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MicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.
MicroRNAs (miRNA) are non-coding single-stranded RNA molecules with 18–24 nucleotides, which are produced from endogenous transcripts of short stem-loop structures. Through binding to the 3′-untranslated region (3′UTR) of different target mRNAs (mRNAs), microRNAs can repress the translation of the target mRNAs (Seitz et al., 2003). As a new class of regulatory molecules, miRNAs have diverse functions in cell proliferation (Shatseva et al., 2011; Viticchiè et al., 2011; Yu et al., 2012), survival (Fang et al., 2012a; Ye et al., 2011), invasion (Deng et al., 2011; Luo et al., 2012; Siragam et al., 2012), cell differentiation (Goljanek-Whysall et al., 2012; Kahai et al., 2009; Rutnam and Yang, 2012b), morphogenesis (Wang et al., 2008a), tissue growth (Shan et al., 2009), angiogenesis (Fang et al., 2011; Lee et al., 2007; Smits et al., 2010; Zou et al., 2012), tumor development (Nohata et al., 2012; Volinia et al., 2006) and metastasis (Huang et al., 2008; Ma et al., 2007; Rutnam and Yang, 2012a). The largest functional group of miRNAs is the one involved in cancer development. Recent studies have shown that miR-24 can inhibit apoptosis of both cancer cells (Qin et al., 2010) and cardiomyocytes (Qian et al., 2011) and can accelerate cell proliferation (Zaidi et al., 2009). Upregulation of miR-24 has been observed in oral carcinoma (Lin et al., 2010) and is one of the most abundant miRNAs in cervical cancer (Wang et al., 2008b). It appears that miR-24 can repress expression of the tumor suppressor p16INK4a (Lal et al., 2008). Overall however, the role of miR-24 in cancer cell invasion and metastasis is still not clear.
Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinases. It is expressed on the cell surface mediating a variety of signals associated with cell proliferation. It has been demonstrated that altered expression of EGFR could result in cancer development (Zhang et al., 2007). Furthermore, breast cancer patients with EGFR upregulation have a less favorable prognosis than those with EGFR-negative tumors (Koletsa et al., 2010). Phosphorylation of EGFR plays a crucial role in the biological functions of EGFR (Magkou et al., 2008). Protein-tyrosine phosphatases can remove the phosphate groups from the phosphorylated receptor tyrosine kinases (Tonks, 2006) and antagonize their activity (Zhu et al., 2008). It has been reported that tyrosine-protein phosphatase non-receptor type 9 (PTPN9) can dephosphorylate EGFR and inhibit growth and invasion of MBA-MD-231 cells (Yuan et al., 2010). The role of other PTPs in EGFR dephosphorylation is not clear. In this study, we demonstrated that miR-24 can activate EGFR phosphorylation resulting in enhanced tumor growth, tumor local invasion and metastasis, which resulted in decreased animal survival. Consistent with this was the activation of EGFR. The activation of EGFR activity was achieved by repressing expression of two phosphatases PTPN9 and receptor-type tyrosine-protein phosphatase F (PTPRF). We confirmed that both PTPN9 and PTPRF were the targets of miR-24.
Increased expression of miR-24 in human breast carcinoma
To dissect the roles of miRNA in breast cancer development, we examined miRNA levels in breast cancer tissues and normal breast tissues. RNAs isolated from the paraffin-embedded tissues were subjected to real-time PCR analysis. The assays showed that miR-24 levels were significantly higher in breast tumor tissue than in the corresponding normal breast tissue from the same patient (Fig. 1A, P = 0.000136). We also isolated RNAs from frozen tumor tissues and the surrounding normal breast tissues. Real-time PCR analysis indicated that of the 23 pair specimens obtained from 23 patients, 15 pairs of samples showed higher levels of miR-24 in the tumors than in the normal tissues, while the other eight pairs showed lower levels of miR-24 in the tumor tissues (Fig. 1B). These experiments suggested that miR-24 may play a role in cancer development.
miR-24 promoted cell invasion, migration, proliferation and cell-cell interaction
We generated a miR-24 expression construct (Fig. 1C) which was transfected into breast cancer cells, and then used cell sorting to isolate a stable pooled cell line. After confirming integration of the construct into the genome (supplementary material Fig. S1A, upper) and expression of miR-24 (supplementary material Fig. S1A, lower), we conducted cell invasion assays. Mock- and miR-24-transfected 4T1 cells were loaded onto a Transwell insert with serum-free Dulbecco's modified Eagle's medium (DMEM) and then incubated at 37°C for 24 hours. The invasive cells were stained and counted in six randomly selected fields under a light microscope. We found that cells transfected with miR-24 displayed higher levels of invasion than the mock-transfected cells (Fig. 1D; supplementary material Fig. S1B). To corroborate this result, we performed the invasion assay in a human breast cancer cell line MT-1 (Fig. 1E; supplementary material Fig. S1C) and found that expression of miR-24 promoted cancer cell invasion. The invasion assay was also performed in the presence of Matrigel and we confirmed that 4T1 cells transfected with miR-24 had higher invasive capacity than the mock-transfected cells (supplementary material Fig. S1D).
Cell migration assays were performed to examine the role of miR-24. Mock- and miR-24-transfected 4T1 and MT-1 cells were cultured until 95% confluence. The monolayer of cells was wounded and the cultures were maintained in the presence of mytomycin to inhibit cell proliferation. Cell migration was photographed under a light microscope every 24 hours. The distances between the wounding center and the front of the migrating cells were measured for statistical analysis. The experiments indicated that expression of miR-24 promoted migration of MT-1 cells (Fig. 1F; supplementary material Fig. S1E) and 4T1 cells (supplementary material Fig. S1F).
We examined proliferation of 4T1 cells transfected with miR-24 or the control vector. The experiments showed that the miR-24-transfected cells had higher rates of proliferation than the vector-transfected cells (supplementary material Fig. S1G). Analysis of cell cycle progression indicated that more vector-transfected cells than miR-24-transfected cells were in the G1 phase suggesting a lower rate of proliferation of the former cells (supplementary material Fig. S1H). Consistent with this was the finding that miR-24-transfected cells formed more and larger colonies than the mock-transfected cells in the colony formation assays (supplementary material Fig. S2A). In sphere formation assays, we observed that the miR-24-transfected cells formed more and large spheres than the mock-transfected cells (supplementary material Fig. S2B). In low serum conditions, the miR-24-transfected cells showed increased adhesion and spreading relative to the mock-transfected cells (supplementary material Fig. S2C). The above characteristics of the miR-24-transfected cells were indicative of metastatic cancer cells. We thus performed drug-resistance experiments by culturing the mock- and miR-24-transfected MT-1 cells in the presence of Docetaxel, Doxorubicin and Epirubicin and found that the miR-24-transfected cells were more resistant to these drugs than the mock-transfected cells (supplementary material Fig. S2D).
To corroborate the role of miR-24 in mediating cell activities, we developed an antisense construct to suppress miR-24 function. In cell proliferation assays, we detected that transfection of the anti-miR-24 construct decreased cell proliferation after four days of culture (supplementary material Fig. S2E). We also performed invasion and migration assays in the MDA-MB-231 cells that expressed higher levels of miR-24 and found that expression of anti-miR-24 significantly decreased the invasive capacity (supplementary material Fig. S2F,G).
Expression of miR-24 enhanced breast cancer tumor growth, local invasion and metastasis
In order to confirm the function of miR-24, three transfected stable cell lines were injected into nude mice subcutaneously. After tumors formed, their sizes were measured regularly until the mice were sacrificed or up to 80 days. The experiments showed that tumors formed by the miR-24-cells (miR-24 tumors) grew faster than the tumors formed by the mock-transfected cells (mock tumors) or the anti-miR-24-transfected cells (anti-miR-24 tumors; Fig. 2A). During the assays, mice were sacrificed when the tumors reached the limit size set by the Animal Care Committee of Sunnybrook Research Institute. The survival curves demonstrated that miR-24 expression decreased survival rates (Fig. 2B).
The tumors were harvested, sectioned, subjected to Hematoxylin and Eosin (H&E) staining. In the mock tumors, a mixture of dead cells (showing nuclear condensation and fragmentation) and live cells were detected. Cell death was reduced in the miR-24 tumors but much higher in the Anti-miR-24 tumors than in the mock tumors (Fig. 2C). In the anti-miR-24 tumors, severe necrosis was detected in the center of the tumor. In addition, significant invasion was observed in the miR-24 tumors, showing the invasion of tumor cells into muscles. Tumor sections were also probed with anti-CD34 antibody to examine angiogenesis. We found that miR-24 expression increased tumor-associated vascularization, while anti-miR-24 expression inhibited vascularization (Fig. 2D).
We further performed tumor metastatic assays. Mock- and miR-24-transfected MT-1 cells were injected into NOD-SCID mice via tail vein, 15 in each group. Six weeks after the injection, three mice in the miR-24 group developed cachexia and four had lung metastasis in necroscopy. Typical metastatic lesions in the lungs are shown (supplementary material Fig. S2H). DNA was isolated from lung tissues and subjected to PCR to amplify the CMV promoter to indicate metastasis of the cancer cells. Expression of miR-24 promoted metastasis (Fig. 2E). Lung sections were obtained and subjected to H&E staining. Nodules were detected indicating metastasis of the tumor cells to the lungs (Fig. 2F). The miR-24-group showed multi-focal metastasis lesions in the lungs (supplementary material Fig. S2I).
miR-24 upregulated phosphorylated EGFR activities
