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The invention describes methods for identifying a molecule of interest, as well as nucleic acid molecules which encode it, and binding partners for it. A cDNA library from a cell expressing the target is prepared, and expressed in host cells. Lysates of the host cells are screened with a sample, treated to remove interfering binding partners. The treatment involves contact of the sample to lysates of untransfected host cells, and host cells transfected or transformed with the same vector used to make the cDNA library. Also a part of the invention are antigens and cDNA identified using the methodology.
1. Method for determining an auto-immunoreactive substance produced by a subject capable of eliciting an immune response comprising:
(a) producing a cDNA library of a cell taken from said subject,
(b) inserting said cDNA library into an expression vector,
(c) transfecting said vector into a host cell to produce a transfected host cell,
(d) culturing said transfected host cell to express said auto-immunoreactive substance,
(e) lysing said transfected host cell to form a lysate,
(f) contacting a sample of a body fluid taken from said subject which contains an immunologic binding partner for said immunoreactive substance with a sample of lysed, non-transfected host cells, to remove any immunologic binding partner from said sample which is specific for said non-transfected host cell, to produce a stripped body fluid sample,
(g) contacting said stripped body fluid sample with a sample of lysed host cells transfected with the same vector into which said cDNA library has been inserted wherein said vector does not contain any library cDNA, to remove any immunologic binding partners specific for vector-produced antigens thereby producing a twice stripped body fluid sample,
(h) contacting said twice stripped body fluid sample to the lysate of (e), whereby any immunologic binding partners specific for said auto-immunoreactive substance bind thereto, and
(i) determining binding in (h) to determine said immunoreactive substance.
2. The method of claim 1, further comprising identifying the transfected host cell expressing said immunoreactive substance, and isolating the cDNA contained therein.
3. The method of claim 1, further comprising isolating any immunologic binding partner bound to said auto-immunoreactive substance.
4. The method of claim 1, wherein said host cell is E. coli.
5. The method of claim 1, wherein said vector is a viral vector.
6. The method of claim 5, wherein said viral vector is a eukaryotic virus based vector.
7. The method of claim 5, wherein said viral vector is a phage vector.
8. The method of claim 7, wherein said phage is lambda phage.
9. The method of claim 1, wherein said host cell is a prokaryote.
10. The method of claim 1, wherein said host cell is a eukaryote.
11. The method of claim 1, further comprising immobilizing said lysate of step (e) on a solid phase prior to contact with said twice stripped sample.
12. The method of claim 1, wherein said body fluid sample is serum.
13. The method of claim 1, wherein said immunologic binding partner is an antibody.
14. The method of claim 1, wherein said auto-immunoreactive substance is an antigen associated with cancer.
15. The method of claim 1, wherein said auto-immunoreactive substance is an autoimmune disease associated antigen.
16. The method of claim 1, wherein said auto-immunoreactive substance is produced by a pathogen.
This invention relates to methodologies for identifying molecules of interest. In particularly preferred embodiments, the invention relates to the identification of molecules associated with pathological conditions such as cancer, (melanoma or renal cancer, e.g.), Hodgkin's Disease, autoimmune diseases and so forth. Also a part of the invention are the isolated molecules found as a result of the inventive method. These molecules include, inter alia, protein-containing molecules, isolated nucleic acid molecules encoding these, and antibodies which specifically bind to the protein-containing molecules. For convenience, the method described herein will be referred to as "serological fishing".
It is fairly well established that many pathological conditions, such as infections, cancer, autoimmune disorders, etc., are characterized by the inappropriate expression of certain molecules. These molecules thus serve as "markers" for a particular pathological or abnormal condition. Apart from their use as diagnostic "targets", i.e., materials to be identified to diagnose these abnormal conditions, the molecules serve as reagents which can be used to generate diagnostic and/or therapeutic agents. A by no means limiting example of this is the use of cancer markers to produce antibodies specific to a particular marker. Yet another non-limiting example is the use of a peptide which complexes with an MHC molecule, to generate cytolytic T cells against abnormal cells.
Preparation of such materials, of course, presupposes a source of the reagents used to generate these. Purification from cells is one laborious, far from sure method of doing so. Another preferred method is the isolation of nucleic acid molecules which encode a particular marker, followed by the use of the isolated encoding molecule to express the desired molecule.
To date, two strategies have been employed for the detection of such antigens, in e.g., human tumors. These will be referred to as the genetic approach and the biochemical approach. The genetic approach is exemplified by, e.g., dePlaen et al., Proc. Natl. Acad. Sci. USA. 85: 2275 (1988), incorporated by reference. In this approach, several hundred pools of plasmids of a cDNA library obtained from a tumor are transfected into recipient cells, such as COS cells, or into antigen-negative variants of tumor cell lines which are tested for the expression of the specific antigen. The biochemical approach, exemplified by, e.g., Falk et al., Nature 351: 290 (1991); and Kawakami et al., Nature 369: 69 (1994) both of which are incorporated by reference, is based on acidic elution of peptides which have bound to MHC-I molecules of tumor cells, followed by reversed-phase high performance liquid Chromatogaphy (HPLC). Antigenic peptides are identified after they bind to empty MHC-I molecules of mutant cell lines, defective in antigen processing, and induction of specific reactions in cytotoxic T-lymphocytes. These reactions include induction of CTL proliferation, TNF release, and lysis of target cells, measurable in an MTT assay, or a 51 Cr release assay.
These two approaches to the molecular definition of antigens have the following disadvantages: first, they are enormously cumbersome, time-consuming and expensive; second, they depend on the establishment of cytotoxic T cell lines (CTLs) with predefined specificity; and third, their relevance in vivo for the course of the pathology or disease in question has not been proven, as the respective CTLs can be obtained not only from patients with the respective disease, but also from healthy individuals, depending on their T cell repertoire.
The problems inherent to the two known approaches for the identification and molecular definition of antigens is best demonstrated by the fact that both methods have, so far, succeeded in defining only very few new antigens in human tumors. See, e.g., van der Bruggen et al., Science 254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495 (1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994), Kawakami et al., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).
It would be desirable to have available a method which can be used not only for detection of tumor-associated antigens, but to determine molecules associated with any abnormal or pathological condition. Such a method would also facilitate the identification of such molecules, thereby enabling their use on the generation of, e.g., antibodies, cytolytic T cells, and so forth.
It is therefore the purpose of the present invention to develop a method for the simple detection and molecular characterization of antigens in human tissues, especially in tumor cells, which are useful in the molecular diagnosis of diseases and/or for immunotherapy and gene therapy of infectious, autoimmune and malignant diseases. The invention is delineated in the disclosure which follows.
FIG. 1 shows the principles of the approach of the invention.
FIG. 2 shows a nitrocellulose membrane with a positive clone derived from the cDNA of a renal cell clear carcinoma that reacts with a 1:100 dilution of the patient's serum.
FIG. 3 shows, in bar graph form, the Northern Blot analysis of clone HOM-RCC-313 in renal cell carcinoma, normal kidney and other human tissues.
FIG. 4 shows the translated region of the gene coding for HOM-RCC-313.
The following disclosure describes a methodology referred to as serological fishing. In it, a cell sample is taken from a subject afflicted with a pathological condition. The cells preferably are exemplary of the pathology. For example, if the subject has melanoma, the cells are melanoma cells. If the subject is suffering from a neural disorder, e.g., then the cells are preferably a sample of the afflicted cells. This approach is warranted because the afflicted cells are most probably the best source of protein-containing molecules of interest, i.e., such molecules which are specifically associated with the pathological condition of interest.
Note that cells representative of pathological conditions are not the only cells which may be used in the inventive method. It is very important, e.g., to ascertain those cellular "markers" associated with differentiation and maturation of cells, for example. The example of hematopoietic stem cells comes to mind. Similarly, the invention contemplates the isolation of, e.g., receptor molecules for specific ligands. In effect, one can assay for the presence of any molecule of interest using this methodology.
The cells chosen are then used to prepare a library of complementary DNA (i.e., "cDNA"). This methodology is well known to the skilled artisan, and need not be reiterated here. It is, of course, based upon the established fact that if proteins are expressed by the cells, then messenger RNA (mRNA) must be present. These mRNA molecules are not long lived, and are unstable, so they are not practical to work with. The stability brought to the molecules when cDNA is used is very helpful to the method.
Once the cDNA is made, it is used to construct a vector library. In short, carrier vectors are treated, such as by cutting and splicing, to receive molecules of cDNA. The choice of vector may vary, as the skilled artisan is well familiar with many such examples.
Especially preferred are virus based vectors. In the case of eukaryotic cells, retrovirus or adenovirus based vectors are preferred. Such vectors contain all or a part of a viral genome, such as long term repeats ("LTRs"), promoters (e.g., CMV promoters, SV40 promoter, RSV promoter), enhancers, and so forth. When the host cell is a prokaryote. Then bacterial viruses, or phages, are preferred. Exemplary of such vectors are vectors based upon, e.g., lambda phage. In any-case, the vector may comprise elements of more than one virus.
The resulting vectors are transfected or transformed into a host cell, which may be eukaryotic or prokaryotic.
Any cell normally used for transfection or transformation may be used in the protocol. Preferred materials include strains of E. coli, CHO cells such as CHO-1, COS cells such as COS-7, and so forth. Similarly, yeast cells, e.g., strains of Saccharomyces, strains of Pseudomonas, such as strains of Pseudomonas aeruginosa, Bacillus bacteria, Spodoptera frugiperda, a well known insect host cell, and so forth, may all be used.
Once the recipient cells receive the vectors, they are cultivated so as to express foreign, protein containing molecules. "Protein-containing" is used herein because, while prokaryotes express only proteins, eukaryotic cells are well known for their ability to post-translationally modify proteins, so as to produce glycoproteins, lipoproteins, e.g. It must also be borne in mind that "protein containing" as used herein, also encompasses peptides, such as the peptides presented by MHC molecules.
The processes now described below take place independently of the process described above, and no chronological relationship between the two facets of the invention is intended.
