Abstract. Glomerular accumulation of extracellular matrix (ECM) is the common pathologic feature following glomerular injury, and the alteration in the synthesis and degradation of ECM may be involved in the glomerular accumulation of ECM. Glomerular fibrin formation occurs in various forms of human and experimental glomerulonephritis, and it may play an important role in progressive glomerular injury. Thrombin, a multifunctional serine proteinase that is generated at the site of vascular injury, has central functions in hemostasis and it also shows various biologic effects. In this study, it is hypothesized that thrombin may alter the production and the degradation of type IV collagen, which is an important component of ECM in the glomeruli. Human mesangial cells (HMC) were cultured, and the levels of type IV collagen, tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-2 (MMP-2) in the culture supernatants were measured by enzyme immunoassay using specific antibodies. MMP-2 activity was also evaluated by zymography using polyacrylamide/sodium dodecyl sulfate gel-containing gelatin. Thrombin increased the production of type IV collagen and TIMP-1 in a dose- and time-dependent manner, but it did not increase MMP-2. Thrombin also stimulated the gene expressions of the type IV collagen and TIMP-1 in HMC in a dose- and time-dependent manner. Thrombin treated with diisopropylfluorophosphate, a serine proteinase inhibitor, did not show any of these effects. Hirudin, a natural thrombin inhibitor, and anti-transforming growth factor-β-neutralizing antibody inhibited the stimulating effect of thrombin. These findings suggest that thrombin may contribute to the excessive accumulation of ECM and progression of glomerulosclerosis through an increase of type IV collagen production and a decreased matrix degradation presumably via a transforming growth factor-β-dependent mechanism.
Glomerular accumulation of extracellular matrix (ECM) is the common pathologic feature of glomerular injury, and the alteration in the synthesis and degradation of ECM may be involved in the glomerular accumulation of ECM. Glomerular ECM has been shown to consist of type IV collagen, laminin, heparan sulfate proteoglycan, fibronectin, and other proteins. Glomerular deposition of type IV collagen has been reported in human glomerular diseases (1,2,) and experimental models (3,4). Matrix metalloproteinase-2 (MMP-2) is an important proteolytic enzyme demonstrated to degrade type IV collagen in in vivo as well as in vitro studies, and it is shown to be synthesized by mesangial cells in vivo as well as in vitro (5,6,7). Mesangial cells also synthesize tissue inhibitor of metalloproteinase-1 (TIMP-1), which can inhibit MMP-2 (8,9). Recent investigations have shown that an imbalance between matrix-degrading metalloproteinases and the inhibitors of these enzymes may contribute to the development of glomerulosclerosis (7,10).
Intraglomerular fibrin formation is commonly observed in various types of glomerulonephritis, and the coagulation process has been suggested to contribute to the progression of glomerulosclerosis (11,12,13). Thrombin, a multifunctional serine protease that is generated in the coagulation process and at the site of vascular injury, has central functions in hemostasis. It is well known that thrombin has various biologic effects besides its role in hemostasis. For example, it has been reported that thrombin stimulates the mitogenesis of cultured fibroblasts (14,15), lymphocytes (16), endothelial cells (17), vascular smooth muscle cells (18,19), mesangial cells (20,21), and glomerular epithelial cells (22). It is reported that thrombin can regulate the production of several components of the fibrinolytic system in various cultured cells (22,23,24,25). Thrombin may therefore have an important role in the deterioration of renal function and progression of glomerulosclerosis. In the present study, we examined whether thrombin could influence the production of type IV collagen, TIMP-1, and MMP-2 by cultured human mesangial cells (HMC).
HMC derived from human fetal kidneys (16 and 18 wk gestation) were kindly provided by Dr. M. R. Daha (University Hospital of Leiden, The Netherlands). The cells were cultured in Dulbecco's modified Eagle's minimum essential medium (Life Technologies Laboratories, Grand Island, NY) with 20% heat-inactivated fetal calf serum (Bioserum, Canterbury, Australia) at 37°C in 5% CO2. The cells were identified as glomerular mesangial cells by standard criteria (26), i.e., they exhibited a typical stellate morphology when subconfluent and typical hillocks were seen at confluence. Staining for actin, myosin, and type IV collagen was positive by immunofluorescence, and the cells were negative for cytokeratin and factor VIII. The cells were used between passages 6 and 9 for the experiments.
The concentrations of type IV collagen and TIMP-1 in culture supernatants were quantified by enzyme immunoassay (EIA) using the Panaassay IV-C and human TIMP-1 assay kits (Daiichi Kagaku Yakuhin, Tokyo, Japan). MMP-2 was measured by a one-step sandwich EIA, as described previously (27). This assay useed a pair of monoclonal antibodies raised against the precursor form of MMP-2 (proMMP-2) purified from the conditioned medium of interleukin-1α-treated human skin fibroblasts (CCD-41SK; American Type Culture Collection [ATCC] no. CRL1505) (28) and against a synthetic peptide corresponding to the N-terminal domain (residues 17 to 35) of proMMP-2. The sensitivity of this EIA was 0.24 ng/ml, and linearity was obtained between 1.0 and 500 ng/ml.
HMC were cultured in 12-well plates (Falcon; Becton Dickinson, Franklin Lakes, NJ) and grown to confluence, which means HMC covered the entire plate. Subsequently, the cells were washed with Hanks' balanced salt solution (Life Technologies) and cultured with Dulbecco's modified Eagle's minimum essential medium containing 0.2% bovine serum albumin (Sigma, St. Louis, MO) for 24, 48, or 72 h. Then type IV collagen, TIMP-1, and MMP-2 in the cell supernatants were measured. The cells in each well were lysed in 1N NaOH, and the protein content was measured by the method of Lowry et al. (29), using bovine serum albumin as a standard. The levels of type IV collagen, TIMP-1, and MMP-2 in culture supernatants were expressed as ng per μg of lysed HMC protein.
