THE public gathered in the Bull Ring to pay respects to those who gave their lives during the First World War.
Sunday, November 11 marked the centenary of the First World War and the remembrance service was well attended.
Cllr Bill Walmsley, mayor of Middlewich, said: "The number of people who attended the Remembrance Service in Middlewich on Sunday was staggering and I must compliment the Royal British Legion and Middlewich Town Council on how well-organised the event was.
"I was honoured to attend as mayor of Middlewich and to lay a wreath on this momentous occasion in our history. It made me very proud of our community.
"Over the course of the last few days, several people have mentioned to me how impressed they were with our display of poppies on lampposts through the town.
"This came from both Middlewich residents and from people I've met with in other towns.
Cllr Ken Kingston said: "That was the biggest turnout we have seen for the service.
"This year we had a special roll of honour made for the order of service to include the new names added to the memorial.
The Act of Remembrance took place at the War Memorial in the Middlewich Bull ring and was led by the Royal British Legion with Churches in Middlewich taking part.


text:
Dokumentenidentifikation EP0482571 03.04.1997
Titel Bestimmung von Substanzen mittels durchfluss Zytometrie
Anmelder Sumitomo Electric Industries, Ltd., Osaka, JP
Erfinder Kishimoto, Toshihiko, c/o Osaka Works of Sumitomo, Konohana-ku, Osaka, JP;
Niwa, Shin-ichiro, c/o Osaka Works of Sumitomo, Konohana-ku, Osaka, JP;
Nakabayashi, Makoto, c/o Osaka Works of Sumitomo, Konohana-ku, Osaka, JP;
Shirai, Toshikazu, Kokubunji-shi, Tokyo, JP;
Hirose, Sachiko, Shibuya-ku, Tokyo, JP;
Tsurui, Hiromichi, Nerima-ku, Tokyo, JP
Vertreter Hoffmann, Eitle & Partner Patent- und Rechtsanwälte, 81925 München
Vertragsstaaten CH, DE, FR, GB, LI
IPC-Hauptklasse G01N 33/546
IPC-Nebenklasse G01N 33/53 G01N 15/14 G01N 33/551
Beschreibung[en]
The present invention relates to an assay useful in various fields of technology including immunology, molecular biology, diagnosis and the like. More particularly, it relates to a method for qualitatively or quantitatively measuring a substance to be detected in a sample by means of flow cytometry, wherein said substance is bound to a microparticle through a second substance which is immobilized to said particle and capable of binding to the substance specifically.
Reliable and efficient assay for a given substance in a sample has been continuously needed in various fields of technology including medicine, chemistry, agriculture and the like, and there have been provided various methods before the present invention. A group of widely known methods employ a specific reaction between a substance to be detected (hereinafter, referred to as an analyte) and a second substance, for example, a reaction between an antigen and its antibody. These methods are generally carried out by immobilizing said second substance to an insoluble carrier, contacting a sample containing the analyte to the immobilized second substance, separating the carrier from the reaction mixture and determining the presence or absence, or concentration of said analyte captured on the carrier through the second substance. Typical assays of this type are immunoassays such as Enzyme-Linked Immunosorvent Assay (ELISA). Outline of such a conventional immunoassay is described below.
A plastic plate having multiple wells is often used as the solid carrier. To each of the wells is added a solution containing an antigen and the plate is kept overnight at about 4°C or for about 3 hours at room temperature. After the solution is discarded, the wells are blocked in order to prevent any unspecific adsorptions. The blocking is carried out by filling the wells with a solution of a blocking agent such as skim milk, standing for several hours at room temperature or overnight at about 4°C, and washing thoroughly with PBS buffer. To the blocked wells is added a sample solution suspected to contain the antibody to be assayed, and the plate is kept for several hours and washed thoroughly, which is followed by the addition Of an antibody labeled with an appropriate signal-producing substance such as enzyme, fluorogenic substance, or the like. The plate is then allowed to stand for several hours. After washing the plate, the signal produced in each well is observed by any of known methods. When the label is a fluorogenic substance, the observation is carried out by adding a suitable buffer to the wells and determining the level of fluorescence with fluorophotometer. When the label is an enzyme, a solution of a substrate is added to each well and allowed to react for a while. Thereafter, the reaction is stopped and the absorption of light caused by the products of enzymatic reaction is measured with spectrophotometer.
As stated above, the existing methods generally use, as a solid carrier, a plastic product in the form of microtiter plate, tube or the like. However, such plastic solid carrier does not always afford reliable data because of its tendency of forming unspecific bindings. Thus, unspecific binding or attachment of various substances to the carrier makes it difficult to establish a uniformity of binding between the carrier and a substance to be immobilized and causes undesirable background effects. Furthermore, when a microtiter plate is used as a solid carrier, each well gives just one datum which differs from well to well, resulting in the inefficiency of the experiment and unreliability of the data.
One solution of the above problems lies in bringing the binding between the carrier and the substance to be immobilized more specific, thereby eliminating the background effects and reducing the variation of data.
The present inventors have now found that the above problems can be solved by contacting a sample solution containing an analyte with particulate carriers to which a second substance capable of specifically binding to said analyte has been immobilized, and qualitatively or quantitatively detecting the analyte bound to the particles through the second substance by means of flow cytometry.
Thus, the present invention provides a method for qualitatively or quantitatively measuring substance A in a sample solution, which in principle comprises the following steps:
immobilizing substance B capable of specifically binding to the substance A to a particulate carrier;
contacting the particulate particle with the sample solution suspected to contain the substance A; and
detecting the substance A bound to the particulate carrier through the substance B by means of flow cytometry.
For the purpose of the invention, as disclosed and claimed herein, the following terms are defined below.
The term "substance A" or "analyte" refers to any substances of interest and can be detected by the method of the present invention. Examples of such substances include chromosome, DNA, DNA fragment, amino acid, protein, enzyme, hormone, antigen, antibody and the like.
The term "substance B " or "second substance" refers to any substances which have abilities of specifically reacting and binding with the substance A or analyte and can bind to a microparticle used as a carrier. Although any combinations of substance A and substance B are applicable to the method of the present invention so long as they react specifically and one of them can be immobilized to a particulate carrier, a combination of an antigen and its specific antibody, especially monoclonal antibody, is preferable.
The term "sample solution" means a solution which is known or suspected to contain the substance A. Examples of sample solutions include blood, urine, body fluid, cultured broth and the like.
The apparatus used for the flow cytometry is commercially available and any of existing devices can be used in the present method. The determination by flow cytometry is effected basically by passing a suspension of particulate substances to be detected, for example cells or chromosomes, through a fluid system at high speed while detecting the photoelectric signals from individual particle on detector. The detection is carried out by irradiating a laser beam to suspended particles moving at high speed and converting the optical properties of each particle into photoelectric signals. The optical property of the particle is generally provided by appropriate signal-producing systems. Such signal-producing systems involve the production of detectable signals such as absorption, scattering or emission of light. Examples of signal-producing substances are fluorogenic or chromogenic compounds such as rhodamin, FITC (fluorescein isothiocyanate), acridine orange, propiodium iodite and the like. Such a signal-producing system can be attached to the microparticles by adding substances containing appropriate signal-producing substance associated to a molecule capable of binding to the substance A. In the preferred embodiment, said labeled substance is an appropriately labeled second antibody.
Thus, the present invention further provides a method for determining the presence or absence, or concentration of substance A in a sample solution which comprises:
immobilizing substance B capable of specifically reacting and binding with substance A to a particulate carrier;
contacting the particulate carrier to which substance B has been immobilized with the sample solution suspected to contain the substance A; and
adding a labeled substance C capable of binding to the (substance A) ― (substance B) ― associated carrier; and
detecting the substance A bound to the carrier through the substance B by means of flow cytometry.
Examples of apparatus for the flow cytometry include FACS series distributed by Becton Dickinson, EPICS series distributed by Nippon Bunko, Ltd., Showa Denko, Ltd., Cor-tar Electronics, Ltd. and the like.
Microparticles to be used as a solid carrier may be any shape but preferably spherical of appropriate diameter for the determination by flow cytometry. The size of particles may vary depending on the size of a nozzle attached to the apparatus for flow cytometry. However, microparticles may be in the range of about 1 nm to 1000 µm, preferably about 1 nm to about 400 µm, more preferably, 100 nm to 100 µm, in diameter. Specifically, the average diameter of particles may be from about 1 nm to about 400 µm when the diameter of nozzle is between about 10 to about 1,000 µm, and the average diameter of particles may be from about 0.1 to about 100 µm when that of nozzle is between about 10 to about 500 µm. Especially preferred particles are those of about 0.1 to 100 µm in diameter.
The microparticles or particulate carriers can be made of a variety of synthetic or naturally-occurring materials. Examples of microparticles include those made of organic synthetic polymers such as cellulose, nitro cellulose, polytetrafluoroethylene, polyethylene and the like; inorganic polymers such us graphyte, porous glass, silica and the like; metals such as aluminium, apataite and the like; ceramics such as alumina and the like. Preferable microparticles are polymers including organic and inorganic polymers, metals and ceramics. More preferably, microparticles are selected from the group consisting of organic and inorganic polymers. However, other materials are also available in the present method provided that they can be applied to the flow cytometry and do not prevent the specific reaction between the substance A and substance B. Although these microparticles may be used without further treatment, they can be modified variously to make the immobilization of substances more stable and uniform. For instance, organicpolymer particles can be modified by chemical or physical treatment such as oxidation, reduction, hydrolysis, plasma-radiation or the like. Particles made of inorganic polymer, metal or ceramics may be subjected to a surface-finishing process by physical and/or chemical treatment, for example, ion-plating method and the like.