In pathological conditions such as cancer and, e.g., autoimmune diseases, there is some immune reaction to molecules associated with the pathology. This reaction can include an antibody response, B cell proliferation, proliferation of specific T cell subpopulations, increases in cytokine production, and so forth. The molecules and cells associated with the response may be found in body fluids of a subject, such as his or her serum. The immune responders will react with the molecule of interest whether it is produced recombinantly or autologously. The problem is to find them. As the examples show, this is done in a unique way. First, the body fluid, or other sample of interest, is reacted with a sample of the same host cells used for transfection or transformation. In this first step, the host cells are not transfected or transformed. The effect of this is to strip any immunogenic binding partners specific for the host cell rather than the targeted molecule. This step is necessary because, as was pointed out, supra, the host cell may be one against which the subject has developed an immune respone at some point. This first stripping removes these immune components.
A second stripping step is then carried out. In this step, the previously stripped sample is now reacted with a sample of the same host cell as was described supra, this time having been transfected or transformed with the carrier vector lacking cDNA from the subject. The reason for this second stripping step is an observation made by the present inventors not reported in the literature. The materials used as vectors, such as phages, viruses, etc., are useful because they naturally infect cells. Thus, E. coli which inhabit the lower intestine of humans are infected with lambda phage. It had not been considered, previously, that the immune response to E. coli includes a response to these infectious agents. Thus, applicants have surprisingly, achieved an ability to remove interfering immune components to an unprecedented degree by carrying out the two stripping steps. As noted, the first is against untransfected or untransformed host cells. The second is against host cells transfected or transformed with a vector which does not carry cDNA, wherein the vector is immunologically equivalent to the vector used to carry cDNA, as described supra.
It is especially preferred to carry out each of these stripping steps using a plurality of similar, but different procedures. The experiments which follow, for example, show absorption on a solid phase column, and then absorption on nitrocellulose paper. Applicants do not wish to be bound by any theory as to why the use of two similar but different protocols produces the results described herein. It is to be borne in mind, hereafter, that whenever "contacting a sample" is used herein, it is not to be limited to one contact step only, but may refer to more than one, preferably different contact protocols designed to remove interfering binding partners from a sample under scrutiny.
It should be understood that these stripping steps may be done completely independently of the steps used to prepare the cDNA library. For example, if the test for an antigen is to be done at day "0", the stripping of sample may be done the day before, a week before, and so forth. One can also "bank" stripped sample from a donor or subject for future use.
The sample used is preferably serum, but need not be. Any sample which contains immunogenic binding partners may be so used.
In the next step of the method, lysed, transfected cells carrying the cDNA and expressing heterologous protein are contacted with the twice stripped sample. This sample should only contain immune components specific for the heterologous protein, and should bind thereto. This binding is facilitated if the cell lysates have been immobilized via contact to, e.g., activated filter paper, a solid phase column, etc., but this solid phase binding is not necessary, as the art will surely recognize that many, varied forms of assays are available for identifying a molecule of interest.
Once the immune component binds to the target molecule, a further step is desirably, but not necessarily, carried out. This additional step involves the use of some binding partner for the first immune component, such as anti-IgG, carrying an identifiable label. The label may be a dye, an enzyme, a gold particle, a radiolabel, or any of the standard labels used in immunoassays.
Once identification is carried out, the immune components are removed, leaving the target molecule. The target molecule is then studied, using any of the standard methodologies in the art.
The artisan will note that the methodology also results in isolation of immune components which bind to the molecule of interest. Thus, in another aspect of the invention one can isolate antibodies, e.g., which are specific binding partners for the molecule of interest.
Yet another immune component which may be identified and isolated following the invention is a cytolytic T cell ("CTL" hereafter), specific for complexes of peptides derived from the identified molecule and MHC molecules to which these peptides bind, forming a complex. It is fairly well accepted that a CTL response involves the identification of complexes of MHC molecules and peptides, generally about 8-12 amino acids in length, but most preferably 9 or 10 amino acids in length, by T cell receptors ("TCRs") on the surface of circulating T cells. The TCRs react by binding to these complexes, setting in motion as it were a series of reactions including the proliferation of CTLs specific for these complexes. One can produce and/or isolate such CTLs using the method of the invention, plus further steps.
As is pointed out in the examples which follow as well as the disclosure in general, one can easily identify cDNA encoding an antigen of interest. Once this is identified, one uses the cDNA to transfect host cells which either already present desired MHC molecules on their surface, or which have been transfected with DNA encoding these MHC molecules. The cDNA for the molecule of interest is expressed, and the molecule is processed to antigenic peptides which are presented by MHC molecules, such as HLA molecules. CTLs directed against the complexes are obtained from lymphocytes, such as autologous lymphocytes. From responder cell populations, long-term CTL clones are then obtained by the well known technique of limiting dilution. Once a positive CTL response is observed, the specific peptides presented to the CTLs are identified using established methods for example, screening the previously identified CTL clones. Alternatively, the more recently described method of studying the sequence of the molecule of interest to identify potential MHC-binding motifs and then analyzing these peptides, first for binding to the relevant MHC molecule and then, if positive for MHC-binding, for their ability to generate CTLs recognizing the peptide MHC complex. Of course the peptides can also be eluted from the cells and sequenced, using well known techniques.
It will also be noted by the skilled artisan that one can correlate the expression of the molecule of interest back to a particular host cell or cells which expressed it. In so doing, one can remove the cDNA which expressed the molecule of interest, sequence it, and so forth. This aspect of the method is another feature of the invention.
Specific embodiments of the invention will be seen in the examples which follow. FIG. 1 depicts the method generally.
For the establishment of a cDNA library from human tissue total RNA was obtained from 0.5 g of a renal clear cell carcinoma and established according to the method of Chomzynski, J. Analyt. Biochem. 162: 156-159 (1987), incorporated by reference. The mRNA was extracted from total RNA with oligo-dT-cellulose. The synthesis of the first strand cDNA was accomplished by the method described by Gubler and Hoffmann, Gene 25: 263 (1983) using RNase H and DNA polymerase I. For adaptation of the cDNA Klenow enzyme, adaptors with EcoRI restriction enzyme sites were ligated to the cDNA ends using T4 DNA ligase (Ferretti L and Sgamerella V, Nucl. Acids Res. 9: 3695 (1981)). Following restriction enzymatic digestion with the enzyme Xhol, cDNA molecules of different length were separated using Sephacryl 400 and transfected into λZAPII phage vectors (Short JM et al., Nucleic Acids Res. 16: 7583 (1988)). The recombinant phage DNA was packed into phages after ligation with packing extracts and used for the transfection of E. coli bacteria. The titration of the library resulted in 1.8�106 recombinant primary clones. The total cDNA library was transfected in E. coli and amplified. The titer of the cDNA library after amplification was 1011 plaque forming units per ml (pfu/ml). These transfected cells were used in experiments which follow.
In accordance with the invention as described, supra, identification of immunogenic material was achieved by using human sera which has been completely depleted of antibodies directed against antigens derived from native and lytic λ phage-transfected E. coli bacteria. To this end, the serum was "stripped" via absorption, as now described.
E. coli bacteria of the strain XL1-blue were cultured in 50 ml LB medium overnight. After achieving an optical density of OD600 =1.0, the bacteria were pelleted by centrifugation, resuspended in 5 ml phosphate buffered saline (PBS), and sonificated by ultrasound. The bacterial lysate was bound onto a matrix of activated Sepharose, which was then put into a column and used for the absorption of the human serum. The serum was run over this column 10 times.
A culture of E. coli XL1 blue bacteria in the exponential growth phase was pelleted by centrifugation, transfected in 0.01 M magnesium sulfate with 106 λZAPII phages without a recombinant insert and incubated in 5 ml LB medium for four hours. The lysate of the transfected bacteria was used in the same manner as the untransfected bacteria, with the human serum described supra being passed through the column an addition ten times.
To complete the depletion of the serum, interfering antibodies from lytically transfected E. coli bacteria were cultured on agar plates and their proteins were blotted onto nitrocellulose membranes after 10 hours of culture at 37� C. Following this, the serum which had been preabsorbed according to the above steps was transferred to the blotted nitrocellulose membrane, and the absorption procedure was repeated five times. The serum, which was processed in accordance with the invention, was totally depleted of antibodies directed against antigens derived from E. coli and phages.
In this, a renal cancer-specific antigen was identified via the following steps. Bacteria of the strain XL1 blue were transfected with recombinant phages derived from the described cDNA library and plated at a density of 4-5�103 plaque forming units (pfu) per plate in LB-medium with isopropylthiogalactopyranoside ("IPTG"). After 12 hours of incubation at 37� C. nitrocellulose membranes were put on top of the cultures and culture plates were incubated for another four hours. This was followed by incubation of the nitrocellulose membrane for one hour in Tris-buffered saline (PBS) with 5% milk powder. After washing the nitrocellulose membranes three times in TBS, the stripped human serum secured following Example 2 was diluted 1:1000 in TBS/0.5% (w/v) milk power and incubated overnight with gentle shaking. After the incubation with the nitrocellulose membrane the serum was removed and kept for additional testing. Following incubation with serum, the nitrocellulose membranes were washed three times in TBS, and incubated with a polyclonal alkaline phosphatase-conjugated goat anti-human IgG serum for one hour. Following this, the nitrocellulose membranes were washed repeatedly with TBS/0.01% (v/v Tween 20). The reaction was developed using nitroblue tetrazolium chloride and bromochloro-indoyl-phosphate in TBS. The binding of human antibodies to the expressed protein became visible by a blue ring-formed color deposit on the nitrocellulose membrane. The efficient preabsorption of the serum made in possible to develop the membrane at 37� C. over several hours without compromising the quality of the test because of background reactivity caused by antibodies against E. coli and phage antigens.
Positive clones were localized on the agar plates, transferred into transfection buffer, and used for a second round of transfection and subcloning. A total of 1.8�106 recombinant clones were subjected to screening and five different positive-reacting clones were identified.