We measured type IV collagen, TIMP-1, and MMP-2 in the supernatants of HMC incubated with 0.5 to 5.0 U/ml α-thrombin (Sigma) for 72 h to determine its dose-dependent effect. The dosage of thrombin was adopted according to a previous report (23). Time effect of thrombin was also examined by incubating HMC with or without 5.0 U/ml thrombin for 24, 48, and 72 h. We also examined the effect of diisopropylfluorophosphate (DFP; Wako Pure Chemical Industries, Osaka, Japan)-treated thrombin (DFP-thrombin) on the production of type IV collagen and TIMP-1. DFP is a serine proteinase inhibitor, and DFP-thrombin has no enzymatic activity. In addition, we examined the inhibitory effect of recombinant hirudin (American Diagnostica, Greenwich, CT), a specific natural thrombin inhibitor. DFP-treated thrombin was prepared by reacting thrombin (200 U/ml) with 10 mM DFP at pH 7.2 for 1 h at 37°C, and then was dialyzed overnight against 0.01 M phosphate-buffered saline at pH 7.4. HMC were incubated for 72 h with 5.0 U/ml thrombin, 5.0 U/ml DFP-thrombin, 5.0 U/ml thrombin plus 10.0 U/ml hirudin, or 10.0 U/ml hirudin, and the levels of type IV collagen and TIMP-1 in the cell supernatants were measured. Finally, we examined the effect of anti-transforming growth factor-β (TGF-β)-neutralizing antibody (King Brewing Co., Kakogawa, Japan), which is a polyclonal rabbit antibody, on the production of type IV collagen and TIMP-1. HMC were incubated for 72 h with 5.0 U/ml thrombin, 5.0 U/ml thrombin plus 50 μg/ml anti-TGF-β antibody, or 5.0 U/ml thrombin plus 50 μg/ml normal rabbit IgG, and the levels of type IV collagen and TIMP-1 in the cell supernatants were measured. The cytotoxicity by thrombin, DFP-thrombin, or hirudin was evaluated by assessing lactate dehydrogenase (LDH) release from HMC. HMC were incubated in 24-well plates for 72 h with 5.0 U/ml thrombin, 5.0 U/ml DFP-thrombin, or 10.0 U/ml hirudin, and the amount of LDH in the cell supernatants was measured. After removing the cell supernatants, HMC were lysed by mellitin (50 μg/ml, Sigma), and the amount of LDH in the cells was also measured. LDH was quantified by a colorimetric method using the LDH assay kit (Sanassay LDH, Sankou Junyaku, Tokyo, Japan). The levels of LDH in the supernatants for 3 wells were 113.3 ± 5.8 Wroblewski units/ml in 5.0 U/ml thrombin, 118.3 ± 10.4 U/ml in 5.0 U/ml DFP-thrombin, 105.3 ± 13.6 U/ml in 10.0 U/ml hirudinin, and 110.0 ± 10.0 U/ml in control. There was no significant difference among LDH levels in the cell supernatants of these groups. There was also no significant difference among LDH levels in the cells of these groups. So it was considered that no cytotoxicity was observed by these agents that we used in this experiment. It is well known that thrombin enhances the proliferation of cultured mesangial cells (20,21). We estimated the proliferation of HMC after confluence by a colorimetric assay using Cell Titer 96TM (Promega, Madison, WI) and by counting cell number. There was not a significant increase of HMC incubated for 72 h with 5 U/ml thrombin.
Sodium dodecyl sulfate (SDS) substrate gels (7.5%) were prepared by including gelatin (DIFCO Laboratories, Detroit, MI) in the standard Laemmli acrylamide polymerization mixture at a final concentration of 1 mg/ml. The samples were solubilized in nonreducing sample buffer and electrophoresed using 25 mA/gel. After electrophoresis, the gels were washed in cold 50 mM Tris/HCl, pH 8.0, containing 50 mM NaCl, 10 mM CaCl2, and 0.05% Brij 35 (Sigma). The gels were then incubated overnight in fresh Tris buffer at 37°C. Thereafter, the gels were stained with Coomassie Blue and zones of lysis were visualized. Prestained protein molecular weight standards (Bethesda Research Laboratories, Gaithersburg, MD) were used for designation of molecular weight. For analytical SDS-polyacrylamide gel electrophoresis, HMC were incubated with 0.5 to 5.0 U/ml α-thrombin for 72 h, and 10 μl of cell supernatants were electrophoresed to examine the molecular weight of their proteinases.
HMC were stimulated with α-thrombin (0.5 U/ml or 5.0 U/ml) for 3 or 6 h, and then harvested and stored in liquid nitrogen until use. Total RNA was extracted from the frozen cells by a single-step method using RNAzol B (Biotecx Laboratories, Houston, TX). Then RNA samples (15 μg/lane) were subjected to electrophoresis on 1% agarose gel containing 2.2% formaldehyde and transferred onto nylon membranes (Hybond-N, Amersham, Buckinghamshire, United Kingdom) by capillary blotting. Membranes were hybridized overnight at 65°C with cDNA probes in the hybridization buffer containing 5× saline-sodium phosphate-ethylenediaminetetra-acetic acid, 5× Denhardt's solution, and 0.5% SDS. The cDNA probes were labeled with (α-32P) dCTP (3000 Ci/mmol; Dupont, Boston, MA) by random primer extension (30). After hybridization, the membranes were washed and exposed to Reflection film (Dupont) at -80°C with an intensifying screen for 6 h to 3 d. The cDNA probes for human type IV collagen-α2, human TIMP-1, and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from American Type Culture Collection (Rockville, MD).
All data are expressed as mean ± SD. Results were compared using one-way factorial ANOVA followed by multiple comparison tests, and two-way repeated-measures ANOVA.
The digested zone of 72 kD of mesangial cell proteinase, which is thought to be MMP-2, was demonstrated by 7.5% SDS-polyacrylamide gel electrophoresis including gelatin, and thrombin did not increase the MMP-2 activity. Even when HMC were incubated with 5.0 U/ml thrombin, no gelatinase of other molecular weight was observed (Figure 1).
Zymography of human mesangial cell (HMC) supernatant in a 7.5% sodium dodecyl sulfate-polyacrylamide gel containing 1 mg/dl gelatin as a substrate. The gelatin digestion zones represent activated matrix metalloproteinase-2 (MMP-2). Thrombin did not stimulate MMP-2 enzyme activity after 72 h incubation when compared with untreated control supernatant. Representative data from one of two separate experiments are shown. Lane 1, molecular marker; lane 2, control; lane 3, 0.5 U/ml thrombin; lane 4, 5.0 U/ml thrombin.
