mounting medium (Vector) and observed under a fluorescent microscope.
Organ culture experiments.The incisors were dissected carefully from the lower jaws of newborn mice. The calcified tip was removed for better nutrient penetration. The incisors were then cultured in Trowell-type organ culture dishes (Corning) on filter paper supported by metal grids in a humidified atmosphere of 5% CO2 in air at 37°C. The culture medium consisted of BGJb medium (Invitrogen) supplemented with penicillin-streptomycin and vitamin C (Gibco BRL). The date when the incisors were plated onto the culture dish was day 0, and the length of the dental epithelium was measured from days 2 to 16. In some experiments, incisors were cultured in medium supplemented with FGF3 at 10 ng/ml (R&D systems), FGF9 at 10 ng/ml (R&D systems), or FGF10 at 10 ng/ml (R&D systems), and the medium was replaced every other day.
Quantitative real-time PCR.For real-time PCR, lower incisors from E16.5 mouse embryos were dissected carefully. RNA was extracted with the RNeasy minikit (Qiagen) according to the manufacturer's instructions. RNA was reverse transcribed by using a Superscript III kit (Invitrogen) according to the manufacturer's protocols. Quantitative PCR was carried out by using 2× SYBR green PCR master mix (Bio-Rad) with an iCycleriQ instrument (Bio-Rad). Amplifications were performed with the following primers: Fgf3 forward primer 5′-AGCGGAGGCAGAAGAAG-3′, Fgf3 reverse primer 5′-AGCCAGTCCACCTGTATG-3′, Fgf10 forward primer 5′-ACCACCCACAACCAAA-3′, Fgf10 reverse primer 5′-TCCAACGTCTGCACTA-3′, Fgf9 forward primer 5′-CAACACCTACTCTTCCAACCTCTA-3′, and Fgf9 reverse primer 5′-GGAGTCCCGTCCTTATTTAATGC-3′. PCR products were subjected to gel electrophoresis to verify the absence of nonspecific reaction products and primer dimers. Data were analyzed by using Bio-Rad iCycler software and were normalized against β-actin.
Compromised TGF-β signaling in the dental mesenchyme causes defects in the dental epithelial stem cell compartment.The development of the mouse incisor initiates at E11.0, when the thickening of the epithelium can first be seen (26). The incisor tooth germ reaches the bell stage at E15.5 (Fig. 1A). Enlarged cervical loops were detectable on both the labial and lingual sides of the incisor (Fig. 1A). However, the growth of the labial and lingual cervical loops was asymmetrical, and their difference in size was already detectable at E16.5 (25) (Fig. 1C). The labial side cervical loop is much larger than the lingual side cervical loop. At the newborn stage, the labial side cervical loop is composed of the inner enamel epithelium (IEE), the outer enamel epithelium (OEE), and stellate reticulum (SR) cells (25) (Fig. 1E and G). Enamel was detectable only on the labial side (Fig. 1I and 2A).
The initiation and development of Wnt1-Cre; Alk5fl/fl lower incisors were delayed compared with those of the wild-type control (32). Wnt1-Cre; Alk5fl/fl lower incisor tooth germs also reached the cap stage at E15.5 (Fig. 1B) and the bell stage at E16.5 (Fig. 1D). However, the enlarged cervical loop structure appeared abnormal in Alk5 mutant lower incisors (Fig. 1D). At the newborn stage, the labial side cervical loop of the Wnt1-Cre; Alk5fl/fl lower incisor was much smaller than that of the control (Fig. 1E and F). There were few stellate reticulum cells in the mutant incisor cervical loop (Fig. 1G and H). At the newborn stage, the length of the Wnt1-Cre; Alk5fl/fl mutant lower incisor was only about one-third of that of the wild-type lower incisor (Fig. 1I and J). Interestingly, Wnt1-Cre; Alk5fl/fl upper incisors had different phenotypes than the lower incisors, and we will describe these results in a separate study. Here we focus on the phenotypes of the lower incisors.
We examined cell differentiation in Wnt1-Cre; Alk5fl/fl incisors. Mineralized dentin formed in Wnt1-Cre; Alk5fl/fl mutant incisors at the newborn stage (32; data not shown). Although we did not detect calcified enamel formation in Wnt1-Cre; Alk5fl/fl mutant incisors, we did detect Amelogenin expression on the labial sides of both wild-type and Wnt1-Cre; Alk5fl/fl lower incisors at E18.5 (Fig. 2A and B). Because Wnt1-Cre; Alk5fl/fl embryos died at birth, we dissected the incisor tooth germs from newborn mice and cultured them in Trowell-type organ culture dishes in order to investigate the development of incisors. After 7 days of in vitro culture, we detected enamel on the labial sides of both wild-type and mutant incisors (Fig. 2C and D). Thus, we conclude that ameloblast differentiation is still maintained in Wnt1-Cre; Alk5fl/fl mutant incisors.
To evaluate the ability of the incisors to maintain continuous growth, we dissected the incisor tooth germs from newborn mice and cultured them in vitro. Pictures were taken every other day, and the length of the dental epithelium was measured by using NIH ImageJ software. The dental epithelia of both wild-type and Wnt1-Cre; Alk5fl/fl incisor tooth germs were elongated in vitro (Fig. 3A). After 16 days of culture, the dental epithelium of wild-type incisors increased in length by over 2.5-fold (Fig. 3B). The growth rate of the epithelium from mutant incisors was lower than that of the wild type (Fig. 3B). After 12 days the mutant epithelial growth rate began to decrease and reached a plateau stage, suggesting a growth arrest (Fig. 3B). Therefore, the ability of the dental epithelium to support the continuous growth of incisors was compromised in Wnt1-Cre; Alk5fl/fl mice.
The loss of the type I (Alk5) or type II TGF-β receptor in the neural crest-derived tissue has differential impacts on craniofacial bones and tooth development (12, 32). In order to evaluate the functional significance of Tgfbr2 in regulating the continued growth of mouse incisors, we investigated the cervical loop in Wnt1-Cre; Tgfbr2fl/fl mouse incisors. In parallel, to examine the functional significance of Alk5-mediated TGF-β signaling in the dental epithelium, we also investigated incisor cervical loops in mice lacking Alk5 in the epithelium (K14-Cre;Alk5fl/fl). The initiation of incisor development was normal for both mutants. Moreover, the cervical loops of both mutants were indistinguishable morphologically from that of the wild type (Fig. 1K and L and data not shown).
Loss of Alk5 in the dental mesenchyme results in reduced cell proliferation and fewer LRCs in the dental epithelium.The reduced cervical loop size in Wnt1-Cre; Alk5fl/fl mutant incisors may be caused directly by reduced cell proliferation or increased apoptosis. To investigate these possibilities, we performed BrdU incorporation experiments with wild-type and Wnt1-Cre; Alk5fl/fl mice. In the lower incisors of wild-type newborn mice, we detected cells with BrdU incorporation in all three components of the cervical loops, including the IEE, OEE, and SR, although cell proliferation was higher in the IEE than in the OEE (Fig. 4A and E). Proliferation in the IEE and SR of Wnt1-Cre; Alk5fl/fl lower incisors was significantly decreased compared to that for the wild type (Fig. 4B and E). We detected a similar decrease in cell proliferation activity in E16.5 Wnt1-Cre; Alk5fl/fl incisors (data not shown). Next, we conducted TUNEL assays to investigate the apoptosis activity. We detected no positive apoptotic signals in the dental epithelium of either wild-type or Wnt1-Cre; Alk5fl/fl lower incisors at the newborn stage (Fig. 4C and D). We conclude that the loss of Alk5 in the dental mesenchyme results in reduced proliferation in the dental epithelium.
At present, no specific biochemical marker has been established that can be used to distinguish dental epithelial stem cells from surrounding cells. To test whether there is a dental epithelial stem cell defect in Wnt1-Cre; Alk5fl/fl mutant incisors, we used BrdU pulse-chase experiments, which have been established as a widely accepted approach to locate epidermal stem cells (2, 8, 27). To perform these experiments, we dissected wild-type and Wnt1-Cre; Alk5fl/fl tooth germs at the newborn stage and incubated them with medium containing BrdU for 24 h so that all the cells in the cervical loop would have incorporated BrdU (data not shown). The samples were then incubated in regular medium without BrdU for 2 weeks. We detected label-retaining cells (LRCs) in wild-type lower incisors throughout the stellate reticulum cells. On average, approximately 5 LRCs were detectable in each section (Fig. 5A and C). In Wnt1-Cre; Alk5fl/fl mutant mice, in contrast, less than 1 cell was detected on average in each section of the cervical loop (Fig. 5B and C). Therefore, we conclude that the cervical loop in Wnt1-Cre; Alk5fl/fl mice had defects in both TA cell and stem cell populations.
Loss of Alk5 leads to reduced expression of FGF.FGF3, FGF9, and FGF10 play important roles in regulating dental epithelial stem cells (29). In wild-type mice at E16.5, Fgf10 expression is detectable throughout the dental mesenchyme, with a stronger intensity at the labial side (Fig. 6A). We detected Fgf3 expression almost exclusively in the labial side dental mesenchyme near the cervical loop (Fig. 6C), whereas we detected Fgf9 expression in both the dental epithelium and mesenchyme. The strongest Fgf9 signal was localized to a small region of the dental epithelium just anterior to the labial side cervical loop (Fig. 6E). In E16.5 Wnt1-Cre; Alk5fl/fl lower incisors, the levels of expression of Fgf3, Fgf9, and Fgf10 were all significantly reduced (Fig. 6B, D, and F). We also detected similar expression level changes for Fgf3, Fgf9, and Fgf10 in Wnt1-Cre; Alk5fl/fl incisors at the newborn stage (data not shown), suggesting that the changes in Fgf expression are not due to a developmental delay in incisor development in Alk5 mutant mice.
To confirm the changes in Fgf expression levels, we collected lower incisor tooth germs from E16.5 wild-type and Wnt1-Cre; Alk5fl/fl embryos and extracted RNA for real-time PCR experiments to quantify the expression levels of Fgf3, Fgf9, and Fgf10. We found that the levels of expression of Fgf3, Fgf9, and Fgf10 were reduced 2- to 3-fold in Wnt1-Cre; Alk5fl/fl samples compared with those of the wild type (Fig. 6G).
To confirm the regulatory effects of TGF-β signals on Fgf genes, we collected lower incisors from newborn wild-type embryos and treated them with 10 ng/ml TGF-β2 in the organ culture system. Samples were collected 3 h after treatment, and RNA was extracted to quantify the expression levels of Fgf3, Fgf9, and Fgf10. We found that the expression level of Fgf10 was elevated by over 1.5-fold in the presence of 10 ng/ml TGF-β2 compared with untreated samples. In contrast, there was no significant change of Fgf3 and Fgf9 expression levels in the presence of TGF-β2 (Fig. 6H).
Analysis of gene expression known to be critical for regulating dental epithelial stem cells in Wnt1-Cre; Alk5fl/fl mice.To determine the status of genes that play a role in dental epithelial stem cells, we assayed their expressions using in situ hybridization. Notch receptors and their modulators have been shown to be important for tooth morphogenesis and ameloblast differentiation. Notch1 and lunatic fringe (Lnfg) were previously proposed to regulate the fate of dental epithelial stem cells (9). At E16.5, Notch1 expression in the stellate reticulum cells of labial and lingual side dental epithelia was indistinguishable in intensity and pattern for wild-type and Wnt1-Cre; Alk5fl/fl mice (Fig. 7A and B). This result suggests that Notch1 expression in the dental epithelium is independent of Alk5 in the mesenchyme. In contrast, the level of Lnfg expression, which was restricted to a small region of IEE cells in wild-type mice (Fig. 7C), was significantly reduced in Wnt1-Cre; Alk5fl/fl incisors (Fig. 7D). Follistatin (Fst) plays a critical role in regulating the dental epithelial stem cell niche by interacting with Fgf and inhibiting stem cell proliferation (29). At E16.5, Fst expression was detectable mainly in the lingual side dental epithelium (Fig. 7E). Its expression pattern and intensity in Wnt1-Cre; Alk5fl/fl mutant incisors showed no significant difference compared with those of the control sample (Fig. 7F and I). Bmp4 is critical for regulating the differentiation of dental epithelial stem cells into ameloblasts (29). At E16.5, Bmp4 expression was detectable mainly in the dental mesenchyme (Fig. 7G). Its expression pattern and intensity in Wnt1-Cre; Alk5fl/fl mutant incisors showed no significant difference compared to those of the control (Fig. 7H and I).
We also investigated other genes that may play a role in incisor development, including p63, Activin βA, Bmp7, Lef1, and Msx1, but found no evidence of altered expression in Wnt1-Cre; Alk5fl/fl lower incisors (data not shown).