Positive clones secured following Example 3, i.e., those which had bound antibodies derived from the processed human serum, were subcloned to monoclonality by repeated rounds of transfection and testing of reactivity With the processed human serum. P-bluescript phagemids with the respective cDNA inserts were cloned by in vivo excision (Hay B and Short JM, Strategies 5: 16-19, 1992) from the λZAPII phage vectors and used for the transfection of E. coli SOLR bacteria. Plasmids were isolated from the bacteria after alkaline lysis with NaOH in a modification of the method of Birnboim HC and Doly J. J. Nucl. Acids Res. 7: 1513 (1979). The recombinant plasmid DNA was sequenced according to the classic method of Sanger (Proc. Natl. Acad. Sci. USA 74: 5463 (1977)) using M13-forward and M13-reverse oligonucleotides. The DNA sequence obtained and the resulting amino acid sequence were checked for in nucleic acid and protein data banks (Gene Bank, EMBL, Swiss Prot). The sequencing of the cDNA inserts was continued using internal oligonucleotides. Analysis showed no homology with any sequences deposited in the data banks. The full length cDNA clone referred to as SK313, which had been cloned with the RACE method (Frohman MA, Dush MK, Martin GR, Proc. Natl. Acad Sci. USA 85: 8998 (1988)), had a carbonic anhydrase domain at the 5' end. The nucleic acid sequence of this molecule is presented in SEQ ID NO: 1. FIG. 2 shows a nitrocellulose membrane with a positive clone from these experiments.
As a follow up to these experiments, RNA was isolated from a spectrum of malignant and normal human tissues according to the method of Chomyznski P, Sacchi N, Analyt Biochem. 162: 156 (1987). After denaturation, the total isolated RNA was separated on an agarose gel containing 1% formaldehyde by electrophoresis (Goldberg DA, Proc. Natl. Acad. Sci. USA 77: 5794 (1980)) and then blotted onto a nylon membrane according to a known method (Seed B, Nucl. Acids Res. 10: 1799 (1982)) Radiolabeled cDNA inserts of the identified clones were used for hybridization. The hybridization was carried out according to a known method (Geoffrey MW, Berger SI, Enzymol. 152: 419 (1987)). The presence of the respective RNA was demonstrated using autoradiography and X-ray films. The analysis demonstrated that the mRNA of clone HOM-RCC-313 was overexpressed in 4 out of 19 renal cell carcinomas compared to normal kidneys. Very weak expression was found only in colonic mucosal tissue and in normal kidney. Expression in other tissues could not be demonstrated.
To determine the incidence of antibodies against antigens which are identified in accordance with the invention, sera from healthy individuals and tumor patients were analyzed. To this end, the sera were processed as described, supra, and depleted from antibodies against antigens derived from E. coli and phages. For the detection of antigen-specific antibodies, phages derived from reactive clones were mixed with non-reactive phages derived from the same cDNA library at a ratio of 1:10 and tested as described supra for reactivity with antibodies in the human test serum. The serum which had been used for the identification of the antigen was used as a positive clone. The non-reactive phages served as a negative control. A serum sample was positive for antigen reactive antibodies, if the expected percentage of the phage plaques showed a positive reaction. In the case of the renal cell carcinoma antigen represented by clone HOM-RCC-313, the analysis of a spectrum of human sera showed that only sera from renal cell carcinoma patients contained reactive antibodies. Sera from healthy controls and patients with other tumors did not contain such antibodies.
The cDNA for clone HOM-RCC-313 was excised from the plasmid DNA by digestion with the restriction enzyme EcoR1, was separated by agarose gel electrophoresis, followed by extraction from the gel. This was then used to create a vector which expresses a fusion protein with the bacterial protein anthranilate synthetase. A relevant fragment in the exact open reading frame was cloned into pATH plasmid vectors (Koerner TJ, Hill JE, Myers AM, Tzagaloff A, Meth. Enzymol. 194: 477 (1991). Induction of protein expression was obtained after transformation of the plasmids into E. coli of strain BL21 as described (Spindler KR, Rosser DSE, Berk AG, J. Virol. 49: 132 (1984)). Expressed, fusion proteins were separated by SDS gel electrophoresis, excised from the gel, eluted and freeze dried. Rabbits were immunized by subcutaneous injection with 100 μg of the lyophilisate dissolved in Freund's adjuvant. Immunization was repeated three times at two-week intervals using Incomplete Freund's Adjuvant. The rabbits were bled and antiserum was obtained. The obtained antiserum was depleted from antibodies reactive with E. coli and phages in the manner as is described supra and tested for reactivity against the renal carcinoma antigen as described for the human serum. Reactivity was detected at dilutions of 1:>100,000.
The protocols set forth in the preceding examples were followed, using biopsied tissue taken from different subjects suffering from (i) malignant melanoma, (ii) astrocytoma, and (iii) Hodgkin's Disease. Table 1, which follows, summarized the results, including those obtained with the renal cancer study, set out in detail in Examples 1-6, supra.
TABLE 1______________________________________Antibody reactivity of autologous sera with recombinantclones derived from human tumor cDNA. cDNA libraries were screenedwith autologous patient serum. Positive clones were subcloned tomonoclonality. Inserts from each clone were amplified with plasmidprimers and separated by agarose gel electrophoresis. Southernblots were performed by cross hybridization with the respective inserts. clones positive differenttumor tested clones inserts______________________________________malignant melanoma 1.0 � 106 40 10renal cell carcinoma 1.8 � 106 7 5astrocytoma 1.2 � 106 49 5Hodgkin's disease 1.0 � 106 14 4______________________________________
Following the analysis of the different inserts observed, the melanoma cells expressed the known tumor rejection antigen precursor MAGE-1 (see van der Bruggen et al., Science 254: 1643-7 (1991), incorporated by reference), as well as a new antigen. A portion of the cDNA sequence of this antigen is set forth in SEQ ID NO: 2.
When the astrocytoma study was completed, the observed insert corresponded to the previously described Tegt gene (Old, Canc. Res. 41: 361-375 (1981), incorporated by reference).
When the Hodgkin's Disease study was completed, a previously unknown antigen was isolated, and cDNA encoding it was identified in the library, using standard methods. The antigen is a newly observed, lectin-like structure, a portion of the cDNA for which is set forth in SEQ ID NO: 3. Also observed were antibodies against restin, described by Bilbe, et al, EMBO J 11: 2103-13 (1992). This is an intermediate filament associated protein, expression of which has been shown to be restricted to Hodgkin and Reed-Sternberg cells, as well as cultured monocytes.
A further study of occurrence of antibodies against the antigens described in Examples 1-7 was carried out. Table 2 summarizes these assays. In these studies, phages from positive clones were mixed with non-reactive phage (ratio:1:10), and then used to transfect bacteria (e. coli). Dilutions of patient sera (1:200), were used, in an enzyme linked immunosorbent assay (ELISA), as described supra. "HOM-MEL-40" refers to the new melanoma antigen (SEQ ID NO: 2), while "HOM-MEL-55" refers to MAGE-1 (van der Bruggen et al., supra). "HOM-RCC 3.1.3" is the renal cancer antigen of SEQ ID NO: 1. "HOM-GLO-30.2.1" refers to the previously identified astrocytoma associated antigen, "HOM-HD-21" refers to the new, lectin-like antigen of SEQ ID NO: 3, and "HOM-HD-397" is the previously identified restin antigen.
TABLE 2__________________________________________________________________________Humeral immune responses against human tumor antigens. Phages frompositive clones were mixed withnonreactive phages of the cDNA-library at a ratio of 1:10 and used totransfect bacteria.IgG antibodies to the clones were detected with an enzyme-linked assayusing1:200 diluted patient sera.n.t. = not testedantigen HOM-MEL-55 HOM-RCC-3.1.3* HOM-GLIO-30.2.1 HOM-HD-21* HOM-HD-397identity/homology HOM-MEL-40* MAGE-1 CAH-like tegt lectin-like restin__________________________________________________________________________melanoma patients 2/11 4/11 n.t. n.t. n.t. n.t.renal cancer patients 0/8 0/8 2/14 0/7 0/7 5/7astrocytoms patients 0/10 0/10 0/11 2/13 0/11 7/11Hodgkin's patients 0/10 0/10 0/17 0/17 10/18 14/17healthy controls 0/12 0/12 0/15 0/20 0/17 12/17__________________________________________________________________________
The fact that antibodies against the tumor antigens, excepting only restin, were detected, albeit at varying rates, only in the sera of patients diseased with the same type of tumor suggests that tumor growth is essential for the development of a humoral response against tumor antigens.
The reason for the presence of restin in healthy controls is not clear. One may speculate that tolerance against respective antigens might be circumvented, because the antigen may have similar sequences to another antigen, the donor may have premalignant cells, or the antigen may be activated in normal cells under non-malignant conditions, such as viral infections, or other inflammatory processes.
In order to determine the expression pattern of the newly identified antigens described herein, Northern blot analysis was carried out, using a variety of human tissues.
RNA was extracted from tissue samples (tumor and normal) using the well known guanidium isothiocyanate/phenol/chloroform method of Chomzynski, et al., supra. The RNA integrity was checked via electrophoresis in formalin/MOPS gels. Then, gels containing 40 ug of RNA per lane were blotted onto nylon membranes. These Northern blots were then probed with the cDNA of SEQ ID NO: 1, 2 or 3. Hybridization was with 32 P labelled probes at 42� C., with formamide. The filters were washed at 65� C., at 1�SSC, 0.2% SDS, and exposed for 16 hours. These are "stringent conditions" as defined hereafter. After exposure, filters were stripped and rehybridized with GAPDH.
Table 3 summarizes these results.
TABLE 3__________________________________________________________________________Expression pattern of tumor antigens in various tissues (selection).Northern-blot analysis was performed with RNA samples from tumor andnormal human tissues matched by hybridization with GAPDH.Expression ratios were calculated after densitometric analysis ofautoradiographs.The signal obtained with the normal counterpart of the diseased tissuewas set to 1.n.t. = not tested;antigen HOM-RCC-3.1.3* HOM-GLIO-30.2.1 HOM-HD-21*identity/homology HOM-MEL-40* CAH-like Tegt Lectin-like__________________________________________________________________________kidney -- 1 1.5 --brain -- n.t. 1 n.t.tonsil -- -- 1 1stomach -- -- 1.5 --colon mucosa -- 0.2 1.5 --breast -- -- 1.0 --renal cancer -- >5 in 4/19 cases n.t. -- ≦1 in 15/19 casesHodkin's tissue n.t. -- n.t. >10astrocytoma n.t. n.t. >5 in 8/12 -- 1 in 4/12melanoma ++ -- n.t. --__________________________________________________________________________
As will be seen the new melanoma associated antigen is strongly expressed in melanoma, but not other tissues. Carbonic-anhydrase-like antigen was strongly expressed in about 20% of renal cell carcinomas, and only weakly in normal renal tissue. Tegt was overexpressed on 8/12 astrocytoma tissues compared to normal brain tissue. The mRNA for the lectin like molecule associated with Hodgkin's disease was increased about ten fold in diseased tonsils as compared to normal tonsils, suggesting that overexpression may be a frequent characteristic of proteins which elicit autologous B cell responses.
As the foregoing shows, the invention relates to a method for determining or isolating an immunoreactive substance. "Immunoreactive substance" as used herein refers to any material which provokes some form of immune response in the subject which produces it. This response may be based upon either a B cell or a T cell response. Such immunoreactive substances include proteins, peptides, glycoproteins, lipoproteins, peptide containing complexes (e.g., MHC/peptide complexes), antibodies, and so forth. To determine such substances, a cDNA library is prepared from cells of a subject, using well known, standard methods. The cDNA is then inserted into an appropriate vector, such as a eukaryotic cell specific virus or a phage (i.e., a bacterial virus), to form a transfecting/transforming library, which is then incorporated into a host cell. The host cells are treated so that they express the library component (cloned cDNA) they receive. The host cells are then lysed, so that the expressed material is available for further treatment.
