text:
|Publication number
|Publication type
|Application number
|Publication date
|Jul 26, 1983
|Filing date
|Aug 19, 1981
|Priority date
|Aug 19, 1981
|Fee status
|Publication number
|Lois B. Wilson, John T. Wilson, Robert F. Geever
|Original Assignee
|Medical College Of Ga. Research Inst., Inc.
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|Patent Citations (1), Non-Patent Citations (7), Referenced by (105), Classifications (17), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The Government has rights in this invention pursuant to Grant Number PCM 7909054 and Grant Number HL 23294 awarded by the National Science Foundation and the National Institute of Health, respectively.
(1) Field of the Invention
This invention relates to a diagnosis of sickle cell anemia and, more particularly, to a direct analysis of human hemoglobin genes by the use of restriction endonuclease Dde I and molecular hybridization procedures to detect sickle cell anemia.
(2) Description of the Prior Art
Sickle cell anemia is a debilitating genetic disorder that affects two of every thousand Blacks born in the United States. The disease is also prevalent in the Mediterranean area, as well as India. It is characterized by general weakness and pains in muscles and joints and can be fatal, frequently at an early age; victims do not live much beyond the age of 30. The disorder is caused by a single nucleotide base mutation in the globin gene which converts the glutamic acid codon (GAG) at amino acid position 6 to one for valine (GTG). That chemical abnormality produces red blood cells which are distorted into an inflexible sickle shape that can clog capillaries, blocking the flow of blood to the tissues.
The current test for sickle cell anemia diagnosis in fetuses is performed on samples of fetal blood obtained by fetoscopy. Through this procedure, a small sample of blood is taken from the fetus through the umbilical cord and then analyzed for sickle cells. The mutant protein migrates differently on the gel than the normal protein. While generally accurate, this diagnostic method is dangerous as it can cause a spontaneous abortion of the fetus 10 to 20% of the time.
Recombinant DNA techniques coupled with blot hybridization analysis have proven to be valuable tools for studying the molecular basis for hemoglobinopathies. Various researchers have used blot hybridization to confirm that δβ-thalassemia and hereditary persistence of fetal hemoglobin are the results of gene deletions, whereas α-thalassemia and β-thalassemia are due to both gene deletions and point mutations. One study has shown that at least one case of δ-thalassemia is probably due to a base mutation.
These studies have also been extended to the clinical setting as methods for prenatal diagnosis of various genetic hematological conditions, generally the thalassemias. In addition, Kan and Dozy have reported, in Proc. Natl. Acad. Sci. USA 75, 5631-5635 (1978), the finding of a polymorphism for a Hpa I restriction endonuclease site in American Blacks 3' to the β globin gene, which was shown to have a 60% association with the sickle cell allele. From their studies, they estimated that blot hybridization using this polymorphism alone could be successfully used for prenatal diagnosis of a sickle cell anemia in 36% of couples at risk. Phillips, et al. have combined the Hpa I analysis with a second polymorphism found in the γ globin genes. In so doing, they have reported an extension of blot hybridization for prenatal diagnosis of sickle cell anemia to over 80% of the couples at risk. Proc. Natl'l. Acad. Sci. USA 77, 2853-2856 (1980). However, their analysis, which is safer than fetoscopies, requires family studies in order to establish the association of the polymorphic sites with the sickle cell allele. The analysis of polymorphisms requires analyzing DNA samples from both the mother and father to get 36% reliability and from both parents and one child already born to get 80% reliability. This limited application is a major disadvantage of these procedures.
A direct analysis of the sickle cell anemia should be possible by use of a restriction enzyme whose recognition sequence is created or eliminated by the sickle cell mutation. This approach would not require family studies, and should be useful for all couples at risk. Dr. A. Nienhuis has proposed such a direct analysis with restriction endonuclease Mnl I. N. Engl. J. Med. 299, 195-196 (1978). However, efforts in various laboratories have failed to attain the sensitivity requisite for the resolution by the DBM filter paper of the small [60-80 bp (base pairs)] fragments generated by this enzyme.