Changes in the concentrations of type IV collagen, TIMP-1, and MMP-2 in the HMC culture supernatants after 72 h of incubation with α-thrombin are shown in Figure 2. The mean level of type IV collagen in four wells was 0.054 ± 0.007 ng/μg cell protein (thrombin 0 U/ml), 0.071 ± 0.006 ng/μg (thrombin 0.5 U/ml), 0.107 ± 0.022 ng/μg (thrombin 2.0 U/ml), and 0.478 ± 0.049 ng/μg (thrombin 5.0 U/ml) (Figure 2A). The mean level of TIMP-1 in four wells was 15.50 ± 4.59 ng/μg cell protein (thrombin 0 U/ml), 17.82 ± 4.57 ng/μg (thrombin 0.5 U/ml), and 27.82 ± 1.45 ng/μg (thrombin 5.0 U/ml) (Figure 2B). The mean level of NMP-2 in four wells was 0.61 ± 0.12 ng/μg cell protein (thrombin 0 U/ml), 0.62 ± 0.16 ng/μg (thrombin 0.5 U/ml), and 0.56 ± 0.03 ng/μg (thrombin 5.0 U/ml) (Figure 2C). Thrombin significantly increased the production of type IV collagen and TIMP-1, but it did not change the amount of NMP-2. Time effect of thrombin was also examined by incubating HMC with or without 5.0 U/ml thrombin for 24, 48, and 72 h. Thrombin significantly stimulated the production of type IV collagen (Figure 3A) and TIMP-1 (Figure 3B) in a time-dependent manner. We also examined the effects of DFP-thrombin, recombinant hirudin, and anti-TGF-β-neutralizing antibody on the production of type IV collagen and TIMP-1. DFP-thrombin (5.0 U/ml) did not stimulate type IV collagen production, and hirudin (10.0 U/ml) inhibited the stimulating effect of thrombin, while hirudin itself did not influence type IV collagen production (Figure 4A). Anti-TGF-β antibody blocked the stimulating effect of thrombin in type IV collagen production, while normal rabbit IgG did not. Anti-TGF-β antibody itself did not influence type IV collagen production (Figure 4B). DFP-thrombin (5.0 U/ml) did not stimulate TIMP-1 production. Recombinant hirudin (10.0 U/ml) inhibited the stimulating effect of thrombin, while hirudin itself did not influence TIMP-1 production (Figure 5A). Anti-TGF-β antibody blocked the stimulating effect of thrombin in TIMP-1 production by HMC, while normal rabbit IgG did not. Anti-TGF-β antibody itself did not influence TIMP-1 production (Figure 5B).
Thrombin stimulated the production of type IV collagen (A) and tissue inhibitor of metalloproteinase-1 (TIMP-1) (B) by HMC, but it did not stimulate MMP-2 production (C). Values are the mean ± SD for four wells, and representative data from one of two experiments are shown, respectively. *P < 0.02 versus 0 U/ml thrombin; **P < 0.001 versus 0, 0.5, and 2.0 U/ml thrombin; ***P < 0.02 versus 0 and 0.5 U/ml thrombin.
Time course of the production of type IV collagen (A) and TIMP-1 (B) by HMC in the absence ([UNK]) or presence (○) of 5 U/ml thrombin. Thrombin stimulated the production of type IV collagen (P < 0.0001) and TIMP-1 (P < 0.005) in a time-dependent manner. Results are expressed as ng per μg cell protein. Values are the mean ± SD for four wells.
Thrombin (T; 5 U/ml) stimulated type IV collagen production by mesangial cells. However, 5 U/ml diisopropylfluorophosphate (DFP)-thrombin (DFP-T) did not stimulate type IV collagen production. Hirudin (H; 10 U/ml) inhibited the stimulating effect of thrombin, but hirudin itself did not influence type IV collagen production. (B) Anti-transforming growth factor-β (TGF-β) antibody blocked the stimulating effect of thrombin in type IV collagen production by mesangial cells, while normal rabbit IgG (nl.Rb.IgG) did not. Anti-TGF-β antibody itself did not influence type IV collagen production. Values are the mean ± SD for three wells, and representative data from one of two separate experiments are shown, respectively. C, control. *P < 0.0001 versus other groups; **P < 0.0001 versus C and T + anti-TGF-β.
Thrombin (T; 5 U/ml) stimulated TIMP-1 production by mesangial cells. However, 5 U/ml DFP-thrombin (DFP-T) did not stimulate TIMP-1 production. Hirudin (H; 10 U/ml) inhibited the stimulating effect of thrombin, but hirudin itself did not influence TIMP-1 production. (B) Anti-TGF-β antibody blocked the stimulating effect of thrombin in TIMP-1 production by mesangial cells, while normal rabbit IgG (nl.Rb.IgG) did not. Anti-TGF-β antibody itself did not influence TIMP-1 production. Values are the mean ± SD for three wells, and representative data from one of two separate experiments are shown, respectively. C, control. *P < 0.0005 versus other groups; **P < 0.01 versus C and T + anti-TGF-β.
Northern blot analysis showed that mesangial cells expressed type IV collagen and TIMP-1 mRNA under basal conditions. Thrombin (0.5 to 5.0 U/ml) upregulated the gene expressions of type IV collagen and TIMP-1 in a dose-dependent manner compared with the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is used as internal control (Figure 6A), and incubation with 5.0 U/ml thrombin for 3 and 6 h upregulated the gene expressions of type IV collagen and TIMP-1 in a time-dependent manner (Figure 6B).
(A) Type IV collagen mRNA and TIMP-1 mRNA expressions of HMC incubated with media alone, media with 0.5 U/ml thrombin, and media with 5.0 U/ml thrombin for 6 h. Type IV collagen mRNA and TIMP-1 mRNA of mesangial cells incubated with thrombin were increased in a dose-dependent manner compared with GAPDH. (B) Type IV collagen mRNA and TIMP-1 mRNA expressions of HMC before incubation, 3 h, and 6 h after incubation with 5.0 U/ml thrombin. Type IV collagen mRNA and TIMP-1 mRNA of mesangial cells incubated with thrombin were increased in a time-dependent manner compared with GAPDH. Representative data from one of two separate experiments are shown, respectively.
The accumulation of ECM is a common feature of glomerulosclerosis in many glomerular diseases. Because type IV collagen is an important component of the glomerular basement membrane and the mesangial matrix, alterations of its synthesis and degradation might contribute to the development of glomerulosclerosis. Glomerular cells have the ability to produce several ECM components, as well as several metalloproteinases and TIMP (10,31). Metalloproteinases can degrade the glomerular ECM and glomerular basement membrane, while TIMP inhibit their activity. Thus, an imbalance in the production of these enzymes may induce the destruction or the impaired remodeling of glomerular architecture (7,8,9,32). Recent investigations have suggested a pathologic role of matrix-degrading metalloproteinases and their inhibitors in glomerular injury (7,32,33). The gene expressions of these enzymes in the glomeruli are often altered in pathologic situations (33).