The lysed material is then contacted with a "stripped" sample believed to contain an immunogenic binding partner for the immunoreactive substance. "Immunogenic binding partner" as used herein refers to any immune system associated material which binds to the target, i.e., the immunoreactive substance. Such binding partners include, but are not limited to, antibodies, T cells, cytokines, ligands, receptors, and so forth, as well as truncated portions of these molecules, complementary nucleic acid molecules, and so forth. Note that for some of these components, such as T cells, further steps including those recited herein are required.
The stripped sample, as indicated supra, has been treated by contact with both (i) non-transfected or transformed host cells, and (ii) host cells transfected or transformed with vectors which do not contain the pertinent cDNA.
The stripped sample is useful for identifying binding partner for the expressed material because many of the immune components which would otherwise interfere with the specific immunological reaction desired have been removed via the absorption steps described herein.
The identification of the expressed material may be followed by isolation of the cDNA encoding it. One can punch holes through a membrane placed on top of Petri dishes containing colonies of host cells thereby giving position on the solid phase, such as nitrocellulose membrane, used in the immune reaction. Each colony is based upon limited cDNA transfection, thereby facilitating isolation and identification of relevant cDNA.
The invention also relates to the isolated nucleic acid molecules of SEQ ID NO: 1, 2 or 3, which encode for molecules which are associated with particular conditions. In addition to their role as coding materials, these molecule can also be used as probes to identify cells expressing the relevant antigens, as it has been shown that these cDNA molecules (SEQ ID NO: 1, 2 and 3) are based upon mRNA which translated to the antigen.
Also a part of the invention are isolated nucleic acid molecules, the complementary sequences of which hybridize to one of SEQ ID NO: 1, 2 or 3, and which encode a protein equivalent to those encoded by SEQ ID NO: 1, 2 or 3. "Stringent conditions" as used herein, refers to conditions at least as stringent as hybridization at 50 μl/cm2 of 3.5�SSC, 1�Denhardt's solution, 25 mM sodium phosphate buffer (pH 7.0), using a 32 P-labelled probe, for 18 hours at 65� C., followed by four washes (one hour, each wash, at 65� C., 2�SSC, 0.1% SDS), and a final wash for 30 minutes at 1.0�SSC 0.2% SDS. The final wash can be changed to 0.5�SSC to 0.2�SSC, or even 0.1�SSC, and SDS can be lowered to 0.1% to increase stringency, if desired.
Other features of the invention will be clear to the skilled artisan and need not be reiterated here.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 3(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2679 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double- stranded(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CGCGAAGATGCCCCGGCGCAGCCTGCACGCGGCGGCCGTGCTCCTGCTGG50TGATCTTAAAGGAACAGCCTTCCAGCCCGGCCCCAGTGAACGGTTCCAAG100TGGACTTATTTTGGTCCTGATGGGGAGAATAGCTGGTCCAAGAAGTACCC150GTCGTGTGGGGGCCTGCTGCAGTCCCCCATAGACCTGCACAGTGACATCC200TCCAGTATGACGCCAGCCTCACGCCCCTCGAGTTCCAAGGCTACAATCTG250TCTGCCAACAAGCAGTTTCTCCTGACCAACAATGGCCATTCAGTGAAGCT300GAACCTGCCCTCGGACATGCACATCCAGGGCCTCCAGTCTCGCTACAGTG350CCACGCAGCTGCACCTGCACTGGGGGAACCCGAATGACCCGCACGGCTCT400GAGCATACCGTCAGCGGACAGCACTTCTCCGCCGAGCTGCACATTGTCCA450TTATAACTCAGACCTTTATCCTGACGACAGNACTGCCAGCAACAAGTCAG500AAGACCTCGCTGTCCTGGGTGCTCTCATTGAGATGGGCTCCTTCAATCCG550TCCTATGACAAGATCTTCAGTCACCTTCAACATGTAAAGTACAAAGGCCA600GGAAGCATTCGTCCCGGGATTCAACATTGAAGAGCTGCTTCCGGAGAGGA650CCGCTGAATATTACCGCTACCGGGGGTCCCTGATCACACCCCCTTGCAAC700CCCACTGTGCTCTGGACAGTTTTCCGAAACCCCGTGCAAATTTCCCAGGA750GCAGCTGCTGGCTTTGGAGACAGCCCTGTACTGCACACACATGGACGACC800CTTCCCCCAGAGAAATGATCAACAACTTCCGGCAGGTCCAGAAGTTCGAT850GAGAGGCTGGTATACACCTCCTTCTCCCAAGTGCAAGTCTGTACTGCGGC900AGGACTGAGTCTGGGCATCATCCTCTCACTGGCCCTGGCTGGCATTCTTG950GCATCTGTATTGTGGTGGTGGTGTCCATTTGGCTTTTCAGAAGGAAGAGT1000ATCAAAAAAGGTGATAACAAGGGAGTCATTTACAAGCCAGCCACCAAGAT1050GGAGACTGAGGCCCACGCTTGAGGTCCCCGGAGCTCCCGGGCACATCCAG1100GAAGGACCTTGCTTTGGACCCTACACACTTCGGCTCTCTGGACACTTGCG1150ACACCTCAAGGTGTTCTCTGTAGCTCAATCTGCAAACATGCCAGGCCTCA1200GGGATCCTCTGCTGGGTGCCTCCTTGTCTTGGGACCATGGNCACCCCAGA1250GCCATCCGATCGATGGATGGGATGCACTCTCAGACCAAGCAGCAGGAATT1300CAAAGCTGCTTGCTGTAATTGTGTGAGATTGTGAAGTGGTCTGAATTCTG1350GAATCACAAACCAACCATGCTGGTGGGCCATTAATGGTTGGAAAACACTT1400CCATCCGGGGCTTTGCCAGAGCGTGCTTTCAAGTGTCCTGGAAATTCTGC1450TGCTTCTCCAAGCTTTCAGACAAGAATGTGCACTCTCTGCTTAGGTTTTG1500CTTGGGAAACTCAACTTCTTTCCTCTGGAGACGGGACATCTCCCTCTGAT1550TTCCTTCTGCTATGCAAAACCTTTAATCTGCACCTTACANACTCGGGGAC1600AAATGGGGACAGGAAGGATCAAGTTGTAGAGNAGAAAAAGAAAACAAGAG1650ATATACATTGTGATATATATTAGGGACACTTTCACAGTCCTGTCCTCTGG1700ATCACAGACACTGCACAGACCTTAGGGAAATGGCAGGTTCAAAGTTCCAC1750TTCTTGGTGGGGATGAGAAGGGAGAGAGAGCTAGAGGGACAAAGAGAATG1800AGAAGACATGGATGATCTGGGAGAGTCTCACTTCGGAATCAGAATTGGAA1850TCACATTCTGTTTATCAAGCCATAATGTAAGGACAGAATAATACAATAAT1900AAGTCCAAATCCAACCTCCTGTCAGTGGAACAGTTATGTTTTATACTCTA1950CAGATTTTACAAATANATGAGGCTNGTTCCTTGAAAANTGTGTTGNNTTG2000CTGTNGTCCNNTGGAGGAGACATGAGTTCCGAGATGACCAACTCNNGCNT2050TGNATNCTNGGAGGNAATANGGCAGAACCAAAATGACTGTAGAACTTATT2100CTCTGTAGGCCAAATTTCATTTCAGCCACTTCTGCAGGATCCTACTGCCA2150ACCTGGAATGGAGACTTTTATCTACTTCTCTCTCTCTGAAGATGTCAAAT2200CGTGGTTTAGATCAAATATATTTCAAGCTATAAAAGCAGGAGGTTATCTG2250TGCAGGGGGCTGGCATCATGTATTTAGGGGCAAGTAATAATGGAATGCTA2300CTAAGATACTCCATATTCTTCCCCGAATCACACAGACAGTTTCTGACAGG2350CGCAACTCCTCCATTTTCCTCCCGCAGGTGAGAACCCTGTGGAGATGAGT2400CAGTGCCATGACTGAGAAGGAACCGACCCCTAGTTGAGAGCACCTTGCAG2450TTCCCCGAGAACTTTCTGATTGCACAGTCTCATTTTGACAGCATGAAATG2500TCCTCTTGAAGCATAGCTTTTTAAATATCTTTTTCCTTCTACTCCTCCCT2550CTGACTCTAGGAATTCTCTCTTCTGGAATCGCTTGAACCCAGGAGGCGGA2600GGTTGCAGTAAGCCAAGGTCATGCCACTGCACTCTAGCCTGGGTGACAGA2650GCGAGACTCCATCTCAAAAAAAAAAAAAA2679(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 931 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double- stranded(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:ACTTTCTCTCTCTTTCGATTCTTCCATACTCAGAGTACGCACGGTCTGAT50TTTCTCTTTGGATTCTTCCAAAATCAGAGTCAGACTGCTCCCGGTGCCAT100GAACGGAGACGACGCCTTTGCAAGGAGACCCACGGTTGGTGCTCAAATAC150CAGAGAAGATCCAAAAGGCCTTCGATGATATTGCCAAATACTTCTCTAAG200GAAGAGTGGGAAAAGATGAAAGCCTCGGAGAAAATCTTCTATGTGTATAT250GAAGAGAAAGTATGAGGCTATGACTAAACTAGGTTTCAAGGCCACCCTCC300CACCTTTCATGTGTAATAAACGGGCCGAAGACTTCCAGGGGAATGATTTG350GATAATGACCCTAACCGTGGGAATCAGGTTGAACGTCCTCAGATGACTTT400CGGCAGGCTCCAGGGAATCTCCCCGAAGATCATGCCCAAGAAGCCAGCAG450AGGAAGGAAATGATTCGGAGGAAGTGCCAGAAGCATCTGGCCCACAAAAT500GATGGGAAAGAGCTGTGCCCCCCGGGAAAACCAACTACCTCTGAGAAGAT550TCACGAGAGATCTGGACCCAAAAGGGGGGAACATGCCTGGACCCACAGAC600TGCGTGAGAGAAAACAGCTGGTGATTTATGAAGAGATCAGCGACCCTGAG650GAAGATGACGAGTAACTCCCCTCAGGGATACGACACATGCCCATGATGAG700AAGCAGAACGTGGTGACCTTTCACGAACATGGGCATGGCTGCGGACCCCT750CGTCATCAGGTGCATAGCAAGTGAAAGCAAGTGTTCACAACAGTGAAAAG800TTGAGCGTCATTTTTCTTAGTGTGCCAAGAGTTCGATGTTAGCGTTTACG850TTGTATTTTCTTACACTGTGTCATTCTGTTAGATACTAACATTTCATTGA900TGACGAAGACATACTTAATCGATATTTGGTT931(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1692 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double- stranded(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GATCCCCCGGGCTGCAGGAATTCGGCACGAGCAAAGGACTTCCTAGTGGG50TGTGAAAGGCAGCGGTGGCCACAGAGGCGGCGGAGAGATGGCCTTCAGCG100GTTCCCAGGCTCCCTACCTGAGTCCAGCTGTCCCCTTTTCTGGGACTATT150CAAGGAGGTCTCCAGGACGGACTTCAGATCACTGTCAATGGGACCGTTCT200CAGCTCCAGTGGAACCAGGTTTGCTGTGAACTTTCAGACTGGCTTCAGTG250GAAATGACATTGCCTTCCACTTCAACCCTCGGTTTGAAGATGGAGGGTAC300TTGGTGTCCAACACGAGGCAGAACGGAAGCTGGGGGCCCGAGGAGAGGAA350GACACACATGCCTTNCCAGAAGGGGATGCCCTTTGACCTCTGCTTCCTGG400TGCAGAGCTCAGATTTCAAGGTGATGGTGAACGGGATCCTCTTCGTGCAG450TACTTCACATCTCGTCATGCCCTGTCCACCGTTGTGGACACCATCTCCGT500CAATGGCTCTGTGCAGCTGTCCTACATCAGCTTCCAGCCTCCCGGCGTGT550GGCCTGCCAACCCGGCTCCCATTACCCAGACAGNNNTCATCCACACAGTN600GCAGAGCGCCCNCTGGACAGATGTCTCTACTCCCGCCATCCCACCTATGA650TGTACCCCCACCCCGCCTATCCGATGCCTTTCATCACCACCATTCTGGGA700GGGCTGTACCCATCCAAGTCCATCCTCCTGTCAGGCACTGTNCTGCCCAG750TGCTCANGAGGTTCCACATCNAACCTGTGCNCTGGGAACCACATCGCCTT800CCACCTGAACCCCCGTTTTGATGAGAATGCTGTGGTCCGCAACACCCAGA850TCGACAACTCCTGGGGGTCTCAGGAGCGAAGTCTGCCCCGAAAAATGCCC900TTCGTCCGTGGCCAGAGCTTCTCAGTGTGGATCTTGTGTGAAGCTCACTG950CCTCAAGGTGGCCGTGGATGGTCAGCACCTGTTTGAATACAACCATCGCC1000TGAGGAACCTGCCCACCATCAACAGACTGGAAGTGGGGGGCGACATCCAG1050CTGACCATGTGCAGACATAGGCGGCTTCCTGGCCCTGGGGCCGGGGGCTG1100GGGTGTGGGGCAGTCTGGGTCCTCTCATCATCCCCACTTCCCAGGCCCAG1150CCTTTCCAACCCTGCCTGGGATCTGGGCTTTAATGCAGAGGCCATGTCCT1200TGTCTGGTCCTGCTTCTGGCTACAGCCACCCTGGAACGGAGAAGGCAGCT1250GACGGGGATTGCCTCCTCAGCCGCAGCAGCACCTGGGGCTCCAGCTGCTG1300GAATCCTACCATCCCAGGAYGCAGGCACAGCCAGGGAGAGGGGAGGNGTG1350GGCAGTGAAGATGAAGCCCCATGCTCAGTCCCCTCCCATCCCCCACGCAG1400CTCCACCCCAGTCCCAAGCCACCAGCTGTCTGCTCCTGGTGGGAGGTGGC1450CTCCTCAGCNCCTCCTCTCTGACCTTTAACCTNACTCTCACCTTGCACCG1500TGCACCAACCCTTCACCCCTCCTGGAAAGCAGGCCTGATGGCTTCCCACT1550GGCCTCCACCACCTGACCAGAGTGTTCTCTTCAGAGGACTGGCTCCTTTC1600CCAGTGTCCTTAAAATAAAGAAATGAAAATNCTTGTTGGCAAAAAAAAAA1650AAAAAAAAACTCGAGGGGCNNCCCNGTACCCAATTCGCCCTA1692__________________________________________________________________________
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Cincinnati Reds (1-2) at New York Mets (0-3), 7:10 p.m.
Probable Starting Pitchers: Cincinnati - Mike Leake (0-0, 0.00) New York - Jenrry Mej�a (0-0, 0 00)
(SportsNetwork.com) - Young Dominican right-hander Jenrry Mejia can continue his momentum from a standout spring training when the New York Mets host the Cincinnati Reds on Friday night in the opening game of a three-game series.
The 24-year-old dueled former Boston ace Daisuke Matsuzaka for the fifth spot in the Mets rotation in March and won the competition after striking out 13 batters and posting a 2.70 earned run average in 13 1/3 spring innings.
He was hit by a comebacker in his final spring appearance and suffered a bruised forearm, but X-rays were negative and he's been cleared to pitch against the Reds.
Mejia will be opposed by Reds veteran Mike Leake, who had his best major- league season in 2013.
The Arizona State alumnus posted career-highs in wins (14), earned run average (3.37), strikeouts (122), innings pitched (192 1/3) and starts (31), then built on that breakout with six scoreless innings in his final spring training start.
He was hit hard in his most recent outing against New York, allowing four runs on eight hits in just 1 2/3 innings of a 4-2 loss in Cincinnati last September, but had registered a 1.31 ERA against the Mets in five previous starts.
Leake is 18-12 in his career on the road and won his lone lifetime decision at Citi Field.
On Thursday in Cincinnati, Jhonny Peralta smacked a two-run homer and Matt Adams went 3-for-5 with two runs scored as the St. Louis Cardinals waited out another long rain delay before posting a 7-6 win over the Reds.
Cincinnati's Homer Bailey (0-1) wasn't effective in his season debut, permitting four runs on seven hits and walking three before exiting after 4 1/3 innings.
The start of the game was pushed back three hours and 42 minutes by the weather. Wednesday's matchup, a 1-0 Reds' walk-off win, was delayed two hours and 40 minutes by rain.
In New York, Ryan Zimmerman went 4-for-5 with a homer and two RBI as the Washington Nationals completed a season-opening three-game sweep of the Mets with an 8-2 win.
New Met Curtis Granderson broke out of an early slump, finishing 2-for-4 with a pair of doubles and an RBI. He had gone 0-for-9 with five strikeouts in the first two games.
Juan Lagares continued his hot start to the season for New York, which is off to its worst opening to a season since beginning the 2005 campaign with five straight setbacks. The center fielder ended 1-for-3 with an RBI and is now batting .455 with two doubles, a homer, two RBI and four runs scored in three games.
The Mets haven't dropped their first four home games since 1992.
New York starting pitcher Zack Wheeler (0-1) suffered the loss, allowing three runs on seven hits in six innings. The right-hander threw 114 pitches, striking out six and walking two.
Cincinnati is 12-5 all time at Citi Field, which included a three-game sweep in 2013 and a 7-1 record there over the past two seasons. The Reds are also 4-0-1 in their last five series in Flushing.