Since activation of EGFR is tightly associated with tumor invasion and metastasis, we analyzed levels of phosphorylated EGFR (pEGFR) and EGFR in both 4T1 and MT-1 cells and observed a dramatic induction of pEGFR levels in both cell types, while the levels of EGFR were similar, suggesting activation of the existing EGFR protein (Fig. 3A; supplementary material Fig. S3A). The cell lines expressing higher levels of miR-24 produced higher levels of pEGFR (supplementary material Fig. S3B). We further analyzed pEGFR level in the tumor sections and found that the miR-24 tumors expressed higher level of pEGFR than the mock tumors (Fig. 3B). The effect of miR-24 on pEGFR expression was confirmed by western blot analysis, which showed higher levels of pEGFR in the miR-24 tumor than in the mock tumor (Fig. 3C, left). In addition, the miR-24 tumor displayed higher levels of pErk than the mock tumor (Fig. 3C, right). Lung sections prepared from mock and miR-24 mice were also analyzed for pEGFR activity. A higher level of pEGFR activity was displayed in the miR-24 lung nodules than in the mock control (Fig. 3D). On the other hand, transfection with anti-miR-24 decreased pEGFR level (supplementary material Fig. S3C).
We employed the pEGFR inhibitor AG1478 to confirm the effect of pEGFR in mediating miR-24 functions. Mock- and miR-24-transfected MT-1 cells were treated with AG1478 followed by analysis of pEGFR levels. We detected downregulation of pEGFR levels in the presence of AG1478 compared with the untreated miR-24-cells (supplementary material Fig. S3D, left). In cell invasion assays, we found that treatment with AG1478 significantly decreased cell invasion in both the miR-24- and mock-transfected cells compared with the untreated controls (Fig. 3E; supplementary material Fig. S3E). In migration assays, we found that treatment with AG1478 significantly reduced the activities of cell migration in both the miR-24- and mock-transfected cells compared with the untreated controls (Fig. 3F; supplementary material Fig. S3F).
miR-24 targeted PTPN9 and PTPRF
To test how miR-24 exerted metastasis-promoting activities, we analyzed potential targets of miR-24 using the software miRBase (http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc = MI0000080). There were a great number of candidates potentially targeted by miR-24. We focused on PTPN9 and PTPRF, two protein tyrosine phosphatases. The potential binding sites appeared at nucleotides 2333–2355 for PTPN9 and nucleotides 6111–6132 for PTPRF (Fig. 4A). We examined the levels of these proteins in 4T1 and MT-1 cells stably transfected with miR-24. In both cell lines, miR-24 expression resulted in decreased levels of PTPN9 and PTPRF (Fig. 4B, left). Similar results were obtained in the miR-24 tumor (Fig. 4B, right). Consistent with this, transfection with anti-miR-24 increased PTPN9 and PTPFR levels (Fig. 4C). Treatment with AG1478 produced little effect on PTPN9 and PTPRF expression (supplementary material Fig. S3D, right).
To confirm the targeting of PTPN9 and PTPRF by miR-24, we performed luciferase activity assays. The luciferase reporter vector was engineered with a fragment of the 3′UTR of PTPN9 or PTPRF harboring the miR-24 target site, producing two luciferase reporter constructs Luc-PTPN9 and Luc-PTPRF (supplementary material Fig. S4A). In addition, the potential target sites were mutated as shown (Fig. 4A) producing two mutant constructs Luc-PTPN9-mut and Luc-PTPRF-mut (supplementary material Fig. S4A). Breast cancer cell line 4T1 was co-transfected with miR-24 and the luciferase reporter construct Luc-PTPN9 or Luc-PTPN9-mut. The luciferase reporter vector containing an unrelated sequence served as a negative control. The presence of the miR-24 target site reduced luciferase activities, an effect that was reduced when the target site was mutated (Fig. 4D, left). As well, co-transfection with miR-24 and Luc-PTPRF displayed significantly decreased luciferase activities compared with the control, an effect that was reversed when the target site was mutated (Fig. 4D, middle). As expected, a mixture of PTPN9 and PTPRF (50% of each) produced similar results in the luciferase activity assays (Fig. 4D, right). We also conducted the luciferase activity assays in a different cell line U343 and similar results were obtained (supplementary material Fig. S4B).
Examination of the target sequences indicated that the miR-24 target sites in the 3′UTRs of PTPN9 (supplementary material Fig. S4C) and PTPRF (supplementary material Fig. S4D) were highly conserved across different species. In all species obtained, only one nucleotide in PTPN9 and two nucleotides in PTPRF contained some deviations, while the rest were 100% homologous.
To confirm that PTPN9 and PTPRF were important in mediating the effects of miR-24 in regulating cell invasion and migration, we introduced siRNA to suppress their expression. Cell lysates prepared from MT-1 cells transfected with siRNAs against PTPN9, PTPRF or a control oligo were analyzed by western blotting to confirm appropriate RNA silencing as compared with the control (supplementary material Fig. S4E). We then analyzed EGFR expression and its activation using lysate from miR-24-transfected cells, which served as a control. Silencing of PTPN9 and PTPRF had no effect on EGFR expression but enhanced EGFR phosphorylation, reaching levels similar to those found in miR-24-transfected cells (Fig. 4E). Treatment with EGF also enhanced EGFR phosphorylation (supplementary material Fig. S4F).
MT-1 cells were treated with siRNAs against PTPN9, PTPRF, or the control oligo. The treated cells were harvested for invasion and migration assays. Silencing PTPN9 and PTPRF by treating the cells with siRNA against PTPN9 and PTPRF, respectively, promoted cell invasion (Fig. 4F; supplementary material Fig. S4G) and migration (Fig. 4G; supplementary material Fig. S4H).
Expression of pEGFR, PTPN9 and PTPRF in human breast carcinoma cell lines and patient specimens
To examine whether there was any correlation in the expression of miR-24 and its potential protein targets, we analyzed expression of pEGFR, PTPN9 and PTPRF in a number of cell lines including human breast carcinoma cells and found that expression of pEGFR was higher in the breast cancer cell lines MDA-MB468, MT-1, and MDA-MB231 than in the other cell lines tested (supplementary material Fig. S5A). Consistent with this was the lower levels of PTPN9 and PTPRF in MDA-MB468, MT-1 and MDA-MB231. Interestingly, the levels of miR-24 were also higher in these cell lines than the other cell lines (supplementary material Fig. S5B), suggesting a potential role of miR-24 in repressing PTPN9 and PTPRF expression in these cells, resulting in promotion of EGFR activity.
We then examined expression of pEGFR, PTPN9 and PTPRF in human breast carcinoma specimens. We found that expression of pEGFR was much higher in human breast carcinoma specimens than in the normal breast tissues (Fig. 5; supplementary material Fig. S5C). On the other hand, expression of PTPN9 and PTPRF was much weaker in the tumor tissues than in the normal tissue (Fig. 5A; supplementary material Fig. S5D,E). Double staining confirmed that expression of pEGFR was stronger than expression of PTPN9 and PTPRF in the tumor tissues, which was opposite to what was found in the normal tissues (supplementary material Fig. S6). The sections were probed for miR-24 expression using the miRCury LNA microRNA ISH Optimization Kit from Exiqon. We detected high levels of miR-24 expression in the cancer cells but not in the normal ductal structure of the breast (Fig. 5B).
PTPN9 and PTPRF rescued the effects of miR-24
To further confirm that PTPN9 and PTPRF played essential roles in mediating miR-24 effect, we conducted rescue experiments. The retroviral vector pBABE was engineered with the coding sequences of either PTPN9 or PTPRF (supplementary material Fig. S7A). MT-1 cells were transfected with PTPN9 combined with miR-24, PTPRF combined with miR-24, miR-24 alone or were mock transfected and then analyzed by western blotting. As expected, transfection with PTPN9 and PTPRF increased their expression respectively, which resulted in decreased pEGFR levels (Fig. 6A).
Invasion assays were performed on MT-1 cells transfected with PTPN9 combined with miR-24, PTPRF combined with miR-24, miR-24 alone or the mock construct. Enhanced cell invasion induced by miR-24 expression was abolished by transfection with PTPN9 and PTPRF (Fig. 6B; supplementary material Fig. S7B). The cells were also cultured to sub-confluence before being utilized in wound healing and migration assays. Enhanced cell migration by miR-24 expression was abolished by transfection with PTPN9 and PTPRF (Fig. 6C; supplementary material Fig. S7C). Ectopic expression of PTPN9 and PTPRF also abolished the effects of miR-24 on cell proliferation (supplementary material Fig. S7D) and colony formation (supplementary material Fig. S7E). The rescue effects of PTPN9 and PTPRF were also confirmed in another breast cancer cell line 4T1. Ectopic expression of PTPN9 and PTPRF reduced activation of pEGFR (supplementary material Fig. S7F), resulting in decreased cell invasion (supplementary material Fig. S7G), cell proliferation (supplementary material Fig. S8A), migration (supplementary material Fig. S8B) and colony formation (supplementary material Fig. S8C).
The MT-1 cells stably infected with pBABE-PTPN9 and pBABE-PTPRF were subjected to metastasis assays. After being sacrificed, the mice were examined for nodules in the lung. Lung tissues were harvested for DNA isolation followed by real-time PCR analysis to quantify ectopic DNA in the lung, which is a sign of metastasis. The experiments indicated that there were significantly higher levels of metastasis in the miR-24-treated mice than that in the mock-treated mice (Fig. 6D). Ectopic expression of PTPN9 or PTPRF decreased metastasis significantly. The tissues were also subjected to sectioning and H&E staining. We found nodules in the miR-24-treated mice but not in the mock-treated or in the PTPN9- and PTPRF-treated mice (Fig. 6E). The lung sections were immunostained for expression of PTPN9 and PTPRF to confirm metastasis of tumor cells (supplementary material Fig. S8D) and activation of pEGFR. We found that ectopic expression of PTPN9 and PTPRF abolished the increased expression of pEGFR by miR-24 (Fig. 6F).
Overexpression of miR-24 enhanced EGFR signaling and its down-stream pathways
We finally investigated the signaling pathway mediating the effects of miR-24. Cell lysates from miR-24- or mock-transfected MT-1 cell were analyzed by western blotting and probed for expression of MMP2, MMP11, ADAM15, TIMP2 and TIMP3. We detected upregulation of MMP2, MMP11 and ADAM15, but downregulation of TIMP2 in the cells (Fig. 7A). Expression of TIMP3 was not affected. To corroborate these results, we transfected MDA-MB-231 cells with anti-miR-24 and mock constructs, followed by western blot analysis of the expression of MMP2, MMP11, ADAM15, TIMP2 and TIMP3. Opposite to the above results, we detected downregulation of MMP2, MMP11 and ADAM15 but upregulation of TIMP2 in the cells (Fig. 7B).