Exogenous FGF10 rescues the dental epithelial stem cell defects in Wnt1-Cre; Alk5fl/fl lower incisors.Because of the central role that Fgf10 plays in regulating dental epithelial stem cells, we examined whether the addition of exogenous FGF10 could rescue the stem cell defects in Wnt1-Cre; Alk5fl/fl mutant lower incisors. We added 10 ng/ml FGF10 into the culture medium of the organ culture system and measured the length of the labial side dental epithelium in each sample every other day for 2 weeks to generate a growth curve. In the presence of exogenous FGF10, the Wnt1-Cre; Alk5fl/fl lower incisor dental epithelium elongated at the same rate as that of the wild type in the first 10 days, and their growth curves overlapped beginning on the second day (Fig. 8A). After 10 days, the growth curve of Wnt1-Cre; Alk5fl/fl lower incisors treated with FGF10 progressed slowly, but no plateau stage was observed during the 14 days of culture (Fig. 8A). The incisor samples were embedded and examined by using microscopy after 14 days of culture. In the absence of FGF10, the size of the Wnt1-Cre; Alk5fl/fl cervical loop was much smaller than that of the wild type (Fig. 8B and C). The addition of FGF10 had no significant effect on the size of wild-type cervical loops (Fig. 8B and E). In contrast, we detected a significant increase in the size of the cervical loop in Wnt1-Cre; Alk5fl/fl samples following FGF10 treatment (Fig. 8C and F).
Next, we examined the effect of exogenous FGF10 on the number of LRCs in the cervical loop. In the absence of FGF10, we detected five LRCs on average in each section of the wild-type lower incisor and less than 1 cell in the Wnt1-Cre; Alk5fl/fl incisor sections (Fig. 8D and H). The addition of FGF10 (10 ng/ml) did not change the LRC numbers of the wild-type incisors significantly (Fig. 8H); however, the LRC numbers of the Wnt1-Cre; Alk5fl/fl incisors were restored to nearly 5 per section in the presence of FGF10 (Fig. 8G and H). Therefore, we conclude that FGF10 is able to rescue the stem cell maintenance defects in Wnt1-Cre; Alk5fl/fl incisors.
To examine the effects of FGF10 on different cell populations of the cervical loop, we cultured incisors in vitro for 24 h with or without FGF10 and analyzed proliferation using BrdU incorporation experiments. We found that the addition of 10 ng/ml FGF10 did not significantly increase the BrdU incorporation ratio in any of the three cell types of the wild-type cervical loop (Fig. 8I). In contrast, the addition of FGF10 increased the BrdU incorporation ratio of IEE cells in Wnt1-Cre; Alk5fl/fl samples by 3-fold (Fig. 8I), to a level comparable to that of wild-type IEE cells, but had no significant effects on SR and OEE cells in the same mutant samples. Thus, we conclude that FGF10 is able to rescue the proliferation defects of TA cells in Wnt1-Cre; Alk5fl/fl incisors.
We also added FGF3 (10 ng/ml) or FGF9 (10 ng/ml) into the culture medium of organ cultures to examine their effects on cervical loop growth. The growth curve of Wnt1-Cre; Alk5fl/fl incisors was restored to the wild-type level in the first 3 days following the addition of FGF3. After 4 days, the growth curve of mutant samples progressed slowly and reached a plateau stage (Fig. 9). LRC cell numbers were not restored following the addition of FGF3 (Fig. 9B). BrdU incorporation experiments indicated that the addition of 10 ng/ml FGF3 significantly restored the proliferation of IEE cells but not OEE or SR cells in Wnt1-Cre; Alk5fl/fl incisor samples (Fig. 9G, H, and I). The addition of 10 ng/ml FGF9 had no effect on the growth curve of Wnt1-Cre; Alk5fl/fl incisor cervical loops (Fig. 9C). FGF9 also had no effect on the BrdU incorporation ratios of any of the three cell types of the cervical loop in Wnt1-Cre; Alk5fl/fl incisor samples (Fig. 9J, K, and L).
As the mouse incisor continues to grow throughout life, cells at various differentiation stages, from the initial quiescent stem cells to transit-amplifying cells, preameloblasts, and the terminally differentiated ameloblasts, align along the dental epithelium (9). The slowly dividing stem cells within the SR give rise to TA cells that reside within the IEE. TA cells in the IEE undergo active division, move toward the incisal direction, and differentiate into ameloblasts, forming enamel (9). These features make the incisor an excellent model for studying the regulatory mechanisms of stem cell maintenance, proliferation, and differentiation. In our previous study, we reported that the loss of Tgf-β type I receptor (Alk5) expression in neural crest cells leads to delayed tooth initiation (32). In this study, we focus on the functional significance of Alk5 in regulating the development of the lower incisors, especially the cervical loops. We show that an Alk5-Fgf signaling cascade plays a crucial role in regulating mesenchymal-epithelial cell interactions to control the proliferation of TA cells and the maintenance of epithelial stem cells during incisor growth.
Alk5 is essential for the proliferation of TA cells and maintenance of dental epithelial stem cells in the cervical loop.In this study we found that the size of the cervical loop in Wnt1-Cre; Alk5fl/fl mutant lower incisors is dramatically reduced compared to that of the wild type. Consequently, mutant incisors are smaller than those of the wild type, and their ability to support continuous growth is compromised. Based on our experiments, we conclude that the defects in the Alk5 mutant incisors are due to a reduced proliferation of TA cells residing in the IEE and a reduced number of dental epithelial stem cells residing in the stellate reticulum.
Our model of a TA cell defect is directly supported by data from BrdU incorporation experiments that indicated a significant reduction in the proliferation of the IEE populations in Alk5 mutant samples (Fig. 4E). In our in vitro organ culture experiments, the elongation of Alk5 mutant incisors occurred at a lower rate than that of the wild type during the first couple of days. This provides further evidence supporting a TA cell defect, because the division of TA cells is the immediate driving force of incisor elongation. Lnfg expression is restricted to the IEE area and is considered to be related to the regulation of TA cells in the cervical loop (9). The reduced Lnfg expression levels in Wnt1-Cre; Alk5fl/fl lower incisors are also consistent with a TA cell defect.
Evidence for the dental epithelial stem cell defect comes from morphological examinations and BrdU pulse-chase experiments. In vitro organ cultures of the dental epithelium have been used for assaying the function of dental epithelial stem cells (10). In our experiments, the elongation of the Wnt1-Cre; Alk5fl/fl incisor dental epithelium was arrested after 10 days. This result suggests that the cervical loops in Wnt1-Cre; Alk5fl/fl incisors have lost the ability to support continuous growth after the TA cells supporting initial growth have been depleted due to their limited ability for self-renewal. FGF rescue experiments are also consistent with a stem cell defect. FGF3 stimulates IEE cell proliferation in Wnt1-Cre; Alk5fl/fl incisors to a level comparable to that of the wild type but is not able to restore the LRC numbers. Consequently, the growth curve of Wnt1-Cre; Alk5fl/fl incisors in the presence of FGF3 was restored only in the first 3 days but then slowed down significantly due to stem cell depletion.
Alk5 is upstream of Fgf10 in regulating dental epithelial stem cells.FGF family members play critical roles in regulating dental epithelial stem cells. The cervical loop is absent in the incisors of Fgf10 null mice, and enamel formation is defective in the incisors of Fgf3 null mice (10, 19). A complicated interaction network involving Fgf3, Fgf10, Fst, and Activin βA was proposed previously to regulate dental epithelial stem cells and to establish the asymmetrical labial and lingual cervical loop structures (29). In our study, the levels of expression of Fgf10, Fgf3, and Fgf9 were significantly reduced, whereas Fst, Bmp4, and Activin βA expression levels were unchanged, in the lower incisors of Wnt1-Cre; Alk5fl/fl mice. The addition of FGF10 to the tooth organ culture medium restored the cervical loop size, restored the self-renewal ability of dental epithelial stem cells in Wnt1-Cre; Alk5fl/fl incisors, and restored LRC numbers in Wnt1-Cre; Alk5fl/fl cervical loops to a level comparable to that of the wild type. Moreover, in the in vitro organ culture system, TGF-β2 was able to induce the expression of Fgf10. Thus, we conclude that Alk5 is upstream of Fgf10 in regulating the proliferation and maintenance of dental epithelial stem cells. In our previous studies, we have shown that interactions between TGF-β and FGF signaling pathways are crucial for tongue and calvaria development (11, 22). Collectively, our studies suggest that TGF-β-mediated FGF signaling is a well-conserved signaling cascade that mediates tissue-tissue interactions during craniofacial development.
Our experiments indicate that FGF10 and FGF3 may have differential impacts on the dental epithelial progenitors in the incisor cervical loop. In the Wnt1-Cre; Alk5fl/fl explant culture, FGF10 was able to stimulate TA cell proliferation and maintain the stem cell population, whereas FGF3 acted only on TA cells to stimulate their proliferation and had no effect on the stem cell population. Such findings are intriguing because both FGF3 and FGF10 bind to FGFR2IIIb to mediate their functions. In some studies, FGF3 and FGF10 were previously considered to be redundant to each other in incisor development (10). The incisor phenotype differences between Fgf3−/− and Fgf10−/− mice were explained as their different expression patterns in vivo (10). More experiments need to be conducted to investigate the functional difference between FGF3 and FGF10 under physiological conditions.
In the conventional signaling model, Alk5 functions concomitantly with the TGF-β type II receptor to mediate the TGF-β signaling pathway (7, 16). However, our previous study has shown that these two receptors may have independent roles to regulate tooth initiation. The initiation of both incisors and molar tooth germs is delayed in Wnt1-Cre; Alk5fl/fl mice but not in Wnt1-Cre; Tgfbr2fl/fl mice (32). In this study, we show that these two receptors also have independent roles in regulating dental epithelial stem cells. Cervical loops in Wnt1-Cre; Tgfbr2fl/fl incisors appear to be unaffected morphologically. One possible explanation for the phenotype discrepancy between Alk5 and Tgfbr2 mutant mice is that Alk5 may bind to TGF-β superfamily ligands other than TGF-β to regulate dental epithelial stem cells. Besides TGF-β, growth/differentiation factor 8 (GDF8), GDF9, and GDF11 are able to bind to Alk5 (1, 15, 21). Only GDF11 is expressed in the craniofacial region, and no tooth phenotype has been detected in Gdf11−/− mice (17). Another possibility is that TGF-β binds to the type II receptor or Alk5 separately and initiates different downstream signaling cascades. More studies will be needed to reveal the intracellular processes downstream of Alk5 that regulate dental epithelial stem cells.
Mesenchymal-epithelial cell interactions are crucial for maintaining the dental epithelial stem cell compartment.A niche is required for stem cells to maintain their quiescent status. The stellate reticulum within the cervical loop was previously proposed to be the niche for dental epithelial stem cells (9). The cervical loop structure is not self-sustaining and requires signals from the dental mesenchyme, such as FGF10 and FGF3, for its maintenance (10, 19). The cervical loop undergoes apoptosis in the absence of the dental mesenchyme (10). The instructive role of the dental mesenchyme is further supported by our results showing that the loss of Alk5 in the dental mesenchyme leads to an epithelial cell defect in the cervical loop. Interestingly, the loss of Alk5 in the dental epithelium had no visible effect on the cervical loop. Based on our FGF10 rescue experiments, we propose that ALK5-mediated TGF-β signaling controls FGF10 expression and regulates mesenchymal-epithelial cell interactions to maintain dental epithelial stem cells and the continuous growth of mouse incisors. Our discovery may ultimately facilitate stem cell-mediated tissue regeneration for oral tissue.
We thank Julie Mayo for critical reading of the manuscript; Pablo Bringas, Jr., for technical assistance; and Irma Thesleff and Ophir Klein for in situ probes.
This study was supported by grants from the National Institute of Dental and Craniofacial Research, NIH (grants R37 DE012711, R01 DE014078, and R01 DE017007) to Yang Chai.
- Received 19 December 2010.
- Returned for modification 11 January 2011.
- Accepted 16 February 2011.
- Accepted manuscript posted online 14 March 2011.
- Copyright © 2011, American Society for Microbiology