subset: commoncrawl


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The members of the Tamil Nadu AITUC Construction Workers' Union observed a day long fast at Somarasampettai on Thursday to press for the provision of pension by the Unorganised Sector Workers Welfare Board to all construction workers beyond the age of 60.
The pension scheme was introduced in 2005 and due to improper functioning of the board and the indifferent attitude of the official machinery it was not implemented properly. A large number of construction workers enrolled themselves as members of the welfare board, but they were denied pension on the excuse that they had not completed five years as members.
Later, the board decided to sanction pension to all those who were its members for four and a half years. But, the labour officials did not implement this decision of the board. After various agitations, the government came out with a GO favouring provision of pension to all the construction workers who had crossed 60 years.
The officials did not implement this GO also. Even those who were sanctioned pension, were given the same after a delay of one year.
The officials were not prepared to implement the scheme of provision of a compensation of Rs. 1 lakh to the family members of the construction workers killed in accident.
They demanded the intervention of Chief Minister to set right the wrongdoings of the officials and render justice to the hapless construction workers.
N. Periasamy, state president of the union, and K. Suresh, district secretary of the union, spoke.
Keywords: AITUC Construction Workers' Union, fast, pension, Unorganised Sector Workers Welfare Board


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Interview: Todd Lincoln - Director (V/H/S: Viral, The Apparition, Gorgeous Vortex)March 26th, 2015Interviewed by: Todd Martin
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Book Review: The Pendle Curse - Author Catherine CavendishReviewed by: Dave Gammon
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Film Review: Prowl (2010)
SYNOPSIS: Amber dreams of escaping her small town existence and persuades her friends to accompany her to find an apartment in the big city. When their transportation breaks down, she and her friends gratefully accept a ride in the back of a semi. But when the driver refuses to stop and they discover the cargo is hundreds of cartons of blood, they panic. Their panic turns to terror when the truck disgorges them into a … Continue reading


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Classic 60s Movies: The Entire SeriesFebruary 3rd, 2015 by Scott
January was Classic 60s Movies month on the blog. Here are 30 movies from that decade spotlighted by our distinguished group of guest posters:
Butch Cassidy and the Sundance Kid
Dr. Strangelove Or: How I Stopped Worrying and Learned to Love the Bomb
The Good, the Bad and the Ugly
They Shoot Horses, Don't They?
Who's Afraid of Virginia Wolf?
Pretty impressive list. How many of these movies have you seen? I have not seen Repulsion or The Loved One, but I intend to remedy that as they go on my Must See list.
And that's really the whole point of these decade by decade retrospectives we've undertaken over the last year or so: To motivate folks to watch movies. This practice, along with read scripts and write pages, are the foundation of any screenwriter's education. Here are the other decades we've covered thus far, over 120 guest posts in total now stored in the GITS archives:
70s Movies
80s Movies
90s Movies
Thanks to everyone who has participated in this effort. You are helping create a resource to guide writers in what movies to watch and analyze, building up their base knowledge as they immerse themselves in the world of cinema.
Next up: The 50s. We'll get to that a few months down the road.
In meantime… WATCH MOVIES!