The disadvantages of the prior art methods are overcome by the present invention which utilizes a restriction enzyme which recognizes the nucleotide base sequence CTNAG (where "N" is any base), such as Dde I, for direct analysis of sickle cell anemia. Briefly, the analysis comprises the steps of either removing an effective amount of amniotic fluid and isolating DNA from the cells present in that fluid, or isolating DNA from peripheral blood lymphocytes, digesting the DNA with the restriction enzyme Dde I, separating the DNA fragments by molecular weight to form a band pattern and visualizing the band pattern or DNA fragments with a hybridization probe. The method may also include the step of transferring the DNA fragments to filter paper prior to hybridization. Normal individuals (AA) have approximately 175 bp and 201 bp blands on autoradiograms of the hybridized DNA segments, whereas sickle cell individuals (SS) have an approximate 376 bp band. Sickle cell trait individuals (AS) have the combination of approximate 175, 201 and 376 bp bands. Thus, the present invention can distinguish between AA, AS and SS individuals.
The procedure uses a restriction endonuclease, such as Dde I, which recognizes the base sequence CTNAG (wherein "N" is any base), for digestion of total genomic DNA. This restriction recognition site is present in normal DNA at codons 5 (CCT) and 6 (GAG), yet is not present in sickle cell DNA (CCT GTG) due to the sickle cell mutation. Following enzyme cleavage, the resulting restriction fragments are separated by molecular weight. One method of separating the fragments is by polyacrylamide gel electrophoresis and electrophoretically transferring the separated fragments to DBM paper. Another method is by high pressure liquid chromatography on reversed phase columns (RPC-5); the separated fragments are then immediately bound to filter paper (either DBM or nitrocellulose) for the sickle cell analysis.
For hybridization, applicants have isolated a probe which is complementary to the 5' end of the β globin gene. This probe is nicktranslated to 1-2�108 dpm/μg of DNA, with hybridization occuring by the Southern blotting method. Southern, J. Mol. Biol. 98:503 (1975)
Besides radioactive labeling, probes for visualizing DNA fragments can be produced by using protein or biotin coupled nucleotides. Protein or biotin coupled nucleotides, once incorporated into the probe, can allow visualization of the β globin gene fragments through the use of fluorescent antibodies binding to the protein or biotin molecules or to non-fluorescent antibodies also bound to such molecules. Such antibodies will bind specifically to DNA fragments to which hybridization has occurred since only hybridized fragments will have the initial protein or biotin mucleotide associated therewith. These complexes can then be visualized by illumination with ultraviolet light.
Applicants' current studies have been concerned with identification of the sickle cell allele (βs gene) within DNA isolated from peripheral DNA as well as amniotic fluid cell DNA for clinical antenatal diagnosis of sickle cell anemia.
FIG. 1 is a restriction map of a portion of the human β globin gene; and
FIG. 2 is a photograph of autoradiograms of sickle cell (SS), sickle cell trait (AS), and two control individuals (AA, and AA2) prepared by the present invention.
Patients with sickle cell anemia (SS), sickle cell trait (AS), and control individuals with no known hematological disorder (AA) were the subjects of the study. Approximately 10-20 ml of blood were collected in vacutainers with EDTA as anticoagulant. The samples were placed in ice and transported to the laboratory for immediate processing. Informed consent was obtained.