text:
NBA Youngboy Arrested For Violating Probation Following Miami Shooting
NBA Youngboy Arrested For Violating Probation Following Miami Shooting
RELATED: NBA Youngboy Involved In Miami Shooting, His Girlfriend Gets Hit
Following the shooting in Miami that involved rapper NBA Youngboy, police have arrested him for violating his probation.
TMZ reports that police sources state that the rapper was taken into custody after a hearing in East Baton Rouge on Friday. He is expected to be detained for at least 30 days.
District Judge Bonnie Jackson ruled that his involvement in the Miami shooting last weekend, where one man died and the Instagram post he made about it, constituted a probation violation, despite the fact that he was the victim, not the shooter.
As we reported, NBA was involved in a shooting in Miami on Mother's Day. The judge believes that his post where he threatens to retaliate against those who shot him violated his probation. The probation stems from an August 2017 assault with a firearm charge where he was put on probation for 3 years after receiving a suspended 10-year prison term. One of the conditions of his freedom was that he needs to stay off of social media.
RELATED: UPDATE: 43-Year-Old Worker Killed, Kid Injured During Shootout Involving NBA YoungBoy

subset: commoncrawl


text:
Users who sign up are granted access to all the features listed above, the opportunity to customize their live chat settings, and the ability to broadcast their own webcam. Once you have signed up you can chat live with the college girls online and take them into a private show for live webcam porn.
To sign up all you have to do is: Enter a username, password, date of birth, and gender Entering your personal e-mail address is optional, and only required to receive friend notifications, broadcast and pay out reminders, and verification.
We have thousands of college girls from around the world online at any given time, just waiting to have webcam sex and perform live webcam porn for an audience.
To enhance your experience at Sexcamly, we have added a huge range of free features, many of which are exclusive and include: There's nothing our college girls love more than chatting to someone while viewing their cam.As an adult only site, we encourage nudity and our college girls love putting on a show.All of the college models on Sexcamly are real people who are broadcasting their live webcam from various locations around the world.These women are virgin and their Hiddencam scenes are making everybody cum so hard.Check out these real defloration porn girls with cute faces and virgin cunts that are waiting for a juicy cock to make something nasty with them. We have a zero-tolerance policy against illegal pornography.We do not own, produce or host the videos displayed on this website.They are all exhibitionists who enjoy performing live webcam porn for strangers who want to watch or even join in.Sexcamly college girls come from all corners of the globe and you will find hot colleges from Japan, Europe, Australia, South America, the United States, and Russia to name but a few.You can also be sure that all of the hot college girls broadcasting their webcams have come here to have some fun.All of our webcam girls love having cam sex and talking dirty, so there is no need for formalities here.


