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Research | Open | Published: 01 July 2015
Novel naphthochalcone derivative accelerate dermal wound healing through induction of epithelial-mesenchymal transition of keratinocyte
Ga Young Seo1,
Manh Tin Ho1,
Ngoc Thuy Bui1,
Young Mee Kim1,
Dongsoo Koh2,
Youngho Lim3,
Changlim Hyun4 &
Moonjae Cho1,5
Journal of Biomedical Sciencevolume 22, Article number: 47 (2015) | Download Citation
Wound healing is an intricate process whereby the skin repairs itself after injury. The epithelial-mesenchymal transition (EMT) is associated with wound healing and tissue regeneration. Naphthochalcone derivatives have various pharmaceutical properties. We investigated the effect of a novel naphthochalcone derivative, 2-(5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol (TDPN), on dermal wound healing in vivo and the migration of keratinocytes in vitro.
We investigated the effect of TDPN on signaling pathway and epithelial-mesenchymal transition through protein and transcriptional expression. The TDPN treatment accelerated dermal closure about 3 days and remodeling of dermis. We found that treatment with TDPN induced the migration of keratinocytes but not cytotoxicity. TDPN induced the phosphorylation of ERK and AKT. TDPN-treated cells showed loss of adherence protein and showed induction of the transcriptional factor Slug, mesenchymal marker, and fibronectin. Moreover, TDPN treatment induced the expression of matrix metalloproteinase-1 (MMP-1), which degrades specific components of the extracellular matrix, thereby providing new substrates that facilitate migration and invasion. MMP expression is considered to be one of the major attributes acquired by cells after EMT.
We propose that a novel naphthochalcone derivative TDPN is capable of promoting keratinocyte migration via the induction of EMT resulting acceleration of wound closure and matrix remodeling.
Skin is the outermost organ of body that provides a barrier function to the body. In the epidermis layer of the skin, keratinocytes are the major cell population, constituting more than 90 % of all the cells [1]. When injuries to the skin occur, the keratinocytes at the wound site undergo morphologic alterations, changing from sedentary cells to migratory cells [2]. The phenotype of the migratory cells changes with respect to cell-cell adhesion and cell-matrix adhesion during the re-epithelialization stage. This phenotype partially resembles the epithelial to mesenchymal transition (EMT) process, which plays a critical role in cancer metastasis [2]. During both re-epithelialization and cancer metastasis, cells that undergo EMT lose contact with each other and the extracellular matrix (ECM) [3]. The ECM may be degraded by matrix metalloproteinases (MMPs), which represent a family of zinc-containing endopeptidases. The relationship between the EMT and MMPs has been clarified. MMP expression is considered to be one of the major attributes acquired by epithelial cells after they undergo the EMT [4]. MMPs, such as MMP-1, MMP-2, MMP-9, and MMP-13, are frequently associated with processes that involve tissue re-modeling and cell migration. Membrane type 1-MMP (MT1-MMP) is spatially and temporally regulated during MCF10A cell migration owing to its mediation of pericellular proteolysis of the laminin-5 (Ln-5) g2 chain [5]. The EMT is influenced by a wide range of regulatory factors. The signaling pathways for PI3K/AKT/mTOR and MAPK (ERK) are reported to be involved in the regulation of EMT [2, 6]. Increased phosphorylation of AKT has been implicated in the induction of the EMT and cell migration [7]. Furthermore, the transcriptional factors Snail and Slug play essential roles in cell migration. Slug expression has been shown to be up-regulated in the wound margins in the in vitro, ex vivo, and in vivo settings [8]. In Slug-null mice, re-epithelialization is reduced, compared with wild-type mice [8].
Pure chalcones, which are extracted from plants, display interesting biological properties, such as antioxidant, cytotoxic, anticancer, and antimicrobial activities [9]. Naphthochalcone is a precursor to a variety of pharmaceutical agents. Naphthochalcone derivatives, which can be isolated from nature or biochemically synthesized, show various pharmaceutical effects [10]. One of the newly synthesized naphthochalcone derivatives, 1-(naphtho[2,1-b]furan-2-yl-carbonyl)-3,5-disubstituted-2,3-dihydro-1H-pyrazole, has been characterized by elemental analysis and in spectral studies. This compound has been evaluated for antimicrobial activity [11]. The compound (hydroxyphenyl) naphthol sulfonamide could be useful in optimizing 17β-HSD1 inhibitors for the treatment of endometriosis [12]. Another derivative, of methyl-1-hydroxy-2-naphthoate, may inhibit lipopolysaccharide-induced inflammatory responses in macrophages via suppression of the NF-κB, JNK, and p38 MAPK pathways [13]. Regarding structural analogues of the flavones, alpha-naphtoflavone promotes pro-collagen production in skin fibroblasts and is suggested to have anti-aging effects [14]. Recently we have screened series of compounds for promoting migration and proliferation of HaCaT cells and found that novel naphthochalcone derivative, 2-(5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol (TDPN) (Fig. 1) had good activity.
Structure of -(5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol (TDPN)
The present study was aimed to examining whether novel naphthochalcone derivative (TDPN) exerts any effects on process of wound healing and, if so, what are the underlying mechanisms responsible for the action of TDPN. A murine excisional wound healing model in vivo and experiments using human keratinocyte cell line in vitro indicated TDPN accelerated wound closure by activating keratinocyte movement via inducing EMT like change. The results of this study provide novel drug candidate of cutaneous skin wound.
The novel naphthochalcone derivative, 2-(5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol (TDPN) (Fig. 1), was synthesized and kindly provided by Professor Youngho Lim (Division of Bioscience and Biotechnology, Konkuk University, Seoul, Korea). TDPN stock solutions were stored as aliquots at −20 °C, and were diluted to the final concentration before use.
The human keratinocyte cell line HaCaT was cultured in RPMI medium that was supplemented with 10 % fetal bovine serum (FBS; GIBCO) and 1 % penicillin/streptomycin (PAA). Cells were incubated in a humidified atmosphere at 37 °C in 5 % CO2.
Scratch wound healing assay
Because the doubling time of HaCaT cells is approximately 24 h, cells were seeded at 50 % confluency in culture dishes 24 h before the wound healing scratch assay. Therefore, the experiment was performed when cells reached monolayer formation. A scratch was made in the cell monolayer by drawing a sterile p-200 pipette tip across the surface of the culture dish. After the scratch was made, the culture medium was supplemented with TDPN. Dimethyl sulfoxide (DMSO; Amresco) treatment was used as the control. At 0 h and 24 h post-treatment, photographs of the cell monolayer were acquired at 4X magnification using the Olympus IX70 microscope equipped with a digital camera. The scratch was measured using the ImageJ software, and the difference between the initial and final width of the scratch was calculated.
Cells were seeded on 96-well plates at 200 μl of cells which have density of 3 × 104 cells/ml for each wells. Cells were treated with TDPN or DMSO for 24 h. MTT solution (10 μl of 5 mg/ml solution; Amresco) was added to each well and incubated for 37 °C for 4 h. Subsequently, the medium was gently removed and replaced with 150 μl of DMSO and incubated for 30 min with shaking to dissolve the precipitate. The samples were measured at an absorbance of 570 nm in a spectrophotometer [15].
Cells were treated with TDPN or DMSO in time-dependent and dose-dependent manners. The cells were harvested by scraping and underwent lysis in RIPA buffer. The BCA method (Thermo Scientific) was used for protein concentration determination. The extracts were analyzed by SDS-PAGE followed by Western blotting with appropriate antibodies.
The following antibodies were used for Western blotting: p21 (catalogue no. 2947), AKT (9272), phosphorylated AKT (9271S), ERK (4695), Slug (9585S), and GAPDH (2118) were from Cell Signaling Technology; E-cadherin (610181) and Zo-1 (610966) were from BD Science Transduction; cyclin E (sc-247), cyclin D1 (sc-246), p53 (sc-126), phosphorylated ERK (sc-7383), collagen I (sc-25974), collagen III (sc-28888), and fibronectin (sc-9068) were from Santa Cruz Biotechnology; and MMP-1 (444209) was from Calbiochem. The secondary antibodies used in the Western blotting were anti-mouse (PI-2000; Vector Laboratories) anti-rabbit (PI-1000; Vector Laboratories), and anti-goat (AP-107P; Millipore). The blots were analyzed using the ImageJ software. The relative change in the ratio of the target protein to the DMSO control was determined.
RT-PCR
Cells were seeded in a 60-mm culture dish and treated with TDPN. RNA from the treated cells was extracted using the Trizol reagent (Invitrogen). Then, 2 μg of total RNA were used to synthesize cDNA synthesis using a Reverse Transcriptase Kit (Promega). The resulting cDNA was used for RT-PCR using the G-Taq kit (Cosmo Genetech, Seoul, Korea) according to the manufacturer's instructions.
RT-PCR was performed using the following gene-specific primers (forward and reverse primers, respectively), selected using the Blast Primer program: for GAPDH, 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGATTTC-3′; for p53, 5′-ACACGCTTCCCTGGATTGG-3 and 5′-CTGGCATTCTGGGAGCTTCA-3′; for p21, 5′-GTCAGTTCCTTGAGCCG-3 and 5′-GAAGGTAGAGCTTGGGCAGG-3′; for MMP-1, 5′-AGGGGAGATCATCGGGAC-3′ and 5′-GGCTGGACAGGATTTTGG-3′; for MMP-2, 5′- AACACCTTCTATGGTGCCC-3′ and 5′-ACGAGCAAAGGCATCACCA-3′; and for MMP-7, 5′-TACAGTGGGAACAGGCTAGG-3′ and 5′-GGCACTCCACATCTGGGC-3′. The results were analyzed using the ImageJ software. The relative change in the ratio of the target protein to the DMSO control was determined.
MMP zymography
MMP zymography was performed according to the method described by Gogly et al. [16], with the following modification: 8 % sodium dodecyl sulfate (SDS) gels that contained gelatin (0.01 mg/ml) were used. The SDS in the gels was removed by incubating the gels twice (30 min each) in 200 ml of 2.5 % Triton X-100 at 4 °C. Thereafter, the gel slabs were incubated at 37 °C overnight in the incubation buffer. The gels were then fixed and stained for 1 h with 0.05 % Coomassie Blue R-250. The molecular mass protein markers were readily visible as stained bands against the lighter blue color of the stained gelatin background. Gelatinase activity was apparent as clear zones of lysis (negative staining) against the blue background. The gels were scanned to create a permanent record of the results.
Transwell invasion assay
The Transwell invasion assay was performed using a commercial Transwell plate (Corning). Cells were harvested by trypsinization and 7x104 cells/well in 100ul of medium were seeded into the wells of the insert. The receiving wells were set up with medium containing FBS as a chemoattractant. After 24 h, media in insert wells was replaced by 100ul of free serum medium containing TDPN, TGF-β1, or DMSO. Invasive cells in the receiving wells were collected by trypsinization and counted by hemacytometer.
Hoechst stain proliferation assay
The Hoeschst stain proliferation assay was performed as previously described [17]. 200 μl of cells at a density of 3 × 104 cells/ml were seeded into the wells of 96-well plates. Cells were treated with TDPN or DMSO for 24 h. Hoechst 33342 solution (1 μl of 10 mM stock solution; Sigma) was added to each well and incubated for 37 °C for 30 min. Cell proliferation was estimated by direct measurement of changes in cell fluorescence using a spectrofluorometer (SPECTRAFLUOR, Tecan). The instrument was equipped with a 365-nm broadband filter for the excitation beam and a 450-nm narrowband interference filter plus a UV blocking filter for the emitted light
ECIS migration assay
HaCaT cells were seeded at a density of 1 × 105 cells/cm2 into ECIS arrays and impedance was measured using an ECIS instrument. Wounding pulses of 2400 mA were applied for 20 s at 6400 kHz. Media was then replaced by media containing TDPN or TGFβ1 or DMSO. The results were exported to the ECIS program.
Trypan blue cell counting assay
5x104 cells/well were seeded on 24-well plates. After 24 h of incubation, cells were treated with TDPN or DMSO in various concentrations for 24 h. All cells in each well were collected by trypsinization. The live cells were identified and counted by trypan blue staining. This experiment was repeated three times.
Animal experiment