The coagulation process, including platelet aggregation and localized intravascular coagulation, has been implicated as either a primary pathogenetic factor or a secondary mediator of glomerular injury in a variety of glomerular and vascular renal disease (11,12,13). Fibrin-like material has been demonstrated in the glomeruli, and fibrin degradation products have been found in the serum and urine of patients with glomerulonephritis. However, the mechanisms responsible for glomerular injury by the coagulation process have not been well established. Depression of blood flow caused by intraglomerular coagulation may injure glomerular cells and coagulation factors and/or platelets may cause glomerular injury by altering glomerular metabolism. Thrombin is the serine protease that converts fibrinogen to fibrin, and is well known to have various biologic activities. Thrombin is mitogenic for lymphocytes, endothelial cells, and other types of cells (16,17,18,19), and it can regulate the production of components of the fibrinolytic system by various cultured cells (22,23,24,25).
In the present study, we found that thrombin accelerated the production of type IV collagen and TIMP-1, but not MMP-2, by cultured HMC. It was also found that thrombin upregulated the gene expressions of type IV collagen and TIMP-1 in HMC. Such changes could lead to the excessive production and reduced degradation of type IV collagen, and cause the glomerular accumulation of type IV collagen. In contrast, a stimulating effect was not seen in DFP-treated thrombin. DFP is a serine proteinase inhibitor and it blocks the enzyme activity of thrombin. The specificity of the effect of thrombin was also tested by addition of hirudin, a natural specific thrombin inhibitor that blocks both the proteolytic and membrane receptor binding functions of thrombin (34,35). At 10 U/ml, hirudin significantly inhibited the stimulating effect of thrombin in the production of type IV collagen and TIMP-1. The enzymatic activity of thrombin has been reported to be necessary for its mitogenic effect on smooth muscle cells (36,37), while inactivation of the thrombin catalytic site by both hirudin and DFP blocks the thrombin-mediated increase of tissue plasminogen activator and plasminogen activator inhibitor-1 (23). The recent cloning of the thrombin receptor on platelets and vascular endothelial cells has revealed a novel proteolytic mechanism of receptor activation (38), in which thrombin cleaves the receptor N-terminal extension site to create a new N-terminal that functions as a tethered ligand and activates the receptor. This notion is supported by the fact that DFP-thrombin did not stimulate the production of type IV collagen and TIMP-1, and hirudin completely suppressed TIMP-1 production and it considerably suppressed type IV collagen production.
To our knowledge, there have been no previous investigations regarding the effects of thrombin on type IV collagen, MMP-2, and TIMP-1 production by cultured cells. Thrombin is generated at sites of glomerular injury by activation of the intrinsic or extrinsic coagulation pathway. Our findings suggest that thrombin may contribute to glomerular sclerosis in part by stimulating ECM production as well as by increasing metalloproteinase inhibitor expression in mesangial cells.
Many in vivo and in vitro studies have shown the effects of cytokines and growth factors on the accumulation of ECM (39,40,41,42,43,44,45,46,47), which may depend on the balance between synthesis and degradation. It is reported that various modulators may influence the balance of metalloproteinases and TIMP within the mesangium and determine the overall direction of matrix metabolism (48,49). Kitamura et al. demonstrated that a high glucose concentration upregulates the gene expression of type IV collagen, TIMP, and laminin, but not that of transin, which is a matrix metalloproteinase (8). They also showed that heparin selectively inhibits expression of the transin gene in cultured mesangial cells (50). Martin et al. reported that HMC constitutively secrete MMP-2 and a small amount of MMP-9, which is a 92-kD enzyme, and when the cells were stimulated by IL-1β or phorbol myristate acetate, MMP-9 was increased in a dose-dependent manner, while MMP-2 remained unaffected. They thought that MMP-2 may have an important role in the regular turnover of matrix, while MMP-9 may become predominant in pathologic situations (51). However, in our study, even when HMC were incubated with 5.0 U/ml thrombin, the MMP-2 activity was not increased and no proteinase of other molecular weight was observed. Lovett et al. demonstrated that low concentrations (0.1 to 1.0 ng/ml) of TGF-β increased the production of TIMP in cultured HMC, while high concentrations of TGF-β increased gelatinase activity (52). TGF-β has also been shown to decrease the production of ECM-degrading proteinases and increase the production of their inhibitors in cultured human fibroblasts (53,54). TGF-β has been thought to play a central role in the accumulation of ECM in glomerulonephritis (55,56,57,58).
In our study, the stimulating effect of thrombin was completely inhibited by anti-TGF-β antibody. Several authors have reported that thrombin can modulate the production of some cytokines (18,59). We have reported that thrombin also stimulates TGF-β production in cultured HMC (60). Taken together, these data suggest that thrombin stimulates the production of type IV collagen and TIMP-1 through a TGF-β-dependent mechanism.
In summary, we have shown that thrombin could stimulate the production of type IV collagen and TIMP-1 by HMC. This effect of thrombin was blocked with anti-TGF-β antibody. Thrombin is released when glomerular injury occurs, and it may play an important role via a TGF-β-dependent mechanism in the progression of glomerulosclerosis.