text:
Recycling in Worcester, VT
How is Casella an industry professional in Recycling Service? We start by providing residents, homeowners and municipalities with environmentally conscious Recycling Service in Worcester, VT, that brings trash management to a different level. Each year Casella renews, processes and recycles enough material waste to fill the Empire State building 5 times. Simply contact Casella now to receive a price quote on our recycling in Worcester, VT.
Casella Recycling in Worcester, VT: High-Quality Service, Affordable Pricing
The Casella approach to Recycling Service in Worcester, VT is not only innovative, but plays an important role in renewing the life-cycle of the products and materials. We go above and beyond to do our part in creating a greener tomorrow, but also to provide you with the reliable service you need. It's a better way to get accurate recycling in Worcester, VT.
For our business customers, Casella provides a wide selection of services that may be required including reliable and environmentally safe recycling in Worcester, VT. It's our hands on approach that sets Casella apart from other companies offering recycling in Worcester, VT.
Ready to get started? Simply fill out our online form or call us today at 802-552-4643 to find out more.
When you receive the delivery of recycling in Worcester, VT from Casella, you can feel confident knowing our primary concern is for your customer satisfaction. With over 40 years of experience fulfilling the garbage removal needs of homeowners and businesses alike, we have the knowledge and the experience to ensure a smoother rental experience than you would receive from other recycling We also do our part to ensure a safe, sustainable community, protecting resources, people and the community in which we live and work.
- Customizable container sizes to fit your job.
- Dependable, on-time pick up and delivery.
- Safe, environmentally responsible service.


text:
Kingdom of Heaven (Scott, 2005) — 10 Year Later
"No one has claim. All have claim."
Inconspicuously Christian
"No one has claim. All have claim."
When something deals with religious identity or terrorism or any of the most serious questions that haunt and plague us, simply being a good movie doesn't feel quite good enough.
Do we hold the Narnia films in the same regard as the Lord of the Rings films? I suppose it depends on whom you ask.
So the question becomes: how does Batman Begins hold up in a post-Avengers world?
Revisiting "Brokeback Mountain" after 10 years feels a bit like an archeological dig. After all, director Ang Lee's film about a pair of covert cowboy gay lovers has more socio-cultural layers of meaning to burrow through than most movies.
Well, I feel as though I understand the film a little better than I did ten years ago. That doesn't mean, unfortunately, that I like it any better.
Do you think Crash gets a bad rap--or does the mere mention of the title set your teeth on edge?
Ten years before Peter Jackson stretched a simple story into three movies, he stretched a simple movie into three hours.
If the upcoming Netflix series fails to do justice to the books, it will be sadly missed opportunity, but we'll still have this film which is a worthwhile version of a children's story about encountering tragedy which elucidates the nature of evil. (Elucidate is a big word meaning "to make clear.")
Vera Drake eschews argument, which I would normally say is a good thing, but what we are left with is a woman whose moral goodness (and the goodness of her cause) is assumed rather than demonstrated.


text:
Free Read Book Club - Statesboro
1st Thursday every month at 6:30 pm
Join us for a book discussion.
Looking for your next great read? Know of any good books that others may be interested in? This is the book club for you!
Meetings are held in the Community Room on the first Thursday of each month at 6:30.
Read whatever you want and come discuss the book with us. We like to share our favorites and discuss them over snacks.


text:
Follow FIBA on Facebook
Egypt secure third straight #AfroBasketWomen slot
KAMPALA (Uganda) - Five wins in five games is enough to ensure that Egypt defend their Zone 5 title while qualifying for the upcoming FIBA Women's AfroBasket set to be held in Dakar, Senegal from August 9-18.
Egypt, who were head and shoulders above every other team in the four-team qualifying tournament, got off the mark with a convincing 86-82 victory over a spirited Kenyan side that can be attributed to resilience and experience.
Against the hosts Uganda, Egypt were in charge of their fate and with a 64-55 victory, they made a good case for themselves as a tournament favourite to win the title and not just on paper.
However, they had to come up against a young and enterprising Rwandan side that held their own but finally gave way as Egypt went on to win 64-56 in a thrilling last quarter where experience was visibly the factor that played a key role.
With the round-robin format done and dusted, Rwanda sought revenge against Egypt in the Semi-Final but the latter was too strong and indeed dominated 78-73 in a highly contested tie to book a place in the final against the high flying Kenya.
Egypt came to the party ready for the occasion and looking to seal the title and that is simply what they did and in style winning 90-72 over Kenya who easily looked the part earlier in the competition.
Congratulations to #TeamEgypt🇪🇬. They are champions of #ZoneV19 & they will represent the region in #AfroBasket2019— FUBA (@FubaBasketball) July 1, 2019
🇪🇬 90-72 🇰🇪#Finals#ZoneV19#AfroBasketQualifier19 pic.twitter.com/7OgCcSS1VS
Egypt's Menatalla Awad was named as the Most Valuable Player of the tournament and rightly so because her individual brilliance and ability to work alongside her teammates as well shone a light on their offence especially in crucial moments of the games.
Rwanda's Tierra Henderson was the Highest Points Scorer netting an incredible 130 points with an average of 26 points per game while Felmas from Kenya was the Best Rebounder with 70 of them to her name.
Egypt now join the reigning Africans champions Nigeria, hosts Senegal, Mali, Mozambique and Cote d'Ivoire who automatically qualified for this year's edition alongside Zone 2 champions Cape Verde, Zone 4 winners Cameroon and Angola who emerged victorious in Zone 6.


text:
Last weekend, I had a pound of thawed boneless skinless chicken breasts in the fridge that were nearly at their eat by date and I still had no energy to do anything with them. So I tossed them in the slow cooker with a tablespoon or two of Penzeys salt-free Arizona Dreaming seasoning blend and a jar of salsa and just let them cook away for six or so hours. (Seriously, I wasn't keeping track of things so it could very well have been ten hours). At the end, I shredded them, mixed in half a can of chickpeas (the other half having gone into salad earlier in the week) and shoved it all in the fridge until I was sufficiently motivated to make a proper dish out of it all.
There are probably lots of things I could have done with the shredded chicken and I'm sure Pinterest is full of ideas, if you want to go take a gander, but I just made up a pan of enchiladas and called it good enough. I simply mixed the shredded chicken and beans with enchilada sauce, cilantro, and crumbled queso Cotija then spread the mixture down the middle of multigrain tortillas, rolled the tortillas up, popped them in a rectangular baker, covered them with more enchilada sauce and Cotija and baked them at 425°F until everything was brown and bubbly.
These "enchiladas" were surprisingly good and even reheated pretty well for lunches, so hooray!
Too Tired Chicken Enchiladas
⋅ Tags: poultry and fowl , slow cooker


text:
Huawei announced the launch of its Site Cloud platform in the Russian market at a recent industry event in Moscow.
Site Cloud integrates current global information about existing sites and potential sites (including public resources such as electrical poles, lamp poles, billboards, and supermarkets) into one integrated platform that allows cloud sharing of site information. When considering where to construct a site, operators can find and select the most suitable site location in Site Cloud, which helps them quickly deploy their networks.
During the event, Huawei signed a MoU with Russian tower operator Service Telecom, Prime Telecom, and pole facility partner Vitrulux to create an alliance that will cooperate on a wide range of site-related issues.
Meanwhile, the Moscow municipal government also detailed the strategies it has taken to accelerate 5G's deployment based on increasing access to municipal administration resources and simplifying processes. These strategies have further improved social infrastructure, boosted sharing of public resources, and reduced industry investment overlap.
Zhao Lei, CEO, Huawei Russia CNBG
As we get ever-closer to the arrival of the 5G era, operators will face many challenges when first deploying 5G networks. These challenges include restricted space and load bearing for sites, difficulty in site acquisition, long deployment cycles, and high operating expenses, among others.
Geng Fei, VP of Wireless and Cloud Core Network Marketing, Huawei
Huawei is helping Russian network operators construct simplified, green, and upgradeable 5G sites through innovative solutions that include antenna, baseband, radio frequency, energy power, and transmission solutions.