6-week old male ICR mice (n = 5 for each group: control and treatment TPDN) were chosen for the experiment. The fur was removed with an electronic hair clipper and removal cream. Dermal wound was placed on the middle of a back using an 8 mm punch instrument. In dermal wound site, we punch out whole dermis including subcutaneous fat layer (deep punch) and appendages of skin didn't preserved. A few days later, granulation tissue and scab were formed at wound and we could confirm the epidermis and dermis were filled from surroundings of wound instead of granulation tissue. 200 μl of TDPN was applied to the wounds of the experimental group in concentrations of 200 μM (dissolved with Vaseline) for 20 days, while the control group was treated with same amount of DMSO (dissolved with Vaseline). Usually in vivo experiment, compounds have treated to animal approximately 100 ~ 200 times higher concentration of in vitro. Because process of the delivery and conversion of ingredients may cause lower of active material to reach to target points. However, in this study, the compound was applied to wound site directly, therefore we only applied just 10 times concentration of TDPN compared to in vitro. The wound was pictured every three day and the rate of wound closure was calculated as relative % of original wound area using Image J program. Skin samples were collected from mice at each of the wounding time points (9, 12, 15 days post-wounded) and fixed in 4 % formaldehyde. We cut formalin-fixed, paraffin-embedded tissues into 3um sections, stains all sections simultaneously by using routine hematoxylin–eosin (H-E) and Masson's trichrome stain methods. The slides were reviewed by an Olympus BX51 microscope (Olympus Corp., Tokyo, Japan) with the apochromatic objective lens and a 0.85 numeric aperture.
TDPN induces the migration but not the proliferation of keratinocytes
Re-epithelialization involves the migration and proliferation of keratinocytes. In the present study, we investigated these two processes using the MTT assay and scratch wound healing assay for keratinocytes (HaCaT cells) treated with different concentrations of 2-(5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol (TDPN) for 24 h. In the migration assay, TDNP promoted HaCaT cell migration dose-dependently up to 20 μM. However, there was little difference between 10 and 20 μM (Fig. 2a). It has been reported that TGF-β can induce EMT and migration of various cell types, including keratinocytes ([20, 31], Rasanen et al., 2010). To compare their abilities to induce migration, we performed migration assay comparing the ability of TGF and TDPN to induce migration, by both the ECIS system (Fig. 2b) and the Transwell invasion assay (Fig. 2c). These results demonstrated that TDPN induced migration and invasion comparably to TGF-β in the same time period of treatment.
TDPN effects on migration and proliferation of HaCaT cell line. Migration of HaCaT cells were measured by scratch wound assay and ECIS system. Scratch wound healing assay of HaCaT cells treated with different concentrations of TDPN for 24 h. Cells were seeded with density 3 × 104 cells/ml 24 h before scratch and treated. Distance of the scratch was measured after 24 h treated with TDPN (a). Cells were subjected to ECIS migration assay in the absence or the presence of TDPN or TGF-β as indicated. Cells were allowed to grow to confluence and initial resistance was measured for few hours. Cells were subjected to high voltage to initiate wound at 3 h resulting in the drop of resistance. Remaining cells were allowed to migrate in the presence or absence of TDPN and resistance changes were measured for 24 h after the creation of wound (b). Represented pictures are shown from 3 independent experiments. Invasion of TDPN treated cells were measured using transwell chambers as described in methods (c). Proliferation of keratinocyte cells were measures three different methods; MTT assay, Hoechst staining, and trypan blue counting assays (d). Cells were seeded for 24 h, then treated with various concentrations of TDPN or DMSO for 24 h. After treatment, cells were collected and counted in Trypan blue assay or incubated with Hoechst 33342 or MTT reagent. Results were measured by flourescence spectrometer or spectrometer. Western blot of cell cycle-related proteins comparing cells treated with 10 μM TDPN and untreated cells. Protein lysate from cells treated with TDPN for 24 h were taken using RIPA buffer. The expression level was measured by comparing the density of the target protein to GAPDH (e). (*)P < 0.05 compare to control group
Since migration rate may be influenced by proliferation rate, we tested cell growth. And keratinocyte proliferation was not affected by TDPN proven by three independent assays such as MTT assay, hemacytometer counting assay and in the proliferation Hoechst staining assay (Fig. 2d). These results also showed no significant changes between treated and untreated cells. To confirm the results, proteins related to the cell cycle were investigated in the treated cells. The levels of cyclin-dependent kinases E and D1, p53, and p21 were not significantly changed by TDPN (Fig. 2e). These data suggest that TDPN induces the migration but not the proliferation of keratinocytes.
ERK and AKT phosphorylation is involved in promotion of keratinocyte migration by TDPN
It is well-known that mitogen-activated protein (MAP) kinase family members, ERKs, as well as the PI3K/AKT pathway, are important for the regulation of cell migration. Therefore, we investigated the expression levels of regulatory proteins related to cell migration, such as AKT and ERK. Keratinocytes were treated with TDPN for different time periods and protein lysates were assayed for phosphorylation of AKT and ERK. We used a stock of TDPN containing approximately 0.05 % DMSO. Each time point, cells treated either DMSO or TDPN in DMSO were collected and assayed by western blot analysis. The relative intensity between TDPN and control group at each specific time point was calculated (Fig. 3a). Within 2 h ERK and AKT signals were activated. These signals were increased dose-dependently at 2 h time point (Fig. 3b) and the treatment of specific inhibitors against ERK and AKT attenuate TDPN induced phosphorylation (Fig. 3c). To identify the TDPN induced migration of keratinocyte was mediated by ERK and AKT signaling, we performed a wound healing assay in which cells were pretreated with a PI3K inhibitor (LY49002, 50 μM) and a MEK inhibitor (PD98059, 50 μM) then treated with TDPN or left untreated. The results demonstrated that inhibiting the AKT and ERK pathways abolished the effects of TDPN on migration (Fig. 3d).
ERK and AKT phosphorylation is involved in promotion of keratinocyte migration by TDPN. HaCaT cells were treated with either DMSO or 10 μM TDPN for the indicated time (a) or the indicated dose for 2 h (b) and harvested for western blot analysis. Band intensity is presented as mean ± SE of relative to GAPDH and ratio between pAKT or pERK of TDPN and pAKT or pERK of control DMSO treatment from least 3 replications. (*)P < 0.05 compare to control group) (c) Cells were pre-treated with PI3K inhibitor (LY49002, 50 μM,) or MEK inhibitor (pd98059, 50 μM,) for 24 h, then treated with TDPN for 1 h and harvested for western blot analysis, to assess the involvement of the ERK and AKT signaling pathways. (d) After co-treatment of TDPN and PI3K inhibitor (LY49002, 50 μM) or MEK inhibitor (pd98059, 50 μM) for 24 h, scratch wound healing assay were performed. Distance of the scratch was measured relative to control after 24 h of treatment with TDPN. (*)P < 0.05)
TDPN induces EMT-like change
It has been reported that EMT occurs in keratinocytes at wound sites during the re-epithelialization stage [18, 19]. Thus, we investigated whether TDPN promotes re-epithelialization through EMT. We treated TGF-β1 (10 ng/ml) and TDPN for 24 h and observed morphological and biochemical changes. On above condition, morphological changes were not dramatic (Additional file 1: Figure S1). It has been reported that TGF-β induces invasion and changes the cell size of several cell types including epithelial cells and keratinocytes [20-22]. However, morphological change of keratinocytes by TGF-β on in vitro varies depending on cell types. HaCaT cells treated with TGF-β1 for 24 h only form actin stress fibers but morphology does not change [23]. However, in cellular level, main EMT markers such as epithelial marker proteins Zo-1 and E-cadherin, and mesenchymal marker, transcriptional factor Slug changed (Fig. 4a). Western blot results revealed that TDPN treatment increased Slug expression and decreased the E-cadherin and Zo-1 in a time-dependent manner (Fig. 4a) and dose-dependent manner (Fig. 4b). This suggests that TDPN treatment causes the loss of cell-cell adhesion. In addition, the level of the mesenchymal marker fibronectin was slightly increased (Fig. 4d).
TDPN induces EMT like change. HaCaT cells were treated with TDPN (10 μM) for the indicated time (a) or at the indicated dose (b) and harvested for western blot analysis. (data presented as mean of relative to GAPDH ± SE from least 3 replications, (*)P < 0.05 compare to control group). (c) HaCaT cells treated with 10 μM TDPN for 24 h. After treatment, the conditioned media from the cells was concentrated using amicon centrifugation. Protein in media was collected and analysed by western blot for Collagen I, Collagen III, Fibronectin, and MMP1. (d)Transcriptional expression of MMPs in HaCaT cells treated with TDPN (10 μM for 24 h). After treatment, total RNA from each cells was taken by Trizol reagent and analysed by RT-PCR for MMP1, MMP2, MMP7. GAPDH was used as a control. (data presented as mean of relative to GAPDH ± SE from least 3 replications, (*)P < 0.05 compare to control group)
In both EMT tumors and migrating keratinocytes, degradation and remodeling of the ECM are needed. The EMT pathway has been reported to involve MMP expression [4]. In the present study, zymography revealed that the levels of MMP-2 and MMP-9 were not changed by TDPN treatment (Additional file 2: Figure S2). However, the Western blot results for TDPN-treated cells showed that the MMP-1 protein level was significantly increased (Fig. 4c). The transcriptional expression pattern showed a similar result (Fig. 4d).
TDPN accelerated dermal wound healing
Full-thickness excisional wound were made on the dorsal of mice and TDPN or Vaseline were applied to wound site topically every day. Hereby, we investigated the effects of TDPN on skin remodeling phase. It could affect scar formation and faster resolving collagen deposit. TDPN treated mice exhibited a faster wound closure then muck treated animals as early as day 9 (Fig. 5a). The average of opened wound area were measured by image program and the rate of closure of TDPN treated was 3 day faster than untreated ones (Fig. 5b). Tissue samples were taken at day 9, 12 and 15 post-wounded and performed H&E staining. On day 9, TDPN treated wound revealed that epidermal closure is complete but dermal closure is not sufficient to have open area (yellow arrow) and inflammation and necrosis is still remain in outer skin (Fig. 6a). Whereas muck treated wound showed that necrosis and inflammation accompanied in wound site and epidermal leading edge is observed (black arrow) but epidermal closure is not complete. The granulation tissue formation in dermis is incomplete and dermal closure is ongoing (Fig. 6b). On day 12, in TDPN treated wound, epidermal and dermal closure is complete and hair follicle start to show but not yet start differentiation. Contraction of granulation tissue squeezes hair follicle to wound direction (Fig. 6c). In muck treated wound, granulation tissue formation is processed and dermal closure is complete, however, the proliferation of epidermis is not sufficient and the epidermal closure is incomplete (Fig. 6d). On day 15, in TDPN wound, epidermal and dermal closure is complete perfectly and glands differentiation is processing throughout the wound site (Fig. 6e). Day 15 muck treated wound still have no sign of a nodule that resembled early sweat or sebaceous glands (Fig. 6f). Collagen deposits were stained by Masson Trichrome (Fig. 6g-j). In TDPN treated animal, until day 12 collagen deposition is still ongoing and distribution of collagen fiber is uneven (Fig. 6g) however on day 15, collagen density is high and evenly distributed as normal dermis (Fig. 6h). In muck treated wound, the collagen deposit did not show much difference compared to TDPN treated (Fig. 6i-j). Results suggested accelerated wound closure may attributed by faster epidermis differentiation.
Wound closure after a full-thickness dermal excision. Male ICR mice (6 weeks of age) underwent a full-thickness 8 mm excisional wounding. They were either muck treated or treated with cutaneous application of TDPN (200 μM) daily. The wound closure was pictured every three day (a). The averages of opened wound area were measured by image program and plotted as relative % of original wound (b). The values were expressed as means ± SE from 6 wounds in each group till day 9, 4 wounds at day12 and 2 wounds at day 15
Histological findings of dermal wound tissues during the healing process. Dermal specimens were obtained from ICR mice either TDPN treated (a, c and e) or muck treated (b, d, and f) as in Fig. 5, at day 9 (a and b), day 12 (c and d) or day 15 (e and f). They were subjected to H-E staining (a-f) or Masson Trichrome staining (g-j). Yellow arrow indicated open area. Black arrow indicated epidermal leading edge. Represented pictures are shown from 2 wounds in each group at each day
TDPN promote epidermis development via keratinocyte activation
We performed Ki-67 antibody staining for detect proliferation (Fig. 7). In both case, the day 9 show highest proliferation index (Fig. 7a-f). In the TDPN group at day 12 and muck treated group at day 15, cells located at the bilayer of epidermis (yellow arrow) show strong reaction as seen in normal epidermis (Fig. 7b,f). However, on day 15, TDPN treated group show high proliferation not only epidermal bilayer but also around nodules that resembled early sweat or sebaceous glands (Fig. 7c). These results suggested that TDPN mainly activated epidermal keratinocyte rather than dermal fibroblast.