High molecular weight DNA was isolated from peripheral blood lymphocytes or cells present in the amniotic fluid by a method of that of Blin and Stafford. Nucl. Acid Res. 3, 2303-2308 (1976). Briefly, 10-20 ml samples were centrifuged at 3,000 rpm and the plasma or fluid fraction removed by aspiration. Residual amounts of plasma were extracted by repeated washes of the cell pellet with 0.9% NaCl. For DNA extractions from blood samples, the reticulocytes and older red cells were hemolyzed by the addition of two cell pellet volumes of sterile H2 O and the lymphocytes collected by centrifugation at 3,000 rpm for 15 min. The lymphocytes and amniotic fluid cells were lysed by the addition of 20 ml of lysing solution (0.05 M Tris pH 7.5, 0.5% SDS, 0.1 M NaCl, 0.001 M EDTA, 100 μg/ml proteinase k, Beckman) and incubated at 37� C. for 48 hrs. Following lysis, the solution was deproteinized by repeated chloroform-phenol (1:3) extractions and dialyzed overnight at 4� C. against 50 mM Tris pH 8, 10 mM EDTA, 10 mM NaCl. Nucleic acids were isolated by the addition of 2.5 volumes of ethanol followed by centrifugation at 9,000 rpm for 15 min. The pellet was resuspended in 10 mM Tris pH 7.5, 1 mM EDTA, incubated at 37� C. for 30 min with 50 μg/ml heat-treated RNAse (RNAse A, Sigma), and chloroform-phenol extracted. DNA was collected by ethanol precipitation followed by centrifugation as above, and redissolved in sterile H2 O at a concentration of about 0.2 mg/ml.
A DNA fragment of approximately 1.8 kb was resolved by digestion of a recombinant DNA clone, which had a 4.4 kb DNA fragment of the β globin gene integrated into the bacterial plasmid pBR 322; with Bam Hl and electrophoresis through 0.8% agarose in TEA buffer (40 mM Tris-HCl, pH 7.8, 20 mM NaAc, 2 mM EDTA). Recovery was attained by electroelution into dialysis tubing (Spectroapor 4) immersed in 1/2�TEA buffer, partial volume reduction by lyophilization, phenol-chloroform extraction and ethanol precipitation. Subsequent restriction enzyme digestions were performed with ten units enzyme per μg DNA (where 1 unit is that amount of enzyme which would digest μg of λ DNA in one hour) and resolved on polyacrylamide gels containing 4% (w/v) acrylamide and 0.2% (w/v) N,N' methylenebisacrylamide. Fragments generated by Hae III digestion of φ�174 RF DNA were used as standards for approximate fragment length determinations.
DNA fragments resulting from restriction endonuclease DdeI digestion can also be separated by high pressure liquid chromatography on reversed phase columns (RPC-5) as set forth in Eshaghpour et al., 5 Nucleic Acids Res. 13-21 (1978). Through this procedure, DNA fragments can be separated and immediately bound to filter paper (either DBM or nitrocellulose) for the sickle cell analysis, thus eliminating the gel electrophoresis and DNA transfer steps. Such separation is also based on molecular weight.
Alu I digestion of pBR 322 β Pst (4.4 kb) produces a 737 bp, 5' specific β globin fragment. This fragment was resolved on and isolated from 4% polyacrylamide gels. On the average, 40% recovery was obtained following the electroelution process described above. Since preparations were contaminated with residual amounts of polyacrylamide, the quantity used as substrate for DNA Polymerase I (New England Biolabs, Inc.) was based on an estimate average recovery. Nick translations were performed using α-32 P-dCTP and α-32 p-TTP (500μCi/mmol) purchased from New England Nuclear. Reaction conditions are those of Rigby et al., J. Mol. Biol. 113, 237-251 (1977), but with the buffer reported in Maniatis et al. Proc. Natl. Acad. Sci. USA 72, 1184-1188 (1975) DNA fragments were labeled to an estimated specific activity of 1-2�108 dpm/μg of DNA. Following nick-translation, an aliquot from each reaction was analyzed by electrophoresis on 4% polyacrylamide gels under denaturing conditions and autoradiographed. Nick translations suitable for subsequent hybridization exhibited a near even distribution of fragment lengths between 100 and 700 nucleotides.