To confirm that the up- and downregulation of these proteins was the consequence of miR-24 targeting PTPN9 and PTPRF and activation of pEGFR, we treated the mock- and miR-24-transfected MT-1 cells with 2.0 µM AG1478. Expression of the above proteins was analyzed by western blot analysis. We detected downregulation of MMP2, MMP11 and ADAM15 but upregulation of TIMP2 in the miR-24-transfected cells (supplementary material Fig. S9A). We confirmed that expression of pEGFR, MMP2, MMP11 and ADAM15 was upregulated while expression of PTPN9, PTPRF and TIMP2 was downregulated in the miR-24 tumors as compared with the mock tumors (supplementary material Fig. S9B). Upregulation of MMP2, MMP11, AMAD15 and pErk, while downregulation of TIMP2 was also detected in human breast carcinoma tissues compared with the normal duct structures in the breast (supplementary material Fig. S9C).
To understand the signaling pathway by which miR-24 functioned, we produced a figure to show how miR-24 possibly acts in breast cancer invasion and metastasis (Fig. 7C). In brief, miR-24 targeted PTPN9 and PTPRF, and downregulated expression of PTPN9 and PTPRF, which in turn activated EGFR (pEGFR). Increased pEGFR activity promoted expression of MMP2 and MMP11, which partly downregulated the expression of TIMP2 and upregulated the expression of ADAM15. As a consequence, increased expression of MMP2 and MMP11 enhanced breast cancer cell migration, invasion and metastasis. Thus, miR-24 represses PTPN9 and PTPRF, which coordinately regulate EGFR to mediate miR-24 functions. In an accompanying paper, we demonstrate that miR-17, producing miR-17-5p and miR-17-3p, represses PTEN, GalNT7 and vimentin. These targets, although functioning in different signal pathways, can mediate miR-17 functions coordinately, leading to the development of hepatocellular carcinoma (see Shan et al., 2013 in this issue of Journal of Cell Science).
Our study demonstrated that miR-24 was highly expressed in human breast carcinoma. This suggested a role of miR-24 in cancer development/progression, which was consistent with previous studies showing that miR-24 was highly expressed in patients with gastric cancer and lung cancer (Xie et al., 2011), cervical cancer (Wang et al., 2008b) and oral carcinoma (Lin et al., 2010). MiR-24 has also been reported to target p16, a tumor suppressor (Lal et al., 2008). We found that expression of miR-24 promoted tumor growth, invasion and metastasis by upregulating EGFR activity due to its suppressive effect on PTPN9 and PTPRF, two phosphatases of EGFR. Rescue experiments expressing these phosphatases reversed the effects of miR-24 on tumor cell invasion and metastasis. Currently reported miR-24 functions include promotion of cell proliferation (Zaidi et al., 2009) and inhibition of apoptosis in cancer cells (Qin et al., 2010) and cardiomyocytes (Qian et al., 2011). This may be due to its capacity in repressing a great number of potential genes (368 candidate targets being predicted at http://pictar.mdc-berlin.de/cgi-bin/PicTar_vertebrate.cgi?action = Search%20for%20targets%20of%20a%20miRNA&name2 = hsa-miR-24). In addition, miR-24 has been reported to target some 'seedless' 3′UTRs (Lal et al., 2009). Initially reported ten years ago (Lagos-Quintana et al., 2001), the roles of miR-24 are only starting to be recognized in a variety of cells and tissues.
To dissect the signaling pathway associated with miR-24 promotion of tumor growth, invasion and metastasis, we examined expression of a number of oncogenes and tumor suppressors and found that EGFR was strongly activated in the miR-24 expressing cells and tumors. As a cell surface receptor, EGFR transitions from an inactive state to an active state by autophosphorylation of its tyrosine residues (Swanton et al., 2008). Phosphorylation of EGFR results in activation of downstream MARK, ERK, Akt and JNK signaling pathways leading to DNA synthesis, cell proliferation and cell migration (Schmidt-Ullrich et al., 1997; Sørensen et al., 2006). Thus, phosphorylated EGFR plays important roles in tumor invasion and metastasis (Arora and Simpson, 2008; Gong et al., 2008). Over-activation of EGFR including constant activation and mutations is often associated with cancer development and progression (Lynch et al., 2004; Walker et al., 2009). Our results showing that EGFR was strongly activated upon miR-24 overexpression appeared to be a consequence of miR-24 repressing PTPN9 and PTPRF expression. Consistent with these results were the activation of Erk, ADAM15, MMP2 and MMP11 and downregulation of TIMP2 levels, which supported a role of EGFR in mediating miR-24 activities.
Since EGFR was not a target of miR-24, we reasoned that miR-24 may have targeted the endogenous inhibitors for EGFR phosphorylation such as the protein tyrosine phosphatases (PTPs). Due to their critical roles in physiology and tissue development, a great number of PTPs were expressed in various cell types, and the activity of each PTP was tightly controlled. PTPN9 is a non-receptor type 9 phosphatase in the cytoplasm, playing a role in intracellular secretory vesicle fusion (Huynh et al., 2004). PTPN9 has been reported to inhibit EGFR signaling by dephosphorylating EGFR leading to impaired growth and invasion of breast cancer cells (Yuan et al., 2010). We showed in this study that miR-24 could promote EGFR functions by repressing PTPN9 expression. Our results confirmed the function of PTPN9 in dephosphorylating EGFR as reported (Yuan et al., 2010) and added an additional layer to the regulation of PTPN9 expression, namely the regulation of PTPN9 by miR-24. This regulation appears to be significant in breast carcinoma patients who express high levels of EGFR. We found that approximately two-thirds of breast cancer patients express high levels of miR-24 and that most of the breast carcinoma specimens displayed high levels of pEGFR. Whether or not these patients expressed high levels of activated EGFR awaits further investigation. In addition, our results demonstrated that in human breast cancer cell lines, both EGFR and miR-24 were highly expressed while PTPN9 was downregulated, suggesting a possible inhibitory role of miR-24 in PTPN9 expression while promoting EGFR activity (e.g. enhanced EGFR phosphorylation).
Furthermore, we demonstrated that PTPRF was another target of miR-24. PTPRF is a receptor-type F protein tyrosine phosphatase. Although the function of PTPRF is not clear, it has been reported that PTPRF plays a role in mesenchymal stem cell self-renewal (Song and Tuan, 2006). There have been no reports on the role of PTPRF in dephosphorylating EGFR. In this study, we showed a reverse correlation between pEGFR and PTPRF levels. These results suggested that PTPRF may dephosphorylate EGFR in these cells. In clinical specimens, we also detected an inverted relationship between PTPRF and pEGFR levels, supporting a role of PTPRF in EGFR dephosphorylation. Knockdown of PTPRF increased pEGFR levels leading to enhanced cell invasion and migration. Ectopic expression of PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis to the lung. These results directly supported the role of PTPRF in the inhibition of EGFR activities. The precise manner by which PTPRF dephospohrylates EGFR, awaits further investigation.
Materials and Methods
The monoclonal antibodies against ERK2, pERK, and the polyclonal antibodies against EGFR, pEGFR, PTPN9, PTPRF, MMP2, MMP11 and ADAM15 were obtained from Santa Cruz Biotechnology. EGF, selective EGFR inhibitor AG1478, mytomycin, and the monoclonal antibody against β-actin used were obtained from Sigma. The polyclonal antibodies against TIMP2 and TIMP3 were obtained from Abcam. Horseradish peroxidase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad. Immunoblotting was performed using the ECL Western Blot detection kit. Cell Proliferation Reagent WST-1 and High Pure PCR Template Preparation kits were obtained from Roche Applied Science. The MT-1 cells used in this study was originally described by Naundorf and co-workers (Naundorf et al., 1992), while 4T1 cells were described by Aslakson and Miller (Aslakson and Miller, 1992).
Construct generation
Plasmid miR-24 and control plasmid were generated which have GFP and selected antibiotics marker respectively. Both plasmids contain a Bluescript backbone, a CMV promoter driving green fluorescent protein (GFP) expression and a human H1 promoter driving pre-miR-24 or a non-related sequence serving as a control. The control plasmid was the same as the miR-24 construct except the pre-miR-24 sequence was replaced with a non-related sequence (5′-atacagtactgtgataactgaagtttttggaaaagctttagttattaa-3′), serving as a mock control. In order to analyze the function of miR-24, the two constructs were transfected into 4T1 cells. After cell sorting through monitoring GFP signal by FACS (fluorescence activated cell sorter), transfected stable cells were obtained and used in this study.
A luciferase reporter vector (pMir-Report; Ambion) was used to generate the luciferase constructs. A fragment of the 3′-untranslated region (3′UTR) of human PTPN9 or PTPRF was cloned by RT-PCR. Two primers, huPTPN9-SacI (5′-cccgagctcctctcctacgaacctcctacc-3′) and huPTPN9-MluI (5′-cccacgcgtggcaaatgctcatcaaacctcccc-3′), were synthesized to clone the 3′UTR fragment of PTPN9, while the 3′UTR fragment of PTPRF was cloned with the primers huPTPRF-SacI (5′-cccgagctcctaccgctcccctctcctccgccac-3′) and huPTPRF-MluI (5′-cccacgcgtggattcgcgaaaggaaaaatcagag-3′). The PCR products were digested with SacI and MluI and the fragments were inserted into a SacI- and MluI-opened pMir-Report Luciferase vector to obtain the luciferase constructs, Luc-PTPN9 and Luc-PTPRF, respectively. Mutations of the miR-24 binding sites were also created, which was derived by PCR approach, using two primers huPTPN9-SacI-mut (5′cccgagctcctctcctacgaacctcctacctgttggccagccttccttaaactaccctggacaccggactgcct-3′) and huPTPN9-MluI to generate a mutant construct Luc-PTPN9-mut, and two primers huPTPRF-SacI-mut (5′-cccgagctc ctaccgctcccctctcctccgccacccccgccgtggggctccggaggggacccagctcctgactgcca-3′) and huPTPRF-MluI to generate Luc-PTPRF-mut. To serve as a negative control, a non-related sequence was amplified from the coding sequence of the chicken versican G3 domain using two primers, chver10051SpeI and chver10350SacI (Shan et al., 2009). We do not expect any endogenous miRNA to bind to this fragment as it is in a coding region.