The immobilization of substance B to particles can be carried out according to known methods in the art. Although the procedure may vary depending on the substance B and the material of the particle, the substance B can be generally bound to particles covalently at a functional group present in the molecule composing said particle, for example, amino group, hydroxyl group, carboxyl group, thiol group or the like. Thus, the immobilization can be accomplished by, for example, reacting an amino group of substance B and a hydroxyl group of carrier after the activation of the hydroxyl group using an activator such as trifluoroethanesulfonyl chloride or the like [R. Axen et al., Nature, vol. 214: pp.1302 (1967); T.H.Finlay et al., Anal. Biochem., vol. 87; pp.77 (1978); I.Matsumoto et al., J. Biochem., vol. 85: pp.1091 (1979); L.Sundberg, et al., J. Chromatogr., vol.90: pp.87 (1974); K.Nilson et al., Biochem. Biophys. Res. Commun., vol.102: pp.449 (1981); and J.Porath, Meth. Enzymol., vol.34: pp.27 (1974)]; reacting an amino or a carboxyl group of substance B and a carboxyl or an amino group of the carrier in the presence of a condensing reagent such as water-soluble carbodiimide (WSC; supplied by Wako Junyaku, Co. Ltd., Japan, and the like) [A. Tengblad, Biochem. J., vol.199: pp.297 (1981); M. Funabashi et al., Anal. Biochem., vol. 126: pp.414 (1982); G. Saccomani et al., J. Biochem., vol. 256: pp.12405 (1981); B. Belleau et al., J. Am. Chem. Soc., vol.90: pp.1651 (1968); and P. Cuartecasas et al., Biochemistry, vol.11: pp.2291 (1972)]; reacting amino group with an aldehyde group to yield a shiff base or a hydrazide and, if necessary, followed by reduction [Y. Ito et al., J. Biochem., vol.97: pp.1689 (1985); and R. J. Baues et al., J. Biol. Chem. vol.252: pp.57 (1977)]; or forming -S-S- bonds using thiol group [K.Brocklehurst et al., Biochem.J., vol.133: pp.573 (1973)]. In a preferable embodiment of the invention, the substance B is an antigen, especially a DNA molecule, and substance A is an anti-DNA monoclonal antibody, where said substance B is bound to a microparticle of silica gel by the terminal-immobilization.
The immobilized substance B is useful in the determination of presence or absence, or concentration of substance A in a sample. Accordingly, in another embodiment of the invention, it is provided an immobilized substance B useful for the qualitative or quantitative determination of substance A by flow cytometry, wherein said substance B can react and bind with said substance A specifically, and the solid carrier is microparticles.
As mentioned above, the present method allows to detect substance A by associating said substance A to a microparticle with the aid of substance B which is immobilized to the particle and is capable of reacting specifically with the substance A, and subjecting the microparticle to flow cytometry. In the preferred embodiment of the present invention, the substance B is immobilized uniformly to a particulate carrier through a covalent bond between functional groups such as amino, hydroxyl, carboxyl, thiol groups and the like. Although any particulate carriers are available in the present method as previously stated, those used for HPLC such as silica gel are preferable because they hardly generate unspecific binding, which results in the reduction of background effects and the improvement of accuracy of measurement.
In comparison with the currently used ELISA, where a microtiter plate is used as a solid carrier, the present assay has following advantages. The present assay is more efficient than ELISA because one particle in the present assay can be regarded to be equivalent to one well in ELISA and almost 10&sup5; of particles can be measured by one trial. Furthermore, abnormal data are omitted from the results and the background effect is reduced as previously mentioned. Thus, the present method affords about 10 &sup4;&supmin;&sup5; times of data with high reliability and less errors within almost the same detection time compared with ELISA. According to the present method, the strength of affinity and reactivity are obtainable as detection patterns as shown in the accompanying drawings, Figures 1 to 8.
In the Figures:
Figure 1 shows the detection patterns of anti-DNA antibodies using a mixture of particulate carriers to which DNA fragments (a) and (b), as described in Example 1, are immobilized as single-stranded DNA, separately. The number of particles are plotted on the ordinate and the level of fluorescence on the abscissa. The solid lines represent the detection pattern obtained in the experiments conducted using antibody-containing samples and the dotted lines represent those obtained in the control experiments conducted using antibody-free samples. The panel A depicts the binding effect of IgG, the panel B depicts that of IgM, and the panel C depicts that of anti-DNA monoclonal antibody, to the DNA-immobilized particulate carriers. The order of the total number of particles shown on the ordinate is 10,000.
Figure 1 indicates that neither of the non-specific antibodies IgG and IgM which lack affinity to the DNA to be detected, causes the shift of peak (panels A and B, respectively), whereas the specific monoclonal antibody causes a significant shift of peak (panel C), demonstrating that the present method allows the detection of specific antibody correctly.
Figures 2 is similar to Figure 1 except that the carriers to which DNA (a) and (b) are immobilized in the form of double-stranded DNA are used.
Figure 3 is similar to Figure 1 except that the carriers to which DNA (c) and (d), as described in Example 1, are immobilized as single-stranded DNA are used.
Figure 4 is similar to Figure 3 except that the carriers to which DNA (c) and (d) are immobilized as double-stranded DNA are used.
Figure 5 is similar to Figure 1 except that the carriers to which DNA (e) and (f), as described in Example 1, are immobilized as single-stranded DNA are used.
Figure 6 is similar to Figure 5 except that the carriers to which DNA (e) and (f) are immobilized as double-stranded DNA are used.
Figure 7 is similar to Figure 1 except that lysozyme is immobilized to particulate carriers and anti-lysozyme monoclonal antibody is used as an specific antibody.
Figure 8 is similar to Figure 1 except that panels A and C show the results obtained using carriers to which DNA (a) and (b), as shown in Example 3, are immobilized as single-stranded DNA, while the panels (B) and (D) show the results obtained using carriers to which double-stranded DNA consisting of (a) and (b) are immobilized. Additionally, the panels A and B depict the binding effect of IgG and the panels C and D depict the binding effect of anti-DNA monoclonal antibody.
In the present assay, the variation of data, that is, the variation of level of fluorescence from particle to particle, is reflected as the breadth of peak. Further, each particle can be regarded to be equivalent to one well of the currently used ELISA. This means that, according to the present method, about 10&sup4; to 10&sup5; times of data can be obtained by only one experiment in comparison with ELISA. The data which are shown as a peak are highly reliable and allow one to evaluate the affinity between two substances, for example, an antigen and antibody, correctly and efficiently.
Although the present assay is useful in various fields, it is especially useful in clinical experiments, immunology, biochemistry and the like for the purpose of detection or diagnosis of various diseases and/or detection of antibody, specific substances and the like.
Following Examples further illustrate and detail the invention disclosed, but should not be construed to limit the invention.
Example 1 DNA-IMMOBILIZED CARRIER 1. Preparation of DNA-Immobilized Particulate Carrier A. Preparation of activated microparticles for the immobilization of DNA
As a solid carrier for the immobilization of DNA, silica gel is activated with trifluoroethanesulfonyl chloride (tresyl chloride: K & K, Co. Ltd.). SIMPACK diol 300 (average particle size = 5µm in diameter; SHIMAZU, Co. Ltd.) (1 g) is put into a No. 5 mesh glass filter and washed with acetone (20 ml), and then dry acetone (20 ml) by filtering with suction. The gel is taken in a 25 ml round bottle flask, and dry acetone (1 ml), pyridine (100 µl) and a small stirrer chip are added thereto. To the flask is added dropwise 100 to 200 µl of tresyl chloride gradually over about 1 minute while stirring vigorously and keeping away from air by introducing dry nitrogen gas. After the round bottle flask is closed with a plug, reaction is continued further 20 minutes under cooling and stirring gently. When the reaction completes, the mixture is filtered by No. 5 mesh glass filter and washed with 25 ml each of dry acetone, acetone/5mM HCl (1:1), 5 mM HCl, and acetone, successively, and acetone is evaporated. Thus obtained tresyl-activated SIMPAK diol 300 are collected in a Kjeldahl flask. After evaporation of the acetone under reduced pressure, the resultant activated silica gel is sealed in the flask until use.
B. Preparation of DNA
The following DNA fragments were synthesized using DNA Synthesizer A-391EP PCR MATE (Applied Biosystems Inc. (ABI)). In the following fragments, (a) is a 27-mer to be immobilized and (b) is a complementary strand thereof, (c) is a 24-mer to be immobilized and (d) is a complementary strand thereof, and (e) is a 21-mer to be immobilized and (f) is a complementary strand thereof.
X: Aminolink-2 (ABI, Co. Ltd.), which is a reagent used in the DNA synthesis to introduce an amino group at the 5'-terminus of a DNA molecule and is shown by the formula:
The synthesis and purification of the above DNA fragments were carried out according to the teaching of the manufacturer.
C. Immobilization of DNA to Carrier
Immobilization of DNA obtained in the above B. to a particulate carrier obtained in the above A. is conducted by a method comprising the following steps:
1) dissolve DNA fragments (a) and (b) (400 µg, each) into 1 M NaCl (250 µl);
2) heat the solution of the above 1) at 90°C on a water bath and allow to stand for about 5 minutes at the same temperature;
3) cool the solution of the above 2) to 35°C over one hour;
4) add 0.4 M NaHCO&sub3; (pH 7.5) (250 µl) to the solution of the above 3) and mix thoroughly;
5) add tresyl-activated silica gel (250 mg) to the mixture of the above 4) and mix thoroughly;
6) stir the mixture of the above 5) at room temperature for 18 hours; and
7) prepare two types of DNA-immobilized particulate carriers in the following manner.
The mixture of the above 6) is divided into two parts and one of them is washed 3 times with a mixture of 0.5 M NaCl and 0.2 M NaHCO&sub3; (pH 7.5) to yield a carrier to which a double-stranded DNA consisting (a) and (b) is immobilized.
The other half is used to obtain carriers to which single-stranded DNA (a) and (b) is attached separately. Thus, the mixture is placed at 95°C for 5 minutes and rapidly centrifuged (12,000 rpm x 1 min) to separate the supernatant. To the solid phase are added 300 µl of 0.5 M NaCl and 0.2 M NaHCO&sub3; (pH 7.5), and the mixture is treated twice by the process consisting of 5 minutes-heating at 95°C and separation of supernatant by centrifugation, to give particulate carrier to which single-stranded DNA is attached. This type of carrier is mostly a DNA (a)-immobilized carrier.
The same procedure as the above is repeated using DNA fragments (c) and (d), and (e) and (f), respectively, and obtained two types of carriers for each pair of DNAs. Accordingly, there obtained six types of DNA-immobilized carriers including three double-stranded DNA-immobilized carriers and three single-stranded DNA-immobilized carriers for three pairs of DNAs, that is (a) and (b), (c) and (d), and (e) and (f).
2. Detection of Anti-DNA Antibody
Effectiveness of the DNA-immobilized particles obtained above in the detection of anti-DNA antibody was evaluated as follows.
As the specific anti-DNA antibody, anti-DNA monoclonal antibody was used. For comparison, non-specific antibodies, i.e., immunoglobulin G (IgG) and Immunoglobulin M (IgM) were used. The control trial was conducted using a sample without any antibodies.
The anti-DNA monoclonal antibody used in the present experiment was obtained by the process disclosed in the literature (for example, T. Koike et al; Immunology Letter 4 93 (1982)) and the details of the process are well known to one of skill. An auto-immune mouse having anti-DNA antibody producing cells is then sacrificed and spleen cells obtained are fused with myeloma cells, which result in hybrid cells referred to as hybridomas. From the hybridomas, a desired clone which secretes a single antibody species to the DNA is isolated so as to obtain the specific monoclonal antibody.
The assay, which uses the above DNA-immobilized carrier (prepared in the above 1., C.), is carried out as follows:
a) add DNA-immobilized silica gel to a 96-well microtiter plate at a concentration of about 5 x 10&sup5;/well;
b) wash the gel with 200 µl of buffer A [1% BSA (bovine serum albumin), 0.05 % Tween 20, 0.1 % NaN&sub3; and 100 mM Tris-HCl (pH 7.4)];
c) add 30 µl of a dilution (1 µg/ml) of anti-DNA monoclonal antibody to each well and allow the plate to stand for 60 minutes at room temperature;
d) wash the plate three times with buffer A;
e) add 20 µl of a labeled secondary antibody, a 1/150 dilution of goat anti-mouse IgG + IgM + FITC (TAGO, Co., Ltd.), to each well and allow the plate to stand for 30 minutes at room temperature;
f) wash the plate twice with buffer A;
g) add 400 µl of 1 % trifluoroacetic acid and 10 mM phosphate buffer to each well and mix thoroughly and transfer the content of the well to a test tube separately to obtain samples; and