Proliferation of dermal wound tissues during the healing process. Dermal specimens were obtained from ICR mice either TDPN treated (a, c and e) or muck treated (b, d, and f) as in Fig. 5, at day 9 (a and b), day 12 (c and d) or day 15 (e and f). Proliferation of cell in wound site was measured by ki-67 antibody staining. Represented pictures are shown from 2 wounds in each group at each day
Our results reveal that the novel naphthochalcone derivative TDPN show faster wound closure and matrix remodeling (Figs. 5 and 6) via promoting the migration but not the proliferation of keratinocytes (Fig. 2). Importantly, we observed promotion of the EMT pathway in the TDPN-treated cells. The expression levels of the adherence junction protein E-cadherin and tight junction protein ZO-1 were reduced in the cells treated with TDPN. Also, the expression levels of the transcriptional factor Slug, as well as those of the mesenchymal marker fibronectin were induced in these cells (Fig. 4). Furthermore, the ERK and AKT signaling pathways were found to be involved in TDPN induction of EMT, as evidenced by the induction of phosphorylation of ERK and AKT which was confirmed by pretreatment AKT inhibitor (LY49002, 50 μM) or ERK inhibitor (PD98059, 50 μM) attenuated effect of TDPN (Fig. 3). As a consequence of the EMT, MMP-1 (but not MMP-2 or MMP-9) was found to be significantly induced. Following the induction of MMP-1 in the TDPN-treated cells, the ECM proteins collagen I and collagen III were significantly degraded.
The TDPN mainly stimulate keratinocyte not fibroblast. Keratinocyte migration, a part of cell proliferation stage, plays an important role during the wound healing [24]. Several approaches have been used to study new compounds and new pathways that promote migration and wound healing [25, 26]. For example, Protection effects of green tea extract (EGCG) against UV were found to be involved in the proliferation of normal human keratinocytes [27]. EGCG-induced Erk phosphorylation and activation of the Akt pathway was found to promote keratinocyte survival [28]. However, in our study, TDPN showed effects on the migration but not on the proliferation of keratinocytes. VPA cutaneous wound healing by increasing the motility of HaCaT keratinocytes through ERK and phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathways [28].
The epithelial-mesenchymal transition (EMT) is one of the major factors that affect cell migration associated with wound healing and tissue regeneration [18, 19]. Loss of cell junctions is considered to be a crucial marker for EMT [17]. Extensive previous studies have focused on how tight junctions are down-regulated in EMT [29, 30, 5]. In addition, during the early stage of re-epithelialization, when cells undergo EMT, reduced cell-cell contacts allow the cells to migrate. EMT down-regulation of the cell junction protein E-cadherin appears to involve regulation by Wnt or TGF-β via inducible activation of kinase pathways that modulate GTPase, Smads, PI3Ks, MAP kinases, β-catenin, and activate transcription factors, including LEF-1, Snail, Slug, and Scatter, ultimately leading to repression of the E-cadherin gene [31]. Apart from the loss of cell junctions, the increased expression levels of Slug and mesenchymal marker act as a marker for EMT [19, 32]. Slug, a key transcription factor is responsible for the down-regulation of E-cadherin in both explanted human skin and primary keratinocytes [33].
The cell migration is regulated by various signaling pathways. The MAPK signaling pathways are associated with cell migration [34], as well as the EMT pathway [35, 36], and the PI3K/AKT signaling pathway has also been implicated in cell migration [6, 37] and EMT [38]. TDPN induced phosphorylation of ERK and AKT in time- and dose-dependent manners (Fig. 3). A previous report showed that transforming acidic coiled-coil protein 3 (TACC3) induced EMT and migration through induction of the AKT and ERK pathways [37]. Furthermore, it has been reported that inhibition of the ERK and AKT pathways leads to inhibition of the induction of EMT by TGF-β1 [39]. Our results revealed that the phosphorylation of signaling pathway proteins AKT and ERK were induced early in TDPN treatment, while changes of EMT markers such as Slug E-cadherin detected later. We suggest that TDPN triggers early signals in the EMT pathway (Figs. 3 and 4).
Matrix metalloproteinases (MMPs), which are extracellular proteases that are highly expressed at wound sites, degrade specific components of the ECM, thereby providing new substrates that facilitate migration and invasion [40]. The relationships between EMT and MMPs have been described. MMP expression is considered to be one of the major attributes acquired by epithelial cells after EMT [4]. MMP-2 and MMP-9 have been reported to play essential roles in the EMT in the avian embryo [41, 42]. Interestingly, our enzymatic activity results do not accord with the results in the previous report, in that the levels of MMP-2 and MMP-9 were not changed in our treated cells. However, MMP-1 (type I collagenase) is needed in the epidermis for re-epithelialization [40]. In human oral keratinocytes, MMP-1 mRNA was expressed while the keratinocytes covered the wound surface [43]. In agreement with those results, MMP-1 was induced in the TDPN-treated cells in the our study (Fig. 4). In vitro keratinocyte culture, collagen I and collagen III were significantly degraded in the TDPN treatment group. Given that MMP-1 is a collagenase, its induction would lead to the degradation of the collagen helping keratinocyte migration at the margin of dermis.
The finding of novel compound – TDPN – to promote wound healing via keratinocyte migration supplies a good tool for studying wound healing mechanism and possible drug candidate.
TDPN:
2-(5-(2,4,6-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol
EMT:
Epithelial-mesenchymal transition
TGF-β1:
Transforming growth factor beta 1
ECM:
MMP:
Matrix metalloproteinases
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Hudson LG, Newkirk KM, Chandler HL, Choi C, Fossey SL, Allison E. Parent and Kusewitt D.F. cutaneous wound reepithelialization is compromised in mice lacking functional Slug (snai2). J Dermatol Sci. 2009;56:19–26.
Batovska DI, Todorova IT. Trends in utilization of the pharmacological potential of chalcones. Curr Clin Pharmacol. 2010;1–29.
Zangade S, Mokle S, Vibhute A, Vibhute Y. An efficient and operationally simple synthesis of some new chalcones by using grinding technique. Chem Sci J. 2011;13:1–6.
Chandrashekhar CH, Latha KP, Vagdevi HM, Vaidya VP. Synthesis and antimicrobial activity of chalcones of naphtho[2,1-b]furan condensed with barbituric acid. Der Pharma Chemica Der Pharma Chemica. 2011;3:365–9.
Henn C, Einspanier A, Marchais-Oberwinkler S, Frotscher M, Hartmann RW. Lead optimization of 17β-HSD1 inhibitors of the (hydroxyphenyl)naphthol sulfonamide type for the treatment of endometriosis. J Med Chem. 2012;55:3307–18.
Zhang JY, Jin H, Wang GF, Yu PJ, Wu SY, Zhu ZG, et al. Methyl-1-hydroxy-2-naphthoate, a novel naphthol derivative, inhibits lipopolysaccharide-induced inflammatory response in macrophages via suppression of NF-κB, JNK and p38 MAPK pathways. Inflamm Res. 2011;60:851–9.
Liao PL, Li CH, Chang CY, Lu SR, Lin CH, Tse LS, et al. Anti-aging effects of alpha-naphtoflavone on normal and UVB-irradiated human skin fibroblast. Exp Dermatol. 2012;21:546–8.
GY Seo, SG Park, JS Huh, MJ Cho. The protective effec of Glycitin on UV-induced Skin photoaging in Human Primary Dermal Fibroblast. J Korean Soc Appl Biol Chem. 2014;57(4):463-468.
Gogly B, Groult N, Hornebeck W, Godeau G, Pellat B. Collagen zymography as a sensitive and specific technique for the determination of subpicogram levels of interstitial collagenase. Anal Biochem. 1998;255:211–6.
Richards WL, Song M, Krutzsch H, Evarts RP, Marsden E, Thorgeirsson SS. Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA. Exp Cell Res. 1985;159:235–46.
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Nicolas FJ, Lehmann K, Warne PH, Hill CS, Downward J. Epithelial to mesenchymal transition in Madin-Darby canine kidney cells is accompanied by down-regulation of Smad3 expression, leading to resistance to transforming growth factor-beta-induced growth arrest. J Biol Chem. 2003;278(5):3251–6.
Ellenrieder V, Hendler SF, Boeck W, Seufferlein T, Menke A, Ruhland C, et al. Transforming growth factor beta1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation. Cancer Res. 2001;61(10):4222–8.
Rasanen K, Vaheri A. TGF-beta1 causes epithelial-mesenchymal transition in HaCaT derivatives, but induces expression of COX-2 and migration only in benign, not in malignant keratinocytes. J Dermatol Sci. 2010;58(2):97–104.
Brown KA, Aakre ME, Gorska AE, Price JO, Eltom SE, Pietenpol JA, et al. Induction by transforming growth factor-beta1 of epithelial to mesenchymal transition is a rare event in vitro. Breast Cancer Res. 2004;6(3):R215–31.
Reinke JM, Sorg H. Wound healing and regeneration. Eur Surg Res. 2012;49:35–43.
Yang HH, Jung B, Kim JR. Identification of plant extracts that inhibit cellular senescence in human fibroblasts, endothelial cells, and vascular smooth muscle cells. J Korean Soc Appl Biol Chem Volume. 2010;53:584–92.
Park SY, Lim HK, Lee S, Hwang HC, Cho SK, Cho M. Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) regulates cell cycle and the fibronectin synthesis in HaCaT cell migration. Food Chem. 2011;132:487–92.
Chung JH, Han JH, Hwang EJ, Seo JY, Cho KH, Kim KH, et al. Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes. FASEB J. 2003;17(13):1913–5.
Aliouat-Denis CM, Dendouga N, Van den Wyngaert I, Goehlmann H, Steller U, Van de Weyer I, et al. p53-independent regulation of p21Waf1/Cip1 expression and senescence by Chk2. Mol Cancer Res. 2005;3:627–34.
Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Invest. 2009;119(6):1438–49.
Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009;119:1420–8.
Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest. 2003;112(12):1776–84.
Bolós V, Peinado H, Pérez-Moreno MA, Fraga MF, Cano MEA. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci. 2002;116:499–511.
Yan C, Grimm WA, Garner WL, Qin L, Travis T, Tan N, et al. Epithelial to Mesenchymal Transition in Human Skin Wound Healing Is Induced by Tumor Necrosis Factor-α through Bone Morphogenic Protein-2. Am J Patho. 2010;176:2247–58.
Huang C, Jacobson K, Schaller M. MAP kinases and cell migration. J Cell Sci. 2004;117:4619–28.
Shin S, Dimitri CA, Yoon SO, Dowdle W, Blenis J. ERK2 but Not ERK1 induces epithelial-to-Mesenchymal transformation via DEF motif-dependent signaling events. Mol Cell. 2010;38:114–27.
Shi J, Zeng X, Zhou M, Chen Q. Activation of ERK-FAK signaling pathway and enhancement of cell migration involved in the early interaction between oral keratinocytes and Candida albicans. Mycopathologia. 2008;10:1–7.
Kandel ES, Skeen J, Majewski N, Cristofano AD, Pandolfi PP, Feliciano CS, et al. Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage. Mol Cell Biol. 2009;22:7831–41.
Ha G, Park JS, Breuer EK. TACC3 promotes epithelial–mesenchymal transition (EMT) through the activation of PI3K/Akt and ERK signaling pathways. Cancer Lett. 2013;332:63–73.
Davies M, Robinson M, Smith E, Huntley S, Prime S, Paterson I. Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGFb1 involves MAPK, Smad and AP-1 signalling pathways. J Cell Biochem. 2005;95:918–31.
Stevens LJ, Page-McCaw A. A secreted MMP is required for re-epithelialization during wound healing. J Biol Chem. 2011;287:41032–43.
Duong TD, Erickson CA. MMP-2 plays an essential role in producing epithelial-mesenchymal transformations in the avian embryo. Dev Dyn. 2003;229:42–53.
Lin CY, Tsai PH, Kandaswami CC, Lee MT. Matrix metalloproteinase-9 cooperates with transcription factor Snail to induce epithelial-mesenchymal transition. Cancer Sci. 2011;102:815–27.
Garlick JA, Parks WC, Welgus HG, Taichman LB. Re-epithelialization of human oral keratinocytes in vitro. J Dent Res. 1996;72:912–8.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ009045) funded by the Rural Development Administration, Republic of Korea.
Department of Biochemistry School of Medicine, Jeju National University, Jeju, 690-756, South Korea
Ga Young Seo
, Manh Tin Ho
, Ngoc Thuy Bui
, Young Mee Kim
& Moonjae Cho
Department of Applied Chemistry, Dongduk Women's University, Seoul, 136-714, South Korea
Dongsoo Koh
Division of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, South Korea
Youngho Lim
Department of Pathology, School of Medicine, Jeju National University, Jeju, 690-756, South Korea
Changlim Hyun
Institute of Medical Science, Jeju National University, Jeju, 690-756, South Korea
Moonjae Cho
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Correspondence to Moonjae Cho.
GY, MT, NT, YM carried out the experiments, participated in the data analysis. DK, YL synthesized and supplied the compound TDPN. CH performed the immunohistochemistry and analyzed the immunostaining data. MC was the corresponding author who designed and gave advices as well as wrote the manuscript. All authors read and approved the final manuscript.
Ga Young Seo and Manh Tin Ho contributed equally to this work.
Additional file 1: Figure S1.
Morphology of HaCaT cell change when treated with TDPN for 24 h. TGF-β1 (5 μM for 24 h) was used as a positive control.
Gelatin zymograph assay using media from HaCaT cells treated with 10 μM TDPN for 24 h. After treatment, the conditioned media from the cells was concentrated using amicon centrifugation.
Naphthochalcone