To generate PTPN9 expression construct, we amplified the PTPN9 coding sequence (access number NM_002833.2, 1782 base pairs) by PCR with pfu polymerase and two primers (Ptpn9-For, 5′-ccccgaattcgagcccgcgaccgcgccc-3′ and Ptpn9-Rev, 5′-ccccgtcgacttactgactctccacggc-3′). The PCR fragment was cloned at EcoRI and XhoI sites in a Myc-tagged pCDNA3.1 expression vector. After confirmation of the correct sequence, the 1782-bp HindIII and XbaI fragment was released from this plasmid and treated with Klenow enzyme to produce blunt end and sub-cloned at SnaBI of pBabe-puro retroviral vector.
To generate PTPRF expression construct, we performed the amplification with two primers (Ptprf-For, 5′-ccccggtaccatggcccctgagccagcc-3′ and Ptprf-Rev, 5′-ccccgcggccgcccgttgcatagtggtcaaa-3′). This 5694-bp fragment was cloned at BamHI and SacII sites in pBS SK vector. After confirmation of the correct sequence, the fragment was obtained with the restriction enzymes KpnI and SacII from pBS vector and sub-cloned at KpnI and SacII sites in pCDNA4/myc-HisB. An EcoRV and AgeI fragment from the PTPRF-pCDNA4/myc-HisB plasmid was released and treated with Klenow enzyme to produce blunt end and sub-cloned at the SnaBI of pBabe-puro retroviral vector.
Production of stable cell lines
The plasmids pBabe-PTPN9 and pBabe-PTPRF were transfected into Hek293T cells to produce retrovirus. Harvested retroviruses in the cultural media were mixed with 6 µg/ml of polybrene (Sigma) and added into culture dishes to infect cells. 24 hours after infection, cells were trypsinized and diluted into 96-well plates and selected with 2 µg/ml of Puromycin (Invitrogen). Individual clones were screened by western blotting with anti-Myc antibody (Sigma).
Cell proliferation assay
The method was as described previously (Deng et al., 2013).
Cell survival assay
The method was as described previously (Yang et al., 2012).
Mammosphere formation
Harvested mock- and miR-24-transfected cells were mechanically and enzymatically dissociated in 0.05% Trypsin-EDTA at 37°C for 10 minutes, followed by resuspension in serum-free DMEM/F12 medium containing 10 ng/ml bFGF, 20 ng/ml EGF, L-glutamine (1∶400) and B27 supplement (1∶50). Single cell suspensions were plated in 60-mm Petri dishes at 1000 cells/well. After culturing for additional 3 days, sphere cells were collected by gentle centrifugation, dissociated to single cells in 0.05% trypsin-EDTA and then cultured to generate mammospheres of the next generation.
Cell migration assays
We performed two different cell migration assays. In the wound-healing experiment, the method was performed as described (Rutnam and Yang, 2012b). The other migration assay was performed with the modified chemotactic Boyden chamber motility assays, which was performed using 24-well cell culture plates and a 3 µm cell culture insert. The lower chamber of the Transwell was filled with 600 µl of 10% fetal bovine serum (FBS)/DMEM and incubated at 37°C for 2 hours. Mock- and miR-24-transfected cells (1×105) in 100 µl serum-free DMEM were gently injected onto each filter insert (upper chamber) and then incubated at 37°C for 4 hours. The filter inserts were removed from the chambers, fixed with methanol for 5 minutes and stained with Harris' Hemotoxylin for 20 minutes. Samples were subsequently washed, dried and mounted onto slides for analysis using a light microscope. Migrating cells were stained blue and were counted in six fields of views/membrane.
Cell invasion assay
Cell invasion assay was performed with the modified chemotactic Boyden chamber invasion assays using 24-well cell culture plates and an 8 µm cell culture insert as described previously (Rutnam and Yang, 2012b).
Colony formation in soft agarose gel
The method was described previously (Deng et al., 2013).
Western blot analysis
Western blotting was performed as described previously (LaPierre et al., 2007).
Cell cycle analysis
Mock- and miR-24-transfected cells were washed and resuspended in cold PBS and incubated in ice-cold 70% ethanol for 3 hours. The cells were then centrifuged at 1500 rpm for 10 minutes and resuspended in propidium iodide (PI) master mix (40 mg/ml PI and 100 mg/ml RNase in PBS) at a density of 5×105 cells/ml and incubated at 37°C for 30 minutes before analysis with flow cytometry.
Luciferase activity assay
Luciferase activity assay was performed using a dual-luciferase reporter system developed by Promega (E1960). This method was performed as described previously (Jeyapalan et al., 2011).
Real-time PCR
This was performed as previously described (Lee et al., 2009). The primers used as real-time PCR controls were human-U6RNAf and human-U6RNAr.
In vivo tumorigenicity in BALB/c mice, local tumor growth and invasion
The method was described recently except that each group contained 10 mice (Fang et al., 2012b). At necroscopy, primary tumors, stromal tissues, lungs, liver, spine were dissected. Half of the organs were fixed in 10% formalin and the other half were frozen in liquid nitrogen for subsequent analysis. The animal experiments were performed according to the guideline approved by the Animal Care Committee at Sunnybrook Research Institute.
In vivo tumorigenicity in nude mice, lung metastasis
The mock- and miR-24-tansfected MT-1 cells were cultured in 10% FBS/RPMI 1640 media at 37°C with 5% CO2. At 70% to 80% subconfluency, the cells were given fresh 10% FBS/RPMI 1640 media 24 hours before being introduced into the mice. Cell viability was determined and cells were suspended with greater than 95% viability without cell clumping. Four-week-old nude mice were injected in the tail vein with the above transfected cells (2×105 cells in 150 µl 10% FBS/RPMI 1640 medium). Each group had 15 randomly selected mice. All mice were sacrificed 6 weeks after injection. At necroscopy, lungs, liver, spines were dissected. Half the organs were fixed in 10% formalin and the other half were frozen in liquid nitrogen for subsequent analysis.
Mouse lung tissues were homogenized and the genomic DNAs were isolated with High Pure PCR Template Preparation kit according to the manufacturer's instructions. In order to estimate tumor burden, we extracted three samples from the above organs of each animal. Tumor burden for each individual tissue was measured using real-time PCR. Primers used were as follows: CMV forward (5′-gtcatcgctattaccatggtgatgcgg-3′) and CMV reverse (5′-agctctgcttatatagacctcccaccg-3′) for genotyping; β-actin forward (5′-ccggcatgtgcaaagccggcttcg-3′) and β–actin reverse (5′-ctcattgtagaaggtgtggtgcc-3′) for loading control.
Tissue H&E staining, immunohistochemistry
Primary tumors or lungs were freshly excised and fixed in 10% formalin overnight, immersed in 70% ethanol, embedded in paraffin, and sectioned. The sections were subjected to Hematoxylin and Eosin (H&E) staining and immunohistochemistry and were de-paraffinized with xylene and ethanol and then boiled in a pressure cooker. After washing with Tris-buffered saline (TBS) containing 0.025% Triton X-100, the sections were blocked with 10% goat serum and incubated with primary antibody in TBS containing 10% goat serum albumin overnight. The sections were washed and labeled with biotinylated secondary antibody, followed by avidin-conjugated horseradish peroxidase provided by the Vectastain ABC kit (Vector, PK-4000). The slides were subsequently stained with DAB followed by Mayer's Hematoxylin for counter staining and slide mounting.
For multiple antigen staining, an avidin/biotin blocking kit (SP-2001) was used to prevent the interaction of the second set of labeling reagents with the first set of labeling reagents. Briefly, the sections were incubated with Avidin solution for 15 minutes and then incubated in Biotin Solution for 15 minutes. After washing with Tris-buffered saline (TBS) containing 0.025% Triton X-100, the sections were blocked with 10% goat serum and incubated with primary antibody to second antigen and followed by each step as the first staining.
Clinical specimens
Breast specimens were collected at the time of mastectomy from previously untreated patients, fixed in 10% formalin overnight, immersed in 70% ethanol, embedded in paraffin and sectioned. Corresponding normal tissue was obtained from the same mastectomy specimen as the tumor, from a different quadrant of the breast. The tissue blocks were confirmed with examination under light microscopy by a pathologist and confirmed to be benign or malignant based on histology of the corresponding H&E stained slide. Sections were subjected to immuno-histochemistry probed with antibodies against EGFR, pEGFR, PTPN9 and PTPRF. The work was conducted following a protocol approved by the Institutional Review Board at the University of Iowa Carver College of Medicine and a protocol approved by the Affiliated People's Hospital of Jiangsu University.
Experiments using freshly frozen clinical specimens were also conducted independently in the Affiliated People's Hospital of Jiangsu University according to a protocol approved by the Clinical Research Ethics Committee at the Hospital. In brief, small RNAs were isolated from formalin-fixed, paraffin-embedded tissue blocks, using the Total Nucleic Acid Isolation Kit (Ambion) or from freshly frozen tissues. Quantification of miRNAs was performed with real-time PCR.
Statistical analysis
The results (means values ± s.d.) of all the experiments were subjected to statistical analysis using Student's t-tests. The level of significance was set at P<0.01.
Author contributions
W.W.D. performed most of the cell biology experiments, western blot analyses and luciferase assays, and was involved in tumor formation assays, data analysis and paper writing. L.F. was involved in animal dissecting analysis, directed pathological analysis and some immunostaining. M.L. helped W.W.D. to initiate the project and was involved in some tumor formation assays. X.Y. performed some immunostaining. Y.L. generated PTPN9 and PTPRF expression constructs, and cell lines. C.P. supervised Y.L.′s work. W.Q., Y.Q.O., R.W.A., S.L.S., J.Q., J.L., provided patient specimens. Z.J., A.J.Y. and M.S. were aware of the experiments and involved in discussion. Z.D. performed real-time PCR. S.W.S. was involved in generating luciferase constructs. C.-H.W. was involved in generating miR-24 expression construct. B.B.Y. designed and supervised the project, analyzed the results, and wrote the paper.
This work was supported by the Canadian Institutes of Health Research [grant numbers MOP-102635 and MOP-111171 to B.B.Y. and MOP-89931 to CP], and a Career Investigator Award from the Heart and Stroke Foundation of Ontario [grant number CI 7418 to B.B.Y.].
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.118299/-/DC1
- Accepted January 24, 2013.
- © 2013. Published by The Company of Biologists Ltd