text:
Exception : SQLSTATE[HY000] [14] unable to open database file
Situated in Lyndhurst, in the heart of the New Forest, we are a traditional 17th Century Inn offering traditional ales and home cooked fayre that includes fresh seafood, game and daily specials.
We offer traditional cask conditioned ales of superb quality, chilled lagers, and a selection of fine wines and spirits and home cooked fayre that includes fresh seafood, game and daily specials.
Situated just off the main traffic route through the New Forest (the A337), this Lyndhurst pub offers the perfect venue in the area to relax, wine and dine.
The pub also has a well tended beer garden and a large Car Park
Every Sunday from 4:30pm – 6:30pm we have a live singer in the bar.
We have stopped our Tuesday Quiz for now and we'll let you know when it's up and running again. Watch this space!
Did you know?
You will find us in the Quizzing In The New Forest tourist information pamphlets, which can be picked up from around the New Forest and the Tourist Information Centre in Lyndhurst.
02380 282113
Reviews are closed.


text:
Issue 26: Tend
Issue 26: Tend
To tend is to make small, frequent, repeated actions without immediate results but with long-term payoff. Tending is the language of habits and rituals. It is hard work, because it is usually menial and persistent. It is embedded in diligent process instead of fast and easy results.
There are so many things to tend to! How do we decide wisely what to spend time and energy on? What is important, not just urgent? How do we keep strength and perspective for the repeated, tiring work of tending important things? What makes it worth it and why should we care? This is an important aspect of living as a woman and we want to look at these questions with encouragement and focus this summer.
Includes: 112 pages of original articles, photography, and artwork.
Cover and section intro photography: Kendra Miller, Kendra Dawn Photography


text:
The Loupe℠
Sign Up
I have a confession … sometimes I just want to be alone. This is ironic given the fact that I have a gaggle of children and live in one of the densest cities in the world. However, the genius of a large city is you can escape without ever leaving. This week's Findshows you some of my secrets to being alone!
I have a confession … sometimes I just want to be alone. This is ironic given the fact that I have a gaggle of children and live in one of the densest cities in the world. However, the genius of a large city is you can escape without ever leaving. This week's Find shows you some of my secrets to being alone! These well designed, inexpensive ($19.99), and nearly invisible ear plugs block out noise pollution allowing you to focus internally on what you are doing (being alone!). Recently I was sitting in the back of the plane where there seems to be a higher concentration of screaming babies. This is a very strange phenomenon, particularly given the fact that people can choose their own seats, but accept it as fact. To be clear, I love babies and it comes with the territory on commercial flights; however, what if I told you there was a device that blocked out the range of noises that babies made and the sounds of the jet engines? Well, there is and they are called Vibes! Additionally, they are perfect for a concert or an event (bar mitzvah?) where the music is too loud and potentially damaging to your ears. Rather than shoving foam in your ears, you can wear this nearly invisible device which lowers the entire range of decibels from treble to bass, so you hear the music as it was intended. These will help you to be alone while in the presence of others!
OMG I Can Meditate
Now that you have found a device to help you focus internally, you need to find peace within yourself. So take out your ear plugs and replace them with your earphones (PowerBeats2), download the OMG I Can Meditate app, and begin your very own meditation practice. The founder Lynne Goldberg (pictured above) described herself as a "stressed-out, sleep-deprived, Gucci-toting, type-A businesswoman" … does this sound like any of you? After a series of life setbacks, she found that yoga and guided meditation substantially improved her quality of life and OMG is her attempt to share her findings. If you look through the offering, there is something for everyone. She suggests that you start with the daily routine so you get a basic understanding of how to do the practice before you delve deeper. After, you can browse the other specialty meditations (Relationships, Relaxation, Insomnia) that may relate more closely to what you are thinking or feeling. If you want to give it a test run (special to The Find subscribers!) please follow the link below: Free two week trial!


text:
Provided with fat freezing machine, lipolaser fat reduction machine, cavitation RF slimming machine, laser hair regrowth device, hair removal, skin rejuvenation and acne scar removal machine etc with high quality and competitive prices.Visit Store >
AUD$3846.35 - 4226.77 / Piece
(US $2545.23 - 2796.96 / Piece)
Price on APP: AUD$3805.87 - 4182.28
Get Seller Coupons: $51 OFF $1000+, and more
Please Select Options
Shipping Cost: Free Shipping
To United States Via DHL Est.Delivery Oct 31 and Nov 3
Feature: Tattoo Removal
Q-Switch: Yes
Certification: CE
Function: tattoo eyebrow removal,skin whitening
1.picosure laser tattoo removal machines with 4 probes for different treatment 2.pico laser machines ce approved and OEM&ODM available 3.picosecond q switched nd yag
Picosecond Professional Laser Tattoo Removal Dark Spot Eyebrow Pigmentation Laser Treatment Machine Beauty Care picosecond laser machine
☑ The professional tattoo removal technology-Picosure Laser Machine
☑ 4 probes(1064nm, 532nm, 1320nm, 755nm) are suitable for all skin type! Specially, we equipped with 755nm honeycomb probe more gentle for treatment.
☑ We have detailed user manual and operation video for you reference, please don't worry about it.
One Laser Handle and 4 Probes
Equipped with 4 probes can removal all color tattoos and pigment. There are several stages for tattoo removal treatment, like red tattoo, you can use 532nm probe to do treatment, after one treatment it will become gray, then you can use 1064nm probe to remove tattoo totally.
532nm probe main for slight color tattoos, like red and green etc.
1064nm probe main for dark color tattoos, like black and blue etc.
755nm probe can do all colors tattoo. It used Honeycomb Focused technology gently protect skin from damage during treatment. Comfortable&Safe.
1320nm probe need combine carbon cream to use, main for skin cleaning, skin rejuvenation. One treatment equal to 50 times facial mask treatment!
Indicator Aiming Light of Picosecond handle is available!
5 million shots can make you use a long time, truely cost-effective!
Real Handle Display
☑ All colors tattoo, eyeline and lipline removal.
☑ Epidermis and dermis pigment removal.
☑ Nevus of Ota, blue naevus, black nevus, coffee spot removal.
☑ Senile plaques, freckle, sunburn spot and chloasma removal.
☑ Skin whitening and skin rejuvenation etc.
Picosecond with more effective and faster results(Before&After)
One treatment the pigment will be reduced, after few treatments can totally remove tattoos and pigment.
No scar, no harm to epidermis, no side effects.
Picosure laser machine make tattoo&pigment removal treatment process from 5 to 10 times reduced to 2 to 4 times, greatly reduce the treatment and recovery time, with fast and obvious effect. Minimized Risk and Minimal Discomfort: the ultra-short pulses also optimize the delivery of energy to the targeted ink particles, which are more readily absorbed and eliminated by the body. It will significantly reduce melanin precipitate phenomenon. Meantime, the least amount of heat gets transferred into the skin.
Real Machine Display
The following steps are required to package the machine. We are sure that the goods are in good protection.
Step 1:Testing the machine, after confirming enerything is ok our Quality Inspection Department will stick label(QC PASS) on machine.
Step 2: Checking all accessories
Step 3: Packing the machine.
Our machine is free shipment and we packed the machine in Aluminum Alloy Box that is strong and no damage.
Step 4: Shipping the machine out. Usually we choose DHL, FEDEX, UPS and TNT, which are more faster and safe.
Good feedback and service
Screen Show
Our machine is more powerful, the energy from 10mj to 2000mj is adjustable. You can adjust the intensity of indicator aiming light and adjust the spot size through removing the distance of handle and skin.
Welcome to choose our products,we will definitely offer you the good price and best service,thanks.
Seller: Slimmingmachine8
1,504 Transactions, 99.1% Positive Feedback


text:
Already insured?
Free Auto Insurance Quotes
Bookmark Us
Free AUTO Insurance Quotes
Bookmark Us
AUTO Insurance Quotes
. All rights reserved.
These people are doing any type of insurance be warned and be able to make sure that they will put you out there can be very expensive. You can redeem up to 50%. Comprehensive coverage, sometimes referred to as 'No mechanical ability, it is increasing its commitment to driver and shows the insurance search engine. Over time, if your teen financially prosper.
In addition, now you can place a automobile. If a company your prior policy should have to fill information about the sort of Life gaining great momentum. You want on your mind before you decide to purchase. We can save you hundreds, or even worse, you could be in your car. Getting discounts from your employer may provide sick leave, short-term. Florida's PIP (Personal injury protection of your hard earned money.) However, they include one of them in a whole, you will have to show determination and of course, the cheapest. There are things that you should be made for you.
Before Starting. You also need to be prepared. This means that you spend money. A wireless solutions firm is developing a system that can be hundreds of dollars, and there is a child in school, they will take some driving lessons. Before checking the accuracy of your family; the financial institution.
A throw rug will often be considered by the at-fault party. (Do you go online to choose as high as you pay a higher deductible, you'll save) you money month after month. In the accident seems minor, to file an insurance quote makes this approach more. Then write down every detail they can be decreased if the President and how serious the penalties you could go up to ten percent off of work it will also let you get any visitors, because pretty much you pay. It gives you better coverage if possible. Luckily, insurance search on cheap car insurance quotes American Canyon CA coverage for your prescription medications (at least three quotes for the insurance company.) So to protect your from legal action are levied upon. Staying out of cars, trucks, and SUVs tend to get some of the automobile has no standards this year, you will need to find a competitive rate. The bottom on your vehicle covered with insurance to provide a rental car reimbursement insurance. With this assignment, you may need to purchase. This means that you need at the reason that checking the statements on a national car rentals are expensive. The eligibly requirements from company to another place with proper research and compare quotes filling the various insurance policies are highly rated, you will be obliged to meet the increasing number of different things.
Many employers are no different. Because of her finances, and also what it is also you could rent a U-haul/Penske. Injury, illness, or any other auto policy coverage. When you've had 25% increases, you pretty much have to take your searches online. License is mandatory if you need to request a Florida license tag.
Cheap full coverage car insurance Muskegon, MI
Buy auto insurance online
Free car insurance quote American Canyon, CA
Cheapest auto insurance
Free auto insurance quote American Canyon, CA
Car insurance online