Joseph P. Rieth, 82, of Zephyrhills, Fla., passed away on Sunday, July 22.
He is predeceased by his first wife, Elizabeth (Trautner) Rieth, and their son, Brian Rieth.
He is survived by his wife of 37 years, Barbara (Stanechewski) Rieth; two daughters, Donna Smith and husband, William, and Joanne Nelson and husband, Thomas; a son, Michael F. Rieth, all of Clinton; two step-daughters, Beverly Jacques, of Webster and Charmaine DaCruz, of Lutz, Fla.; two sisters, Caroline Dowd of Clinton and Geraldine Fitch and husband, Stephen, of Sterling; daughter-in-law, Lynn Rieth of Athol; 15 grandchildren, 23 great-grandchildren, numerous nieces, nephews and many dear friends.
Mr. Rieth was born in Clinton to the late Everett and Mary (Hastings) Rieth. He attended local schools and was a graduate of the Clinton High School, Class of 1948. He began his career with the former Clinton Supermarket and later was a supervisor for Victory Supermarket before opening his own business. He owned and operated IGA Supermarket Stores throughout northern Worcester County. Upon retirement, he and his wife settled in Florida and have enjoyed extensive travel.
A graveside service will be held at 11:30 a.m. on Thursday, Sept. 6, in St. John�s Cemetery, Parker Road, in Lancaster. Contributions may be made to the Alzheimer�s Association of Massachusetts, 311 Arsenal St., Watertown, Mass. 02472. Online condolences may be placed at www.philbincomeaufh.com.