In my dream, it was daytime. The full moon had rainbow colors on it, not around it like a halo. I looked at it in wonder in my dream, calling others over to see. When I awoke, I wondered what it must mean. This is a typical occurrence since my Haudenosaunee great grandmother began coming to me in dreams a few years ago. I look to old teachings to try to find the answers. Sometimes they are there and sometimes they are not.
Many days, I feel my spirit being called back to my homelands, where I can learn from my elders what these things mean. Where I can be reminded that everything has a voice, a meaning, and a power…Orenda. My tongue yearns to speak the language taken away from my great-grandmother and my grandmother in the boarding schools created to strip them of their culture.
Whether trauma is ancestral or in our current lives, it can serve to create a disconnection from who you are. And yet, behind all of the things in life that can create disconnection, we have a stronger drive toward reconnection. This is our innate ability to heal. It is encoded within us. The reconnection could be as simple as a dream, a meditation, locking eyes or holding hands with your sisters and brothers. This is where healing begins.
Kunolùkwa, I love you. In the spirit of reconnection, may we share this good medicine with one another.


英文摘要 Typhoon is the most common natural disaster in Taiwan. August 6, 2009, Typhoon Morakot invaded Taiwan and battered southern Taiwan, resulting in a very serious disaster. The disaster scale is formed in Tainan, Pingtung, Kaohsiung and southern counties of the village. Those areas were destroyed by landslides, causing considerable casualties, is called 88 Flood. However, when the disaster approaches, the Central Emergency Measure Center which is organized by the government was not work. In 88 Flood, the governmental management of comprehensive disaster was not very well. The most criticized by people is the information system about disaster not working.
Relative to government communications issues, people cannot get information about disaster from the government and also cannot update information to the government, they starting to have their own online information platform for disaster mitigation and recruiting volunteers, materials and it showed the information network is so fast and it has far-reaching power. The government has been engaged in promote E-government for over ten years, it also has a very good public praise in the world. However, we can't see any advantage of e-government in 88 Flood. What is the problem? Furthermore, In the 88 Flood of rescue communications, some of private web sites formed a special rescue forces and links for information release in a few minutes. Is it a good way for the government and non-profit organizations to set up a partnership of disaster mitigation in the future? How the disaster mitigation information platform by E-government should be done? All of these are the issues in this study.
This paper reviews a variety of related literatures, online observation and depth interviews to probe into 88 Flood for the case study. There are two major findings of this thesis. First, Comparing to private sector, the government lacked of using new IT, like web 2.0, and if the government creates partnership with private sector immediately would be a better way for disaster management.
Second, the government should stress the importance of information platform, and modify the disaster management system. The NSTCDR(National Science and Technology Center for Disaster Reduction) should play the central role.
Annan,K.A.,(2004),living with risk, a global review of disaster reduction initiatives, International Strategy for Disaster Reduction (ISDR),United Nations.
Thurow, L. (1999), Creating Wealth: The New Rule for Individuals, Companies and Countries in a Knowledge-Based Economy, London:Nicholas Brealey.
Hernon, P. (1996). Government Information Policy in Time of Uncertainty and Change. In Federal Information Policies in the 1990s: Views and perspectives. Norwood, New Jersey: Ablex Publishing Corporation, 1-18.
Gore, A. (1993). Creating a Government that Works Better and Cost Less: Reengineering through Information Technology. Washington, D. C.: Government Printing Office.
West, D. M. (2007), Digital Government, NJ: Princeton University Press.
Donaldson, J. F., & C. E. Kozoll (1999). Collaborative program planning: Principles, practices, and strategies. Florida: Krieger Publishing.
Mattessich, P. W., & B. R. Monsey (1992). Collaboration: What makes it work. MN.: Amherst H. Wilder Foundation.
Ansell, C., & A. Gash (2008). Collaborative governance in theory and practice. Journal of Public Administration Research and Theory, 18, 543-571.
Thomson, A. M. & J. L. Perry. (2006). Collaboration process: Inside the black box. Public Administration Review, 66(6), 20-31.