In some instances, hybridization can occur directly on the gel, thereby eliminating the DBM transfer step. As to that step, details for preparing DBM paper and transferring DNA are as given in Bittner et al., Ana. Chem. 102, 459-471 (1980), with the exception that aminobenzyloxymethyl (ABM) paper was made according to Levy et al. Gene 11, 283-290 (1980). For these studies, 20 μg of cellular DNA was digested with four-fold excess Dde I restriction endonuclease (1 hour units) overnight under conditions suggested by the manufacturer (New England Biolabs). Samples were phenol extracted, precipitated with ethanol, and resuspended in TEA buffer before electrophoresis on 5% polyacrylamide TEA gels at 90 V for 18 hrs at room temperature. The DNA was denatured at 4� C. for 20 minutes in 0.2 M NaOH and 0.6 M NaCl, then equilibrated for 1 hour with several changes of transfer buffer (25 mM sodium phosphate buffer, pH 6.5). Electrophoretic transfer to diazobenzyloxymethyl (DBM) paper was conducted as a currect flux of 2 amps for 90 minutes.
Conditions for pretreatment and hybridization to DBM-paper are given by Walh et al. Proc. Natl. Acad. Sci. USA 76, 3683-3687 (1979). However, formamide was omitted, the incubation temperature was increased to 55� C., and the probe was allowed to hybridize for 48 hrs. After hybridization, the DBM paper was washed once in 6XSSC, 0.5% SDS at room temperature, followed by successive washing in 6�SSC, 3�SSC, 1�SSC, and 0.3�SSC. Each of the latter steps was performed twice at 55� C. for 30 minutes and all solutions contained 0.5% SDS. The DBM paper was dried and exposed to Kodak XRP-5 X-ray film using DuPont Lightning-Plus intensifying screens for 2 to 8 days. Experiments involving recombinant DNA were conducted at P2-EKl containment in accordance with NIH guidelines.
In addition to the radioactive probes to facilitate autoradiographic localization of the hybridization sites, as described above, probes for visualizing DNA fragments can be produced by incorporating protein or biotin coupled nucleotides into a hybridization probe, in accordance with the work of Langer et al. at the Yale University School of Medicine. Once incorporated into the probe, those nucleotides can allow visualization of the β globin gene fragments through the use of fluorescent antibodies to the protein or biotin molecules, the antibodies being coated on the filter paper. Such antibodies will bind specifically to DNA fragments to which hybridization has occurred and, in turn, can be visualized by illumination with ultraviolet light.
Hb S has been shown to be the result of a single nucleotide base mutation in the β globin gene which converts the glutamic acid codon (GAG) at amino acid position 6 to one for valine (GTG). Marotta et al., J. Biol. Chem. 25, 5040-5043 (1977). As shown in Table 1, the A to T transversion within the β globin gene sequence affects a restriction endonuclease recognition site for both enzymes Mnl I (GAGG) and Dde I (CTNAG, where N=any base).
TABLE I______________________________________A COMPARISON OF THE SICKLE CELL MUTATIONSITE IN Hb A AND Hb SType of Amino Acid Sequence (Codons 5, 6 and 7)Hemoglobin Corresponding Nucleotide Sequence______________________________________Hb A pro glu glu ##STR1## ##STR2## GAGHb S pro val glu CCT GTG GAG______________________________________
The direct analysis of the present invention was first approached by digestion of genomic β globin clone β Pst with restriction endonuclease Dde I. This study allowed a mapping of the expected fragments for the normal β globin gene. In addition, a probe was isolated which was specific for the 5' end of the genomic β globin gene. This probe simplifies the analysis by restricting the studies to DNA fragments complementary to the probe and, therefore, to the 5' region of the β-like genes.
FIG. 1 shows a restriction map or an illustration of where restriction enzymes cut that part of the β-globin region complementary to the Alu I (727 bp) probe. The representation of restriction sites within and around the human β globin gene was reproduced in part from Lawn et al. Cell 21, 647-652 (1980). For consistency, their method for numbering restriction sites from the intragenic reference point (R) for Bam Hl cleavage has been maintained. The protein encoding regions (filled areas), intervening sequences (open areas), noncoding regions (hatched areas), and the sequenced portion of the 5' intergenic region (wavy line) are indicated. The approximate location of additional restriction sites is so indicated (˜). Fragment lengths generated by Dde I cleavage of normal β and the sickle cell βs alleles within the region of probe complementarity are given below in the expanded portion of the restriction map. Numbers represent base pair fragment lengths.