text:
The damage to patient access caused by proposed 340B Drug Pricing Program guidance would dwarf any clarity the guidance could add to program requirements and harm vulnerable patients, America's Essential Hospitals said in Oct. 27 comments on the 340B Drug Pricing Program omnibus guidance.
The proposed guidance, which the Health Resources and Services Administration (HRSA) released Aug. 28, threatens access to care "by potentially restricting the definition of a patient, adding requirements for hospital outpatient facility eligibility, and narrowing the interpretation of which drugs qualify as covered outpatient drugs," the association said.
The association also noted that the guidance runs counter to the direction in which the health care delivery system is moving: toward integrated, coordinated care. "While government initiatives in other programs encourage care coordination across different settings, many components of the guidance, such as the prohibition on the use of 340B discounts for discharge medications, push the delivery system in the opposite direction," America's Essential Hospitals said. "Not only does the proposed guidance threaten to limit access and quality but it will also raise overall health care costs by increasing avoidable admissions."
The association called on HRSA to "pull back its proposal to dramatically narrow the definition of a patient to whom a covered entity may provide 340B discounted drugs" and make other changes to clarify the guidance before moving forward with a final version.

subset: commoncrawl


text:
- Did you mean
- urban agricultural garden soil less
Search results
Registration is Now Open for Leadership in the City!
Are you interested in Extension in urban areas and ready to improve your knowledge, skills, and ... of entrepreneurial theory and urban Extension practice and will build upon existing leadership ... Navigate as a leader working in the urban and university contexts. Implement elements of entrepreneurial ...
The Evolution of Agritourism: Current Legal Issues and Future Trends
It wasn't that long ago that "agritourism" was an unfamiliar term to in the agricultural ... laws and legal issues. Join OSU Extension Educators Eric Barrett and Rob Leeds and Agricultural Law ... Legal Issues and Future Trends," hosted by the National Agricultural Law Center. We'll examine ...
July 2020 Highlights
the Ronald P. Lynch Dean of the College of Agriculture and Life Sciences (CALS) at Cornell University. ... Annual Meeting can be found here. Ohio State News featured Dr. Melvin Pascall and the study he is leading ... Agriculture, the National Institute of Food and Agriculture, and Pressure BioSciences Inc.to create a video ...
Oats as a Late Summer Forage Crop
contribute to significant soil in the harvested crop, leading to higher ash content in the feed. If weeds ... Jason Hartschuh, Extension Agriculture and Natural Resources Educator, Crawford County; Al Gahler, ... Extension Agriculture and Natural Resources Educator, Sandusky County; and Dr. Bill Weiss, Professor and ...
Late Summer Establishment of Perennial Forages
Science and Mr. Rory Lewandowski, Agriculture and Natural Resources Extension Educator, Wayne County, The ... given the low soil moisture status across many areas. The decision to plant or not will have to be made ... for each individual field, considering soil moisture and the rain forecast. Rainfall/soil moisture in ...
Rory Lewandowski Retirement
Chris Zoller, Agricultural Extension Educator, Tuscarawas County, Ohio State University Extension ... Rory Lewandowski, Extension Educator, Agriculture and Natural Resources, Wayne County, is retiring from ... Ohio State University Extension on July 29, 2020. Rory has served agricultural clientele in Guernsey, ...
Short-Season Forages for Late Summer Planting on Dairy Farms
cover year-round protects the soil from erosion and contributes to building soil organic matter over the ...
Work in the Time of COVID: Balancing Family, Work, and a Pandemic
time as well. Technology has helped him bridge gaps in and outside the office. The CFAES lead fiscal ...
Milk Prices, Costs of Nutrients, Margins and Comparison of Feedstuffs Prices
was $4.53/lb, leading to a higher Class III milk price. The Class III future for July is nearly double ...
ODA Warns Not to Plant Unsolicited Seeds
The Ohio Department of Agriculture (ODA) has been notified that several Ohio residents have ... and protect Ohio agriculture. All foreign seeds shipped to the United States should have ...


text:
Keywords: Apache Solr - Google Cloud Platform - Technical issue - PermissionsDescription: my VM instance was disabled due to billing issues, i have rectified it and activated it back once more.
But i cannot seem to connect to the external IP, even after enabling thee http/https firewall rules it refused to connect. But connects via ssh
find screenshot of enabled http/https traffic
Hi @ibenemevitalis,
Regarding to our documentation, it seems you need to add the firewall rule to your instance. To do so:
I hope it helps.
i have created the firewall rule, kindly find attached.
I have also contacted google support, i was told this
"Using "nmap" just confirmed that ports 80 443 are closed in your instance (attachement) however, firewall rules look correct so it might be some misconfiguration that happened after the instance was suspended"
How do i open these ports?
It seems the configuration is ok. However, the services seem to be stopped. Could you please check it by accessing via SSH and executing the command below?:
sudo /opt/bitnami/ctlscript.sh status
You can start the service with:
sudo /opt/bitnami/ctlscript.sh start
This topic was automatically closed 14 days after the last reply. New replies are no longer allowed.


text:
Along with falling leaves, colder weather and comfy sweaters, autumn also brings the flu, and while the U.S. Centers for Disease Control and Prevention said last year's season was one of the worst on record, only time will tell how serious this season will be.
Despite the prevalence of the influenza virus and availability of vaccines, the virus still remains deadly on an annual basis. The CDC reported an estimated 80,000 people in the U.S. died from health complications related to influenza during the 2017-18 season, the highest fatality rate compared to any contemporary season on record since first published in 1976.
Of those deaths 183 were children, the most since 171 died in the 2012-13. Approximately 80 percent of those children who died did not receive a flu vaccination, according to the CDC.
The 2017-18 flu season yielded 30,453 influenza-related hospitalizations from October 2017 through April 2018. People 65 years or older accounted for the majority of those hospitalizations, according to the CDC. Overall hospitalization rates were also the highest on record.
Influenza viruses are hard to pin down, as they come in several forms which can require different vaccinations. The influenza A virus was the preeminent strand throughout the 2017-18 season, though influenza B viruses showed up in different parts of the season.
The CDC report for 2017-18 said the flu shot was only 25 percent effective against the H3N2 virus and 65 percent against H1N1, both type A viruses. Meanwhile it was 49 percent effective against B viruses. The report estimated the overall vaccine effectiveness at 40 percent, meaning it reduced a person's overall risk of having to seek medical care for flu illness at that rate.
The CDC still strongly recommends vaccines as the best way to prevent contracting the virus, but especially for children at least 6 months old, and people aged 50 and older. Children aged 6 months through 8 years who require two doses should receive the first vaccination as soon as possible, and their next dose four weeks later, according to the CDC. For those looking to travel this season the CDC recommends a vaccination two or more weeks before departure.
The new vaccines being rolled out for the 2018-19 flu season will contain agents to specifically target the A(H1N1) and A(H3N2) viruses along with the usual B viruses.
The CDC recommends everyone 6 months or older gets a shot before the end of October. Flu shots are available at most primary care physicians, but also in CVS Pharmacy, Rite Aid and Walgreens stores free with most insurance plans. The shot is also available in pharmacies in local Stop & Shop, Walmart, Target and Kmart stores. Many colleges, such as Stony Brook University, are offering flu shots to its students. Call your doctor or local pharmacy to ask whether they currently supply flu shots.


text:
North Port Real Estate Jobs
North Port Real Estate Job Search Results
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Data Entry Clerks
Description: We are actively seeking customer service and appointment setting representatives. Your primary responsibility is to supply specialized assistance to our current client base. In this position, you will (more...)
Company: Morani Enterprise
Location: Sarasota
Posted on: 2020-08-03


text:
At various occasions throughout my life, I have settled on an individual decision to not have a sweetheart, yet have kept on observing escorts.
© 2020 Yarabook™
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This mouthwatering sweet potato casserole will satisfy lovers of crunchy pecans and cornflakes as well as marshmallows.
How to Make It
Preheat oven to 400°. Bake sweet potatoes at 400° for 1 hour or until tender. Let stand until cool to touch (about 20 minutes); peel and mash sweet potatoes. Reduce oven temperature to 350°.
Beat mashed sweet potatoes, granulated sugar, and next 5 ingredients at medium speed with an electric mixer until smooth. Spoon potato mixture into a greased 11- x 7-inch baking dish.
Combine cornflakes cereal and next 3 ingredients in a small bowl. Sprinkle over casserole in diagonal rows 2 inches apart.
Bake at 350° for 30 minutes. Remove from oven; let stand 10 minutes. Sprinkle marshmallows in alternate rows between cornflake mixture; bake 10 minutes. Let stand 10 minutes before serving.


text:
Coming soon to a church near you (specifically, Christ Church Stannington, on February 6th)....
Christian Basics is a weekly group that will meet in the Vicarage on Wednesday's from 2 - 3:30pm to talk about what Christianity is, what it means to be Christian, and why do we even need a God anyway?
While this is aimed at those new to the faith, or who are even thinking about Christianity, we welcome all those who are interested - even if you've been going to church for years - it is always good to have a 'refresher course' where you look at the basics of faith and return to your core foundations.
Looking forward to seeing you there!
If you would like to join us, do let us know on 0114 2324490 or firstname.lastname@example.com - or leave a message on the website.
Vicarage address:
214 Oldfield Road
(the office is at the end of the drive on the left)
Not got a Login? Register here »


text:
Been meaning to post this for a while, and a discussion in class reminded me to do so…
It's really important that you collect and view a number of sources as reference for your own product. It's not a case of encouraging you to mindlessly ape what others have done, it's about giving your research enough depth and breadth so you have options as you consider your scripts. It will also give you ideas for shots and editing when you start filming and then panicking as you get nearer your deadline. (Never underestimate the power of a good panic!)
To that end, here are a few documentaries and 'mockumentaries' that may prove useful/entertaining/thought-provoking. Some are available on Netflix or Amazon, others I have and will let you borrow if you think they'll be useful. Happy researching!
First up is one of the most horrific films/documentaries I've ever seen: The Cove (2009). It's an Oscar™ winning film about the annual killing of dolphins in Japan. It is not for the squeamish. It is also not immune from criticism for its portrayal of the Japanese whaling industry and the methods used to make the film. Here's the trailer:
Another serious documentary worth watching is Michael Moore's Bowling for Columbine (2002). It addresses America's infatuation with guns, and is notable for many things, not the least of which is that the oft reviled Marilyn Manson comes across as an articulate and intelligent commentator on the horrific shootings that he was partially accused of causing. Here's the trailer:
The next one is short, and jaw-droppingly beautiful, but switch off the 'awe factor' section of your brain and switch on the 'how and why did they film that' bit (Hint: There's a 'making of' video). Welcome to Danny MacAskill: The Ridge
And finally (for now… as it's late… and I've got school tomorrow…)
The ultimate Rock music (spoof) documentary… it's far too close to the truth, and it goes up to 11: This Is Spinal Tap!
Watch lots, and think "How did they do that?", "Why did they do that?", "Do I want to do that?", and "How can I do that?"