h) detect the signal present on particles in each sample by flow cytometry using FACS tar (one of FACS series).
The above procedures were repeated using IgG or IgM instead of anti-DNA monoclonal antibody in the step c), and in the absence of antibody. Results are shown in the accompanying drawings, Figures 1 to 6.
In the figures, the number of particles are plotted on the ordinate and the level of fluorescence on the axis. The solid lines represent the detection pattern obtained in the experiments conducted using antibody-containing samples and the dotted lines represent those obtained in the control experiments conducted using antibody-free samples.
Figures 1 and 2 show the results obtained using carriers to which DNA (a) and (b) are immobilized as single-or double-stranded DNA, respectively. Figures 3 and 4 show the results obtained using carriers to which DNA (c) and (d) are immobilized as single- or double-stranded DNA, respectively. Figures 5 and 6 show the results obtained using carriers to which DNA (e) and (f) are immobilized as single-or double-stranded DNA, respectively. Throughout the Figures 1 to 6, the panel A depicts the binding effect of IgG, the panel B depicts that of IgM, and the panel C depicts that of anti-DNA monoclonal antibody. The order of the number of particles shown on the ordinate is 10,000.
When the DNA immobilized on a particle is effectively bound to antibody to be detected, the level of fluorescence of said particle increases, and the peak, as the total fluorescence of all the particles detected, should shift to the right. The extent of the shift indicates the facility or strength of binding between the immobilized substance (antigen) and other substance to be detected (antibody), in other words, it indicates the affinity of these substances.
From Figures 1 to 6, it can be easily seen that neither of the non-specific antibodies IgG and IgM, which lack the affinity to DNA, causes the shift of peak (panels A and B, respectively), whereas the specific monoclonal antibody causes a significant shift of peak (panel C). This demonstrates that the present method is effective for the detection of substance A which is specific to an immobilized substance B on microparticles.
In the present assay, the variation of data, that is, the variation of level of fluorescence from particle to particle, is reflected as the breadth of peak. Further, each particle can be regarded to be equivalent to one well of the currently used ELISA. Therefore, according to the present method, about 10&sup4; to 10&sup5; times of data can be obtained by only one experiment in comparison with ELISA. The data expressed as a peak are highly reliable and allow one to evaluate the affinity between two substances, for example, an antigen and antibody, correctly and efficiently.
Example 2 LYSOZYME-IMMOBILIZED CARRIER 1. Preparation of Lysozyme-Immobilized Particulate Carriers
A. As a solid carrier for the immobilization of lysozyme, SIMPACK diol 300 was activated with tresyl chloride in the same manner as described in Example 1, 1. A. The tresyl-activated silica gel was stored in a Kjeldahl flask until use.
Lysozyme (Funakoshi & Co. Ltd.) was immobilized as follows:
1) dissolve lysozyme (1 mg) into 500 µl of 0.5 M NaCl and 0.2 M NaHCO&sub3; (pH 7.5);
2) add 250 mg of tresyl-activated silica gel to the solution of the above 1) and mix thoroughly;
3) stir the mixture of the above 3) at room temperature for 18 hours;
4) wash the resultant silica gel three times with 0.5 M NaCl and 0.2 M NaHCO&sub3; (pH 7.5);
Thus obtained lysozyme-immobilized microparticles is then used for detecting the anti-lysozyme antibody as follows.
2. Detection of Anti-lysozyme Antibody
The evaluation of the effectiveness of the lysosyme-immobilized particulate carriers in the assay of anti-lysozyme antibody was conducted in the similar manner as described in the above Example 1.
The lysozeme-specific monoclonal antibody was prepared from hybridoma obtained by fusion of spleen cells of lysozeme-immunized mouse and mouse myeloma cells. Immunoglobulins G (IgG) and M (IgM) were also used and the control trial carried out using a sample without any antibodies.
The assay was carried out in accordance with the procedures described in Example 1. Thus, to a 96-well microtiter plate was added lysozyme-immobilized silica gel at a concentration of about 5 x 10&sup5;/well and washed with 200 µl of buffer A. After addition of 30 µl of a solution (1 µg/ml) of anti-lysozyme monoclonal antibody into each well, the plate was allowed to stand for 60 minutes at room temperature. After washing the plate with buffer A (x3), 20 µl of a secondary antibody solution, 1/150 dilution of a goat anti-mouse IgG + IgM + FIFTC (TAGO, Co., Ltd.) was added to each well and the plate was allowed to stand for 30 minutes at room temperature. The plate was then washed twice with buffer A. To each well were added 400 µl of 1 % trifluoroacetic acid and 10 mM phosphate buffer and mixed thoroughly. The content of each well was transferred into a test tube to give samples. Each sample was subjected to flow cytometry for the detection of signals present on the micropraticles using FACS tar.
The above procedure was repeated using IgG or IgM instead of anti-lysozyme monoclonal antibody, and in the absence of antibody. Results are shown in the accompanying drawing, Figure 7.
In Figure 7, the number of particles are plotted on the ordinate and the level of fluorescence on the abscissa. The solid lines represent the detection pattern obtained in the experiments using antibody-containing samples and the dotted lines represent those obtained in control experiments using an antibody-free sample. The panel A depicts the binding effect of IgG, the panel B depicts the binding effect of IgM and the panel C depicts the binding effect of anti-lysozyme monoclonal antibody. The order of number of particles shown on the ordinate is 10,000.
When the lysozyme immobilized on a particle is effectively bound to antibody to be detected, the level of fluorescence should increases, and the peak, as the total fluorescence of all the particles detected, should shifts to the right. The extent of the shift of the peak indicates the facility or strength of binding between immobilized lysozyme (antigen) and anti-lysozyme antibody, in other words, it indicates the affinity of these substances.
From Figure 7, it can be easily seen that neither of non-specific antibodies IgG and IgM causes the shift of peak (panels A and B), whereas the specific anti-lysozyme monoclonal antibody causes a significant shift of peak (panel C). The variation of data, that is, the variation of level of fluorescence from particle to particle, is reflected as the breadth of peak. Thus, the present assay, where each particle can be regarded as a well in ELISA, gives data as many as almost 10,000 times of those obtained by ELISA. The data obtained by the present assay are highly reliable, and it is possible to evaluate the affinity between an antigen and antibody more correctly.
The Example also demonstrates that the present method gives data with high reliability and is useful for the detection of a substance in a sample and evaluation of the affinity between an antigen and antibody correctly and efficiently.
Example 3 DNA-IMMOBOLIZED CARRIER 1. Preparation of DNA-Immobilized Particulate Carriers
As a solid carrier for the immobilization of DNA, a commercially available particulate organic polymer, Tresyl 5PW (TOSO, & Co. Ltd.), was used.
The following 27-mer DNA fragment (a) to be immobilized and its complementary sequence (b) were synthesized using DNA Synthesizer A-391EP PCR MATE (ABI, Inc.).
Immobilization of the above DNA fragments were conducted in the same manner as described in Example 1, 1. C. except that Tresyl 5PW (250 mg) was used instead of SIMPACK diol and two types of DNA-immobilized carriers, a carrier to which double-stranded DNA consisting of (a) and (b) have been immobilized and a mixture of carriers to which the single-stranded DNA (a) and (b) are immobilized, separately.
The evaluation of the effectiveness of the DNA-immobilized particulate carriers in the assay of anti-DNA antibody was conducted in the same manner as described in the above Example 1, 2.
The DNA-specific monoclonal antibody was prepared as mentioned above. As non-specific antibody, IgG was used.
The assay was carried out in accordance with the procedures described in Example 1, 2. Thus, to a 96-well microtiter plate was added DNA-immobilized silica gel at a concentration of about 5 x 10&sup5;/well, and the plate was washed with 200 µl of buffer A. After addition of 30 µl of a solution (1 µg/ml) of anti-DNA monoclonal antibody into each well, the plate was allowed to stand for 60 minutes at room temperature. After washing the plate with buffer A (x3), 20 µl of a secondary antibody solution, 1/150 dilution of a goat anti-mouse IgG + IgM + FIFTC (TAGO, Co., Ltd.), was added to each well and the plate was allowed to stand for 30 minutes at room temperature. The plate was then washed twice with buffer A. To each well were added 400 µ1 of 1 % trifluoroacetic acid and 10 mM phosphate buffer, and they were mixed thoroughly. The content of each well was transferred into a test tube to give samples. Each sample was subjected to flow cytometry for the detection of signals present on the micropraticles using FACS tar.
The above procedure was repeated using IgG instead of anti-DNA monoclonal antibody. Results are shown in the accompanying drawings, Figure 8.
In Figure 8, the number of particles are plotted on the ordinate and the level of fluorescence on the abscissa. The panels A and C show the results obtained using carriers to which DNA (a) and (b) are immobilized as single-stranded DNA, while the panels (B) and (D) show the results obtained using carriers to which double-stranded DNA are immobilized. The panels A and B depict the binding effect of IgG and the panels C and D depict the binding effect of anti-DNA monoclonal antibody. The order of number of particles shown on the ordinate is 10,000.
When the DNA immobilized on a particle is effectively bound to antibody to be detected, the level of fluorescence should increase, and the peak, as the total fluorescence of all the particles detected, should shift to the right. The extent of the shift of the peak indicates the facility or strength of binding between immobilized DNA (antigen) and anti-DNA antibody, in other words, it indicates the affinity of these substances.
From Figure 8, it can be easily seen that the non-specific antibody IgG does not cause the shift of peak (panels A and B), whereas the specific anti-DNA monoclonal antibody causes a significant shift of peak (panels C and D). The variation of data, that is, the variation of level of fluorescence from particle to particle, is reflected as the breadth of peak. Thus, the present assay, where each particle can be regarded as a well in ELISA, gives data as many as almost 10,000 times of those obtained by ELISA. The data obtained by the present assay are highly reliable, and it is possible to evaluate the affinity between an antigen and antibody more correctly.
Anspruch[en]
A method for qualitatively or quantitatively measuring substance A in a sample solution, which comprises:
contacting the particulate carrier to which the substance B has been immobilized with the sample solution suspected to contain the substance A; and
detecting said substance A bound to the particulate carrier through the substance B by means of flow cytometry.
The method of Claim 1 wherein the substance A is an antibody and the substance B is an antigen.
The method of Claim 2 wherein the substance B is a DNA and the substance A is an anti-DNA antibody.
The method of Claim 1 wherein the particulate carrier is made of polymer.
The method of Claim 4 wherein the particulate carrier is made of organic polymer.
The method of Claim 4 wherein the particulate carrier is made of inorganic polymer.
The method of Claim 1 wherein the particulate carrier is made of metal.
The method of Claim 1 wherein the particulate carrier is made of ceramics.
The method of Claim 1 wherein the particle size of the particulate carrier is from about 0.1 to 100 µm in diameter.
The method of Claim 3 wherein the DNA is immobilized at the terminus.
The method of Claim 1 wherein the particulate carrier is silica gel.
The method of Claim 1 which comprises:
immobilizing substance B capable of specifically binding with substance A to a particulate carrier;
contacting said particulate carrier to which the substance B has been immobilized with a sample solution containing the substance A; and
The method of Claim 12 wherein the substance B is an antigen, substance A is an antibody thereto and substance C is a secondary antibody thereto.
A particulate carrier to which substance B capable of binding to substance A to be detected is immobilized.