text:
Антоний Реман (, 13 мая 1840, Краков — 13 января 1917, Львов) — польский учëный-географ, геоморфолог, путешественник. Признанный специалист по проблемам геоботаники и физической географии, основатель львовской школы географов. Профессор Львовского университета им. Яна Казимира.

Биография

В 1860—1863 годах изучал естественные науки и географию в Ягеллонском университете в Кракове. В 1864 году получил степень доктора философии в области ботаники.

Был членом Физиографической комиссии Польской Академии знаний (с 1865), Польского научного общества естествоиспытателей им. Коперника во Львове (в 1888—1889 — президент общества).

В 1910 году ушел на пенсию.

Научная работа
Возглавлял научные геоботанические экспедиции в Карпаты, Молдавию, на Подолье, Кавказ, Южную Америку (1865—1882 гг.)

В 1865 году исследовал степи Подолья на юго-западе Украины, совершил ряд поездок на берега Днестра, в район наивысшего горного хребта в украинских Карпатах — Черногора.

В 1866—1867 г. прошел специализацию по курсу анатомии и физиологии растений под руководством профессора К. Негели в Мюнхене.

В 1868 году Антоний Реман отправился в путешествие на юг России. В 1869 году в Ягеллонском университете прошел процесс хабилитации в области анатомии растений.

В начале 1880-х годов во Львовском университете была открыта кафедра географии, которую возглавил инициатор её создания профессор Антоний Реман), уже известный работами по физической географии Карпат, которой руководил на протяжении 28 лет.

Антоний Реман был инициатором создания в 1911 г. при этой кафедре Географического института.

В 1884—1897 г. Реман, кроме того, преподавал ботанику в Академии ветеринарной медицины во Львове.

В 1873—1874 году побывал на Кавказе и в Крыму, в 1875—1877 и 1879—1880 — в Южной Африке, исследовал территории населенные бушменами, готтентотами, зулусами и группой народов банту.

Антоний Реман описал много новых таксонов, в частности, в 1868 году — Полыни горькой (лат. Artemísia absínthium) в еë пьенинской разновидности (Artemisia absinthium L. var. calcigena Rehm.), которая в настоящее время считается эндемиком в Пьенинах и включена в «Красную книгу растений и грибов Польши».

Избранные труды
 Szkice z podróży do południowej Afryki (1881);
 Echa z południowej Afryki (1884);
 Tatry pod względem fizyczno-geograficznym (1895).

Примечания

Литература
 Piotr Köhler: Botanika w Towarzystwie Naukowym Krakowskim, Akademii Umiejętności i Polskiej Akademii Umiejętności (1815—1952). Kraków 2002.

Ссылки
 История Львовского университета им. И. Я. Франко
 Україна та її географічні дослідження. Реман Антоній (1840—1917)

Ботаники Польши
Географы Польши
Путешественники Польши
Выпускники Ягеллонского университета
Преподаватели Львовского университета
Члены Польской академии знаний
Ботаники по алфавиту
Геоморфологи Польши

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text:
Eastern goes 1-2 against Belmont
Dillan Schorfheide, Assistant Sports Editor
Foster Anshutz was perfect going into the weekend series against the Belmont Bruins with a 5-0 record as a starter in Eastern's rotation, but Belmont ruined that with a 5-1 win over Eastern Saturday.
Anshutz gave up four earned runs in his 6.2 innings on seven hits during the final game of the series, while also allowing three walks and striking out three batters.
He was not the only Eastern pitcher who could not stymie the Belmont batters, though, as the Bruins scored more than five runs in each game.
Eastern (16-16, 8-7) took the first game of the series Friday, 9-6, but Belmont (15-16, 10-5) outscored the Panthers 14-8 in the final two games to close out the series with two wins.
So continues the trend for the Eastern baseball team, as the Panthers look for consistency from its high-scoring offense and its pitching staff that is prone to giving up big games to opponents' hitting.
That has been a big issue plaguing Eastern this season.
The Panthers bring in 6.65 runs per game and give up 5.97 to opponents.
Even though Eastern has the upper hand in that respect, it cannot always make up for the games where the pitching staff gives up five or more runs, which it has done in 18 games.
All three of the games against Belmont factor in to that overall 18.
The Eastern offense was able to perform like it is capable of in the first two series games, unlike the third game where the Panthers only brought in one run to support Anshutz.
Eastern scored 16 runs in the first two games, and in the lone victory, the Panthers scored six runs in a three-inning stretch to pull away for the victory.
Keith Kerrigan started Eastern's big day Friday with a double in the second inning, which scored Christian Pena to give Eastern a 1-0 lead.
Belmont tied the game up right away in the bottom of the second, but Eastern countered in the third inning with two runs as Hunter Morris and Ryan Knernschield were credited with those RBIs.
Both sides were quiet until the fifth inning, which is when Belmont scored two more runs to tie the game again at 3-3.
Eastern's relief staff has had some trouble in the middle innings in the past, but Friday, starter Michael YaSenka went seven innings, only giving up those three earned runs on six hits, one walk and eight strikeouts.
YaSenka got the win Friday, improving to an even 3-3 on the year.
The Panthers' offense also gave YaSenka backup, scoring the final six runs in the sixth through eighth innings.
Matt Waznis flied out in the sixth inning, scoring Knernschield to put Eastern up 4-3.
An inning later, Knernschield reached first base on a fielder's choice, scoring Jimmy Govern.
Pena followed that up with a groundout, but Grant Emme was still able to score.
Rounding it out in the eighth inning was Govern, who hit a two-run home run followed by Knernschield who, despite not getting a hit again, reached on an error and scored Emme for Eastern's ninth run.
Belmont gave Eastern a scare in what looked like a for-sure Eastern victory, as Chandler Adkins hit a three-run home run in the eighth inning to give Belmont its sixth run.
But Alex Stevenson pitched the final 1.1 innings and to close out Eastern's lone win, earning his second save on the season.
After the road trip, Eastern now has a five-game home stand over the next week, starting Tuesday when the Panthers host Southern Illinois at 3 p.m.
Dillan Schorfheide can be reached at 581-2812 or [email protected]
Dillan Schorfheide, Sports Editor
I'm Dillan, I am currently the Sports Editor at The Daily Eastern News. I have played and loved sports my whole life, so why not cover them...
Baseball team to host Tennessee-Martin
Eastern blows out Robert Morris 13-2 at home
Eastern baseball team defeats Eagles on Friday
Eastern drops doubleheader to Eagles
Eastern beats Chicago State 6-3 at home
Baseball finishes home stand Tuesday
Eastern goes 1-2 against Eastern Kentucky
Eastern hosting Colonels for 3 games
Eastern loses 9-8 to Salukis
Eastern clobbers Illinois College 11-1