I would like to start with a short story of which a few versions are known. Here, I am using Leo Tolstoy's version.
At conferences we have a tendency to accumulate knowledge. We all know the joys and pitfalls to be found in knowledge. In our kindergartens and schools we want to enable young people to learn in such a way that their knowledge becomes personalised, becomes part of their being.
If you want to remember the conference in the next few weeks or months, just take the prism which you have carried around with you this past week. Look through the glass. Depending on how and in which light you are holding it, you will see colours. Colours appear through the interplay of light with the glass. The glass is a kind of obstacle for the light, and this obstacle makes the colour appear. Has your world also become a little more colourful at this conference? Did you also have to overcome some obstacles during the conference and has this also given you new courage?
I suggest you do a simple exercise within the next few days or weeks: Find a quiet place inside yourself and let the events of this week pass before you. Perhaps you'll see the week in a new light, in new colours. Maybe the first, loud impressions move to the background and the quiet, seemingly insignificant events light up. Experiences change in retrospect and over time. Usually, we don't know at first what kind of power lives in them. With this small exercise we free ourselves from the first impression of an experience and increasingly approach its true meaning.
This week, a question has lived in all the workshops and lectures: How do we develop the courage for a free spiritual life or how do we create a healthy We?
The culture of the We deserves our attention. The We enables a lot, even troublesome things. What kind of We lives in our teaching, in the work with our colleagues, in the school community or even in society? Are we aware of the process of how the We is born?
We can create a replacement for the supervision of the School Board as we form this preparatory course, and, through the work, receive what unifies the school. We can achieve that sense of unity through this course if we work with all diligence.
2. The mantra describes what is meant by "working with all diligence": It is to study The Foundations of Human Experience and to connect the content with the spiritual world. The fruit of our labour are not meant for us but for others, for the children and the colleagues.
I would now like to show you the three steps of the mantra in a picture.
Imagine you are sitting on a chair, you sit there all alone and start to plan your lesson. You remind yourself that the child's individuality wants to show itself in the present and that you are its servant. Essentially, this is about the human being, it is about knowledge and relationships. As a teacher you are responsible for the atmosphere in which the child learns. As teachers we play with time and atmosphere like artists; we do it for the children and we should do it for ourselves, too.
Unfortunately, we do not give the preparation for lesson planning much attention. Try to get into the right mood, connect yourself with the children or teenagers you are planning the lesson for. Study The Foundations of Human Experience for a moment. Then, follow it up by studying the content of your lesson. Connect the various elements of what you want to teach and then ask yourself: What does all this have to do with me as a human being? What do an engine, an elephant or silica have to do with me? Please pay attention to your relationship to the world. Does the world have a place within yourself or do you feel separated from it? On the one hand, there is the little human being who faces the world, planting his or her feet firmly on the ground. But there is also the large, cosmic human being who encompasses everything. We are both, the large and the little individual or the day person and the night person. We are teaching for both of them. Whatever has been absorbed during the day will be digested in the night. Real learning happens at night. The large human being who lives at night embraces the spiritual world and in particular the world of the angels. Together with the angels we prepare the next day because the night leaves an impression in us.
What happens when we add another chair and face a partner? In 2010, Marina Abramović sat on a chair at the MoMA in New York and did nothing other than look into the eyes of the people who sat opposite her. She did this for 90 days, six days a week, for seven hours at a time, without taking a break, without any food or drink, without talking. She called her piece: The Artist is Present.
The crucial element here is, what one can give to the other. This is the source of trust, friendship, You, love and courage.
Let us now place yet another chair here. We have only just got used to the person opposite and so a further chair causes a small tremor. The cosy duo is properly shaken up by the addition of a third person. Triads are challenging. We have two relationships and in one of them we are a mere observer. This is where the interplay of the group starts.
A group of three is somewhat manageable, however, its complexity increases with the addition of each new member. Suddenly, we are faced with the challenges of community and the question arises, which kind of closeness is created by the group.
In a college of teachers we are faced with a lot of different people. And, against better judgement, a few assumptions never die. For example, we presume that the wellbeing of the community is more important than the wellbeing of the individual. At the moment, politics experiences a revival of nationalistic feelings of We, which have mainly to do with the integration into a group and the exclusion of the so called "foreign". The longing for identity, which everybody has, can easily lead to the misunderstanding that the individual is unimportant and that only the group counts. If individuals sacrifice their own needs for the group, they weaken their power for self-responsible action.
The awareness of a healthy We leads to a different set of criteria. The American Anthropologist Margaret Mead said, "Human diversity is a resource, not a handicap." This is an important statement which values diversity as a positive factor. The individual is the basis of the group. Steiner's view of community is also based thereupon.
In a true teachers' republic we will not have the comfort of receiving directions from the Board of Education. Rather, we must bring to our work what gives each of us the possibility and the full responsibility for what we have to do. Each one of us must be completely responsible.
My dear friends, we can accomplish our work only if we do not see it as simply a matter of intellect or feeling, but, in the highest sense, as a moral spiritual task.
No doubt, only a deep trust in ourselves and our fellow beings can be the foundation of community. This trust encourages us to step towards diversity. We have to solve the question of how to be united in our heterogeneity. It is common to be afraid of our fellow human beings. However, our feet lead us towards the others. If we do not stop our feet, we create room for encounters, thus we allow life to meet us, we welcome diversity. We try to cope with the differences, even to accept them as creative tension.
A new community can only be lived in the reality of daily life. It contains the potential to provide orientation for the individual. The new community does not demand sacrifices but it is important that each individual overcomes the fear of their own courage.
What Steiner described as "unity" can only appear if we create a We. It is a gift. We prepare ourselves, we get ready by creating a We.
The main entrepreneurial risk for any Waldorf institution today and tomorrow is to create a We in which the individual's strength and courage to meet another human being lives. This We recreates itself again and again and is the source of leadership. This is a truly modern concept of leadership. It was created one hundred years ago and has been tried and tested in many places all over the world. Its particular quality is the conscious attention to the connection with the spiritual world. If we are thus prepared to include the help of the spiritual world in our work, we will receive its gifts. The three monks practiced just that. They included the spiritual world completely in their work.
We are three. You are three. So may it be.
"We wish to form our thoughts in such a way tat we may have consciousness: Behind each one of us stands his Angel, gently laying his hands on the head of each. This Angel gives you the strength which you need.
Above your heads there hover the circling Archangels. They carry from one to the other what each has to give to the other. They unite your souls. Thereby you are given the courage of which you stand in need. Out of this courage the Archangels from a chalice.
The light of wisdom is given us by the exalted beings of the Archai, who are not limited to the circling movements, but who, coming forth from primal distances (Urfernen). They reveal themselves only in form of a drop (of light) in this place.
(3) Steiner, R. (1996) The Foundations of Human Experience. Anthroposophic Press. p.30. Opening Address given on the Eve of the Teachers' Seminar.