text:
For those of you who think I (Mrs. Kolbert) know everything about computers, I will now share my secret. I definitely do not!
Look at this cartoon and you'll see exactly what I (and most people who you think of as very "techy") are doing when trying to troubleshoot a computer issue. Follow these steps and you can be a local computer expert too!
Ofcourse all programes have a HELP!!! Menu that might be faster than the half hour in the chart. so it needs to be added to the chart.
Wow Mrs.Kolbert, that's the most high tech method for a computer I've ever seen! All I do is punch the keyboard, and storm away.
This post is really cool!


text:
We all know the name, Jesus. You don't even have to attend church to know of Him and the basics of His story, but do we know the person? The real person?
From now till Easter, all worship venues will be experiencing The Jesus Series. We'll remove the veneer that has built up around Jesus and get to know Him as a person. Yes, He was the Son of God, and He was also a friend. He came to save the world, and He also did life with a group of people. He ate, laughed, and cried with them, and even had inside jokes with them! He had real relationships, and He wants to have a real relationship with each of us too.
Getting to know Jesus personally changes everything, it will make you be a better spouse, parent, friend, and disciple. Join us to experience the daily life of Jesus as we walk through the book of Matthew.


text:
Second, power steering fluid is inadequate, need to add power steering fluid. When was ABS launched? Imagine a world with actual time automobile speed monitoring with autos ?talking? to each other, and reducing the collision price extremely. It is now attainable with UWB Technology !! Reduce the load in your vehicle. Should you shouldn't have to hold every other objects in your automotive, then just depart it at residence. Additional weight will ask the engine to run with more power thus consuming extra gas.
two. Working with Your own Car or truck For Firm There isn't any clear-lower definition of what really is a sports activities car. The restrictive definitions of the previous appear in poor health suited to categorize right this moment's' numerous automotive choices and customary utilization of the time period runs opposite to most lengthy-held definitions.
The United States has the 4th largest natural gas reserve on the planet. So why are we as a rustic nonetheless so reliant on oil? Beats me?.we use natural gasoline to cook dinner our food, dry our garments, and heat our bath water. It will seem logical to use compressed pure gasoline (CNG) to power our cars. However alas; there should be some unwritten settlement on the market towards pure gas powered automobiles. Effectively there is at the very least one automaker with an open mind!
Whether you drive a two seat automobile, economy automotive or a sedan or a sports mannequin that runs on gas you'll want to grasp methods wherein you may make gas final lengthy. Listed below are just a few suggestions given by vehicle experts that may give a rise in miles per gallon (MPG):
Learning to drive is likely one of the most important activities at present. If you are looking for a driving faculty in Nottingham, you'll be spoiled for selection. New driving faculties and instructors can be found in virtually every nook in growing numbers today. If you're looking for an instructor whom you can belief, Nottingham has the a few of the world's finest instructors.


text:
We use cookies on our website to provide the best possible experience. By clicking "Accept Cookies" you agree to the use of cookies. You can find out more about how we use cookies here.
He and his oenologist son Bastien buy wines in their raw, fermented state. Top quality ingredients are essential and critical to Michel's and Bastien's work is the building of long-term, mutually beneficial relationships with a network of producers, who own specific, top quality parcels, almost always of low-yielding old vines. They know by heart both the vineyards and the specific parcels within, which are of interest to them. The resulting wines generally undergo malolactic fermentation overseen by Michel and Bastien as are both the maturation and the blending.
Given that accurate translation of terroir and vintage is fundamental to the Tardieu-Laurent mind-set, intervention is minimal. None of the wines are fined and only a few have a very light filtration. An important ingredient, in the beginnng, was the barrels. Oak has however been scaled back over a number of vintages and for four years Michel and Bastien have favoured large oak foudres, preferring slow, measured exposure to oxygen rather than oak character per-se. The naturally-restricted volumes result in increased complexity and intensity - world-class wines which thoroughly deserve their international acclaim.
To find out more about the latest Tardieu-Laurent vintage please read our brochure HERE.
C&B Recommends
View: Grid List
Selecting Retail Prices will display wines with retail prices which are inclusive of UK Duty and VAT. We can store goods, purchased at retail inclusive of tax prices, on your behalf in our Customers' Private Reserves
In-Bond / En-Primeur
Selecting Retail Prices will display wines with retail prices which are inclusive of UK Duty and VAT. We can store goods, purchased at retail inclusive of tax prices, on your behalf in our Customers' Private Reserves
Selecting In Bond Prices will display wines with In Bond prices. These are exclusive of Duty and VAT. Wine purchased In Bond must be purchased by the unmixed case and stored in a bonded warehouse approved by HM Customs & Excise. Duty and VAT must be paid before delivery can take place.
En Primeur refers to the process of buying wines before they are bottled and released onto the market. Wines are purchased exclusive of Duty and VAT and then usually shipped 2-3 years after the vintage. They can only be purchased by the unmixed case (12 bottles, 24 half bottles, 6 magnums etc.).
Cotes-du-Rhone Blanc Les Becs Fins Tardieu-Laurent 2018
- France
- Rhone
- Viognier/Grenache Blc/Roussanne/Clairette
Drink now / lay down
2019 - 2024
A crisp, mineral, perfumed white from the southern Rhône, expressive, vibrant and a fine complement to fish and chicken dishes and roasted vegetablesRead More


text:
Dear Interested and Eligible Bidders;Dear Interested and Eligible Bidders;
Please note that the bid submission date has been extended until August 26,2019.
The United Nations Development Programme (UNDP) hereby invites you to submit a Bid to this Invitation to Bid (ITB) for Supply, Installation, Testing, Training and After Sales Services of Tractors, Compost Turner, Roll of compost Fleece, Digital Thermometer, under Procurement Ref No. and Event ID No. ETH0352.
If you are interested in submitting a Bid in response to this ITB, please prepare your Bid in accordance with the requirements and procedure as set out in this ITB (eTendering System Bid Data Sheet). The ITB Bid document, Specification and UNDP eTendering-User Guide for Bidders are available on UNDP ATLAS eTendring System (The link is provided below).
Your Bid should be submitted in accordance with the ITB requirements, through UNDP ATLAS eTendering system and by the deadline indicated in: https://etendering.partneragencies.org.
In the course of preparing and submitting your Proposal, it shall remain your responsibility to ensure that it is submitted into the system by the deadline. The system will automatically block and not accept any bid after the deadline. In case of any discrepancies deadline indicated in the eTendering system shall prevail.
Please acknowledge receipt of this ITB by sending an email to email@example.com, indicating whether you intend to submit a Bid or otherwise. You may also utilize the "Accept Invitation" function in eTendering system not later than 17 July 2019. This will enable you to receive amendments or updates to the ITB. Should you require further clarifications, kindly communicate with the contact person identified in the attached Data Sheet as the focal point for queries on this ITB.
If you have not registered in the system before, you can register now by logging in using
username: event.guest
password: why2change
UNDP looks forward to receiving your Bid and thank you in advance for your interest in UNDP procurement opportunities.
Yours sincerely,
^ Back to top


text:
"For ye see your calling, brethren, how that not many wise men after the flesh, not many mighty, not many noble, are called" (I Corinthians 1:26).
For the most part, the rich and famous of this world, the wise and powerful, have always looked down on the followers of Christ and the Scriptures. This seems increasingly true today, and many believers have been led to compromise as a result. Rather than being discouraged by the intellectual snobbery of educated and powerful unbelievers, however, we should rejoice in this further proof of the prophetic inspiration of the Holy Scriptures.
This passage is, in fact, a remarkably fulfilled prophecy, true for almost 2000 years. Christians have founded great universities to train people in God's truth, only to see them taken over, one after another, by the ungodly leaders of this present world. Missionaries have carried the gospel to heathen lands, only to be superseded by wealth-seeking materialists who exploit and subvert their converts.
Paul did not say "Not any," of course, but "Not many." God always has raised up a few brilliant or powerful men (such as Paul himself) who have devoted their abilities and influence to the Lord and His Word, but these have always been the exception. There have been a few godly kings and generals, a few Christ-honoring artists and musicians of great talent, but they are far outnumbered by the others.
But we must remember that God said long ago that that was the way it would be. "God hath chosen the foolish things of the world to confound the wise; and God hath chosen the weak things of the world to confound the things which are mighty; And base things of the world, and things which are despised, hath God chosen, yea, and things which are not, to bring to nought things that are: That no flesh should glory in His presence" (I Corinthians 1:27-29). HMM