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DEFINITION of 'Announcement Effect'
The impact on markets from the news that a change will occur at some future date. It can be used as a general term for the reaction to any development that affects trading, such as a change in dividend policy or a stock split. It is most often used, however, to describe investor reactions to changes in monetary policy, such as a hike or cut in a key interest rate level.
Also known as a "signal effect."
INVESTOPEDIA EXPLAINS 'Announcement Effect'
Stock traders eagerly await the announcement of changes in Federal Reserve policy, and stock volumes are notably higher on so-called Fed days; depending on the investment environment, volatility may be substantially heightened as well. Researchers have also found that trading on the day preceding announcements of Fed policy is relatively calm.
ECB AnnouncementAn announcement by the European Central Bank (ECB) Governing ...
Dividend PolicyThe policy a company uses to decide how much it will pay out ...
Announcement Date1. The date at which a company will announce the details regarding ...
Monetary PolicyThe actions of a central bank, currency board or other regulatory ...
Federal Reserve System - FRSThe central bank of the United States. The Fed, as it is commonly ...
DividendA distribution of a portion of a company's earnings, decided ...
Why do stock prices change following news reports?Stock prices move up and down every minute due to fluctuations in supply and demand. If more people want to buy a particular ... Read Full Answer >>
Trading Strategies
The Daily Routine Of A Swing TraderFrom pre-market to after hours, see what you need to do to capture gains quickly.
Active Trading Fundamentals
Identifying Market TrendsThe success or failure of your long- and short-term investing depends on recognizing the direction of the market.
Forex Education
How To Trade Forex On News ReleasesWhen economic data comes out, it can have a marked impact on the currency market. Find out how to profit.
Trading Strategies
Momentum Indicates Stock Price StrengthMomentum can be used with other tools to be an effective buy/sell indicator.
Options & Futures
Minimize Risk With The Long CollarThink your favorite stock is on the way down? This simple option-trading strategy can help you manage your risks without selling the stock.
Stock Analysis
How Realty Income Became A Top Dividend Stock?Real-estate investment trusts have gained in popularity over the years as income investors look for reliable stream of cash from their portfolios.
Trading Strategies
American Express: Headwinds and TailwindsAny investors considering a position in American Express need to know these important facts.
Using the Dividend Discount ModelThe dividend discount model is a way of applying net present value analysis to estimate the future dividends a stock will pay. Those dividends are then discounted back to their present value. ...
Due Diligence On DividendsUnderstanding dividends and how they work will help you become a more informed and successful investor.
Active Trading Fundamentals
Finding Value In A Sideways MarketWhen the market is non-directional, your profit potential need not go sideways too.


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Steelers Place LS Greg Warren On IR, WR Limas Sweed On Reserve/Non-Football Illness List
The Steelers announced today that long snapper Greg Warren and wide receiver Limas Sweed will miss the remainder of the season. Warren has been placed on injured reserve with a right knee injury after tearing his ACL on the final play of Sunday's game against the Green Bay Packers. Wide receiver Limas Sweed has been placed on the Reserve/Non-Football Illness list. The Steelers will add two players to complete their 53-man roster prior to Sunday's 1 p.m. regular season home finale against the Baltimore Ravens.
← Previous Story 2009 Week 15 Game Recap: Steelers 37 Packers 36


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16 U.S. Code § 111c - Revision of boundaries; vested rights; administration
The boundaries of Mesa Verde National Park are hereby revised to include the following described lands, which, subject to valid existing rights, shall be administered as a part of the park in accordance with sections 1, 2, 3, and 4 of this title, as amended and supplemented:new mexico principal meridian, colorado Township 36 North, Range 14 West
Section 29: All portions of the south half and the southeast quarter northwest quarter lying south and west of the right-of-way of United States Highway 160.
Section 32: Those portions of the section lying south and west of the right-of-way of United States Highway 160, except the north entrance road to the park, the southeast quarter southwest quarter, and the southeast quarter northeast quarter southwest quarter.
Section 33: That portion of the northwest quarter northwest quarter, more particularly described as follows: Beginning at a point on the west line of section 33 which is 456.5 feet south of the northwest corner of section 33, thence running south along the west line of section 33 for a distance of 373.0 feet, thence running east for a distance of 516.8 feet, thence running north for a distance of 132.7 feet, thence running north 65 degrees 06 minutes west for a distance of 570.0 feet along the southwesterly right-of-way of Highway 160 to the point of beginning.
Source(Pub. L. 88–235, § 1,Dec. 23, 1963, 77 Stat. 473.)
LII has no control over and does not endorse any external Internet site that contains links to or references LII.


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Be a part of a unique online community that connects post production professionals and film academics worldwide.
You'll have access to personalize your news feed, access to Live Post Talks and much more. Contribute to the community by posting interesting post production content.
March 28, 2015, 3:38 pm
How to morph 3D shapes to music in After Effects, using Mettle's ShapeShifter Ae and Trapcode SoundKeys.
March 28, 2015, 3:37 pm
What was the 13th year like for the post-production team? A real can of worms! At the SXSW 2015 panel, Boyhood Post-Production: 13th Year of a 12 Year Movie, members of the post-production team explained how they dealt with the process, and here are the top five takeaways.
March 28, 2015, 3:37 pm
Many people find getting started with Pro Tools a bit daunting and so I thought it would be useful to have a series on starting out with Pro Tools, taking you through setting up your PT system, creating your first session, some tips & tricks and finally some basic troubleshooting advice. But first lets take a look at specifying your first system.
March 28, 2015, 4:18 am
2014 ACE Eddie Awards Student Editing - 2014 ACE Eddie Awards Opening Sequence American Cinema Editors
March 28, 2015, 4:18 am
GUESTS Greg Boren, Product Marketing Engineer, Marshall Electronics Jim Tierney, President & Chief Executive Anarchist, Digital Anarchy Jessica Sitomer, President, The Greenlight Coach Nicholas Pisarro, President, NP Associates, LLC
March 28, 2015, 4:18 am
Many professionals tend to believe that editing is one of the most important aspects of the creative process, the one that actually picks up the pieces and brings the story to life. On the other hand, editing can be really overwhelming and intense, especially when you don't have enough experience and haven't built some type of efficient and time-tested workflow.
March 28, 2015, 4:17 am
Hybride produced a total of 50 VFX shots including large-scale crowd simulations and a virtual replica of the Berlin stadium for the 1936 Olympic Games.
March 28, 2015, 4:17 am
Tech giants, Intel (link is external) and Toshiba (link is external), this week announced a new technology which will allow higher capacity solid state disk drives. The new "3D NAND" memory chips differ from current single plane memory chips by allowing stacked chips to pack in more data.
March 28, 2015, 4:17 am
Let's share a Nightmare. In this dark dream, you are not a talented, creative artist. Instead you are an accountant. You work at the very same studio, in a very similar cube but probably with fewer action figures.
March 28, 2015, 4:16 am
2014 ACE Eddie Awards Golden Eddie Award - Paul Greengrass - Montage. 2014 ACE Eddie Awards Opening Sequence American Cinema Editors...
Middleton, Wisconsin, March 25, 2015/PRNewsire/ – Sony Creative Software is debuting its newest media production packag ...
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Linux Pro PHP Articles Online
ROSE Blog: Rikki's Open Source Exchange
Don't forget: PHPWomen are sponsoring an article-writing contest. The winners will receive a free 1-year subscription to Linux Pro magazine and the option to have their articles posted on our site, too.
Check out the Linux Pro February 2008 PHP issue for these PHP articles:
PHP Scripting, by David Sklar
Tools and techniques for PHP with Linux
PHP Roundup, by James Mohr
Choosing a PHP integrated development environment
Treasure, by Ken Hess
Working with PHP online script archives
Controlled Eclipse, by Alex Legler and Peter Kreussel
PHP programming in the Eclipse IDE
Taking It Easy, by Tobias Schlitt
Easy PHP with the eZ Components library
Issue 174/2015
Buy this issue as a PDF
Microsoft Frees Code for .NET Build Engine
MSBuild is now just another GitHub project as Redmond continues its path to the light.
Researchers Demonstrate Hack by Heat
Malware could pass data and commands between disconnected computers without leaving a trace on the network.
Kernel Developers Adopt Code of Conflict
New rules emphasize collegiality in coding.
Ubuntu Switches to systemd
Upstart lands in the dust bin as a new era begins for Linux.
Old Vulnerabilities Are Kept Alive Through Bad Configuration
HP's annual Cyber Risk report offers a bleak look at the state of IT.
Linus Torvalds Announces Linux 4.0
But what do the big numbers really mean?
Microsoft Frees CoreCLR
.NET Core execution engine is the basis for cross-platform .NET implementations.
New Trojan Attacks Linux Servers
The Xnote trojan hides itself on the target system and will launch a variety of attacks on command.
Cisco Releases Annual Security Report
Spammers go low-volume, and 90% of IE browsers are unpatched.
Zero Day Exploits Target Flash
Adobe scrambles to release patches for vulnerable Flash Player.


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Autumn Jubilee Collection 17 in. Square WreathItem # HN-FLW601
- Quince slices, cedar, salal, wheat, eucalyptus, and more
- Blend of handsome fall colors
- Best for indoor decorative use only
- Unique square design
- Measures 17W x 17H inches
Similar Wreaths
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Precision Cape Cod Chicken Coop
Hayneedle Celestron AstroMaster 114 EQ Reflector Telescope
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Breathtaking Performance of Note To God by Charice - Music Videos
Powerful Cover Of Hillsong Young And Free's 'Gracious Tempest' Will Touch Your Heart - @theremix
00:07:09 1123 views
Blake Shelton And Miranda Lambert Surprise Fans With Duet Of 'God Gave Me You!' - @theremix
00:05:05 8271 views
'When All Is Said And Done' – Beautiful Worship Song Asks What Legacy You Will Leave - @theremix
00:02:45 1372 views
12-Year-Old Lexi Walker Sings Stunning Version Of 'How Great Thou Art' - @theremix
00:02:13 28416 views
Embed theremix 48276 views 2012-05-01T00:00:00 Charice has captured hearts from around the world with her powerful and encouraging performances. Born in the Philippines and raised by a single mother, Charice began singing as a child to help support her family. Her inspirational story is a testament
to how God will bless us beyond our wildest dreams. Watch this stunning performance of Note To God to see just what all the buzz is about!
to how God will bless us beyond our wildest dreams. Watch this stunning performance of Note To God to see just what all the buzz is about!
More Music Videos
- Spectacular Performance Of 'The Old Rugged Cross' Will Give Your Goosebumps Gooseb...Music Videos
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- 2 ADORABLE Toddlers Sing He Arose - It Will Melt Your Heart!Cute Videos
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Hep C Untreated Prognosis
Here's a taste of what TheBody.com has to offer on this topic:
Ask the Experts
Hep C long term prognosisI found out I have hep c in 1994,have done inter/ribo treatment one full year, failed, did a daily for 6 months and had a platelet disorder requiring stopping. now 3 yrs later I have a viral load of 1,190,000 and an alt of 103 and an ast of 70. I...
Response from Barbara McGovern, M.D.
Tufts University School of MedicineI am sorry to hear that you have not responded to treatment. However, I want to assure you that there is NO correlation between your level of virus and the amount of liver disease you have. I have seen patients with extremely low levels of virus you... Read more »
Hep C prognosisMy wife was recently diagnosised with Hep C which would have been dormant for 30 years. She has a... Read more »
Transmission of Hep C? (HEPATITIS C TRANSMISSION VIA SALIVA, 2010)Dr., I so appreciate your honesty and yet I feel you are a bit naive. Transmission is a lot more... Read more »
This Month in HIV: Update on Hepatitis C/HIV Coinfection... Welcome to This Month in HIV. I'm Terri Wilder . This month's topic is HIV and hepatitis C coinfection. Up to one third of people with HIV are estimated to also have hepatitis C, and living with both viruses is no picnic. Read more »
Hepatitis C and HIV... What Is Hepatitis C? How Is It Diagnosed? How Is Hep C Treated? Can Hep C Be Prevented? Hep C and HIV Together The Bottom Line What Is Hepatitis C? The hepatitis C Read more »
HIV and Hepatitis C Co-Infection... Are you HIV positive and newly diagnosed with hepatitis C (Hep C)?Do you have one virus and think youmight be infected with the other? If so, this brochure answers somebasic questions Read more »
Safer Injection Guidelines That Can Reduce Health Risks Including Getting Infected With HIV or Hep...hardier than HIV, and therefore more difficult to destroy. It has not been proven that using bleach for 2 minutes also kills the Hepatitis C virus. It's possible that blood containing Hepatitis C can remain infectious outside of your body for up... Read more »
Connect With Others
Hepatitis C
Posted by hopefulindy, 1 Reply...possible exposure. I have on going health issues and no answers. I am waiting on blood work to see if I have been exposed to Hep C. If someone is infected with Hep c and HIV at the same time would that slow the production of HIV antibodies? That... Read more »
Hepatitis C
Posted by hopefulindy...Does anyone know? If you contract HIV and Hep C at the same time will the Hep C cause a delay in production of Hiv antibodies due to weakened immune system? Read more »
Hepatitis C and Coinfection
Posted by statusCunknown, 1 Reply...opportunity to introduce myself. My name is Shari and I am not infected with HIV, (this by shear luck only). I AM infected with Hepatitis C and the virus has caused severe damage to my liver. Over 5 million Americans in this country are... Read more »
Periodo en caso de coinfeccion con la hepatitis c?
Posted by Anonymous, 1 ReplyAlguien me podria decir si con 6 meses seria suficiente? Read more »
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Scottish border guards would force Englishmen to squeal like a pig
AN independent Scotland would have border checkpoints where guards would force English tourists to squeal like pigs.
America spied on Yorkshire 'for about five minutes'
AMERICA'S National Security Agency tapped the phone calls of Yorkshire for five minutes before throwing its headphones across the room in horror.
Nobel Prize for YouTube comments shortlist announced
THE Nobel committee has selected the potential winners of this year's YouTube comments prize.
UN unveils capitalism opt-out region
THE UN has given people who don't like capitalism the option to try something else instead.
Abandoned Washington DC becomes ape habitat
THE abandoned capital of the United States has been colonised by apes after just three days of a government shutdown.
Britain still gripped by every twist and turn of Syria story
BRITAIN cannot hear enough about the Syrian conflict and has urged the media to report it in greater detail.
Syria vote was based on one of my other massive fuck-ups, says Blair
TONY Blair has insisted it was the way he turned post-invasion Iraq into an utter hellhole that stopped MPs from backing war in Syria.