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text:
Timeline created by maikonardell
The Origin of Darwin
Darwin was born in 1809, into a wealthy, well-educated family that lived in Shrewsbury, England. He was quiet and well mannered, however curious and loved natural history and exploring nature (Browne& Wyhe) & (Galapagos Conservation Trust). Photo retrieved from (Jane Austen's London).
School starts
Starts attending a boarding school in Shrewsbury.
(Browne & Wyhe)
Admission to University
Darwin is sent to the University of Edinburgh to study medicine.
(Browne & Wyhe) Photo retrieved from (Stewart)
Dropping Out
Being bored with academic content and aversion to blood, he disliked life in university. He was more fascinated by the diversity of the natural world according to actual experience through childhood experiences in nature and ended up leaving the university in 1827 without getting a degree and without getting a degree.
Darwin enters Christ's College in Cambridge by his father's wish. He learns theology, classics, and mathematics. Darwin devoted himself to Natural History and insect collection (Galapagos Conservation Trust) & (The Biography.com)
Graduation and Bon Voyage!
After graduating from Cambridge University in 1831, his teacher Henslow introduced him to HMS Beagle, a British Navy survey ship destined to South America for a survey, and also made multiple stops for exploration while sailing around the world. Darwin first observed volcanoes and nature in Cape Verde on the way to North America and started keeping his voyage journal (Browne & Wyhe) & (The Biography.com). Photo retrieved from (McNamara)
The Galapagos island
During sailing all over the world, Darwin discovers fossils, witnesses different characteristics, patterns, and diversity of organisms that are well adapted to the geological features and environment. He also notices that the shape of bird beaks differ from island to island (Browne & Wyhe) & (Galapagos Conservation Trust). Such discoveries and observations lead to the proposal of a revolutionary hypothesis. If characteristics of earth change, so do life on earth.
Return to home land
After returning to England in Oct 1836, Darwin moves to Cambridge to organize his collection and rewrite his voyage journal for publication (The Biography.com). He felt that throughout this voyage, the fauna and flora across the Southern Hemisphere created the species independently, which cannot be thought of as immutable since then. Changes in the continent have created new habitats, and have come to the idea that organisms can adapt to the changes(Galapagos Conservation Trust).
The Voyage of the Beagle
Publishes The Voyage of the Beagle. Work reference:
Darwin, Charles, 1809-1882. The Voyage of the Beagle. London : New York :Dent; Dutton, 1959.
Publication of 'On the Origin of Species'
'On the Origin of Species' advocates that the theory of evolution as the most powerful scientific explanation of natural diversity. This book gained unexpected popularity. Reference of one of his major works:
Darwin, Charles. On the Origin of Species: By means of natural selection. London, John Murray, 1859. Photo retrieved from (St. John's College University of Cambridge).
2nd Edition of The Origin of Species
2nd edition of the book is published, followed by 3rd edition in the following year. Additional editions are to be published every 3-4 years. He also publishes many other scholarly books, almost every year (Browne & Wyhe).
Darwinism on the rise
Darwinism focus on the idea that all organisms evolve with natural selection. The evolution originates from mutations, and some of the mutations are inherited to their offspring, bringing advantages to survival, reproduction, and adaptation to the environment (The Biography.com) & (Galapagos Conservation Trust) . Natural selection occurs when individual organisms compete for limited resources and repeat efforts to continue to exist. In other words, only the strong survive.
Video Files, Darwinism
"Darwinism- Darwins Theory of Natural Selection." Youtube, uploaded by Zoologist Ammar, https://www.youtube.com/watch?v=DyAe-jG8jtk "Charles Darwin - The Theory Of Natural Selection." YouTube, uploaded by Chris Mochinski, 11 May 2015, https://www.youtube.com/watch?v=vnktXHBvE8s
Social Darwinism
The idea of Darwinism can be also applied to society, called 'Social Darwinism', which was developed by a sociologist Herbert Spencer. It is often used to explain social and economic issues and emphasizes the importance of fitness and compatibility within society, and often rationalize discrimination, imperialism, disparities, and inequality as well as artificial selection (The Biography.com).
He proposed that natural selection is a process behind evolution and established evolutionary biology based on his observations, and experiments and presented evidence. His idea and concepts were also supported and further developed by fellow scientists and philosophers such as Alfred Russel Wallace and Herbert Spencer (Browne & Wyhe) & (The Biography.com), which then spread and was widely accepted to the world, triggering a paradigm shift.
The Passing of Darwin
Darwin passes away at 73 due to heart disease. Old Darwin was suffering from chronic fatigue and weakness, however, he never stopped his research and publications until his later years (Galapagos Conservation Trust) & (Browne & Wyhe). His experiments and observations on evolution covered a broad spectrum of plants and marine/terrestrial animals, achieving significant and noteworthy results in scientific history.
Eje cronológico 1833-1874
7B/Rhys ~ Independent Reading #1 "Me Before You" by Jojo Moyes
Test 1 and 2 - 3000 BCE to 200 BCE - Elizabeth Rosa (Western Civilization)
EVOLUCIÓN DE LA PSICOLOGÍA
Historia de la Microbiología
IDEAS CENTRALES LINEA DEL TIEMPO
Vanvittig Verdenshistorie
Antecedentes del Derecho Colectivo de Trabajo
SIGPAS
El Garcianismo
L'Histoire des réseaux sociaux
Jamming in space
Cronología Siglo XX.
línea de tiempo con la evolución de la administración de recursos humanos
1776-1861 Timeline (American Revolution-Civil War)
Roman Expansion
Telephone Timeline
Greek Theatre History
Technology in the 1960's
LINEA DEL TIEMPO ARQUITECTURA
Inventos Segunda Revolución Industrial
20 hechos mas importantes de la historia de la humanidad
LA EVOLUCION DEL TRABAJO
Exposure to Traditional and New Media
Evolución del Turismo en México y su desarrollo
Enlightenment Age
Makeup History
La Evolución de la Automatización
Linea de tiempo de la evolución de los sistemas operativos
The Evolution of how we listen to Music
Tractors Through History


text:
Q: Is there a way to write a pytest test case to test the current api url? I am developing a webapp with Django, PostGres, Docker and a GraphQL API.
I am attempting to write a pytest test that will look at the current server address (localhost or not), send a request to the GraphQL url at that address, and assert the result. The test relies on a notification fixture, which simply creates a record in the Django model. My GraphQL query is working (the query receives results from the API using a browser), but the same query used in the test results in the following error:
"FAILED api/tests/test_api.py::test_get_all_notifications - requests.exceptions.ConnectionError: HTTPConnectionPool(host='localhost', port=2448): Max retries exceeded with url: /graphql ..."
Here is what I have so far:
import requests, json

def get_current_server_url():
    # TODO: <---- THIS FAILS, EVEN THOUGH THIS IS THE WORKING API URL
    return "http://localhost:2448/graphql"

def test_get_all_notifications(db, notification):
    query = """{
      allNotifications {
        id
      }
    }"""
    url = get_current_server_url()
    request = requests.post(url, json={'query': query })
    data = json.loads(request.text)['data']
    assert len(data['allNotifications']) == 1

Please let me know if I can clarify my question in any way.
This StackOverflow question is helpful, but only if I was attempting to get the URL from inside the view, not from inside pytest.