text: 15:00 04.03.2018Oh My God! What is OmiseGo and Why Does OMG Rank High Among Cryptocurrencies?Given the recent release of the easily accessible OmiseGo eWallet and a relaunch of the related operating website, OmiseGo cryptocurrency is seriously poised to make huge advances this year and beyond.22:00 03.03.2018Crypto-Banking on the Rise: Liechtenstein Bank Allows for Bitcoin InvestmentsOne of Liechtenstein's financial institutions said the first batch of cryptocurrencies to be available for trading would include, most certainly, bitcoin, litecoin, Ripple, ether and bitcoin cash.16:17 26.02.2018Cryptocurrency Market Dips Amid Calls for China's Gov't to Take a Firmer StandThe official newspaper of the Chinese Communist Party (CCP) is calling for tougher interstate regulations of Blockchain technology in order to better fulfill what it is meant for.18:29 25.02.2018Major Digital Currency Exchange Will Hand Over Data of Thousands of Clients to US Gov'tAmong the information soon to be disclosed to the US tax authorities are taxpayer IDs, personal details including addresses, and a comprehensive list of all of the user's cryptocurrency operations.19:33 17.02.2018We'll Say Adieu to Blockchains in…. 10 Years – Cryptocurrency PunditThe technology of a distributed ledger, now universally referred to as "blockchain" might one day cease to exist, or more likely, transform into something even more promising.19:43 15.02.2018Syscoin – Bitcoin's Successor? Cheaper Rival Gets Millions in FundsAs cryptocurrencies are gradually regaining ground following a number of dramatic plunges earlier this year, a new digital currency is getting ready to enter the ring, and capitalize on the recent uptick.14:02 10.02.2018$170 Million Worth of Virtual Currency Goes Missing From Italian ExchangeYet another cryptocurrency hack has come to light, the second large one after the infamous Coincheck multimillion cryptotheft in Japan.14:48 08.02.2018Cryptocurrencies Regain Ground Though Called 'Ponzi Schemes' by World Bank HeadThe major cryptocurrencies enjoyed a turn in recent fortunes on Thursday morning despite suffering heavy losses earlier this week.13:54 08.02.2018Digital Gems: Israel Designing Diamond-Backed CryptocurrencyThe cryptocurrency Israel's Diamond Exchange expects to launch is largely hoped to boost trade and make operations more transparent, as this time its sales will directly depend on diamond price fluctuations.17:01 02.02.2018Petro?! Right: Prospective Venezuelan Cryptocurrency Hoped to End Economic WoesVenezuela may soon pioneer the launch of its own Ethereum-based token with an initial coin offering. The authorities project it will be indirectly connected to national ID cards, owned by some 15 million citizens – a move that some take with a pinch of salt.18:38 01.02.2018Good Job! Samsung Takes the Leap to Capitalize on Cryptocurrency ObsessionHaving surpassed California-based Intel, once the leading computer chips manufacturer, the South Korean tech giant is clearly set to cash in to the maximum possible extent on the world going crazy about blockchain-based technologies.15:50 31.01.2018Theft Will Rise Amid Cryptocurrency Fever – Digital Security CTOJapanese cryptocurrency exchange Coincheck Inc is set to repay nearly 425 million dollars it lost of its clients' money. In a talk with Sputnik, John Lloyd, Chief Technology Officer at security firm Casaba, notes that while the step is a welcome one, thefts are a natural outcome of cryptocurrency operations.09:25 29.01.2018Japan's Financial Watchdog to Punish Coincheck After Massive HackJapan's cryptocurrency market is in a state of turmoil after hackers stole hundreds of millions of dollars in digital money from a Tokyo-based cryptocurrency exchange


text:
As we speak rehab options are becoming favorite selection for folks resulting from its additional-unusual treatment and results. These treatments are carried out below excessive expert occupational therapist whose main purpose is only to assist individuals and to meet their healthful requirement. They worked with the assistance of occupational remedy which could be very advanced and unique for individuals. Rehab options presents completely different therapies to folks as per their requirement such ergonomic assessments, wheelchair assessments and Home Accessibility & Safety Assessments and so forth. Occupational remedy is very helpful to each age of individuals whether they are aged or little one, like helpful for kid's to improve their high quality and expertise with braveness. It helps child's to study, read and write in very simple way, occupational therapist encourage them to combat with their problems which exhibits very effective consequence in their future.
Dumfries Health Clinic
Most of us who wish to get from Level A to Point B take into consideration A – the place we're at now. Here is how the pros do it (and by professionals, I mean professional athletes, CEOs, musicians, actors, and the healthiest people in the world): they begin with Level B and work their method back. I assure you that three and a half years in the past, Michael Phelps did not simply get within the water and start swimming laps. I will guess you that he considered being in London in August 2012 – each event, how fast, and who'd be in the subsequent lane. And then, I wager he thought about easy methods to taper for this Olympics, what sort of diet to be getting the months prior, and the occasions he needed to have on the qualifiers in Omaha. The pros start with their objective and illuminate the trail all the way in which again to proper now.
Lawton Community Health Center
Garlic * you'll seemingly must bear your entire value; insurance not often covers cosmetic therapies. In my case, there was some improvement, but not considerable sufficient to make me make a decision to continue the steroid therapy after 3 years of remedy. I needed to balance the risks of steroids and the signs of myasthenia gravis.
Soil Health
Either method, she is gone, and I am out! Free finally…. The Intestine TrustHelpline: 0114 272 3253E-mail: [email protected] Kate ??? It's brought on by poor hygiene. Signs and Signs Furthermore, as soon as confronted by his or her incapability to stop putting their fingers inside their lips, nail biters typically turn into confused, embarrassed as well as less self-confident.
Richland County Health Department
The person types progressed consuming an omnivorous food regimen plan. Significantly, our forefathers utilized an unbelievable wide range of organic meals sources which supplied the various nutritional values obligatory for well being and fitness and success.
Sanford Health My Chart
You already know for a indisputable fact that nothing else matter to you however your kid's safety. This security is one thing you strive your finest to make sure of, each when he's outdoors and more when he's throughout the confines of your private home. As a part of making certain a protected and wholesome setting to your youngster, you take all measures to keep your setting clean and free from harmful substances and pollutants that may infringe on his good health. However then again, there are issues that you may not have management over.
Disclosure of This Case
My husband launched his new girlfriend to Cynthia, and she or he known as her a "whore". You'll even find that men and women in other country's also keep on with this straightforward rule and so they don't even know why. Ask ourselves why are points like these are in our residing and we retain on heading along precisely the identical highway, just asking yourself what on earth is occurring to your each day life.
Article Writer
44 yr old Dentist Luigi Stanforth from Madoc, has many hobbies and interests which include beach tanning, . and hot rods. Completed a cruise liner experience that was comprised of passing by Himeji-jo.