redpajama_set_name: RedPajamaCommonCrawl


text:
La Tirrena-Adriàtica 1969 va ser la 4a edició de la Tirrena-Adriàtica. La cursa es va disputar en cinc etapes, la darrera d'elles dividida en dos sectors, entre l'11 i el 15 de març de 1969, amb un recorregut total de 946,3 km. El vencedor de la cursa fou l'italià Carlo Chiappano (Sanson), que s'imposà en la general amb 31" sobre el segon classificat, el belga Albert van Vlierberghe (Ferretti). L'italià Giuseppe Fezzardi (Sanson) acabà en tercera posició.

Etapes

Classificació general

Referències

Enllaços externs
 Web oficial

1969
Competicions ciclistes del 1969

redpajama_set_name: RedPajamaWikipedia


text:
The Australasian (Melbourne, Vic. : 1864 - 1946)
Sat 9 Dec 1876
Sat 9 Dec 1876 - The Australasian (Melbourne, Vic. : 1864 - 1946)
Page 26 - Family Notices
ANNOUNCEMENTS.
BIRTHS.
AGNEW.—On the 1st inst., at Tennyson-villa, Tenny-
son-street, St. Kilda, the wife of Andrew Agnew of a
daughter.
ARKINS.—On the 20th ult., at Avoca, Mrs. P. R.
Arkins of a daughter.
BLACK.—On the 12th ult., at The Cedars, Queensland,
the wife of Maurice Hume Black of a daughter.
BOSTOCK.—On the 29th ult., the wife of Robert Bostock
Line 0.10.0
of a son.
BOSTOCK.—On the 29th ult., at Upton-house, Chapel-
street, East St. Kilda, the wife of Robert Bostock of
a son.
BALL.—On the 29th ult., at No. 101 Drummond-street,
Carlton, Mrs. Charles Ball of a daughter.
BLASHKI.—On the 2nd inst., at 120 Bourke-street east,
the wife of Mr. P. Blashki of a daughter.
CHRISTIE.—On the 27th ult., at 19 Lyon-street, Bal-
larat, Mrs. L. S. Christie of a son.
COPPEL.—On the 30th ult., at 7 Victoria-buildings,
Queen-street, the wife of E. Coppel of a daughter.
DICKINS.—At Erin-street, Richmond, the wife of Mr.
Robert Dickins of a son.
DICK.—On the 1st inst., at No. 5 Burlington-terrace,
Albert-street, Mrs. Alexander Dick prematurely of a
daughter, stillborn.
DREDGE.—On the 4th inst., at North Fitzroy, the wife
of T. J. Dredge of a son.
DE GROOT.—On the 22nd ult., at 30 Simpson's-road,
Mrs. F. A. De Groot of a daughter. Both doing
FINK.—On the 3rd inst., at 2 Albert-terrace, Albert-
street, East Melbourne, the wife of B. J. Fink of a
GRICE.—On the 1st inst., at Longfield, South Yarra,
Mrs. James Grice of a son.
JAMES.—On the 1st inst., at 40 Union-street, Windsor,
the wife of Henry James of a son.
JOURNEAUX.—On the 26th ult., at Burwood-road, Haw-
thorn, Mrs. J. B. Journeaux of a son.
KNEEN.—On the 15th ult., at Mount Pleasant Creek,
the wife of Thomas D. Kneen of a daughter.
KEMP.—On the 1st inst., at her residence, 133 Nelson-
road, Emerald-hill, Mrs. Alfred Kemp of a son.
MORGAN.—On the 29th ult., at Moonee Ponds, the
wife of Wm. Morgan, formerly of Sandridge, of a
MACDONALD.—On the 3rd inst., at the Manse, Toorak,
the wife of the Rev. M. Macdonald of a son.
MURRAY.—On the 27th ult., at Kilmore East, the wife
of D. Murray of a daughter.
O'SHEA.–On the 25th ult., at No. 22 Davis-street,
South Yarra, wife of Mr. John O'Shea of a son.
Wagga papers please copy.
PICKERING.–On tbe 10th ult., at Pound-cottage.
Smeaton-road, Clunes, the wife of A. Anderson
Pickering of a son. " Hobart Town Mercury" please
copy.
ROSE.–On tbe 30th ult., at Williamstown, Mrs. A. R
Rose of a daughter.
STAINER.–On the 4th inst., at Tarcoola-cottage, Wilson
street, South Yarra, the wife of Mr. Frank Stainer
SALTER.–On the 29th alt, at Mair-street, Ballarat,
the wife of Mr. C. Salter of a daughter.
SLATTERIE.–On the 4th inst., at Burwood-road, Haw
thorn, the wife of A. Slatterie of a son.
TEMPLETON.–On the 22nd ult., at Walhalla, the wife
of A. Templeton of a daughter.
WRIGHT.–On the 9th ult., at Millswyn-street, South
Yarra, the wife of George Robert Wright of a son.
MARRIAGES.
BAIRD—HASTIE.–On the 18th ult., at the Scots' Pres
byterian Church, Collins-street, Melbourne, by
the Rev. Charles Strong, Mr. William Thomas
Baird, of Glasgow, to Mary Ann Jane, youngest
daughter of the late George Hastie, of Glasgow,
BLAKE—JONAS.—On the 23rd ult., at the residence of
the bride's mother, by the Rev. B. Connebee, Wil
liam Adams Blake, of Ivanhoe, to Emmeline, the
eldest daughter of the late John Jonas, of Burwood-
road, Hawthorn.
COOK—PEACOCK.–On the 2nd inst., at St. Peter's
Church, Melbourne, by the Rev. Canon Handfield,
Charles Henry Herbert Cook, M.A., Fellow of St.
John's College, Cambridge, and professor of
mathematics, Canterbury College, Christchurch,
New Zealand, to Emily Denman Peacock, of Watford.
Herts, England.
DIXON—M'CALL.–On the 25th ult., by Mr. H. L.
Geeslin, Frederick, eldest son of Levi Dixon, of Col
lingwood, to Agnes Mitchel, eldest daughter of John
McCall, of Flemington-road.
EDWARDS—ARNALL.–On the 2nd inst., at the resi-
dence of the bride's brother, Clifton-street, Rich
mond, by the Rev. Spencer Williams, Peter Henry
Edwards, late of Guernsey, to Jemima Elizabeth,
only daughter of the late George Arnall, of Camel
pond, Cornwall. Home papers please copy.
HUME—RAE.–On the 22nd ult., at St. Paul's Church,
Melbourne, by the Rev. Canon Dickinson, M.A.,
Thomas, second son of the late Andrew Wilson
granddaughter of the late John Rae, captain 72nd
Regiment Highlanders.
KENYON—SCOTT.–On the 9th ult., at the resi-
dence of the father of the bride, by the Rev. James
Lindsay, of Chalmers Church, Henry Kenyon. of
Liunceston, son of tbe late Henry Kenyon, Esq., of
the Park, Alston, Cumberland,to Clementina, second
daughter of James Scott, Esq, J.P., MTLA., of
Bowhill, near Launceston, Tasmania.
M'LEOD—FLYNN.–On the 28th ult., at St. Patrick's
Cathedral, by the Rev. P. Aylward, Francis M'Leod
to Mary Flynn, third daughter of Denis Flynn.
OFFICER—BESNARD.–On the 28th ult., at St. Peter's
Church, East Melbourne, by the Rev. H. H. P.
Handfield, Charles Myles Officer, of Mount Talbot, to
Ellen Agnes Bernard.
WALLDER—TRENOWETH.–On the 22nd ult., at Kilmore,
by the Rev. J. W. Tuckfield, Charles Edward
Wallder, to Bessie, eldest daughter of William Tre
noweth, Burnside, late of Heidelberg.
WILSON—FULLER.–On the 14th ult., at Gore-street,
Line 0.100.0
Fltsroy, by tbe Rev. James Taylor, Henry, second
son of Mr. G. G. Wilson, of Melbourne, to Flora
Ellen Bourke, eldest daughter of the late Captain
James Fuller, of Sydney. Sydney papers please
DEATHS.
AYLING.–On the 9th ult., at her residence, North
Bow, London, Jane, wife of Captain T. B. Ayling,
ship Aviemore.
BRENNAN.–On the 24th ult., at Melbourne Hospital,
John Brennan, of Williamstown, aged 62 years,
brother of the late Rev. J. D. Brennan, of Sydney.
BUNNEY.–On the 26th ult., at Alexandra, Adam
Bunney, storekeeper, Gobur, aged 80 years. Home
papers please copy.
BAINBRIDGE.–On the 5th inst., at Everton-cottage,
Brunswick-street, North Fitsroy, John, the infant
son of William Henry and Eliza Emma Bainbridge,
aged two days.
BAXTER.–On the 5th inst., at St. Kilda, of apoplexy.
William Baxter, aged 66 years.
BLUNDELL.–On the 8th inst., at his residence, Church-
street, Brighton, James John Blundell, sen., aged
DAVIDSON.–On the 20th ult., at Warbreccan, in his
69th year, Walker Rannie Davidson, formerly Sur-
veyor-General of New South Wales.
DEERING.—On the 28th ult, at Longwood Railway
Station, William John, eldest son of John Deerlng,
aped nine years, accidentally drowned. Dublin
DOUGLAS.–On the 26th March, at the Glebe, Sydney,
John Casement, fourth son of William Casemeut
Douglas, Isle of Man, nephew to the late Major
General Sir W. Casement, K.C.B., H.E.I.C.S.,
Bengal, aged 72.
EASTHAM - On the 15th ult., of typhoid fever, at
Wheatsheaf Hotel, Dunolly, Daneil Eastham, of
Berlin and Burke's Flat, aged 48 years.
GOLDING.–On the 30th ult., at Melbourne, Daniel
Golding, aged 24 years.
HOSKING.–On the 3rd inst., at St. Kilda College,
Bobe-street, of congestion of the brain, Annie
Louise, the only child of Alfred and Carolina
Frances Hosking, aged nine mouths.
HEALY.–On the 9th ult.. at the residence of her
aunt, 47 Lyons-street, Ballarat, of congestion of the
lungs, Annie Healy, of Swanston-street, Melbourne,
relict of the late James Norman Healy, of Ballarat.
HOLT.–On the 17th ult., at Ledcourt Station, near
Stawell, John Holt, Esq , aged 47 years.
LEVINSON.–On the 2Sth ult., at the residence of her
son-in-law, E. Steinfeid. Esq., J.P., Drummond
street, Carlton, Bertha, relict of the late M. Levin
son, Esq., of Sheffield, England, aged 60 years,
deeply regretted.
LEE.–On the 30th ult., at the residence of her
brother, P. M. Lee, Westport-terrace, Reilly-street,
Carlton, Miss Abigail Mary Lee, aged 26 years, late
of New Zealand, second youngest daughter of John
and Mary Lee. New Zealand papers please copy.
LEVER.–On the 1st inst., at his late residence, 58
Bouverie-street, Carlton, Benjamin Robert Lever,
much regretted.
MILLER.–On the 4th inst., at her residence, corner of
Cardigan and Queensberry streets, Carlton, Eliza
Ann, the beloved wife of Mr. David Miller, and
daughter of the late William Mills, County Armagh,
Ireland, aged 31. Home papers please copy.
MARLOW.–On the 5th inst., at 1 Earnbank-terrace
Cardigan-street, Carlton, suddenly, of apoplexy
Douglas Marlow, aged 69 years.
O'ROURKE.–On the 29th ult., at her residence, 63
Rose-street, Fitzroy, Mary, the beloved wife of Mr.
Michael O'Bourke, aged 49 years. B.LP.
O'DRISCOLL.–On the 1st inst., at the Silver Gate
Hotel, Emerald-hill, Mary, the beloved daughter of
William and Hannah O'Driscoll, aged 13 months.
PRUEN.–On the 23th ult., at Wangaratta, Charles
Augustus Pruen, of dysentery, after six days' illness,
aged four years and 10 months.
RONALD.–On 11th ult., at sea, on board the Antiope.
Mary, the beloved wife of R. M. Ronald, late of
Karyrie Station, Morton-plains, aged 30 years.
RICHARDS.–On tbe 2nd inst., at her late residence, 14
Lewisham-road, Prahran, Eliza, the beloved wife of
tbe late John Richards, aged 75, formerly of Hull,
Yorkshire, and Nottingham, England. Home papers
please copy.
STEWART.–On the 29th ult., at No. 6 Barkly street,
Carlton, after a long and painful illness, of pul
monary consumption, Henry Thomas Stewart,
youngest son of the late Mr. M. Stewart, C.B , H.M.
Coastguard Service, Stoke-road, Gosport, Hants, and
brother of L. Stewart, Lygon street, Carlton, aged
STEWART.–On the 1st inst., at his residence, Pasco
street, Williamstown, Mr. Alex. Stewart, ship
chandler, aged 36 years.
SMITH.–On the 29th ult., at his residence, Inkerman
street, St Kilda, William Stokes Smith, of con
gestion of the lungs, aged 38. Beaufort papers
TURNER -On board mission ship John Williams, off
New Guinea, Mary, the beloved wife of Rev. W. Y.
Turner, of the New Guinea mission, son of Rev. G
Turner, LL.D., of Samoa, and brother-in-law of
Rev. S. J. Whitmee. News received by telegram.
Particulars not yet arrived.
Family Notices (1876, December 9). The Australasian (Melbourne, Vic. : 1864 - 1946), p. 26. Retrieved January 21, 2022, from http://nla.gov.au/nla.news-article142995573
"Family Notices" The Australasian (Melbourne, Vic. : 1864 - 1946) 9 December 1876: 26. Web. 21 Jan 2022 <http://nla.gov.au/nla.news-article142995573>.
1876 'Family Notices', The Australasian (Melbourne, Vic. : 1864 - 1946), 9 December, p. 26. , viewed 21 Jan 2022, http://nla.gov.au/nla.news-article142995573
{{cite news |url=http://nla.gov.au/nla.news-article142995573 |title=Family Notices |newspaper=[[The Australasian]] |volume=XXI, |issue=558 |location=Victoria, Australia |date=9 December 1876 |accessdate=21 January 2022 |page=26 |via=National Library of Australia}}
The Australasian (Melbourne, Vic. : 1864 - 1946), Sat 9 Dec 1876, Page 26 - Family Notices