CKM specializes in transforming data into actionable insights. Since 2011, our goal has been to change the way companies make operational and strategic decisions. We believe the power of data can be used to inform, improve, and enable our clients' key management decisions. From top Fortune 500 firms to NGOs, we work with organizations across the globe to provide impactful recommendations and applications.
At CKM, we are at the forefront of applying data science to inform strategic decision-making and drive operational improvement. Grounded in data and driven by the client's needs, our solutions encompass highly attuned machine learning algorithms as well as application-based analytical engines.
Our success is driven by our team-centric approach which seamlessly combines the brightest minds in data science, business and development. Bringing these capabilities together makes our teams adept at tackling the many unique challenges our clients face.
Clients trust us to employ advanced methods of ingesting and analyzing data towards the goal of solving a business problem. To that end, our teams translate analysis into business action through cutting-edge visualizations, recommendations, and data science applications. In striving for complete transparency, openness and reusability in our work, we never offer up a "black-box" solution.


text:
Deh-e Jan or Deh Jan or Dehjan () may refer to:
 Deh Jan, Gilan

redpajama_set_name: RedPajamaWikipedia


Superb goaltending played a major role in lifting the Anaheim Ducks as high as second in the Pacific Division early last week Jay Beagle Jersey Womens , but they'll need to rely on their offense more than ever beginning Saturday night against the Arizona Coyotes at Honda Center.The Ducks lost No. 1 goalie John Gibson to an upper-body injury during a 4-2 loss at the San Jose Sharks on Thursday night, their fourth loss in a row. Anaheim was already without backup goalie Ryan Miller, who sustained a knee injury on Dec. 9 and will be sidelined another four weeks.Through the first 35 games, Gibson and Miller combined to lead the Ducks to a 19-11-5 mark. The Ducks (19-15-5) still own the top save-percentage in the NHL at .918 while facing the second-most shots in the league at 34.8 a game.Article continues below ...Anaheim claimed goalie Chad Johnson off waivers from the St. Louis Blues on Dec. 11, and the eight-year veteran gave up two goals on 23 shots in a 3-1 loss at the New York Rangers on Dec. 18, the start of the current four-game losing streak.He then allowed two goals on 22 shots after replacing Gibson on Thursday."You never know in this league what's going to happen," Johnson told reporters after the loss to San Jose.Arizona (16-19-2) is coming off a 2-1 loss at the Los Angeles Kings on Thursday. The Kings are the lowest-scoring team in the NHL at 2.29 goals a game Curtis McKenzie Jersey Womens , the Coyotes the third-lowest (2.49), and the Ducks are in between (2.41).The Ducks and Arizona met twice in the first full week of this season.Gibson had 41 saves in a 1-0 win in Phoenix on Oct. 6. The Coyotes came back with a 3-2 shootout win four days later in Anaheim. Antti Raanta made 23 saves in that win, but he's been out the past month with a lower-body injury with no timetable for his return.Darcy Kuemper has taken over as the No. 1 goalie for Arizona since returning from a lower-body injury on Dec. 11 and he's been solid. The offense, however, has been held to one goal in four of his past five outings.Kuemper stopped 24 of 26 shots against the Kings, but the Coyotes couldn't muster a second goal and lost 2-1."We didn't win the game, so we get back to the drawing board at practice and Nick Holden Jersey Womens , you know, keep grinding away," Arizona coach Rick Tocchet told reporters after the game.The Coyotes, who last made the postseason in 2012, were just seven points out of the final playoff spot entering Friday.Not only have the Ducks dropped four in a row, but the Edmonton Oilers have also lost four straight, the Minnesota Wild five in a row and the Dallas Stars six of their past nine Paul Stastny Jersey Womens , leaving the Coyotes' well within reach heading into the second half of the season.The Ducks were second in the Pacific Division when their four-game losing streak began. They head into Saturday fourth in the division and just three points from dropping out of a wild-card spot."If we can continue to play simple, we'll get out of our skid here soon," Ducks rookie right wing Kiefer Sherwood told Fox Sports West after scoring his fifth goal of the season Thursday. Washington Capitals forward Tom Wilson's suspension for his preseason hit to the head of the St. Louis Blues' Oskar Sundqvist has been reduced from 20 games to 14, according to Sportsnet's Elliotte Friedman.NHL Player Safety NHLPlayerSafetyWashington鈥檚 Tom Wilson suspended twenty games for an Illegal Check to the Head on St. Louis鈥?Oskar Sundqvist. ojQ4yN6oFNThe league released an official statement on the ruling:NHL Public Relations PR_NHLNHL and NHLPA statement on Tom Wilson suspension.Complete arbitrator decision: 5JXYsGsYbx 8s5l6LtxXJWilson is eligible to play in Tuesday night's game against the Minnesota Wild. The team wasted little time in welcoming him back:Washington Capitals CapitalsWelcome back, tom_wilson! #4锔忊儯3锔忊儯 #ALLCAPS S6pHZm4e10He has already missed 16 games, and while he can't get those two contests back, he can recoup that salary.As ESPN.com'sGreg Wyshynskinoted at the time of the initial ban Max Pacioretty Jersey Womens , a 20-game suspension would have cost Wilson$1,260,162.60. While that total will be lower, it will still be significant.Meanwhile, the hit keptSundqvist off the ice for the start of the regular season. He missed St. Louis' first eight games because of upper-body injuries.Wilson has been in trouble with the league office several times over the past year-plus. He has been suspended four times since September 2017, including athree-game banfor breaking an opponent's jaw during the 2018 Stanley Cup playoffs.His hit on Columbus Blue Jackets center Alexander Wennberg during the first round of the 2017-18 postseason was also reviewed but did not garner a suspension. Wennberg miss timed as the result of a concussion sustained during the hit.Wilson's return should provided a needed boost for Washington, which has gone 7-6-3 (17 points) in his absence. The 24-year-old recorded 14 goals and 21 assists for 35 points in 78 games last season Nic Dowd Jersey Womens , helping the Capitals win the Stanley Cup with five goals and 10 assists in 21 postseason contests.


text:
Table of Contents » Title 56. Public Service Companies » Chapter 15. Telegraph and Telephone Companies » Article 1. Erection of Lines; Rights-of-Way; Eminent Domain, Etc. » § 56-468.2. Reimbursement for relocation costs
§ 56-468.2. Reimbursement for relocation costs.
A. After July 1, 1998, certificated providers of telecommunications services shall receive reimbursement for eligible relocation costs incurred at the direction of a locality that imposes by ordinance the Public Rights-of-Way Use Fee or the Department of Transportation for new installations as defined in § 56-468.1 in any public rights-of-way in accordance with §§ 56-458 and 56-462 on the basis of age and according to the following schedule. Such reimbursement shall be received from either (i) the locality that granted the permit or franchise to use such right-of-way or (ii) the Commonwealth Transportation Board if the road or street is in the primary or secondary state highway system:
1. For the first three years after the completion of the installation, the certificated provider of telecommunications service shall be reimbursed 100 percent of the eligible cost for the relocation of facilities installed in the public rights-of-way.
2. For the fourth through sixth year after the completion of the installation, the certificated provider of telecommunications service shall be reimbursed 50 percent of the eligible cost for the relocation of facilities installed in the public rights-of-way.
3. Beginning in the seventh year, the certificated provider of telecommunications service shall be responsible for the cost of relocating facilities installed in the public rights-of-way.
Such reimbursement shall be received from either (i) the locality that granted the permit or franchise to use such right-of-way or (ii) the Commonwealth Transportation Board if the road or street is in the primary or secondary state highway system.
B. The amount of relocation reimbursement in any fiscal year to be reimbursed under this section shall not exceed the amount of Public Rights-of-Way Use Fees received by that locality either directly or through its secondary highway fund apportionment in the preceding fiscal year. For facilities relocated in 1998 and 1999 at the direction of the locality or the Commonwealth Transportation Board, this limit on relocation reimbursement shall be the estimated annualized fees to be collected in that locality in 1998 for 1998 relocations and in 1999 for 1999 relocations. If the relocation reimbursement limit will be exhausted on a relocation project where two or more certificated providers of telecommunications service are eligible for relocation reimbursement, then the moneys available under the cap shall be shared by those eligible providers by prorating the reimbursement based on the reimbursement to which each provider would be entitled absent the limit.
1998, cc. 742, 758; 2015, c. 256.