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text:
The Problems of the Preservation of the Historical and Cultural Heritage. Materials of the International Conference. 25th - 27th of September, 2013
Yerevan: "Service for the Protection of Historical Environment and Cultural Museum" NCSO, 2013.
Under the general editorship: A. Piliposyan.
Задачи и практика составления ежегодного государственного доклада о состоянии объектов культурного наследия (памятников истории и культуры) в Российской Федерации /The tasks and practice of drawing up the annual state report about the state of the cultural heritage in the Russian Federation (monuments of history and culture)
Веденин Ю. А., Штеле О. Е., Шульгин П. М. В кн.: The Problems of the Preservation of the Historical and Cultural Heritage. Materials of the International Conference. 25th - 27th of September, 2013. Ер.: "Service for the Protection of Historical Environment and Cultural Museum" NCSO, 2013. С. 35-36.
Research target: Philosophy, Ethics, and Religious StudiesArt History
Priority areas: humanitarian
Keywords: cultural heritage Historical EnvironmentPreservation
Regional Development Models Using Cultural Heritage Resources
Abankina T. International Journal of Culture,Tourism and Hospitality Research. 2013. Vol. 7. No. 1. P. 3-10.
Purpose – The purpose of this paper is to introduce findings of comparative analysis and various models based on cultural heritage resources to foster regional development.
Design/methodology/approach – Comparison of operational schemes, market positions and branding of three successful cultural heritage centers in Germany, Great Britain and Russia demonstrates a variety of regional development models based on cultural resources and tourism development, and reveals their advantages and disadvantages.
Findings – The paper evidences the potential of cultural resources and the tourism sector as drivers for regional development, and helps formulate basic recommendations for the Russian situation requiring elaboration of adequate financial and social instruments.
Originality/value – The paper provides a complex analysis of different operational models in three European countries with regard to specific national situations and specificity of heritage operational management.
Historical and Cultural Heritage //Историко-культурное наследие
Edited by: A. Piliposyan Вып. 1. Ер.: Service for the Protection of Historical Environment and Cultural Museum-Reservation. NSCO, 2014.
I Russi ad Amalfi. Suggestioni mediterranee e storie di vita
Kara-Murza A. A., Zhukova O. A., Talalay M. G. Iss. 9. Amalfi: Amalfi: presso la sede del Centro, 2015.
O. A. Zhukova, A. A. Kara-Murza and M. G. Talalay are well known as researchers in the Russian and Italian culture. New work, written in collaboration, is dedicated to Russian writers, philosophers, artists, travelers, who had visited the old Amalfi in southern Italy. The relics of St. Andrew the Apostle are a spiritual treasure of the city for over 800 years.The basis of historical and philosophical program of the book is studies of the intellectual biographies of prominent figures of Russian literature, philosophy and art of the nineteenth and twentieth century. They had inspired by the beauty of Amalfi, embodying it in his works.
The Space of the City and Literary Imagination: the Legacy of Nikolai Antsiferov
Stepanov B. ENTHYMEMA. 2014. No. 11. P. 7-24.
This paper discusses the legacy of Nikolai Antsiferov (1889–1958), a Russian historian who suggested a unique approach to urban studies in which literature played the key role. In the first section of this paper, the genesis of Antsiferov's conceptions of the study of urban history and the image of the city are outlined. The second section provides an analysis of his ideas on the literary image of St. Petersburg and the theory of literary-themed guided tours, which were articulated in his works of the 1920s. The finalsection of the article sheds light on the reception and legacy of Antsiferov's intellectual ideas in the modern humanities and assesses its significance in the modern context of interdisciplinarity.
Combination of the tangible and intangible in cultural events organization
Dedova M. In bk.: Geomorphic Processes and Geoarchaeology: from Landscape Archaeology to Archaeotourism. International conference held in Moscow-Smolensk, Russia, August 20-24, 2012. Extended abstracts. M.; Smolensk: 2012. P. 67-69.
In present extended abstract cultural heritage, both tangible and intangible, is considered as a facilitator of cultural and event tourism development in rural territories.
Creative tourism in Saint Petersburg: the state of the art
Gordin V. E., Matetskaya M. V. Journal of Tourism Consumption and Practice. 2012. Vol. 4. No. 2. P. 55-77.
Tourism development in St. Petersburg, which is a major cultural centre, has improved in terms of the tourist flow; both tourism types and tourist products have become more diverse. These improvements give ground for a fairly optimistic prognosis for the tourist industry in St. Petersburg. At the same time, there are a number of factors which tend to endanger sustainable development of tourism in St. Petersburg. The current situation calls for a more flexible and innovative approach to industry development. Among these factors we can single out the pronounced seasonal character of tourism, short-term visits of most of the tourists, the rather conservative, academic image of the St. Petersburg culture, which compromises the city's appeal as a tourist destination for certain tourist segments. Apart from that, the critical limitation imposed on the development of cultural tourism in general and of creative tourism in particular is the low involvement of the population in cultural and tourist events held in the city. All in all this makes it relevant to look for new approaches for creative tourism development in St. Petersburg as an important tool for the sustainable development of the industry.
The article aims at considering the existing and potential competitive advantages of St. Petersburg as a tourist destination on the basis of creative tourism development.
In the footsteps of Rurik
Selin A. A., Carlsson D. Helsinki; Copenhagen: The Northern Dimension Partnership of Culture (NCDP), 2012.
The main objectives of the preprint was to map and give an account of the Viking Route heritage sites located in Russia, to reveal the most important of them and to analyse their status today when it comes to maintenance, marketing and open up for tourism. This book is edited within the Framework Contract: Study on the Viking Route Heritage Sites in Russia financed by the European Commission and implemented by HTSPE and EuroTrends. The idea behind this book is to highlight to the public the deep interaction that were at hand between the Scandinavian countries and the states on the eastern side of the Baltic Sea in the Viking Age, and point out our common history.
Cultural festivals in St. Petersburg as an example of introduction of the creative industries
Ivanova J. V. In bk.: Izaicinajumu un iespeju laiks: problemas, risinajumi, perspektivas. II Starprautiska jauno petnieku un studentu zinatniski praktiska konference // Time of challenges and opportunities: problems, solutions and perspectives. II International scientific-practical conference of young scientists and students // Время вызовов и возможностей: проблемы, решения, перспективы. II Международная научно-практическая конференция молодых ученых и студентов. Rezekne: SIA "Petrovskis un KO", 2012. P. 178-182.
The aim of this article is to study an influence of various cultural festivals in St. Petersburg on development of the creative industries in the city. The definition of prospects of the development of culture of «Russia's Northern Capital» demands the analysis of an existing scientific and administrative discourse concerning interaction in a city on Neva the rich cultural heritage and new creative industries. The situation of St. Petersburg as а large European cultural center and one of the important cities of the Russian Federation allows to define prospects of its development as «creative city». It includes also the analysis of cultural, social and economic consequences of the development of festival movement.
Культурное наследие и продвижение брендов территорий
Копацкая С. А. В кн.: Туризм и гостиничный бизнес. Стратегии взаимодействия образовательных учреждений и работодателей. Материалы II Международного форума 23-26 ноября 2011 г.. СПб.: СПбГУСЭ, 2011. С. 113-115.
Popular Culture And History: Representations Of The Past In British Popular Music Of The 2000s
Kolesnik A. Working Papers of Humanities. WP. Издательский дом НИУ ВШЭ, 2014. No. 54.
This paper investigates the basic mechanisms of representation of the past in British popular music in the early 2000s. Changes in the music industry associated with the emerging and wide dissemination of new media has affected the search for new musical decisions, reformatting attitudes to the past in general, and to the musical past in particular. In this regard, questions of historical representations in popular music and their relationship with cultural heritage are closely interrelated. This paper analyses the mechanisms and formats of 'working' with the past in British popular music of the 2000s using examples from the London rock band, the Libertines. The author draws conclusions about structure of historical representations in popular music and their typology. The use of popular music studies approach is suggested to analyze mechanisms of representation of the past and broaden the concept of popular music.
Clothing and fashion play significant role in the process of ageing, and help put bodily experiences in the context of culture. It is especially important for young people as social subjects, who are starting to become independent but do not have sufficient experience and social competences to do this. This paper explores how youth of middle and working class in Russia use clothing to denote their social identity and construct boundaries.The paper is based on materials of 81 IDI with young people from St. Petersburg.
Poisson groups and differential Galois theory of Schrodinger equation on the circle
Semenov-Tian-Shansky M., Marshall I. Communications in Mathematical Physics. 2008. No. 284. P. 537-552.
Л.Н. Толстой: Просветитель, преодолевший Просвещение
Климова С. М. Философские науки. 2019. Т. 62. № 2. С. 109-126.
The article considers the Views of L. N. Tolstoy not only as a representative, but also as a accomplisher of the Enlightenment. A comparison of his philosophy with the ideas of Spinoza and Diderot made it possible to clarify some aspects of the transition to the unique Tolstoy's religious and philosophical doctrine. The comparison of General and specific features of the three philosophers was subjected to a special analysis. Special attention is paid to the way of thinking, the relation to science and the specifics of the worldview by Tolstoy and Diderot. An important aspect is researched the contradiction between the way of thinking and the way of life of the three philosophers.
Tolstoy's transition from rational perception of life to its religious and existential bases is shown. Tolstoy gradually moves away from the idea of a natural man to the idea of a man, who living the commandments of Christ. Starting from the educational worldview, Tolstoy ended by creation of religious and philosophical doctrine, which were relevant for the 20th century.
Гуманитарные чтения РГГУ-2010. Теория и методология гуманитарного знания. Россиеведение. Общественные функции гуманитарных наук. Сборник материалов
Edited by: Е. И. Пивовар М.: РГГУ, 2011.
The Irony of Heidegger
Haas A. L.; NY: Continuum, 2007.
This important new book offers the first full-length interpretation of the thought of Martin Heidegger with respect to irony. In a radical reading of Heidegger's major works (from Being and Time through the 'Rector's Address' and the 'Letter on Humanism' to 'The Origin of the Work of Art' and the Spiegel interview), Andrew Haas does not claim that Heidegger is simply being ironic. Rather he argues that Heidegger's writings make such an interpretation possible - perhaps even necessary.
Heidegger begins Being and Time with a quote from Plato, a thinker famous for his insistence upon Socratic irony. The Irony of Heidegger takes seriously the apparently curious decision to introduce the threat of irony even as philosophy begins in earnest to raise the question of the meaning of being. Through a detailed and thorough reading of Heidegger's major texts and the fundamental questions they raise, Haas reveals that one of the most important philosophers of the 20th century can be read with as much irony as earnestness. The Irony of Heidegger attempts to show that the essence of this irony lies in uncertainty, and that the entire project of onto-heno-chrono-phenomenology, therefore needs to be called into question.
История философии для экономистов
Сорвин К. В. учебное пособие. учебное пособие. ГУ ВШЭ, 2000
Entries: "biconditional", "contingent", "sentence"
Schang F. Key Terms in Logic. ISBN-10: 1847061141. Bloomsberry Academic, 2010
A definition of three logical concepts: "biconditional", "contingent", and "sentence".
Свобода техники? К пониманию техники у Эрнста и Фридриха Георга Юнгеров
Михайловский А. В. Философские науки. 2013. № 7. С. 79-95.
The article is concerned with the notions of technology in essays of Ernst and Friedrich Georg Jünger. The special problem of the connection between technology and freedom is discussed in the broader context of the criticism of culture and technocracy discussion in the German intellectual history of the first half of the 20th century.
Материалы XLIII Международной научной студенческой конференции «Студент и научно-технический прогресс»
Т. Востоковедение. Новосибирск: Новосибирский государственный университет, 2005.


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The J. Paul Getty Museum's estimated 1.3 million visitors annually make it one of the most visited museums in the US.
On the Architectural and Garden tour, we will explore Richard Meier's Getty Center's unique design elements, beautiful gardens, and open spaces.
The collection of the J. Paul Getty Museum on display at The Center Center includes pre-20th-century European paintings, drawings, illuminated manuscripts, sculpture and decorative arts; and 19th and 20th-century American and European photographs.


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The student registration process is a stressful time for families and school administrators. Watch this webinar recording as we explore the simplicity of streamlining and automating the enrollment management process.


text:
Lärarhögskolan i Stockholm (LHS) var en svensk lärarhögskola, senare högskola med huvudinriktningen att utbilda lärare.

Historik
Högskolan startade 1956 och låg i Marieberg i Stockholm främst i området Campus Konradsberg. I samband med högskolereformen 1977 avskaffades officiellt begreppet lärarhögskola och Lärarhögskolan i Stockholm sammanslogs då med Gymnastik- och idrottshögskolan och bildade Högskolan för lärarutbildning i Stockholm (HLS). Idrottshögskolan i Stockholm utbröts 1993 åter som egen högskola och då återtogs det gamla namnet Lärarhögskolan i Stockholm.

Huvudbyggnaden för Lärarhögskolan, Konradsberg, användes för psykiatrisk vård fram till 1995 och togs därefter över av Lärarhögskolan i Stockholm. Från och med 1 januari 2008 infogades verksamheten vid LHS i Stockholms universitet. Även före denna sammanslagning läste dock lärarstudenterna många av sina ämneskurser vid Stockholms universitet.

Inom Lärarhögskolan fanns flera centrumbildningar med egen styrelse, bland dem Centrum för barn- och ungdomsvetenskap som verkade för att sprida aktuell forskning om barn och ungdomar och som utgav tidskriften Locus.