If you want to breathe the purest air, photograph deer, and learn the importance of the páramo for Bogotá, you have to go to Chingaza Natural National Park. If you're lucky enough, you might meet the most popular guy in the area: the Andean bear.
The purest air, joyful deer, and stunning páramo landscapes can all be found an hour and a half or so from Bogotá. Where exactly? At the Chingaza Natural National Park. If you are thinking of a full day plan to connect with nature or you want to add some ideas to your "Things to do near Bogotá" list, I truly recommend this visit to you.
Here are four reasons why you should book a getaway to this place as soon as you finish this article.
When visiting the páramo zone, you feel like you are embraced by the mountains. The flora is rich and diverse, the color of every landscape varies depending on the light and rain, and the immenseness and beauty of the place really grounds you. It happens especially at the Alto de la Mina de Palacio, one of the highest points in the park.
Don't you like to see cute animal videos? At Chingaza, you can watch them live and in person. The most common ones to find are the playful deer that graze and frolic through the páramo. Different kind of birds can be seen as well.
If you are lucky enough, you could meet the most popular guy in this zone: the imposing Andean bear. Like many other species, this wonderful animal is endangered; one of the reasons Chingaza is a Natural National Park is to protect it.
© Photograph by Giovanny Pulido – Minga Expeditions.
You probably love Bogotá, but that doesn't mean you won't want to escape from its fast pace sometimes; which is normal for everyone who is accustomed to big cities. At Chingaza, you will find peace. Its silence and the breeze that sways every plant and every flower will reconnect you with your inner self. It's a relaxing experience in more than one sense of the word. It calms not just your brain, but your soul.
An important part of the water we get in Bogotá comes from Chingaza. It's an interesting fact even when you don't see it, but when you are there and realize how important this place is for our daily life, you get to appreciate it even more.
Maintaining responsible consumption of the water is one way to take care of Chingaza. Since I went there, I can't avoid thinking of this place every time I open the tap or take a shower. Every experience that makes us more aware about our environment is sure a great experience, right?
There is no public transportation to the park. If you want to go by yourself, you must do it in a private car. You can get there from Bogotá through La Calera.
If you want to go by yourself, you have to do an entrance request 15 days in advance before going to the park. You should write an email to [email protected] or call 3532400 ext. 3011 and 3012. You can also go the office (Calle 74 # 11-81) in Bogotá.
If you want to stay at the hostel in Chingaza or go camping (or take food or use tour guide services), please communicate with Corpochingaza, the community ecotourism corporation of the park (cell phone number: 3508044199).
I went with Minga Expeditions on its #DíaDeVenados (#DayOfDeer) tour. They take care of everything (transportation, food, park entrance, tour guides, etc.). Their approach to the place is based on respect and care. I loved the experience and truly recommend it!
It's important to wear warm and comfortable clothes. If you have trekking boots, that would be best, although a good pairs of sneakers will work too. The altitude can affect certain people, so Minga Expedition recommends to have some snacks, such as chocolate or cereal (I took fruits, water, and sports drinks in my bag). It might be rainy, so a raincoat might be helpful as well. Sunscreen is also vital!
In any case, the best tip I can give you for your visit is the following: do not leave any kind of trash at the park! Take everything with you when you leave and throw it away in your trash can when you get back to Bogotá.
Are you excited about Chingaza? Let me know if you decided to go after reading this article (it would make me really happy)! Have you already been there? Please tell me about your experience in the comments section below!
Keep visiting Colture for more information on other spectacular places to visit near to Bogotá!


MINGUS AT CHICAGOLAND… Billy Boat Motorsports NASCAR Camping World Truck Series (NCWTS) driver Mason Mingus is behind the wheel of his No. 15 Call 811 Before You Dig Chevrolet Silverado this weekend at Chicagoland Speedway.
RACE RECAP… The 20-year-old was last on track in the NCWTS at the Canadian Tire Motorsports Park on August 30. The No. 15 Call 811 Before You Dig Chevy finished 14th, capturing the 4th top-15 finish in the 2015 NCWTS season.
CALL 811… Common Ground Alliance is the leading association created specifically to work with all industry stakeholders in an effort to prevent damage to underground utility infrastructure and ensure public safety and environmental protection. Along with the new 811-number, CGA and its 1,400 members and sponsors have launched the national "Call Before You Dig" campaign to increase public awareness about the importance of using 811, having utility lines marked before digging and protecting America's vast underground infrastructure of pipelines, conduits, wires and cables. Learn more at www.call811.com and www.commongroundalliance.com.