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Konvertor FM 5.02.2
Konvertor FM is a fast and reliable two-panel, multi-tabs file
manager for Windows. It has an intuitive interface, offers viewers for all popular file formats as well as uncommon and very rare ones (4205 in this version).
Konvertor FM is a way to work with files more efficiently.
(Konvertor FM does not change any settings in Windows, and does not hook itself into anything inside Windows.)


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Case Studies
- World Resources Institute (WRI)
- Climate Victory
- Greenwood Energy
- Advanced Biofuels Association (ABFA)
- Solar Energy Industries Association (SEIA)
Silvio is an unusual talent. He is quite adept at combining his deep sense of strategy with an understanding of modern communications tools and technologies. He's also easy to work with - he will meet your budget and exceed your expectations. We had a tricky timeline and had to do a lot of juggling to make our production schedule work; Silvio adjusted on the fly and kept his cool through the whole process.
Matt Lewis, Director of Communications
Center for the Next Generation
Twitter: @silvio_marcacci
- RT @ValerieVolco: #Obama admin officials assure countries #US #climate pledge on strong footing; @GOP warns it can collapse http://t.co/Niy… about 10 hours ago from TweetDeck
- RT @AllanMargolin: Panel says seas could rise up to 10.6 inches on NC coast by 2045 http://t.co/AcMowu2w3y via @theobserver #climate #seal… about 10 hours ago from TweetDeck
- RT @JustinGerdes: New solar generation made up for 83% of California's lost hydro production in 2014, says @EIAgov: http://t.co/O2WmHVFF0W … about 10 hours ago from TweetDeck
- RT @rebleber: Congress is Losing Top Environmentalists, But Democrats Have a Deep Bench to Take Over http://t.co/0qqwrCeYn4 via @tnr about 10 hours ago from TweetDeck
- RT @petedanko: From local forecaster Mark Nelsen, record warmth in 5 of past 12 months in #PDX. http://t.co/ACvoUpxv58 about 10 hours ago from TweetDeck


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Is this an elm with tiny leaves?
Our builder planted this tree in front of our House in Maryland in 2012. They claim that it is an American Elm, but the leaves just seem too tiny compared to other elms that I have seen in person and in pictures. Bases on this picture does this tree look like a variety of American elm?
Thank you
That is definitely not an American elm. It does however look like to be a hybrid Elm which has many similarities and smaller leaves. I am guessing that it is probably a frontier elm tree. Do you know what color the leaves turn in the fall? The other elm it kind of looks like it is a new horizon elm, but the leaves look to be a little small and not dark green enough. See link for other possibe varieties you may want to google...... nice tree!
Here is a link that might be useful: Elm Cultivators
After looking at the pictures more, I think it may be a Chinese elm which is also known as a Lacebark elm. They have very small leaves and are very disease resistant. they are less American elm like than the hybrid cultivators, but are usually very free of problems and have a nice shape, although much smaller leaves and mature size than an actual American elm.
Thank you for the great answers. The builder told me that it was an American Elm last year, so I was certainly researching the wrong tree. Since then, they have planted several real American Elms that certainly look fuller with much larger, darker leaves. Now I know that I have a Chinese Elm. Hopefully the bark gets as interesting as the pictures that I see online.
Here's 1 more picture of the leaves.
Thanks for the stake advice. It had two stakes, but one broke last year and the tree was leaning a little in one direction, so I naively thought that a year staked to one side would help.
It was planted in June 2012, so the stakes can probably come up.
Our soil is pretty heavy with a reddish clay, so the ground is solid.


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Based on 1 review
Help make it easier for other Avvo users to choose the right lawyer by sharing your experience with this attorney. It's fast, simple, and safe.
Review this lawyer
Lawyers: Use the Peer Endorsements section to provide input about other attorneys.
Used him - fired him - he then tried to keep other attorneys from taking the case - dishonest - doesn't know what he is doing.


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28 year old man | Brooklyn, NY, USA | Seeking women 21-30 within 50 miles
Just enjoying the search of new and interesting experiences one day at a time while avoiding the weight of our modern world.
Confused with what to write. Not sure if I need to exploit some musical talent ( which I don't have, but am willing to learn ) or what. I like to think of myself as a creative individual. I've revolved much of my adulthood, which I hope hasn't been that long, around urban art and technology. I went to an engineering college for its equivalent of an art degree. So hopefully a nice split between the smarts of the engineering type but with the ability to not be totally awkward in social situations. I grew up and went to college in the south but shortly after graduating helped start a creative agency and get it off the ground which we moved here to NYC. Really no expectations and just testing the water.
Book club , Coffee and conversation , Cooking , Museums and art , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing cards , Playing sports , Political interests , Volunteering
Basketball, Bowling, Cycling, Dancing, Other types of exercise, Running, Swimming, Walking / Hiking, Weights / Machines, Yoga
I exercise 3-4 times per week.
I have Dogs
I like Birds, Cats, Exotic Pets, Fish, Horses, Other
Catching a good show at Music Hall of Williamsburg, a delicious cocktail at the richardson or maison premiere. If I'm feeling ambitious, biking up the wes side highway to The Cloisters.
Music: Nicolas Jaar, John Vanderslice, Murder by Death. Reading anything that can twist my perception of reality. Biking / Running somewhere isolated and in nature.
See a show, read, have a cocktail. Often I work on personal endeavors or travel.
The Hidden Reality by Brian Greene and Jitterbug perfume by Tom Robbins.
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The Georgian House
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- Free parking
Photos of The Georgian House
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TripAdvisor Reviews The Georgian House Glasgow
Travel Blogs from Glasgow
Referendum day
... a loud and boisterous pro-independence rally in Glasgow's George Square, complete with saltires, pipers, loudhailers and the shouting down of a small Better Together group.
Within Helensburgh though a reasonably civilised atmosphere has prevailed, with both 'Yes' and 'No' campaign stalls peacefully sharing the newly reopened town square at the weekend. The lamp posts on the road into town alternate 'Yes' and 'No' signs, or else sport both, giving a somewhat ...
...but they shall never take our freedom!!!
... how each religion deals with these milestones. When the museum closed and I had to leave I travelled back to the McGuires for another treat - a home cooked meal! On my second full day in Glasgow I visited the Kelvin Grove Art Gallery and Museum which houses some very interesting displays, most of which with relevance to Glasgow. Such as the room devoted to the Glasgow Boys (a group of painters from Glasgow) and the displays of design. I enjoyed the natural history ...
... tour and we received a complimentary half pint of anything on tap whilst we waited for the others to arrive (the website said we got a pint and the tour cost £7 but we assumed we pay at the end and got another half pint after). There ended up only being 3 of us in the tour, we were told about the brewery as it has been in the same location since 1556 and survived the lows of the wars. We set off in our hats, glasses and safety vests as we explored the brewery. ...
Day 20: killing time in Santa Monica.
... before a ten hour flight :( About 2.30 we head back to LAX finally check in and discover we've been allocated two seats together in the middle four. SUCKS. Despite our best pleading and wheedling tho, this can't be changed so we resign ourselves to it. We charge our phones, but a bunch of sweets and treats and water, and nap. Then board at 4.30 for the longest leg of our journey. London, here we ...
The "small" trip to William Wallace then Glasgow!
... arrive at the William Wallace monument, ensure you are wearing comfortable shoes as it is one heck of a hike! It's a rough terrain, few railings and unpaved pathways. Have a bottle of water handy and take frequent stops. It may not be too bad for the young visitors, but for the older, just take your time getting up there. Once there, it is worth the view and entering inside.
The views of the Scottish countryside were beautiful. There were ...


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Ms. Gretchen Conley
Endocrinology, Diabetes & Metabolism
- 5230 E Stop 11 Rd Ste 150
Indianapolis, IN 46237
- 2 Locations
- (317) 865-5904
Ms. Conley works in Indianapolis, IN and 1 other location and specializes in Endocrinology, Diabetes & Metabolism and Diabetology. Ms. Conley is affiliated with Franciscan Saint Francis Health and Franciscan St Francis Hospital. Ms. Conley speaks English and Spanish.
Gender: Not Available
Languages Spoken: English, Spanish
Endocrinology, Diabetes & Metabolism, Diabetology
Medical School: Not Available
*This physician has multiple office locations. See Office Information for details.