text:
People eat hummus like the world is ending! It seems to be everyone's favorite dip. The sad thing is that most people just buy a cheap container of this dip at the store. While this is fine when in a hurry, homemade hummus is just on another level!
If you have been living under a rock, hummus is a Mediterranean bean dip made from garbanzo beans. It is surprisingly simple to make. It revolves around chickpeas, olive oil, tahini, and spices. Hummus is traditionally served with more olive oil on top, paprika, parsley, and pine nuts. I typically leave out the pine nuts because those are not in my budget. This is a great recipe to make if you are trying to empty out your pantry. I know I always have extra cans of chickpeas lying around.
This hummus is: creamy, rich, savory, and never disappoints me. I say these things about every recipe, but I'm never going to give you guys a recipe that is meh.
Summer is a time when people have parties, picnics, and BBQs. I think this is an awesome thing to make for those summer events. Most people enjoy hummus and this caters to vegans and omnivores. You can serve this with a variety of foods such as carrots, celery, pita, tortilla chips, etc.
Here is what you will need:
- Blender or food processor.
- Garbanzo beans (I used canned).
- Extra virgin olive oil.
- Tahini.
- Paprika.
- Ground Cumin.
- Parsley (fresh).
- Minced garlic.
- Lemon juice.
- Cayenne pepper (optional).
- Salt.
I basically throw everything into the blender when I make this. You can soak the chickpeas if you want the hummus to be easy to blend and super creamy. This can get kind of oil-heavy. You can do half olive oil and half water if you are avoiding high-fat foods. You can always change up the flavor profile as well! I used cumin and cayenne pepper to give the hummus some heat, but you can switch those for milder flavors. Also, you might have to pulse the mixture for a while to get rid of any chunks. Other than that, there is really not much to it!
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See the recipe card below for specifics!
Simple & Flavorful Hummus
This hummus is: creamy, rich, savory, and never disappoints me. It only takes about 10 minutes to make and is a crowd pleaser. Perfect for summer BBQs and gatherings!
- 1 15oz can of garbanzo beans (drained and rinsed).
- 3 tbsp tahini.
- 1/3 cup extra virgin olive oil (divided).
- Juice from 1/2 a lemon.
- 2 tbsp minced garlic.
- 1/4 tsp salt.
- 1/4 tsp paprika.
- 1/2 tsp ground cumin.
- 1/8 tsp cayenne pepper.
- Small handful of parsley (for garnish).
Drain and rinse chickpeas.
Place chickpeas in a food processor or blender. Add in tahini, oil, lemon juice, minced garlic, salt, paprika, cumin, and cayenne pepper. Reserve some oil for garnish.
Blend together the ingredients until the mixture is smooth.
Place hummus on a plate with more olive oil, a sprinkling of more paprika, and parsley.
Thanks for visiting! I hope you enjoy this recipe and feel free to leave a rating or a comment! I'm sorry I didn't get this post up sooner, my laptop charger stopped working and I've been busy outside of food blogging. You can still expect weekly content though. Have a nice weekend and we will catch up next week!
- 7Shares


text:
Stockfeed and pet food supply specialist, Eastern Distributors was given an MX-13 PACCAR engine in a Kenworth T409 to evaluate, for a week. Four years later they haven't looked back.
A long-standing relationship with PACCAR dealer Kenworth DAF Hallam prompted what would become an unlikely and advantageous decision by Eastern Distributors to take on a 13-litre MX PACCAR engine into its fleet of Kenworth trucks.
Based out of Dandenong South, the Victorian company supplies stockfeed and pet food supplies across most of the state. According to director Joey Addamo, his previous experiences with a 24-volt truck were not memorable. Although he was less than thrilled by the prospect, he reluctantly agreed to trial a Kenworth T409 with the MX engine suggested by dealer Daniel Crawford. It would come with Kenworth backing and perform, he was assured, as well, if not better, than the three T409SAR models he was operating without incident. His best driver Trevor Green would be behind the wheel.
"Being it was smaller than a 16-litre truck and given our longer runs to Bairnsdale and Albury weekly in single trailer application it didn't sound like such a bad idea," Joey recalls. "Knowing that it had the Kenworth support behind it I decided to trial it."
As a one-way freight company running back from deliveries empty, Eastern Distributors had good reason to scrutinise the performance in fuel economy and highly touted torque, especially across the hilly terrain. Assigned a regular intrastate run on Monday to Albury and Tuesday to Bairnsdale it immediately impressed.
"We soon noticed it was a very responsive engine especially down low," Joey says. "On a lighter load the MX engine is noticeable for how it's not reliant on having to drink fuel. It was a revelation and we haven't looked back since."
By the end of the week Trevor relayed his impressions of the demo Kenworth, equating the power and smooth ride with a Harley, as the gears clicked through with ease and the MX would keep pulling with gusto on each subsequent change. The initial doubts were soon proven unfounded.
"Trevor told me it was very good down low where it showed great torque and it also demonstrated the best fuel efficiencies he had seen on that particular run," Joey says. "He's been driving it a long time."
Having a 13-litre engine that was unburdened by lack of payload made the drive smoother on the four-hour return journey.
"It had 500hp when you need it and yet it's a lot smoother than the Cummins engine which doesn't like being light. It always wants to be under load," Joey says. "The MX doesn't seem to worry at all."
Having been with the business since it commenced operations, Joey says Trevor is now the sole driver of the Kenworth T409, which ranges from Sale to Shepparton on the intrastate task.
"He's a great operator and one of the reasons is he's very easy on the gear and he knows how to get the best out of the truck."
In 2002 Joey with two other company directors purchased Eastern Distributors from the Tudor family who had resigned to sell it after 40 years at the helm. His family until that point had been heavily involved in fruit and vegetable distribution. After about a year the new group had made it work, appreciating the greater power they had over wholesale products in which the shelf life was 12 months at a minimum.
"The waste you can control and you can control your buying," Joey recalls. "It's much different to fruit and veg which is usually on harvest and reliant on time. We could see a lot of potential in the business and it kept growing."
In the beginning they started out with a 12-pallet truck, working three to four days a week. They have 47 staff. Earlier in the year the business moved into its new facility in Dandenong South. The 7,500 sq. metre building sits on 28,000 metres of land with lots of room to expand.
"Now we run eight trucks all day and night just about," he says.
Eastern Distributors covers the entire state of Victoria traveling as far as Horsham and Warrnambool. On rare occasions the company will farm out work to a subcontractor for a job across the border but for the most part it wants its own drivers engaged with the customers. As a company that sells 6,500 lines of pet food and stock food it's important for it to control as many aspects of the business as possible says Joey. The high associated costs of operating commercial vehicles has given the company pause to consider other business models in which they ultimately sub contract the freight task to bigger fleets. A trial run, however, suggested it would not work.
"The customers like that same driver and familiar face every week," Joey says. "To us freight is a necessary cost to our business given trucks are obviously not cheap to run but it's also a convenience and that's the way it should stay." He adds, "We've done the exercise a number of times and we can get a handful of subbies in to do the work but we don't want to lose contact with our customers."
As it has a number of sales representatives on the road, Eastern Distributors likes to stay in close contact with its customers. That way a driver can relay feedback from a customer should they want to make suggestions on how to improve the service, share complaints and offer praise. Joey says his drivers need to be personable, clean-shaven and presentable at all times.
"The driver is the face of your company. He really is," he says. "A lot of our customers have our drivers' phone numbers so they know where they are. It might not be the most efficient way for us to do business having our own trucks but we like to have them because ultimately it helps us grow."
Wholesale freight, according to Joey, might not be the biggest headache in his business but it certainly throws up some challenges. Pet produce stores, for one, aren't always easily accessible for heavy vehicles. Very few contain a loading dock on site. This requires many of his drivers to back in from the street, load from the side of the road or put the truck in forwards and turn it around inside the car park. It's not ideal for a semi-trailer but the Kenworth according to Joey has excellent vision enabling the driver to see right around the truck.
"There's nothing obscured and the Kenworth T409 works well getting in and out of these locations," he says. "It's a very versatile truck to manoeuvre. Because we deliver to stores we need a manoeuvrable truck."
The first of the Kenworths Eastern Distributors purchased was a SAR following a cold call from Daniel Crawford. That was in 2011. Five trucks later and Joey says he's still buying Kenworths from him.
"The support is great. I know everyone from service to parts to sales," he says. "I don't want any aftermarket stuff. I want it serviced at the dealer and all genuine parts put back on it. I don't mind spending the money on them. Our motto is whatever the trucks need they get. I can't have any downtime. We can't let the customers down as a delivery transport company. It's happened in the past with some other trucks and that's why we've gone to Kenworth."
As far as fuel efficiencies go, Joey confirms the MX engine has been delivering outstanding numbers. It's sitting, in terms of the latest fuel burn numbers, around 2.5 km per litre on average.
"You notice when you come out of inclines and roundabouts it just pulls itself out. It's a clean motor. The 24-volt thing that I worried about initially, I have not encountered one problem. They've done that very well. I would have thought when they did the conversion and when you plug in trailers there would have been an issue. It's streamlined itself. I've never had a hiccup with it. We've connected a couple of different trailers and we've had no dramas at all."
Joey says, by way of morning ritual, all the trucks are started up together at the same time while the drivers get their coffee. The Cummins engines are loud.
"With the MX you wouldn't even know it's on. It looks like a Kenworth. But when you turn the key it doesn't sound like a Kenworth. People look around and say the sound doesn't match the look of the truck."
The servicing of 30,000 kilometres also helps. Despite the additional 10,000 on top of the previous arrangement Eastern Distributors was accustomed to for servicing, Joey likes the fact the business gets the trucks back every 30 instead of 20.
"It just helps with downtime," he says. "When I order the next Kenworth it'll have one of those MX engines in it."
Article reproduced with permission from Prime Mover. Original article published here: http://www.primemovermag.com.au/featured/article/running-on-empty


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We guess that less than 1% of people understand the main cause of poverty, which remedies might work and which can't, and why. Our mission is to increase that number, and we'd like to welcome you to the group.
The basic Progress & Poverty course provides the information you need. This term we'll have a Tuesday section, starting January 19, and a Thursday section starting February 11. For each, you're welcome to attend the first session without obligation. If you choose to enroll, the fee is only $25 for the entire course and it can be waived in exchange for labor. A summary of what you will learn and what you won't learn is here, course description here, schedule here. Pre-registration is optional.
Questions? Just email us or phone 312 450 2906.
"Nothing will fully interest men unless it also interests women."
– Henry George