The temperature is swinging wildly from warm to cold these days.
These pix were taken next to the parking of LL Shihoya Arai School on Sunday, March 24th. This is "Agricultural Water and Erie".
This water was once, when Yoshy was a child, not made by three-sided concrete and… I remember that my mom was washing clothes, Yoshy was taking pond-snails, American-crawfish and so on with my friends in that warming water.
And I think that time was flowing leisurely.
There is already only one scenery which hasn't changed yet; it might be "Myoko mountain range in which we can now see Black Prancer", mightn't it?
RSVP by Fri. April 5th; we want you to e-mail to Chairman, Futoshi Hosaka's:foshim10@yahoo.co.jp , please. Hosaka is now checking your newest e-addresses; be sure to reply to him, please.


text:
Players To Watch This Spring — Part One
The Rays rode a bus (or two) all the way from Port Charlotte to Sarasota yesterday and played their first game of the year. They got their head handed to them by Baltimore, 12-2. Because it's Spring Training, and, after all it's the Rays, we aren't too interested in the outcomes of these games.
On the other hand, as Bulls fans, several players who will be showing up in the lineups are of interest to us. So we'll be checking the box scores to see how they are doing.
Here's the beginning of my list of position players worth watching. I'm not going to try to speculate on pitchers, too many variables.
By "worth watching" I mean that there's some chance that we could see players on this list in Durham this year. Each has a shot at the Rays 40-man, or may currently be on it. Some have already gotten some attention on the web and I'll provide a link or two in that event. I'm not going to dig into contractual stuff — I don't understand it all that well and sometimes the information is just too hard to get to.
I'll start with the "non-roster invitees", that is, players who are in camp and playing with the team, but not on the Rays' 40-man roster.
Ryan Shealy, 31, 1B, minor league contract, with the Royals and their AAA team in Omaha last year. Stats.
J. J. Furmaniak, 31, infielder, minor league contract. We saw him playing for the Phillies' AAA team Lehigh Valley last year. Stats.
Joe Dillon, 35, infielder, minor league contract. Super-solid player with the Bulls virtually all of last year. Trying to extend his range by working in the catcher position this year. Stats and more.
Chris Richard, 36, 1B, minor league contract, NRI. Chris is a stalwart of the Durham Bulls. He got a few at-bats with the Rays last year, but we can expect to see him back in Durham this year — and he's very, very welcome. Stats.
Angel Chavez, 29, 3B, minor league contract. Played for Boston AAA affiliate Pawtucket last year. Stats.
Tim Beckham, 20, SS. Top prospect making his way up through the system. Possible he might show up in Durham near the end of the season, but Rays will put an emphasis on keeping him healthy and developing his skills. Stats.
Craig Albernaz, 28, catcher, NRI. Albernaz spent virtually all of 2009 with the Bulls, but didn't get a lot of time behind the plate. Exceptional arm. Stats.
Nevin Ashley, 26, catcher. Ashley is a prospect working his way up through the Rays system. He spent last season with the Rays' AA affiliate, the Montgomery Biscuits and the Charlotte Stone Crabs, the Rays' A affiliate. Stats.
Alvin Colina, 29, catcher. Colina is a veteran AAA ballplayer who was with the Braves' affiliate, Gwinnett, last year. Stats.
For more on prospects Ashley and Beckham, check out the Rays Prospects website.
Next: Players on the 40-man.
Posted by Chris W. at 9:24 AM
Pitching Phenom
Pitchers Named for Saturday Exhibition Game
How They're Doing — 5
Durham Bulls Basics — 4 — The 40-Man Roster
Durham Bulls Basics — 3 — The Schedule
Bulls Basics — 2 — The International League
Durham Bulls Basics — 1 — AAA Baseball
Watching The Rays
Fernando Perez, and More
Bats, Etc
How They're Doing
Players to Watch This Spring — Part Two
Rays/Yankees on TV Friday (March 5)


text:
Oral History Interview with Raoul Harmelin
Raoul Harmelin, born September 11, 1924 in Boryslaw, Poland (Boryslav, Ukraine), describes being the only son of a doctor; receiving both a secular and a Jewish education; his pre-war life in Boryslaw; life under the Russian occupation; life under the Germans after June 1941, including the pogroms, anti-Jewish measures, attitude of the local population, and formation of forced labor battalions organized by the Judenrat (Jewish council); a series of Aktions (roundups and mass murders of Jews) from November 1941 to 1943, and the murder of 600 Jews in Doly; how some Aktions were conducted by a German Vernichtung Kommando under General Katzman; how Polish and Ukrainian locals, Austrians in the Schutz Polizei, and Reiterzugpolizei, the Polish Kriminalpolizei, and Jews in the Ordnungsdienst all helped to round up Jews; the transfer of Jews to a camp at Ulica Janowska in Lwow (L'viv, Ukraine) or forced labor in local industry; how most Jews were transported to and murdered in camp Belzec; escaping from a roundup and witnessing the murder of an infant and a young girl; his father continuing to work because Jewish doctors were needed to treat the citizens of Boryslaw; hiding with his mother with the help of one of his patients; the creation of a ghetto, which was liquidated after a forced labor camp for Jews was opened in 1943; how Jews who could not hide were murdered or worked as slave laborers in the Zwangsarbeitslager in Boryslaw; working in connection with the war effort and having some degree of protection; getting news from London via radio and from an underground paper published by the Armia Krajowa (Polish Home Army); how an Ukrainian acquaintance hid 13 Jews, including Raoul and his parents from March 13, 1944 to August 8, 1944, when the Russians came back; his postwar life under the Russian occupation, including two arrests and escape to Breslau (Wroclaw, Poland); deciding with his parents to leave Poland after a pogrom in Kielce; staying in Paris, France, aided by HIAS; going to Sidney, Australia in November 1947; bringing his new wife and her parents to Australia later; his life in and adjustment to Australia after a very difficult beginning; his relatives on both sides of his family who were killed or survived; and the actions of non-Jews during the Holocaust.
Interviewer: Edith Millman
Interviewee: Raoul Harmelin
Subject: Forced labor.
Hiding places--Ukraine.
Holocaust, Jewish (1939-1945)--Ukraine--Personal narratives.
Infanticide--Ukraine--Boryslav.
Jewish councils--Ukraine--Boryslav.
Jews--Legal status, laws, etc.--Ukraine.
Jews--Persecutions--Ukraine.
Jews--Ukraine--Boryslav.
Mass murder--Ukraine.
Massacres--Ukraine.
Pogroms--Ukraine.
World War, 1939-1945--Conscript labor.
World War, 1939-1945--Deportations from Ukraine.
World War, 1939-1945--Underground literature--Ukraine.
Location: Boryslav, Ukraine
Permalink: https://hoha.digitalcollections.gratzcollege.edu/item/oral-history-interview-with-raoul-harmelin/


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This part of IEC 61280 is applicable to the measurement of attenuation and optical return loss of installed optical fibre cable plant using single-mode fibre. This cable plant can include single-mode optical fibres, connectors, adapters, splices and other passive devices. The cabling may be installed in a variety of environments including residential, commercial, industrial and data centre premises, as well as outside plant environments.
This standard may be applied to all single-mode fibre types including those designated by IEC 60793-2-50 as Class B fibres.
The principles of this standard may be applied to cable plants containing branching devices (splitters) and at specific wavelength ranges in situations where passive wavelength selective components are deployed, such as WDMs, CWDM and DWDM devices.
This standard is not intended to apply to cable plant that includes active devices such as fibre amplifiers or dynamic channel equalizers.


text:
The Circle Voice
Faculty and Student News
Lectures and Events
Artist Of The Issue
Athlete Of the Issue
News and Commentary
Vol. 50 Number 1
Revisiting Trump's Letter in the Wake of Midterm Elections
Powers Trigg '20, Assistant Sports Editor|November 10, 2018
Zenande Mdludlu '21
Groton has always had a history of public service. There is perhaps no greater emblem of that tradition than the letters and portraits from the last nineteen presidents of the United States that line the halls of the History Wing.
Since the election of Donald J. Trump in the fall of 2016, a debate has ensued about whether a letter from the 45th President should hang in the Schoolhouse. We learned in recent weeks that the letter has arrived although the picture frame has not. As we await final placement of the letter, the timing is ripe to revisit the underlying issues that have animated a months-long debate.
Endicott Peabody saw public service as part of a larger Groton mission, Cui Servire Est Regnare. He was close to Teddy Roosevelt and officiated at Franklin Roosevelt's wedding to Eleanor. Over the decades, Groton alumni have made public service part of their post-university pursuits, with three graduates re-elected this week as members of the House of Representatives: Jim Cooper (D-TN), George Holding (R-NC), and Bobby Scott (D-VA).
However, the environment in which leaders like Cooper, Holding and Scott serve is an increasingly toxic and divisive one. The nonpartisan Pew Research Center has conducted a survey on political values every year since 1994. In 1994, Pew found the average ideological divide to be 15 points. In 2018, the gap has more than doubled to 36 points.
At its ugly worst, this division leads to extremism and even violence. We saw it in the recent pipe bombs mailed to prominent national Democrats, and in the shooting of Republican Rep. Steve Scalise by James Hodgkinson, a left-wing activist, during a practice for a congressional baseball charity event.
While polarization at Groton is nonviolent, the Trump election exposed a stark contrast in opinion within the school community. Zizi Kendall '17, then head of the Young Democrats, called first for students to wear black in protest of Trump's election, then championed a statement of protest to be hung opposite Trump's letter. Sully Hamadan '17 responded to Zizi's call for a "statement of values" by claiming it was a case of selective outrage, as Groton has portraits of other controversial leaders gracing its walls.
Now, over 700 days since Trump was elected, his letter and portrait are set to be placed in the Schoolhouse. What are the lessons to be taken away from the debate?
First and foremost, Groton needs to be a place where opinions can be shared and debated. In his famous Four Freedoms speech, FDR called free speech one of the "four essential human freedoms." Over Parent's Weekend, Headmaster Maqubela followed in that tradition by reading the contents of the letter from President Trump aloud. He didn't hide the letter in a desk drawer or worry that students would be traumatized by its content (which was entirely appropriate to the office and in keeping with the tradition). He read it in full.
In addition, Groton must be a place where ideas can be shared independent of where they fall on the political spectrum. As Frederick Douglass said, a failure to hear the thinking of all violates "the rights of the hearer and those of the speaker." In the spring of 2017, a survey of Yale University students found "42 percent are 'not comfortable' sharing their ideas on politics, race, religion or gender, either in class or on campus." Moreover, 76 percent of conservatives "felt intimidated to share [their] ideas, opinions and beliefs in class." The free exchange of ideas must be bedrock of the Circle.
Finally, as we move from the 2018 midterms to the onset of the 2020 campaign, Groton should continue to fulfill Peabody's founding belief in the value of public service. On Tuesday, according to the exit polls, 18-to-29-year-olds were 13 percent of the total electorate, up from 11 percent in 2014. They voted Democrat by 37 percentage points, the widest margin of any age demographic. As the saying goes, "You can either vote by voting or vote by not voting. It's math." While the blue wave that was predicted for this election was not recognized in full, with Republicans gaining seats in the Senate and keeping their majority, the mobilization of young voters was a key factor in the Democrats winning back the House. This balance of power in the U.S. legislature suggests that we will see either see more civil discourse across party lines or extreme polarization and demonization of the opposing side. I hope Congress, and Groton School, choose the former.
When the next presidential letter and portrait arrive at the Schoolhouse and are ready to be placed in the History Wing, let it still be said that Groton is an institution where inclusion spans the ideological spectrum, where all viewpoints are respectfully heard and where students not only have opinions about the political process, but take the time and energy to shape it.
Powers Trigg '20, Editor in Chief
Erosion of Democracy
Should We Livestream Chapel?
Why I Would Send my Daughter to Groton
Time to Abolish Intervis
Peer Counseling: An Analysis
Civil Rights For The LGBTQ Community
In 2013, Gerald Bostock was fired for "conduct unbecoming" after he joined a predominantly gay softball league that was created as a safe space fo...
Piracy of Privacy: FBI Subpoenas
Not long ago, times were simpler –– at least technologically. Personal information was kept in cards, logs, and fiche film, and digital privacy la...
Protecting British Democracy: Boris Johnson Should Resign
On September 24, the Supreme Court of the United Kingdom unanimously ruled that Prime Minister (PM) Boris Johnson's suspension of Parliament was unl...
Should the Hong Kongers Keep Protesting?
On September 4, after months of protests, the Chief Executive of Hong Kong Carrie Lam withdrew the controversial extradition bill that allowed Hong Ko...
Destruction of the Presidency: Anderson's Authority
It is an impressive feat to fully extinguish all the prestige and tradition of an office, especially such a vaunted one as the presidency, in only thr...
Buddhist Sangha Fails to Meet Student Needs
Ecocide in Groton's Backyard
Two Views: Should the Headmaster Be Able to Overrule the D.C.?
Finding Your Space in Third Form
Should Senior Prefects Campaign? Two Views.
The Student Newspaper of Groton School