To determine the feasibility of using Dde I successfully, the known β globin gene sequence was first analyzed for Dde I cleavage sites within the 5' end of the β globin gene up to the Bam Hl site (FIG. 1). This analysis showed Dde I cleavage sites 33 bp, 122 bp, 210 bp, and 411 bp from the Bam Hl reference site, with the 411 bp site being affected by the sickle cell mutation. An analysis of the known DNA sequences within the intergenic region, 103 bp 5' to the β globin gene, (where 5' is defined as that region leftward of the reference point and encompassing all DNA base sequences transcribed before reaching the sickle cell mutation site) failed to indicate any Dde I cleavage site. Therefore, digestion of the normal β globin gene with Dde I, followed by blot hybridization with 5' specific probe, should reveal two fragments over 100 bp: one fragment of 201 bp representing the DNA restriction fragment 3' from the sickle cell site (where 3' is defined as all DNA base sequences following the sickle cell mutation suite with respect to transcription) and another fragment 170+ bp (103 bp intergenic, 53 bp untranslated and 14 bp translated region) representing the region 5' to the sickle cell mutation site. For the purpose of discussion, these fragments will be referred to as the 3' or 5' fragment.
The identification of the 5' fragment was made by digestion of the 1.8 kb Bam Hl fragment (see Materials and Methods) with Dde I and Dde I+Hae III. A digestion of the 1.8 kb Bam Hl fragment with Dde I produced approximately 460, 285, 210, 201, 175, 170, 105, 88 and 89 bp fragments. The 201, 88 and 89 bp fragments were expected from the gene sequence and were known not to be the 5' fragment. Since the minimum fragment size expected was 170+, the 105 bp Dde I fragment was also eliminated. Base sequence analysis also showed that within the 5' fragment, there is a Hae III site 143 bp from the Dde I site. Therefore, the 5' gene fragment was identified by redigestion with Hae III and the subsequent assay for the fragment which was converted to 143 bp. A double digest (Dde I+Hae III) showed that the 460, 210, and 170 bp fragments were not cut by Hae III, and that the 285 bp fragment was converted to a 265 bp fragment. Therefore, the 175 bp fragment that was cut by Hae III to 143 bp, must include the 5' end of the β globin gene. Consequently, Dde I analysis of the normal β globin gene would be expected to show two fragments, 201 and 175 bp, complementary to the specific probe. Digestion of homozygous Hb S DNA should show a single fragment of 376 bp complementary to the specific β globin probe.
A fragment approximately 855 bp, specific to the 5' region of the β-globin gene was expected from the restriction map presented according to Lawn et al. Cell 21, 647-652 (1980). When subsequent redigestions with Hae III were performed, only one fragment gave the predicted pattern (FIG. 1). However, a slight discrepancy in the length of one Hae III fragment was observed which indicates the existence of an additional Alu I site in the 5' intergenic region. This may be due to the creation of new Alu I site during the construction of this clone. As a consequence, however, the actual length for the Alu I probe is 737 bp, rather than the anticipated 855 bp. Furthermore, blot hybridizations using 32 P-Alu I (737 bp) hybridized to Hae III and Dde I digests of cloned β-globin gene have also given the expected banding patterns.