Rektorer

Rektorer för Lärarhögskolan i Stockholm
 1991–1997 Bengt Börjeson, professor i socialt arbete
 1997–2006 Eskil Franck, docent i teologi
 2006–2008 Ingrid Carlgren, professor emerita i pedagogik

Referenser

Noter

Tryckta källor
Blix, Erik & Arfwedson, Gerhard, Lärarhögskolan i Stockholm 1956-1996. HLS Förlag, Stockholm. 1996.  Libris

Ej längre existerande universitet och högskolor i Sverige
Högre utbildning i Stockholm
Marieberg
Utbildningsinstitutioner bildade 1956
Utbildningsinstitutioner upplösta 2008


text:
GRAPHICA
Hot new reads, with TEEN appeal
Street Angel : Superhero for a Day
by Jim Rugg
What it's about: Jesse 'Street Angel' Sanchez is the deadliest girl alive - a teenage homeless orphan skateboarder, fighting ninjas and trying to pass 7th grade! Jesse knows the pressures of being the girl who always saves the day and she's okay with it. It's just who she is. But when she and her friends stumble across a powerful alien artifact - they soon realize that being a superhero isn't all fun and games.
What's that buzz? "Street Angel stars my favorite comic-book hero of the 21st century, and features some of the coolest artwork I've ever seen." --Brian K. Vaughan
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Heavy Vinyl
by Carly Usdin
What it's about: Starry-eyed Chris has just started the dream job every outcast kid in town wants; working at Vinyl Mayhem. It's as rad as she imagined; her boss is BOSS, her co-workers spend their time arguing over music, pushing against the patriarchy, and endlessly trying to form a band. As events unfold, she is surprised to find out that her co-workers share a secret: they're all members of a secret fight club that take on the patriarchy and fight crime!
Fun fact: Writer Carly Usdin is a film and TV director, known for Suicide Kale and RuPaul's Drag Race.
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What it's about: Now that high school is over, Ari is dying to move to the big city with his ultra-hip band―if he can just persuade his dad to let him quit his job at their struggling family bakery. But while interviewing candidates for his replacement, Ari meets Hector, an easygoing guy who loves baking as much as Ari wants to escape it.
What's that buzz? Bloom is a charming and compelling look at the first summer after high school, with the angst of choice and decision looming and the blush of first love. (Plus: there's baked goods!)"—Melissa Fox, Watermark Books
"This is the best kind of romantic tale: innocent and unfussy, yet overwhelming. Author Panetta's story is baked to perfection: a crush that turns to infatuation—and later love—in the confines of a professional kitchen bristling with hot stoves, stuffed shelves, and perfectly shaped spanakopita." —Los Angeles Review of Books
Fearless Females : The Fight for Freedom, Equality, and Sisterhood
by Marta Breen
What it is: A beautifully illustrated graphic novel that tells the stories of fearless females who have fought, and continue to fight, for the rights of women. This engaging book contains a comprehensive introduction into the history of women's circumstances over the past 150 years.
Starring: Harriet Tubman, Malala Yousafzai,Tahirih, Elizabeth Cady Stanton, Lucretia Mott, Sojourner Truth, Olympe De Gouges, Mary Wollstonecraft, Clara Zetkin, Rosa Luxemburg, Margaret Sanger, and Sappho.
You might also like: Watch Us Rise by Renee Watson and Ellen Hagan, and The Three Escapes of Hannah Arendt: a tyranny of truth by Ken Krimstein.
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MudRiver / BugBite
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What it's about: In "Mud River," Eddy makes a foolish mistake, causing a sweet, innocent Amazon to bonk her head, turning her into a very impressionable automaton. Of course, Eddy can't resist taking advantage of this unexpected development, even as a river of mud approaches. In "Bug Bite," Eddy has brought his family on a vacation to Europe, but he's soon distracted by a series of manifestations of his own obsessions.
Fun fact: Mudbite marks the first new graphic novel by fan favorite Dave Cooper in more than 15 years, marking a welcome return to the medium that he made his name.
Godhead. Volume one
by Ho Che Anderson
What it's about: When a powerful multinational corporation creates a machine that can communicate with God, does it spell humanity's salvation or the end of days? This sprawling contemporary saga, ranging from intimate domestic drama to globalist corporate intrigue, explores the collision courses among science, religion, and corporate greed.
What's that buzz? "Ho Che Anderson's Godhead Volume One is a work of borderline-brilliant dystopian sci-fi, a breathtakingly cinematic action spectacle, a compelling tale of sleazy boardroom intrigue, and a fascinating character study." — Your Chicken Enemy
"Godhead is a serious meditation on technology, God and inequality, spiced with high action." — The Globe and Mail
Super Sikh 1
by Eileen Kaur Alden
What it's about: In "Super Sikh", secret agent Deep Singh is overworked and exhausted from destroying the Taliban at night and maintaining a cover job by day. He's a big Elvis fan, and he decides to take his dream vacation to visit Graceland (Elvis's home). Unfortunately, a crazy Taliban group decides to follow him to America and get rid of him once and for all. But Deep Singh and his team are not going down without a fight!
Also starring: Janelle Williams, an African-American woman with her own "ass-kicking" skills, and Gurpreet Kaur, Singh's cousin, a "STEMinist" (women in the fields of science, technology, engineering and math) who supplies him with gadgets.
Millennium : The Girl Who Danced With Death
by Sylvain Runberg
What it's about: In this sequel to the novel trilogy by Stieg Larsson, Lisbeth Salander prepares to expose the Swedish secret service's files, and must seek out the help of Mikael Blomkvist when her friend Trinity is kidnapped.
For fans of: Stieg Larsson; the story continues!
Rated: Mature
Whitchurch-Stouffville Public Library
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wsplibrary.ca


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Quality built and well designed 1-Bedroom Plus Den in prestigious area close to multi-million dollar estates. High end concrete lowrise built by reputable Intracorp. The spacious floor plan featuring open kitchen with granite island opens onto dining & living rooms. All appliances included plus 1 underground parking stall & 1 storage locker. Close to all the amenities & U-Hill Elementary/Secondary Schools.
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Incredible value for this beautiful 2 Bedroom apartment in quiet Hampton Place. Suite looks on courtyard and fountain. This unit is newly painted and completely clean and move-in condition. This complex has lounge, library, sauna, exercise room and guest suites. Close to University Hill Secondary and Norma Rose Elementary School. It is walking distance to Pacific Spirit Park, Save-On-Foods and UBC facilities. Bright east facing 2 bedrooms at "The Bristol". Steps to UBC and Pacific Spirit Park. Bedrooms separate across living room for privacy. Open house Sunday July 9, 1-3 pm.
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I have sold a property at 105 6063 IONA DR in Vancouver.
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I have sold a property at 102 5632 KINGS RD in Vancouver.
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LS Power Elk Run Energy Station was a proposed coal plant to be built in Waterloo, Iowa that was cancelled in January, 2009.
LS Power proposed a 750-megawatt pulverized-coal power plant five miles outside of downtown Waterloo, Iowa. Iowa census numbers indicate some of the state's highest poverty rates are in Waterloo; East Waterloo, the neighborhood nearest the plant, has a large African-American population and high asthma rates. The Waterloo development economic agency heavily courted LS Power for the project. In June 2007, LS filed for a draft air quality permit with the Iowa Department of Natural Resources. Several groups organized in opposition to the proposal; in May 2007, several hundred local residents turned out to oppose the annexation of land for the plant.
On October 11, 2007, the Iowa City Development Board voted 3-2 to reject an application by the City of Waterloo to annex 345 acres of farmland, belonging to nonconsenting owners, for the construction of the plant. Given the ICDB's rejection, the application must now be approved by both the Iowa Utilities Board and the Iowa Department of Natural Resources in order to go through. Community Energy Solutions is pressuring the Black Hawk County Board of Supervisors to reject any attempts by LS Power to move the plant site outside city limits.
A Nov. 2007 independent report -- authored by William Stigliani, director of the University of Northern Iowa's Center for Energy and Environmental Education -- found that the plant could jeopardize the health of area residents, countered LS's arguments that new coal technology is safe, and noted that African-American residents may suffer disproportionately from the plant.
In Dec. 2007, the mayor of Waterloo announced that the city will appeal the ICDB's decision. In January 2008, the Black Hawk County Health Board called for a state moratorium on permits for coal power plants and stricter standards for state air pollution.
In March 2008, the Iowa Department of Natural Resources denied a construction permit for the plant because the developers had not secured zoning approval for the project. Elk Run Energy planned to appeal the decision.
In June 2008, Iowa's Department of Natural Resources received LS Power's revised air permit application.
In May 2008, Waterloo's City Council approved an ordinance to annex and rezone 260 acres of land for the Elk Run plant. The Iowa Department of Natural Resources still must approve the plan and will likely include public hearings in the final decision process.
In December 2008, Dynegy CEO Bruce Williamson announced that the company was reevaluating its role in developing new power plants, including Elk Run. Williamson cited the tightening credit markets and construction of several new coal plants as reasons for reconsidering its involvement in the siting, permitting, financing and constructing at least six new projects. Other plants include the Longleaf plant in Georgia, Midland Power Plant in Michigan, Plum Point Energy Station in Arkansas, Sandy Creek Plant in Texas, West Deptford Project in New Jersey, and White Pine Energy Station in Nevada. As an alternative, the company will look at adding generation to its existing sites in the Northeast, Midwest and Western U.S.
On January 2, 2009, Dynegy agreed to dissolve its development venture with LS Power, in part because of the credit crisis. On January 6, LS Power announced that because of the economic downturn, it was cancelling plans to build the Waterloo plant.
↑ "Power Plant Foes Ask County to Oppose Project", WCF Courier, October 17, 2007.
↑ "Report Calls for Moratorium on New Coal Plants", Electricity Forum News, November 2007.
↑ "Iowa Agency Rejects Proposed Coal Plant", Associated Press, March 6, 2008.
↑ Jim Polson, "Dynegy to Dissolve Development Venture With LS Power," Bloomberg, January 6, 2009.
↑ Perry Beeman, "Proposal for Waterloo coal plant is scrapped," Des Moines Register, January 7, 2009.
This page was last edited on 13 March 2015, at 21:18.


Correcting this error will require you to access the phone's web interface (GUI) from a web browser on a computer on the same network as the phone.
On the phone, press Options > Phone Status > IP & MAC Addresses.
Using a computer on the same network as the phone, open a web browser and enter the phone's IP address.
Log in with the username admin and the phone's administrator password (typically 22222 or 54321).
To access the check boxes for the problematic options, check the box for Validate Certificates.
The check boxes for the problematic options should now have a white background. Proceed to un-check the boxes for both Check Certificate Expirations and Check Certificate Hostnames.
To validate whether this resolution worked for you, observe the phone the following morning, after configuration and firmware sync have occurred. The Error 1 message should no longer appear.
The phone's certificate settings are not properly configured, causing the error message to appear at night when the phone syncs with the configuration servers.


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