Find Ridgeville, South Carolina SC local builder, building companies, and building contractors near you for residential and commercial building repair or new building. Get free estimates, bids, and quotes.
Palmetto Custom System Homes Inc.


Aditya Varadarajan (Dulquer Salmaan) is a video game developer who accidentally meets Taara Kalingarayar (Nithya Menen) at his friend/colleague's wedding. He later finds out she is planning to pursue her studies in Paris. They soon begin to call each other often, go out together, and start to fall in love during a holiday they take together. They agree that marriage is not for them and decide to have a live-in relationship instead, and Taara moves into Adhi's place, rented from Ganapathy (Prakash Raj). They wish to spend their time with each other before they go their separate ways for their respective careers. Taara and Adhi live happily, being playful with each other.
One midnight, Tara has a conversation with Ganapathy. She tells him that she might get her visa approved for going to Paris, and that it's hard for her to leave Adhi and go far away. The next day Taara goes to Jaipur for her business, and Aditya starts to miss her terribly. Upon seeing the love and patience his houseowner Ganapathy has for his wife, Bhavani (Leela Samson), an Alzheimer's patient, who one day even forgets the way to her own house, they begin to understand the importance of each other's presence in their life.
Soon, Adhi is selected to develop his skills in video games and to go United States. He tells his colleague that it's hard for him to leave Taara. The next day, both of them make an agreement to enjoy themselves to the maximum for the 10 days left before they separate. Nearing the last day, they begin to have little fights and arguments, which don't last long.
One day, Bhavani goes missing again, as she did when she forgot her house address. While looking for her, they continue arguing about their relationship and finally Adhi proposes to Taara and they decide to get married. Post marriage, both of them continue to accomplish their own dreams, one in Paris, another in US. And they live happily, married and in love.


So the stork has delivered the precious package and guess what? There is a new permanent guest in the house—your baby! So now what happens when your old college friends come from far away to celebrate baby's arrival? Or when, for instance, Grandma and Grandpa want to come stay and help you with the wonderful new addition to the family? While some people have the luxury of a guest bedroom in addition to their nursery, others have to get a little more creative and carve out a double duty use of the space, for baby and house guests alike to share.
When BabyCenter asked Project Nursery to help this couple with their specific design dilemma as part of the DIY Dream Nursery 4-Part Video Series we were confident we could help!
While the baby occupies the room most of the time, it's still nice to know you can offer your visitors a place to sleep. We started our work by looking at the footprint of the room to figure out the best layout for a nursery while keeping a sleeping option for guests an option.
Some of the tips included keeping the changer in the closet to create more space and also to choose decor items like a chalkboard, so you can customize your messaging either to baby or to adults. For a full list of our tips on how to successfully create a multifunctional nursery, watch this video and read the full article here. You might just get inspired to try to do the same thing in your house. Watch out, you'll have visitors knocking down your doors in no time!


Looking for a way to plan your Hot Docs viewing? We're breaking down some of the festival's 11 film programmes for you, one by one.
The human faces of America's broken health care regime.
A gangbanger father threatens to saddle his kids with some serious sins.
Like The Wire, but with wheelies.
From Palestinian yearnings, a subtle poetry.
Because Toronto's more movie obsessed than a Quentin Tarantino screenplay (yuk yuk), Torontoist brings you Now on Screen, a weekly roundup of new releases.
A pale shadow of Hitchcock's Shadow from Park Chan-wook.
Robert Carlyle on song in a rare star turn.
A last-minute guide to potential winners of the top prize at this year's Oscars.
Yet more louche douchery from Charlie Sheen.
A hard film to endorse.
A wide-eyed odyssey through nocturnal New Orleans.
Misanthrophy masquerading as criminal mischief.
This fourth feature doc on the West Memphis Three offers yet more maddening revelations.


text:
West Delray Collision Center > NEWS & UPDATES > Local NEWS > Senate moves forward with red tide research | Local News Shared By West Delray Collision
April 9, 2019 westdelray
TALLAHASSEE, Fla. — A Senate panel moved forward Tuesday with a proposal that would direct $3 million a year to a red-tide research initiative between the state and Mote Marine Laboratory, despite arguments that the proposal fails to fully address human involvement in the spread of the toxic algae.
The Senate Agriculture, Environment and General Government Appropriations Subcommittee on Tuesday unanimously supported the proposal (SB 1552), which comes after Gov. Ron DeSantis called for addressing red-tide outbreaks.
The Senate also has proposed $6.6 million a year for red tide research as part of its proposed $90.3 billion spending plan (SB 2500) for next fiscal year.
Bill sponsor Joe Gruters, a Sarasota Republican whose area along the Gulf Coast was inundated by the harmful algae last year, said the goal is to get research dollars in place before the next outbreak.
Shared Local News Channel 12:
https://cbs12.com/news/local/senate-moves-forward-with-red-tide-research
Tags:Local NEW Channel 12, Local News, West Delray Collision Center
5 Star Yelp Review by Daniel D. | West Delray Collision Center


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Capt. Kimberly Williams was the first black woman to reach the rank of captain at the Cincinnati Police Department.
Capt. Kimberly Williams, the first black woman to reach the rank of captain at the Cincinnati Police Department, died Aug. 21 surrounded by family and friends. She was 54.
Williams was the youngest of eight children and raised by her aunt and uncle, John and Catherine Robinson, after her mother died young, according to her family.
She graduated from Mount Healthy High School and Xavier University. She earned a masters degree in criminal justice from the University of Cincinnati.
Williams joined the Cincinnati Police Department in 1988, was promoted to sergeant in 1995, lieutenant in 2002 and captain in 2016.
Sgt. Olivia Greer joined the police force a year after Williams. Greer said they were fast friends, nearly sisters, as the rose through the ranks. They were called "aunt" by each other's children.
Williams retired Aug. 16 and died from cancer five days later. She would have marked her 30th year on Cincinnati's police force on Oct. 2.
Williams was first assigned to District 1 covering Downtown, Over-the-Rhine and West End.
In 1990, she was transferred to the street corner unit designed to combat street-level drug offenses often utilizing undercover operations. She then went to the vice unit and the investigations unit, all before the end of her third year on the force.
Greer said Williams worked with Job and Family Services gaining social work experience before joining the force. Williams brought a special family-focused method of policing to the streets of Cincinnati, Greer said.
"She had a knowledge and a wisdom, a lot of insight, into how to resolve problems," Greer said. "She would talk with a level of authority that didn't degrade a person, but let them know that she understood."
Over her career, she worked in all five police districts in Cincinnati along with the criminal investigations section, youth services unit and internal investigations.
Shortly after Officer Sonny Kim was fatally shot in the line of duty, Williams was moved to District 2, where Kim worked. She cooked a huge meal for the officers who had to work on the anniversary of his death.
Greer said an officer there, who is not easy to impress, wrote a condolence recently saying Williams "made the district feel like a home, like he was part of the family."
She also helped run a slew of programs for Cincinnati's youth: nighttime basketball, tennis and golf programs, City Camp for parents and children and the cadets program for teens.
She co-founded the emergency search for missing and endangered team, called ESME, named after 13-year-old Esme Kenney who disappeared and was found murdered in 2009, a victim of serial killer Anthony Kirkland.
Williams was a member of Golden Leaf Baptist Church where she taught Sunday school and served with the hearing impaired ministry, the kitchen committee and the church party committee.
"I could not get her to stop talking about work," Greer said. "She lived and breathed her job and she took pride in sharing policing with her community and her church."
Williams was also a graduate of the Police Executive Leadership Class, the FBI National Academy, and the Senior Management Institute for Policing.
She is survived by her son, Robinson Perry, a senior at Walnut Hills High School. Her family said the teen is "her greatest gift to the world." Williams is also survived by her brother Governor Williams and sisters Carol Samuel, Kathy Chapman, Valerie Larkin and LaVerne D'Armond. She was preceded in death by a sister, Bobbie Jean Simpson-Wallace, and brother, David B. Williams.
Visitation was held Saturday at Lincoln Heights Baptist Church in Woodlawn. A Fraternal Order of Police service preceded the funeral. Williams' body was interred at Spring Grove Cemetery.
Donations can be made to a memory account in Williams' name at Cincinnati Ohio Police Federal Credit Union. The funds will be provided to her son.