Following the preliminary studies, blot hybridization analyses were performed on Dde I digested lymphocyte and amniotic fluid cell DNAs. Lymphocyte DNAs was obtained from normal, sickle cell and sickle cell trait individuals, whereas amniotic fluid cell DNA was obtained by amniocentesis on a woman carrying a fetus at risk for sickle cell anemia. Autoradiograms of DNA samples isolated from peripheral blood lymphocytes are presented in FIG. 2. As can be seen, DNA from control individuals (AA) show both the approximate 175 and 201 bp bands whereas sickle cell DNA (SS) shows the 376 bp band. DNA from sickle cell trait individual (AS) shows the expected combination of 175, 201 and 376 bp bands. The presence of larger additional bands may be the result of a slight contamination of the probe with other DNA fragments. Such contamination is most probably due to the difficulty in isolating the 5' specific probe free from adjacent DNA fragments. Repeated experiments with DNA obtained from different individuals have shown these larger bands to be consistent for AA and SS individuals. These bands are also present in blot hybridizations from Dde I digestions of the β Pst (4.4) plasmid (data not shown). Purification of the 5' specific probe by recombinant DNA techniques (see the following discussion) should eliminate these bands.
The above analysis of Dde I digested normal, sickle cell and sickle cell trait DNAs are consistent with expected results. A potential complication in this analysis would result from cross hybridization of the probe with other β-like genes. It has been shown that under stringent conditions, cross-hybridization of β globin genes does not occur. However, due to the close similarity of nucleotide sequence of the δ and β globin DNA, cross-hybridization of the probe with the δ globin gene is detectable even under stringent conditions. A survey of the known globin sequence shows that the 5' δ globin gene fragment would not be 175 bp, but a minimum of 189+ bps. Even though this fragment has not been identified, it does not seem to interfere with the analysis, and should be present in both normal and sickle cell DNA. The known δ globin gene sequence shows also a 199 bp fragment similar to that of the 3' β globin gene fragment. Therefore, the 201 bp band in the analysis probably represents a contribution of restriction fragments from both the δ and β globin gene in the normal DNA, but only the δ globin gene in sickle cell DNA. Since the 201 bp band could only represent the δ globin gene in sickle cell DNA, it would be expected to bind only 1/2 as much radioactivity in those individuals, compared to normal individuals.
The data presented herein is from analyses which were performed on DNAs isolated from three unrelated sickle cell individuals and two unrelated control individuals. In all cases, the 376 bp band has been present in association with the sickle cell allele. However, in comparing autoradiograms from the same individual and autoradiograms from different individuals, the 201 bp fragment in sickle cell DNA is often not present, or when present, it is very faint. This probably reflects the 12-16% divergence of the δ globin and the β globin gene nucleotide sequence in that region of the DNA complementary to the probe. Since the 201 bp fragment is sometimes present in sickle cell DNA, then the essence for diagnosis of sickle cell anemia is the absence of the 175 bp fragment and the appearance of the 376 bp fragment. Diagnosis of the sickle cell trait is based on the presence of the 175 and 376 bp fragments.
Fetuses heterozygous for Hb S and other hemoglobin disorders, such as Hb C or nondeletion type β-thalassemia, would give blot hybridization patterns identical to sickel cell trait individuals. Naturally, these could be excluded by family studies. Moreover, these disorders are usually less severe than sickle cell anemia.
A feasible approach for detection of sickle cell anemia has been presented. This analysis was conducted on DNA isolated from peripheral blood lymphocytes and DNA isolated by amniocentesis on a woman carrying a fetus at risk for sickle cell anemia. The analysis on the lymphocyte DNA and the amniotic fluid cell DNA accurately portrayed the person's genotype as showing the predicted pattern for the sickle cell allele. Therefore, this invention is applicable to the prenatal or postnatal diagnosis of sickle cell anemia.
Applicants have observed in blot hybridization experiments with humna genomic hemoglobin genes that the use of recombinant DNA molecules, as opposed to isolated restriction fragments, increases the sensitivity of the procedures. This is probably the result of enhanced specific activity and the formation of more extensive hybridization networks. Experiments have been performed in order to insert the 5' specific probe into recombinant DNA molecules. The use of this cloned probe is expected to increase the sensitivity of this method. Also, this resulted in a purification of the 5' specific probe, which eliminated the occurence of additional high molecular weight bands.