redpajama_set_name: RedPajamaCommonCrawl


International food standards offer the opportunity to provide guidance in preventing and mitigating food safety risks, while enhancing convergence of food regulatory measures adopted around the globe. The Codex Alimentarius Commission has achieved tremendous progress in developing standards, guidance and codes of practice that address various food safety hazards. These standards have guided food regulators worldwide in shaping their food safety requirements for foods produced domestically or imported. Despite this progress, several challenges have been identified in relation with the application and implementation of Codex standards by domestic jurisdictions. Given that Codex standards tend to be outcome-based to ensure more flexibility and their possible adaptation to various scenarios encountered by food regulators and food industry operators around the world, operationalizing such measures can be challenging for the food sector and/or regulators in low and medium economy countries. Helping the implementation of Codex standards is therefore a key area of food safety capacity building efforts, recognized in the Strategic Plan of the Codex Alimentarius Commission 2014-19. International standards have also to remain relevant for Codex membership, if they are to serve members' needs. Codex should therefore be able to adapt and address emerging issues resulting from changes in food safety and nutrition priorities, such as the expanding international trade in foods and food ingredients; the multiplication of free trade agreements at the bilateral and regional level where food and food products continue to be a centrepiece of most traded commodities; the requirements for resource optimisation in economically uncertain times and the importance of innovation to improve value-added of food products. This paper briefly reviews some accomplishments of Codex in the development of international food standards. It also attempts to identify areas of priority investments in capacity building initiatives aimed to propagate the use of these standards. It discusses synergies that can be found to optimize the use of resources and to enhance convergence of food safety regulatory measures, in support of consumers' health protection and reduction of impediments to international trade.


The hands-on learning approach is about providing students with opportunities to experiment with their surroundings as a form of problem solving through coding. It is about creativity and collaboration, motivation and self-direction. It is about improvisation and discovery, and playful learning experiences with real-world relevance.
The unique combination of building with the familiar LEGO bricks, using easy-to-use coding software and engaging curriculum relevant STEM challenges, makes coding fun and relevant for primary and secondary school students. Basically, everything you need to help every student to succeed at STEM through coding.
Children learn best when they are actively engaged in constructing something that has a personal meaning to them – be it a poem, a robot, a sandcastle, or a computer program.
Teach coding, brick by brick. Through our simple and intuitive drag-and-drop interface, primary students develop 21st-century skills through computational thinking.
Get hands-on with the power of coding. With real-life STEM challenges and engaging physical and digital creation, we enable secondary school students to develop their 21st-century skills through computational thinking.


text:
Huawei Mate 40 series may launch with 90Hz refresh rate display
Huawei Mate 40 series is rumored to be released in Q4 2020. According to a Twitter tipster, @RODENT950 the Mate 40 series will follow the on-screen specifications of the P40 series, which is about 1200P + resolution and 90Hz refresh rate.
The Mate 40 series continues the P40 series display specs aka strange resolution + 90Hz. pic.twitter.com/rBbesCwcp1
— Teme (特米)😷 (@RODENT950) June 26, 2020
An earlier report suggests that the Huawei Mate 40 pro may use UTD screens (Ultra Thin Display) Specially designed for folding and ultra curved display phones and the Huawei Mate 40 will have 2 variants. At the same time, Mate Watch smartwatches running Hongmeng OS may also be released.
Huawei Mate 40 Series Key highlights:
5 nm SoCs, with the Kirin 1000 and Kirin 1020 (More powerful Kirin chipset).
"Freeform lens" designed to improve the fidelity of ultra-wide-angle photos. This lens may be exclusive to the Mate 40 Pro.
108-megapixel lens may be features whose optical performance has been greatly improved and said to be available in Mate 40 and Mate 40 Pro.
It will use a 9P lens. Due to the manufacturing method of the lens, the ability of the lens to process images is also greatly improved. Compared with other standard lenses with 108 MP sensors on the market.
Mate 40 series also consists of TOF which can be used for temperature measurement (similar to Honor Play 4 Pro).
Related Items:Huawei, Huawei Mate 40, Huawei Mate 40 90Hz, Huawei Mate 40 Pro, Huawei Mate 40 Series, Mate 40, Mate 40 Pro
Honor X10 Max retail box video leaked ahead of launch
Huawei new 5G phone WKG-AN00 passes 3C certification


text:
function hndls = plot(d,colors)
% GMM\PLOT

% Copyright October, 2006, Brown University, Providence, RI.
% All Rights Reserved

% Permission to use, copy, modify, and distribute this software and its
% documentation for any purpose other than its incorporation into a commercial
% product is hereby granted without fee, provided that the above copyright
% notice appear in all copies and that both that copyright notice and this
% permission notice appear in supporting documentation, and that the name of
% Brown University not be used in advertising or publicity pertaining to
% distribution of the software without specific, written prior permission.

% BROWN UNIVERSITY DISCLAIMS ALL WARRANTIES WITH REGARD TO THIS SOFTWARE,
% INCLUDING ALL IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY
% PARTICULAR PURPOSE. IN NO EVENT SHALL BROWN UNIVERSITY BE LIABLE FOR ANY
% SPECIAL, INDIRECT OR CONSEQUENTIAL DAMAGES OR ANY DAMAGES WHATSOEVER
% RESULTING FROM LOSS OF USE, DATA OR PROFITS, WHETHER IN AN ACTION OF
% CONTRACT, NEGLIGENCE OR OTHER TORTIOUS ACTION, ARISING OUT OF OR IN
% CONNECTION WITH THE USE.

% Author: Frank Wood fwood@cs.brown.edu

if(nargin < 2)
    colors = {'k','r','g','b','m','c'};
end

nc = d.mixture_model.num_components;
hndls = zeros(nc,1);
invp = 1.96;
hold on
for(i=1:nc)
    g = d.mixture_model(i);
    g = g{1};
    mu = g.mean;
    sigma = g.covariance;

    [vec, val] = eig(sigma(1:2,1:2));
    axes = invp*sqrt(svd(val));
%     angles = -atan2(vec(1,:),vec(2,:));

    t= linspace(0,2*pi);
    ellip = vec*invp*sqrt(val)*[cos(t);sin(t)] + repmat([mu(1); mu(2)],1,100);
    ellip = ellip';
    axes = axes';

    hndls(i) = plot(ellip(:,1),ellip(:,2),colors{i});
end
hold off

redpajama_set_name: RedPajamaGithub