The picture featured here is of a cairn Shane and I stacked together. A cairn is simply a stack of rocks, that resembles a Stupa a mound like structure that are used as places of meditation for Buddhists. These stacks of rocks are can be found throughout Tibet. According to our native Tibetan guide, they are frequently left along trails and roads to wish passers-by safe travels and good fortune. Back home, erecting this cairn would have been a simple matter of moving a few heavy rocks. At this altitude there is much less oxygen than at my home in the USA and was certainly the highest we had ever been. We huffed and puffed as we dragged and lifted the rocks into the stack you see here.
To me, this cairn represents achieving a dream long in the making. It wasn't easy to put together but after it was done, it was really something to be proud of. We built the cairn near the camp we would set out towards Mt. Everest from the scenery here is stunningly surreal. Peaks rise up on the horizon of this alpine expanse. Up here the clouds hang low, unable to raise any further, it seems you can almost reach up and touch them. Looking out beyond the cairn, I wondered what lay beyond the first small set of hills. Limited by the effects of the altitude, we didn't venture too far.
Looking back, I wish I had taken more time to contemplate the scenery. In the moment splitting headaches, dehydration, and overall fatigue make it difficult to comprehend where you are. I suppose this is the toll visitors pay to stand among the magnificent mountains. Along with the bitter cold and wind, the altitude is their self defense mechanism, just like thorns on a rose. I think back to my time in Tibet almost daily, the people there endure harsh elements both natural and man-made. Despite the obstacles, the way the hold fast to their culture and traditions inspires me to live a life filled with deep meaning and great purpose.


Anime Coloring Sheets is one of special image reference about coloring hobby. This coloring hobby has been created with excellent idea and follow trend of inspiring coloring ideas.Anime Coloring Sheets was posted in October 24, 2018 at 2:02 pm and has viewed by 171 users. Anime Coloring Sheets reference also have Tags: anime animal coloring sheets, anime boy coloring pages printable, anime coloring book pages, anime coloring pages animals, anime coloring pages apk, anime coloring pages app, anime coloring pages boy, anime coloring pages chibi, anime coloring pages cute, anime coloring pages deviantart, anime coloring pages easy, anime coloring pages fairy tail, anime coloring pages for adults, anime coloring pages for adults online, anime coloring pages for free, anime coloring pages games, anime coloring pages girl, anime coloring pages hard, anime coloring pages hatsune miku, anime coloring pages hd, anime coloring pages images, anime coloring pages naruto, anime coloring pages online, anime coloring pages pdf, anime coloring pages pinterest, anime coloring pages printable, anime coloring pages printable pdf, anime coloring pages sailor moon, anime coloring pages soul eater, anime coloring pages sword art online, anime coloring pages that you can print, anime coloring pages to print, anime coloring pages to print for free, anime coloring pages tumblr, anime coloring pages websites, anime coloring pages wolf, anime coloring sheets free, anime coloring sheets pdf, anime coloring sheets printables, anime coloring sheets to print, anime colouring in sheets, anime colouring pages for adults, anime colouring pages pdf, anime fairy coloring pages for adults, anime girl coloring pages free, anime girl coloring pages printable, anime girl coloring pages to print, anime wolf coloring sheets, chibi anime coloring pages printable, cute anime coloring pages printable, cute anime coloring sheets, cute anime girl coloring sheets, emo anime coloring pages to print, fairy tail anime coloring sheets, free anime coloring pages for adults, free printable anime coloring pages for adults, japanese anime coloring sheets, printable anime coloring pages, printable anime coloring pages for adults for your convenience in searching this reference more specific.
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It was a pretty ugly session for equities, with indices roughly down 1.5% across the board. Implied volatility was also on the ascend with the infamous VIX back above 20%. Corporate bonds were also under pressure as the market had to digest more new issues that added to the recent busy calendar. It was all the more surprising to see the muted - to say the least - reaction of the synthetic market. Credit indices held very well with iTraxx Main and CDX IG only 1.5bp and 1bp wider at 51bps and 55bps respectively. Outright activity on single name CDS was light, and flows were largely limited to position squaring and investors rolling positions from December 2022 to June 2023 maturity. There was very limited need to hedge portfolios, and whatever need there was was met by people willing to take profit of short risk positions. It looks as if everyone is paralised by tomorrow's roll and uses it as an excuse not to express any bearish view. The only name that keeps running away is COFP ( Casino Guichard Perrachon SA ), which was another 10bps wider at 255bps. Its risk premium has increased by 50% since the company released its results early March.


Hey guys, today I've tried to install the 1.3.0 client.
Can you restart computer and try installing again or redownloading it?
So is there anything else I can do? Or will I get problems in the near future because I use the 1.2.1 version of the Client?
Looks like your connection to Problem with Ark Client 1.3.0 was lost, please wait while we try to reconnect.


The 960 Grid System has been around for a while now... but it's been mostly used on the coding side of projects as an HTML/CSS framework. Today, we're going to discuss the advantages of using grid systems like 960GS at the very start of a project, before you even open up Photoshop or Fireworks. If you haven't used a framework before, or you just want a good refresher, this is a great place to start!


ILKE Homes, a British modular homes company , which specializes in off-site manufacturing techniques, is creating 150 new jobs at its first factory in Harrogate.
The new hires will support the business as it delivers up to 2,000 modular homes a year in an effort to help solve the UK's housing shortage.
The business plans to grow staff numbers to 800 over the next three years.
With operations due to commence at the new 270,000 sq ft factory in March at Harrogate, the jobs will initially consist of 100 people in the manufacturing and supply chain team, supported and enabled by a large team of designers, engineers and other professionals.
The manufacturing team is expected to grow to around 300 over the next 12 months.
The new team will create high-specification modular homes using the latest precision manufacturing processes to ensure durable, high-quality homes every time.
Homes will have flexible layouts with over 100 possible variations, from two-story terraced houses to three-story semi-detached properties, and blocks of apartments of up to four stories.
As mentioned before, 68% of Britain's builders embrace modular and offsite construction and that number will continue to grow.


Seymour man injured at work wins millions in case against BNSF Railway Co.
A Greene County jury awarded James Mitchell the money after a week-long trial.
Businesswoman voices concerns about practice. BNSF spokesman says it's safe.
A Seymour man won more than $5 million last week in a case against BNSF Railway Co. after suffering injuries on two separate occasions in the company's Springfield rail yard.
A Greene County jury awarded former conductor James T. Mitchell the sum after a weeklong trial in the Greene County Circuit Court.
BNSF plans to appeal the decision, said Andy Williams, director of public affairs at the railroad company.
"While we respect the work of the jurors, we are disappointed with the result," he said in an email Monday.
The jury determined that BNSF was responsible for the handrail on a rail car ladder that broke while Mitchell was trying to climb down, causing the conductor to fall to the ground and land on his back on Aug. 27, 2015. A derailment on a neighboring track had damaged the handrail earlier that day.
According to the lawsuit, Mitchell required surgery on his lower back and shoulder throughout the next 16 months. He returned to work in 2017.
The lawsuit goes on to state that on Feb. 23, 2017, Mitchell suffered further injury while operating a defective pin-lifting lever to separate rail cars in a train in the Springfield rail yard. Mitchell received multiple spine injections but had ongoing pain. He is unable to return to work due to his injuries, according to the lawsuit.
BNSF denied liability in the case, which was brought under the Federal Employers' Liability Act. The act provides a remedy for railroad workers injured on the job.
"Jamie Mitchell was a loyal railroad worker. But when he was injured due to unsafe working conditions, BNSF denied any responsibility and blamed him," said Mitchell's attorney, Nelson G. Wolff of Schlichter, Bogard & Denton in St. Louis. "This verdict restores his reputation as a good worker and provides him with money needed to pay for significant medical needs and to take care of his family."
An orthopedic surgeon, Dr. Paul Olive, testified that Mitchell will require several spine infusions in the future and that he is no longer able to perform railroad work, Wolff said.


text:
Q: How can a regular user circumvent a restriction on running ls imposed by root? Suppose a root user denied all permissions to use the 'ls' command  for a specific regular user in a system. How can that regular user overcome this denial and how can he use 'ls' command with out any 'sudo' command?

A: I would say talk to the root user. That user either accidentally did something wrong, or they are a real scumbag (in which case talking to their manager is the better option).

Suppose the user is only wanting to view what files and directories are in the current directory, without using ls.  echo * would do that.
Suppose a user would like to see the ordinary ls -l output for the files and directories in the current directory.  find . -maxdepth 1 -ls would do that.

A: Perhaps the simplest way would be to copy the ls binary
from some other system — e.g., over the network, or on a USB thumb drive
— and put it into his private bin directory.
Another workaround is to use stat with wildcards.

redpajama_set_name: RedPajamaStackExchange


Geraldine is a fluidic handwriting font with thin strokes. It's suitable for brand logos, as decorative text, or as a font choice for font pairing design with another typeface designs that complement it.
Geraldine is free to download and use for personal projects. Full version can be purchased too.


I am John Salmons, the host of Takeoff Tech and a residential construction estimator serving home builders and lumber companies since 1994 dba Construction Research Group.
For the ten years prior to becoming a professional estimator, I swung a hammer installing almost everything I estimate today. During this time as a home builder and remodeler, I found myself strapping on the tool belt and framing most of my projects. I also installed my own roofing, drywall, interior trim and just about everything else that I could get my hands on. Having this hands-on field experience allows me to truly understand building materials and how to accurately estimate each of them today.
3D framing plans and lumber takeoffs using VisionREZ Building Information Modeling (BIM) software. This allows me to create a digital prototype on-screen and help resolve potential problems long before the first nail is driven.
Material takeoffs for all other building components using PlanSwift. My takeoffs are generated by tracing 2D architectural plans on-screen using the three fundamental measurements of estimating - Area, Length and Count .
And finally, my software of choice since the beginning – Microsoft Excel. Like most estimators, I've created some highly customized Excel Estimating Templates over the years. With Excel, I can easily re-tool my core templates based on the specifics of each builder or lumber company that I serve.
My mission for this site is simple: To share my passion for estimating, the takeoff software I use to earn a living and my skills that I'm continually improving.