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A Disputation on the Magnitude of Abuses, the Incompetence and the Cronyism of Florida's Child-Welfare Services better known as The 48 Theses.
By Brown Voltaire
Out of deep concern and the desire to see the proper management of Florida's child care operations, I call out the following entities: Broward County Sheriff's Office (BSO)/Child Protective Investigation Services (CPS), the Department of Children and Families (DCF), its subcontractors and the community-based providers. The aim of these 48 theses set forth on this paper is to persuade said entities to revamp its child-welfare services. The bureaucracy is stifling and insipid. They perpetuate misery. They have spun their web of influence into almost every fabric of Florida's society in order to continually secure its aimless mission, objectives and fieldwork; they lobby very hard and spread their manipulative tentacles throughout other agencies and branches of government.
Florida prefers to maintain a sunny disposition rather than looking at the grim truth and manage its child-welfare program better. The irony here is, kids in despair and tragic circumstances are removed from parents. But in foster care their conditions are negatively amplified. Most kids in foster care learn how to be aspiring criminals. The few who make it out of Florida's foster care system unscathed are those whose extended family played a vital role in their lives. I challenge the public and the media to have a debate on the wantonness and egregious actions of DCF and its subcontractors. I challenge the state to show evidence in which a large percentage of foster care kids leave the system with a great outlook. I challenge the state to counter the harsh reality of most foster kids who end up in the system who later commit gross criminal acts and get incarcerated.
The state cannot confidently boast of having a caring child-welfare system. They relinquished their responsibilities to private entities. The citizenry is still waiting to witness a practical exit strategy for kids leaving the system. It is simple; the state ought not to put kids in the system if it cannot ensure their safe arrival to adulthood. Yes, there are some bright spots out there. Some succeed. Thank God though the people who watch over the country's nuclear arsenal are not as reckless and indifferent as child-welfare administrators. Said administrators act like reforming the foster care system is a pesky reality. Self-preservation is not a virtue. Child-welfare administrators should be swift to champion reform over self-preservation.
1. The state of Florida must abandon its blasé attitude regarding child-welfare services. The scale of inefficiency is too high. Most child-welfare entities favorite catch phrase is 'My hands are tied.' If their hands are indeed tied, then, how will they get them loose in order to better themselves?
2. "Thirty percent of the homeless in America and some 25 percent of those in prison were once in foster care" (ABC.go.com). Those numbers are troubling and administrators should worry less about their tenure and more about the above statistics.
3. Child-welfare agencies must refrain from massaging the statutes of Florida. They must strictly adhere to them.
4. Parents' constitutional rights are not a figment of their imagination.
5. DCF and some community-based lead agencies attract way too many lawsuits. Their negligence resulted in enormous financial setbacks and several pending lawsuits.
6. Florida law makers believe that privatization is the way to go. These same law makers stumble for words when the public raise concerns about the lack of oversight. Solutions to manage community-based providers effectively continue to evade lawmakers.
7. Community-based providers and DCF should stop putting children in legal limbo. Neglect is a common reoccurring behavior among most DCF staffers and subcontractors.
8. Two dynamics involved in raising children: one, to consistently mold their character, two, to equip them with life skills to live a content life. So far, the entities mentioned have been missing the mark.
9. The ancient Greeks came up with the word hypocrite which denotes someone who is an actor. The entities mentioned have done a great job acting the part. A wise preacher once said, "Acting [sic] does not paint life in practical pictures."
10. There are different kinds of resources. Most people are generally obsessed with money. Some of the others are: time, natural, talents and information. Said entities are consumed by money. They want more or they don't have enough. They also say don't have any time. They are burned out and cumbered with responsibilities. Sadly they hardly lack the talent to act judiciously but find it easily to act hasty and do sloppy investigations. Further, they are not good at handling information. The management of the abuse hotline is chaotic. Thankfully plans are now in place to streamline it into one database.
11. Child-welfare administrators are vindictive towards natural parents but are very accommodating towards foster parents. Case in point the Barahonas and Mr. McGuigan. The public only hear about foster parents when the department finally runs out of white-out.
12. Community-based providers like 4Kids of South Florida should abandon their quasi-religious affiliation and remember what the gospel of Mark says: "It will be terrible for people who cause even one of my little followers to sin. Those people would be better off thrown into the ocean with a heavy stone tied around their necks." Contemporary English Version (CEV). If they can provide a spotless record please continue but join the queue behind those who are entrusted to take care of the little ones but don't.
13. Child-welfare administrators should expend the same energy tackling child-welfare challenges as they do to prolong their influence and that of their friends.
14. According to the Administration of Children and Families, around 408,000 children were placed in foster care as of September 2010. These days, studies show adoption works out better for the kids. Most kids despise group homes or shelters. Florida needs to be swift to put kids with families or find suitable adoptive parents.
15. This nation created a bureau called the U.S. Department of Health and Human Services. The ACF championed child-welfare services because the good ole' days were terrible for children. But it later ballooned into a bureaucracy that only pumps money to states and collect data.
16. In order for DCF, ChildNet and its subcontractors to be reformed, it must start from the federal level.
17. Some child-welfare judges automatically believe the witness of investigators who stand before them with the state's petition. Natural parents rarely stand a chance because if there is an allegation against them, then they are always culpable.
18. The lawyers with DCF who work to reunite families must stick to doing that and stop changing up petition to scare parents when they don't want to cooperate.
19. The timeline of incident reports should match the specific events. The court should never accept any paperwork submitted several months after a report was made.
20. Newsflash DCF, ChildNet and Broward County, cronyism is not a virtue.
21. DCF oversees a wide range of Florida's social and economic spectrum. Yet it fails the citizenry miserably in the child-welfare department. DCF cannot go on planting weeds but expect to harvest real crops.
22. It is a shame when serious cases of abuse and gross official misconduct occur; child-welfare officials believe the best course of action is to relieve the guilty supervisors or reshuffle them. Criminal prosecution is a rarity. They should own up to their moral failure.
23. Most news outlets are partial towards DCF. Said outlets are sanguine toward its subcontractors too.
24. Police officers should never accompany investigators without first verifying all the legal channels.
25. Investigators should have a keen mind, a big heart, a thorough work ethic, pragmatic job-related skill set and most importantly a sharp intellect.
26. When child-welfare entities and the child-welfare court band together to push through complex cases hastily; that is not a sign of great team work.
27. It is toxic when child-welfare officials say: "The system is broken." A broken system perpetuates brokenness and the children cannot escape this. It is time to stop overlooking America's greatest resource—the American people. There are capable people who are both resourceful and qualified to tackle said system.
28. At some point child-welfare administration officials must one day break down and admit; it is too hard to take care of children. Enact initiatives to make it solely the duty of parents/guardians or legal non-relatives.
29. The cost of Medicaid stems primarily from the services ACF provides. Child-welfare entities benefit from these services. Policy makers are talking about reforming Medicaid but they are not looking at what actually drives up the cost.
30. Unfortunately no one influential will rise to the occasion and reform child-welfare entities. It is after said entities implode and the public lose trust in them; it is then folks will pay attention.
31. Ethics is a foreign word to child-welfare entities. Cronyism is the social currency of operation.
32. The sum of all fears for Florida's child-welfare administrators is fixing the systemic problems within child-welfare entities. They assume if they do; the domino house will crash. They resort to Band-Aid solutions instead.
33. Some Child-welfare administrators misapply the federal government's intention to grant the state of Florida the Title IV-E Waiver initiative. It seems they resort to snatching up kids from their home and placing them in foster care in order to continue the steady stream of federal funding. On the other hand, new directives compel lead agencies to reunite families in order to receive said funding. Most of the times parents' don't have to comply with their case plans but most agencies use guile to force them.
34. The Guardian Ad Litem Program must stick to its original stance—be there for the kids not DCF.
35. Florida expects a miracle to happen in the lives of foster children. Most foster parents are profane and scrupulous, yet they expect the kids to become outstanding individuals.
36. Allegations against foster parents should receive the same amount of attention as allegations against natural parents.
37. Child Protective Investigation Services (CPS) workers must have good diction and excellent writing skills. Shoddy paper work should never be admissible in court.
38. CPS workers must refrain from obtaining background checks on alleged abusers if they do not have court approval.
39. Foster caretakers must be above reproach.
40. Foster parents are not in the witness protection program. Natural parents who do not pose any risk should be allowed to visit their child/children.
41. Community-based providers like ChildNet and 4Kids employ draconian methods to neuter persistent parents. They abruptly cancel mandated visitations. They turn a blind eye to foster parents who don't take calls from parents. They take children to state assistance programs to receive services without any court order. They remain mute in court so that non-dependent kids can remain in legal limbo and they can produce a case plan and receive their filthy lucre.
42. All aforementioned entities should seek out and implement new ideas to revamp the child-welfare system.
43. Child-welfare entities should resist employing a one-size-fits-all approach to their cases. Yes, the job is enveloped in misery; the workers see and expect misery. So a case where there is no misery has to have misery.
44. Child-welfare entities must admit they enjoy milking Medicaid, Social Security and any other Federal funding.
45. The dependency court must cease and desist from sheltering children who are not dependent. It must stop extending professional courtesy to DCF's lawyers to build fruitless cases against persistent parents. It must send the kids home. Judges should never band with DCF to teach persistent parents a lesson.
46. The CEO's of community-based lead agencies need to justify their over the top pay checks.
47. Child-welfare entities are a massive handout program lacking a systematic foundation. ACF should implement constructive assistance geared towards empowering child-welfare programs. ACF should stop writing blank checks to states with horrendous child-welfare background.
48. Community-based lead agencies offer services that are mediocre and elementary. Rarely do the services offer any practical or educational stimulation. The services should be practical and they should match the background of each client.


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PRINCE RUPERT, B.C. - A First Nations grandmother living on B.C.'s north coast is literally diving into the fight against cancer, with a 60 kilometre swim through frigid ocean waters between Prince Rupert and her village of Lax Kw'alaams (lacks-qwah-lambs).
Dayna McKay is no stranger to the challenging currents, powerful tides and unpredictable swells of the North Pacific, because she swam the route last summer, but in reverse.
She's dubbed this leg of her Into The Sea Cancer Free trek as The Journey Home, reflecting her commitment to the battle against all cancers and her support for her uncle, Bill Sampson, who is diagnosed with the disease.
McKay's original journey from Lax Kw'alaams south to Prince Rupert spanned two days and required a dedicated support crew, and she expects her return swim will also be a nearly 24 hour marathon.
The mother of four and grandmother of two intends to dive into waters off Prince Rupert at 6 p.m. on June 28, and — with the help of a 7mm full body wet suit, fins and a support crew of five boats — plans to reach her home shore by 1 p.m. on June 29.
McKay hopes her journey will not only raise awareness about cancer but will also help First Nations youth understand that anything can be accomplished through hard work and desire.