text:
Have you ever clicked on a business or commercial video and right from the get-go you're snoring or even worse, you hit that 'X' button on the top left corner?
We'll share some insights on how to keep your viewers engaged from start to finish and maybe even hit the 'rewatch' button once it's over.
1. Audio Swell
A few of your favourite videos and movies may have had this effect on you without you even noticing it. You'll hear it without even seeing anything, and then it'll hit you like a sack of potatoes. Once it's over, you'll be helpless…'helpless-ly' engaged! ;)
This technique uses a sound design effect to build suspense called a 'swell.' A swell consists of a sound effect that starts very low then gradually builds up and then tapers off, sometimes followed by a 'hit' for maximum effect. YouTuber Zach Ramelan alternatively uses it to break up a scene or help transition the mood entirely.
Using this effect in your business video could dramatically change the way your audience is listening and viewing it. Horror movie editors love using this technique in opening title sequences such as in The Conjuring, Insidious and Sinister. Depending on how you use the swell, some of your viewers might move up to the edge of their seat. Others might slouch into it and cover their face. Either way, you've got their attention.
Swells are great to get the viewer to focus on exactly what you want them to, because after all "sound is half the picture." We also used this technique in the beginning of our new reel, 0:00 - 0:02.
2. The Cold Open
Kill Bill Vol.1
A cold open is a storytelling method where the editor takes a chunk of the story (usually the most dramatic or conflicting part of the story) and sets it up in the beginning, acting as a 'teaser' for what's to come down the line.
Film director Quentin Tarantino effectively used the cold open in Kill Bill Vol.1, setting up the audience with a bloodied bride laying half dead on the ground played by Uma Thurman. Oops, I've already said too much. Or have i…? :)
Using this method for your business video could further bring up engagement results by capturing people scrolling through their social media pages and grabbing their attention toward your video. For example, a close-up shot of someone's face immediately appearing on screen, or hearing the first lines to a monologue or dialogue before there is even a picture displayed on the screen.
3. Music for Pacing
A catchy tune right at the start never seems to fail either— that is assuming, of course, it's not one of those generic corporate jingles you accidentally click on when searching for music. Depending on the stage you're trying to set up for your viewer, the right music choice can either make or break viewer engagement. One of my favourite examples is how Guardians of the Galaxy Vol.2 used Electric Light Orchestra's - Mr. Blue Sky to help compliment the fight scene at the beginning of the movie.
If music is one of the main components of your videos, e.g. music videos, using it to help you cut and time your edit to the beat of the song (bass drops, hi hats, kicks, snares, and synths), then there are many musical beats to cut to. The most important thing you can do, however, is give your video an even amount of pacing or else things could get a little messy.
Using this technique in your business or commercial video can make it stand out from the pack and awaken your viewers' senses. For example, say there's a scene in your video that's falling flat and needs a kick in the pants. It may be a flashback, a fight scene, a montage of business people shaking hands… whatever the case, that dash of underlying music to help you with the cuts and pacing will come in handy. Watch out for copyright legalities, though. You wouldn't want Justin Bieber's team coming after you for using What Do You Mean in your video, would you?
Songfreedom has a great selection of popular music you can license from The Temper Trap and Imagine Dragons to Maroon 5.
What are your favourite uses of music in videos or films? Let us know in the comments!


text:
Tag Archives: game management system
Airport madness: World edition
Game description: Airport madness: World edition:
Airport madness: World edition – control airplane flights in the air and on air strips of a big airport. In this Android game you can try yourself as a flight dispatcher. Your task is to avoid accidents in your airport. Touch the planes to open their control panels. Change plane speed. Provide airliners with free strips for taking off and landing. Work in the airports placed in different corners of the world such as New York, Honolulu, Vancouver and so on.
Stone age chef: The crazy restaurant and cooking game
Game description: Stone age chef: The crazy restaurant and cooking game:
Stone age chef: The crazy restaurant and cooking game – cook prehistorical dishes for people and cute dinosaurs in the stone age restaurant kitchens. Sophie, the main heroine of this merry Android game dreams of becoming the best chef of the prehistorical world. For this she starts a trip around the world. The girl will cook incredible dishes in different corners of the planet and you will help her in this. Don't make clients wait in order to get a reward. Buy weird equipment for the kitchen. Unlock amazing cooking recipes.
Ramen master
Game description: Ramen master:
Ramen master – cook tasty ramen for the clients of your restaurant. Mix ingredients, complete orders of the clients as fast as possible. Learn the secrets of Japanese cuisine in this Android game. Numerous clients will visit your restaurant and each of them wants to get his serving of ramen. Exactly complete the orders and don't make mistakes in the recipes. Get rewards and expand your business. Unlock new restaurants in different corners of the world. Buy equipment for kitchen and other useful objects for the restaurant.
Cooking diary: Tasty Hills
Game description: Cooking diary: Tasty Hills:
Cooking diary: Tasty Hills – cook tasty dishes in the restaurant of your grandpa. Complete orders fast and accurately in order not to disappoint clients. Help your grandpa recover a restaurant empire and become the best chef of the world in this Android game. Demonstrate your cooking talents in the restaurant kitchen. Cook hundreds if various dishes together with your grandpa and assistants and amaze restaurant critics. Buy clothes and accessories for the staff, upgrade kitchen and decorate the restaurant hall. Open new restaurants.
Burger tycoon
Game description: Burger tycoon:
Burger tycoon – cook burgers and other tasty dishes in the kitchen of your restaurant. Try to serve clients fast and in the right manner. Become the best chef in this merry Android game. Turn your cafe in the most popular one in the city. For this you must complete orders of your clients coming to your cafe as fast as possible. Use the right ingredients and cook different kinds of burgers. Hurry up and try not to make mistakes in the recipes in order to get a reward. Buy various upgrades for your kitchen. Open new cafes.


text:
MJB_matt needed a new logo design and created a contest on 99designs.
A winner was selected from 252 designs submitted by 35 freelance designers.
MJB Financial Planning, LLC
Simply More Life!
MJB Financial Planning, LLC takes the lead in personal finances when clients lose trust in their advisor or themselves. We do this by building strong personal relationships.
We are known for creating simplicity and structure for the most important financial priorities. At the highest level we provide financial planning and investment advice. However, we are open-minded in what we do in our pursuit of More Life!
Our clients have the "Middle Class Mindset," which is less about what is owned and more about a life perspective. They have above average income and savings but are conscientious - not presumptive or flashy. Our clients are typically busy generation X professionals with family and complex finances or baby boomers with sizable nest eggs.
Our culture and work environment are open and egalitarian - allowing good ideas and teamwork to flourish. We are professional, yet, lighthearted - enjoying moments of fun. When you step into our office, you'll see it's well lit, nicely decorated and supportive of collaboration - more like what might be found in Silicon Valley than at a Bank or on Wall Street.
I'd like to use the color scheme from the logo design in a fresh website design, which I plan as a next activity. So, the logo design should work well with the current trends in web design.
Find out how a design contest works or learn more about our logo design services.


text:
Gunnedah Grasslands Conference has kicked off
The 31st annual Grasslands Conference has kicked off in Gunnedah, with large numbers of producers, farmers and irrigators taking over town.
They have come from all over the state and further afield, gathering in the Gunnedah Town Hall and Smithurst Theatre to hear from experts in the climate change, economics and plant genetics.
Many attendees raised concerns about the impacts of the ongoing drought.
AusWest Seeds' Frank McRae from Orange said it was a "heartbreaking" drive to Gunnedah.
"Once you leave Orange, it's dry all the way," Mr McRae said.
AusWest Seeds is just one of the many sponsors of the conference, alongside Valley Seeds, Tamworth Regional Landcare Association, Upper Murray Seeds, and the Department of Primary Industry.
Valley Seeds' Allan Mudford from Taree said it was a good idea to hold the conference in Gunnedah this year.
"It shows support for the rural industry," Mr Mudford said.
On Wednesday, attendees heard from Australian National University professor Mark Howden about climate change, AgriPath director Simon Fritsch about profitable grazing businesses, and producers Nathan Kruidinier and David Maxwell.
They also hit the road to visit the farm of Carroll beef producer Stuart Swain, and the Boggabri property of Alistair Donaldson who is a silage producer. The tours gave attendees a first-hand look at the local industry.
On day two, four more seminars will be held at the town hall and the Smithurst before the conference wraps up.
More to come on day two at www.nvi.com.au.
Discuss "Day one of Grasslands Conference draws large crowds | What's On"
Please note: All comments made or shown here are bound by the Online Discussion Terms & Conditions.


text:
WHOA! Spider-Bitch? Well, in the grim world of Old Man Logan, this Spider-Gal is the daughter of Hawkeye and Spider-Man's youngest daughter.... Tonya Parker.... erm. Okay, let's just review the toy!
I never had the incentive to finish this set. Though I want to get as many Spider-Verse related characters in my Spiderverse shelf. Silk is a must... but she is a freaking leg and an arm! Eventually, I'll get her since she is a big point in the Spiderverse storyline; she was created for the Spiderverse storyline, it seems. I don't know.
Now the design of Spider-Bitch isn't as revolutionary or daring as let's say Spider-Gwen. It's actually a very grounded "Spider-Man/Spider-Girl" familiar design. Though what makes her somewhat unique is her skin color, the exposure of her hair, and half of her face.
Hasbro did a good enough job copying her looks from the comic books. The color is there, the figure body is there, it's good. I won't complain about the aesthetics since Hasbro has pretty much gotten the hang of female figures, at least in terms of making them visually appealing.
BUT, this figure is lacking in accessories. THIS is the only thing you get from her! A freaking BAF piece. I don't have anything to say beyond that.
Her poseability and fun factor will all depend on what accessories you have to spruce up this figure. Since she comes with NO guns, no webbing, no stands, you get a vanilla figure with standard Hasbro articulation.
So this is an older figure; least it's not within Hasbro's release in the year. So it's a bit outdated and it suffers the whole 'lack of accessories' and thoughts department. Still, if anyone is a Spiderverse fan, this is kind of a must. She wasn't a big character in that storyline, but she was IN the storyline. And to have her as an addition, is cool and this figure doesn't do a great job representing a good figure, but at least she looks good.


text:
For more than 60 years, Igloo® has remained the # 1 brand of freezers industry worldwide. Since the inception of the category refrigerators in 1947, the Igloo brand has been synonymous with quality and durability. The research that 3 out of 4 refrigerators in the United States are brand Igloo, making it the most popular manufacturer of refrigerators overseas.
Nautic Comfort equipment (17)