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BEST soaker hose
What is the best soaker hose that will LAST for a few years?
i would like one that i can leave down and not have to replace half way thru the year..
You can buy rubber soaker hoses and they last longer. You'll pay for that.
I've never had to replace a soaker hose, unless I hit it with the weed whacker or else don't get it drained before the first hard freeze.
Some of mine are under mulch and some drip line is right out in the sunlight and it is several years old and still good. Sunlight here is intense, so it's not like the hose has only been exposed to filtered light.
I had a problem with the soaker hose getting holes that would just squirt. I'd cut that section out and use a coupler to fit the hose back together rather than replace it.
Then I found out the reason...my water pressure was way too high. I'd turn the faucet on and let it run to get the water out in the shortest amount of time and then move on. Once I found I was shooting myself in the foot and started using less pressure, the hoses quit rupturing.
You can buy a pressure regulator at garden supply stores or from drip irrigation suppliers.
Speaking of drip irrigation, dripline tubing is very long-lasting. I've had some in use for five years.
I'm having trouble with those black ones from HD. They crack and fall apart in less than a year.
This year I'm going to see what an irrigation store can offer that can be attached to a regular hose attachment but hopefully made to last longer.
I got tired of the black soaker hoses. They use to be good, then they started making them cheaper and cheaper. Even with reduced pressure, they would break or split. I ended up spending more money on couplers than if I would have just replaced the hoses.
I cut out my couplers and threw the hoses away and went for drip tape. By far a much better product.
I bought it from Berry Hill Irrigation. I went with the Aqua-Traxx. I liked it because of the blue lines, they go on top. It made installation really easy for my helpers (my 9,7,4, and 2 year old). I bought the 7,500 foot roll. It was $170 and shipping. Just buy the connector ( I would suggest the ones with the valves, didn't do that the first time) some patch connectors ( tillers, hoes and drip tape don't mix). Make sure you get a filter and regulator. They have kits, maybe check those out too. I would my the header line at a local place and save on shipping.
Here is a link that might be useful: Berryhill Irrigation
I used drip irrigation a couple a years ago and it worked ok. However it is time consuming to set up. But the real problem is that if you rotate your plan things every year, the system is configured to last year's plantings and it is very difficult to reconfigure..
My soaker hose is years old and still running fine. It has a few holes that will fountain, but I just proped a rock against it at that spot to keep the water in the garden. Last year I used it under plastic mulch, so it was no problem. I assume I got it from a big box, but I have no idea which or what the brand name was. Drip lines are better, but like someone said, if you change your layout every year, a drip line makes no sense. My soaker is perfect for my 4 foot wide, 15 foot long plot.
Not knowing what kind of gardening you do, but if you are a sqaurefoot or box gardener I'd reccommend the Garden-Grid irrigation system. It partitions everything perfectly and is really durable from what I have experienced. A small company in Florida makes them, I love it! I posted their website below. They also make cedar garden boxes that are beautiful!
Here is a link that might be useful: Garden in minutes - products
Farmington- The "one inch per week" "rule" is a very vague guideline at best. To put it another way, it's completely unreliable. How much you need to water depends on the specific crop, rainfall, heat, your type of soil, sun exposure, if your soil is mulched or not, etc. Nobody can say definitively that your plants need x-amount of water. Sorry, I realize that doesn't help much.
Water until your soil is moist. Not just on the surface but deep down. Certain plants like consistent moisture. Some like to dry out a bit between waterings. If it's been hot, you'll need to water more. If it's been cool you might not need to water for a while. Sandy soil dries out quicker than clay. Mulched soil retains moisture whereas unmulched dries out quick in summer. You just have to get a feel for it.


text: The girl was gorgeous, the kind of girl Clary would have liked to draw—tall and ribbon-slim, with a long spill of black hair. Even at this distance Clary could see the red pendant around her throat. It pulsed under the lights of the dance floor like a separate, disembodied heart.


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Neb. Incident Draws Police Probing Utah Kidnapping
SALT LAKE CITY - Police said today they were investigating a report that a distraught girl about the same age as Elizabeth Smart was spotted in a minivan in Nebraska, possibly being held against her will.
Lincoln, Neb., police issued a nationwide alert of a possible abduction last week after witnesses at a gas station convenience store there reported seeing a crying girl, between 12 and 14, with light hair.
But authorities said they have not connected the sighting to Smart, 14, who disappeared from her home June 5, nor have Salt Lake City police.
"We don't have a whole lot of information," Salt Lake City police Sgt. Fred Louis said today. "It's not clear what they have."
Witnesses said the girl stared at the store with tearful eyes until she was pulled over a seat and out of sight.
"They said they saw a pair of arms come from the back and pull her into the back, but nobody got a look at that person," said Lincoln police Sgt. Larry Barksdale.
Barksdale said police know who owns the van, bearing a Pennsylvania license plate, but don't know the vehicle's whereabouts. He said the driver and a male passenger took their time buying gas and cigarettes, and were not acting suspiciously.
Police have arrested the Smarts' former handyman, Richard Albert Ricci, 48, on theft charges and consider Ricci a possible suspect. He denies any role in Elizabeth's disappearance.
Copyright 2015 Associated Press. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.
Recommended Investigations
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Add an animation to your web projects with a CSS sprite sheet, in this quick, project-based course.
Learn how to create a preloader from an animated GIF, using After Effects, Photoshop or Fireworks, and Adobe Edge Animate.
Shows how to create interfaces, systems, and devices revolved around user behavior.
"My biggest problem is figuring out what I want to watch next. Just when I think I'm all set, I find something ELSE that is fascinating." —John O.
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Comment: No kidding. Darwinism put it well: evolution comes down to reproduction. It is ever clear that Hispanics will eventually took over America and Indians will conquer China, Russia dies on its own from Vodka before then. And we ain't gonna hear much about Japan and Germany, which have birth rate of 0.2 per couple.
East Indian and West Indian, that's it and that's all. Taco, cumen and intercourse out last all others, especially after the nuclear war!
Comment: Before long, they will be in California, where you can buy a house to obtain a citizenship, or Vancougver Canada, or Sydny, Australia. Communists billionaires leave and poor peasants stay.Recommend
Comment: American market has always been open to Americans, if do not do well, Americans can always blame it on others, Iran, China or North Korea, BP, France, etc. A win-win situation.Recommend
Comment: 1. At the end of WW2, people were tired of fighting, killing and dying. They took a break in the mid of 1945, until resuming all of that in WW3.
2. "Wars are started by people in their 50's and 60's and are fought by people in their late teen's and twenty's. Everybody in between dies."
3. We get what we wish for.
Comment: Commonly, there are three chronic conditions in that one loses control of bowel movements:
1. In infancy, as we all know.
2. In dementia, as the cerebral cortex atrophies, which we don't know since we don't go to nursing home to visit our parents.
3. After trauma, such as after homosexual play or jail rape, which we like to know.
One cannot actually say Whooowee in case 1 & 2. And change of formula won't make a difference in 2 & 3.


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Book Search
Disaster Risk
Series: Critical Concepts in the Environment
Especially in an era of rapid global environmental change, questions and issues about and around natural hazards and disasters are dizzying in their complexity—and urgency. Answering the need for an authoritative reference work to make sense of this fast-moving area, and its vast and...
To Be Published April 7th 2015 by Routledge
Disaster Management
International Lessons in Risk Reduction, Response and Recovery
There is a perennial gap between theory and practice, between academia and active professionals in the field of disaster management. This gap means that valuable lessons are not learned and people die or suffer as a result. This book opens a dialogue between theory and practice. It offers vital...
Published September 21st 2014 by Routledge
Handbook of Hazards and Disaster Risk Reduction
The Handbook provides a comprehensive statement and reference point for hazard and disaster research, policy making, and practice in an international and multi-disciplinary context. It offers critical reviews and appraisals of current state of the art and future development of conceptual,...
Published December 21st 2011 by Routledge
Disaster Diplomacy
How Disasters Affect Peace and Conflict
When an earthquake hits a war zone or cyclone aid is flown in by an enemy, many ask: Can catastrophe bring peace? Disaster prevention and mitigation provide similar questions. Could setting up a flood warning system bring enemy countries together? Could a regional earthquake building code set the...
Published August 30th 2011 by Routledge
A Practical Guide for Teachers and Other Professionals
This book provides essential information and guidance about stammering for those working in educational settings. The highly experienced team of authors demonstrates how early intervention is essential if children who stammer are to have the best chance of recovery; the practical strategies that...
Published September 20th 2001 by David Fulton Publishers


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Panorama City is a populated place LOCATED WITHIN the City of Los Angeles. In turn, the City of Los Angeles is located within Los Angeles County.
Panorama City is located at latitude 34.225 and longitude -118.45. The elevation of Panorama City is 817 feet above mean sea level.
Since Panorama City is located within the City of Los Angeles, all of the census and demographic data for residents of Panorama City are included as part of the information reported for City of Los Angeles in the "Data" tab above.
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Not so long ago I published a post about the challenges that products that need cold temperatures to be preservedface, and the different aspects that make the cold chain a very particular type of Supply Chain and probably the fiddliest in terms of transportand reliability (you can find the post here).
Continuing with this topic, this post is an approach to what probably is the most challenging type of delivery and hence the one that entitles most complications. Organ transplant.
Organs for transplant are one of the most critical shipments. To ensure viability of the organs, transportation and logistics must be flawless and perfectly coordinated, quality and speed are the key factors.
Organ transport requires an extraordinary level of 24/7 readiness over a wide geographic area. Some organs, such as a heart or lungs, can stay viable for only 4-6 hours, the cold ischemia time, (the maximal time that the organ outside the body can be kept in a storage solution), making their delivery to the operating room a true race with time.
The transplantation process starts in the hospital when a potential donor is notified. The organ is then prepared and stored before it can be transported.
Considering the limited time for transport and the fact that the smallest mistakewill probably hinder the success of the transplant, reliability in transport is a must in these cases.
Thinking of organ donation as an end-to-end Supply Chain we should also consider a few facts that make things even more complicated:
- You never know when your "product" will become available
- You have multiple products coming from the same "supplier", which are going to different destinations, with a limited shelf life.
- You need to obtain the "product" from the "supplier" with specialists and each extraction requires a different approach. Furthermore, until the "product" is actually extracted, its viability is unknown.
- When it is known that a "product" is becoming available, there is a search to find who the customer is who is most in need. The customer could be a few (or a hundreds)kilometres away.
- As the customer is identified, there is a need to assemble specialists at the "customer's" location for "installation" of the part.
- And during this entire time, the actual logistics for the transport will need to be put in place in a last minute scenario, with transportation "experts" accompanying the "product"
If you though your Supply Chain was challenging and stressful and your customers too demanding, I guess it's time torecap!