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Momyer Creek: The Long Way to San Gorgonio Peak
Every trail to the summit of San Gorgonio is long and hard. This one is longer and harder than all the rest.
Momyer Trail Quick Facts
Permit required
Most hikers overnight on this trip because of the distance
San Gorgonio elevation 11,503′
5,390′ elevation gain
18.5 miles up and back
Momyer is quite a bit further and more of an elevation gain. We wanted to do a loop, so we figured we'd start up Momyer and come down Vivian Creek, making about a 25-mile round trip. We started out on Saturday, May 20th, which was a beautiful morning. The stream had a lot of runoff, which made crossing it tricky, and once we were passed it, it was quite a bit of trudging.
Dollar Lake Saddle
At about the three-quarter point, we came to Dollar Lake Saddle, which is a box canyon. In the distance you can see a meadow, and you wonder how you'll ever get out of this giant bowl with mountains surrounding you on three sides. Well, you basically just come straight out of it, and it's pretty steep. That's when we got to the snow.
Into the Snow
It was May and the snow drifts above Dollar Lake Saddle were 6 feet thick. The snow was hard compacted ice that was heavily sun cupped. It was incredibly difficult to walk on. There were a few moments when I thought, "You know what? I don't know if this is worth trudging through. It's wicked steep, we don't have crampons, and we are all really tired." We rested for a bit, then decided we would push on since the summit was "just over the top of that mountain" in front of us. We had snow spikes, which were just barely adequate for the task.
The trail disappeared completely and the terrain got steep. I wondered about the safety of the situation. We didn't have ice axes so a slide could lead to serious injury. Some really stupid kids hiked past us up the mountain, so we decided to follow. What made them stupid is that they didn't even have boots or spikes, they had soaked trail runners! I wondered if they were going to be OK when the temperature dropped to freezing that night. In the back of my mind I made contingency plans for how we might help.
We ascended what felt like 1,000 feet over a vertical snowfield. At the top, we hoped to find San Gorgonio just around the corner, but we didn't.
The mountain we climbed was Jepson Peak (11,209 feet). We were pretty exhausted at that point and San Gorgonio was still a long way off. We trudged onward. We all felt close to our cardio limit, but we were so close. We pushed on. At that point, my 50lb pack felt like 100lbs. Surprisingly, little Liz seemed to be outperforming Wes and I from an endurance perspective. She is a lot tougher than she looks!
We finally reached the summit at about 6 pm. It had been a full 12-hour day of hiking. We arrived just in time for the sunset, which we watched while making a hot meal. The sunset cast a huge pyramid shadow down the valley so that it seemed to stand right next to San Jacinto Mountain across the valley toward Palm Springs.
A Bad Night's Sleep and a Once-in-a-Decade Sunrise
I didn't sleep well that night. I've never had a situation where I've gone from 1,500 feet to over 10,000 feet and slept well. My body just can't adjust that quickly.
I woke up at 4am and stuck my head out of my bivy. Wow. It was cold and there was only a light breeze. There was a brilliant moon and the oranges and reds of first light were beginning to glow. First light lasted for 90 minutes with the moon visible. I wasn't sleeping well anyway, so I stayed up and watched the long, beautiful sunrise. You only see a sunrise like this a few times in your life. In moments like these, you completely forget you had a crappy night's sleep. It's as if the beauty of the moment you are in replenishes all of what the long night drained from you.
In moments like these, you completely forget you had a crappy night's sleep. It's as if the beauty of the moment you are in replenishes all of what the long night drained from you.
It took Wes and Liz some time to get up that morning, so I took a lot of pictures. After we ate breakfast, we packed up camp and went to the summit marker to take a picture. We were so tired when we arrived that taking a summit picture was the last thing on our minds.
Descending nearly 12 miles to the car was pretty grueling. We were all pretty tired. When we got to the Vivian Creek trailhead (usually where we left the car) we still had three more miles of walking on asphalt road in 90-degree heat to get to the Momyer trailhead. We were totally spent by the time we got to our car.
Still, the funniest thing was when Liz exclaimed, "That was the most awesome adventure ever!" Most of the trip involved a lot of suffering, carrying a heavy pack up steep slopes, and wondering if we had the strength to get to the next steep slope. Yet when you arrive at the car, something magical happens and you tell yourself, "That was an amazing adventure."
Most of the trip involved a lot of suffering, carrying a heavy pack up steep slopes, and wondering if we had the strength to get to the next steep slope. Yet when you arrive at the car, something magical happens and you tell yourself, "That was the most amazing adventure."
I slept in my bivy sack that night. The bivy usually does well, but I sold it after that trip. It was an Outdoor Research Alpine Bivy. It was okay, and it kept me warm and safe from the wind, but it was so confining, almost like sleeping in a body bag. I'm going to opt for a bit more room in my sleeping arrangement on the next trip.
Trail Map
Buy the Map of this Area on Amazon.com Download GPX File
Elevation Profile
You can get a permit in person at the Mill Creek Visitor Center, or by submitting a permit application at least 5 days in advance by fax or email. You can download a permit application at the links below. Permit availability is subject to quotas. The phone number for the Visitor Center is (909) 382-2882.Download Day Hike Permit Download an Overnight Hiking Permit


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Many on-line pet provide retailers take as a lot as every week simply to pack and ship your order to you, however that's not the case here at Arcata Pet Supplies. We course of orders seven days per week and orders placed by 2:30 PM Pacific Time on enterprise weekdays are shipped the same day. Our pet supply stock is actual-time, and you may see the quantity on the market of any product.
However aside from the gems issue, this sport is really enjoyable and pleasurable, it's effectively made and there may be truly an vital number of animals. The weather is certain to heat up for us finally! We wait for sharing our enthusiasm for outside ponds and water gardens with you. If you are trying to arrange a pond, or have questions on your beforehand established pond, come see us!
Worth to your cash is what you want, not primarily the underside value. That is the place our unbiased product testing and expertise helps. Many merchandise simply do not work as claimed. Low-value merchandise typically price additional in the long term. For example, if you buy cheap grocery retailer cat litter, nonetheless have to use twice as loads as our advisable litter, you paid an excessive amount of. When you feed low-cost dog food, you'll have to feed more to get barely ample vitamin, and you will have additional poop to pickup, further shedding fur, and doubtless extra vet payments.
In December 2012, Pet Store Boys carried out a free concert within the just lately opened MediaCityUK BBC facilities accompanied by the BBC Philharmonic Orchestra and the Manchester Chamber Choir. They performed largely songs that hadn't been performed earlier than together with 'Miracles' and 'The Survivors'. A bit entitled 'He Dreamed of Machines'—taken from the band's mission related to Alan Turing —was additionally carried out for the first time.
Coming to you from the center of the Redwoods in Arcata, California, we are an actual pet present retailer, not a division of some bigger firm. Our retailer has been open since 1972, and we now have been selling pet provides on the internet constantly since 1997. We are proud that so many pet householders from everywhere on the planet return to us many times for their provide wants, every thing from specialty pet foods to collars, grooming supplies, dog and cat toys, and so forth, and we hope you'll change into one amongst them! The thought of saving time from procuring is an effective one. As a result of many specialised pet foods and pet objects come from specialty shops, doing shopping for on your pets means an extra journey. The alternative is shopping for regular meals from your grocery retailer and the business meals accessible will not meet the needs of your pet adequately. The consolation of ordering pet items on-line is growing because of this.
Toys and mirrors are additionally good ideas.
Sunil: This blue banded pet mongoose likes to perform magic suggestions for his Littlest Pet Retailer buddies, however his concern of bugs could also be exhausting to beat. The bigger facility at fifty one Notre Dame Ave 95113 has more room for merchandise and homeless pets and affords free buyer parking and straightforward highway entry. In your comfort, hours are 10 a.m. to eight p.m. day-after-day. Name us at (408) 297-0840.
The Adoption Middle may be a 501(c)three non-profit firm with a board of directors. The Adoption Center would work with non-revenue rescue groups to get pets adopted, arrange volunteers to handle pets within the coronary heart, host adoption festivals, current pet proprietor training and educational courses. Andy's Pet Store would continue in the same constructing as a for-revenue enterprise promoting pet supplies for the following six months. On the end of that time, we'll choose to proceed as separate businesses or to transfer the pet supplies enterprise to the 501(c)3 non-revenue.
New targets to help your greatest prospects.
Dogs and puppies are often relatively extra energetic than cats, and time outdoor is critical to a dog's well being. Collars and leashes are a necessity till a dog has its own home to run in. As with cats, ID tags are should-have objects. Decide how a lot food you need to feed your dog in a traditional meal and choose relevant meals and water bowls. The food should have all of the vitamins a rising puppy and dog needs in its meals regimen.
A youthful lady named Blythe Baxter strikes to downtown metropolis in an residence above Littlest Pet Shop. She shortly discovers she can perceive animals. She goes on some crazy adventures with the pets. On 10 June 2009, Pet Store Boys began the summer time leg of their Pandemonium Tour in Saint Petersburg, Russia and ended on 21 July 2009 in Tel Aviv, Israel , enjoying the Manchester Apollo and a bought-out gig at O2 Enviornment on 18 and 19 June 2009 respectively. The second leg of the tour started on 29 August 2009 at the Metropolis, in Montreal , Canada.
Convey your dog in for a gourmand taco from Pawsitively Gourmand. Free taco with every $5.00 dog merchandise purchase. Within the warm weather months we suggest taking your 4-legged good buddy right down to Pablo's Mexican restaurant in Previous Metropolis and having fun with tacos of their exterior seating space. Support native and pamper your pooch!


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At its mid-September meeting, the Vermont Medical Society Council considered and voted to support six policy resolutions and a proposed bylaws change. All six resolutions will now proceed to a vote at the VMS annual membership meeting, 9 to 11 a.m. on Saturday, Nov. 4th, at the Woodstock Inn in Woodstock, Vt.
The following resolutions will be put forward for debate and vote:
- Ensuring a Health Professional Member of the Green Mountain Care Board (submitted by the VMS Executive Committee);
- Ensuring an Adequate and Effective Health Care Workforce to Meet the Increased Demand for Health Services from an Aging Population (submitted by the VMS Executive Committee)
- Vermont Medical Society Policy on End of Life Care (submitted by Fred Crowley, MD)
- Improving the Safety of the Medical Marijuana System (submitted by Catherine Antley, MD, David Rettew, MD & John Hughes, MD)
- Legalizing Recreational Marijuana in Vermont (submitted by Catherine Antley, MD, David Rettew, MD & John Hughes, MD)
- Implementation of VDH Opioid Rules (submitted by Ed Haak, DO)
In addition, the Council voted to support a bylaws change that will remove a term limit from the elected VMS secretary-treasurer position.
For more informaiton about the 204th VMS annual meeting, click here.