In conclusion, additional studies are necessary to determine the degree to which polymorphism in the natural population will interfere with the proposed method. The occurence of polymorphisms that create or eliminate Dde I recognition sites could change the expected blot hybridization pattern. However, since a probe of only 737 bp is being used to assay fragments of 175 and 201 bp, the occurence of a specific polymorphism is expected to be minimal. To date, over 30 individuals have been analyzed by this method and no such polymorphisms have been identified.
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|Affymetrix Inc
|Method of comparing nucleic acid sequences
|Dec 17, 1999
|May 20, 2003
|Affymetrix, Inc.
|Very large scale immobilized polymer synthesis
|Apr 24, 2000
|Aug 26, 2003
|Affymetrix, Inc.
|Support bound probes and methods of analysis using the same
|Dec 6, 2001
|Oct 7, 2003
|Affymetrix, Inc.
|Methods of synthesizing a plurality of different polymers on a surface of a substrate
|Mar 15, 2002
|Nov 11, 2003
|Affymetrix, Inc.
|Nucleic acid reading and analysis system
|May 15, 2002
|Dec 9, 2003
|Affymetrix, Inc.
|Apparatus for polymer synthesis
|Sep 19, 1994
|Mar 30, 2004
|Massachusetts Institute Of Technology
|Oncogenes and methods for their detection
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|Affymetrix, Inc.
|Methods for polymer synthesis
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|Affymetrix, Inc.
|Polypeptide arrays
|Dec 14, 2001
|Oct 18, 2005
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|Apparatus comprising polymers
|Dec 28, 2001
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|Affymetrix, Inc.
|Nucleotides and analogs having photoremovable protecting groups
|Nov 24, 1998
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|The Board Of Trustees Of The Leland Stanford Junior University
|Substrates comprising polynucleotide microarrays
|Jul 17, 2001
|Dec 1, 2009
|The Board Of Trustees Of The Leland Stanford Junior University
|Methods for constructing subarrays and subarrays made thereby
|Apr 4, 2002
|Oct 12, 2010
|Oxford Gene Technology Limited
|Analysing polynucleotide sequences
|Feb 6, 2004
|Feb 15, 2011
|Oxford Gene Therapy Limited
|Analysing polynucleotide sequences
|Nov 14, 2001
|Nov 28, 2002
|The United States Of America , Represented By The Secretary, Department Of Health And Human Services
|Methods for the indentification of textual and physical structured query fragments for the analysis of textual and biopolymer information
|Apr 4, 2002
|Aug 21, 2003
|Edwin Southern
|Analysing polynucleotide sequences
|Feb 6, 2004
|Dec 23, 2004
|Edwin Southern
|Analysing polynucleotide sequences
|Oct 22, 1999
|Jan 27, 2005
|Oxford Gene Technology Limited
|Analysing polynucleotide sequences
|Oct 22, 1999
|Feb 10, 2005
|Oxford Gene Technology Limited
|Analyzing polynucleotide sequences
|Feb 6, 2004
|Jun 4, 2009
|Edwin Southern
|Analyzing polynucleotide sequences
|Jan 9, 1984
|Jul 18, 1984
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Mar 2, 1988
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Jul 18, 1984
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Mar 2, 1988
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Jul 18, 1984
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Oct 12, 1988
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Jul 18, 1984
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Oct 12, 1988
|Fuji Photo Film Co., Ltd.
|Signal processing method in autoradiography
|Jan 9, 1984
|Aug 15, 1984
|Fuji Photo Film Co., Ltd.
|Method for determination of base sequence of DNA or DNA fragment
|Jan 9, 1984
|Oct 17, 1984
|Fuji Photo Film Co., Ltd.
|Method for determination of base sequence of dna or dna fragment
|Jun 25, 1984
|Jan 9, 1985
|Molecular Diagnostics, Inc.
|Testing DNA samples for particular nucleotide sequences
|Jun 25, 1984
|Oct 5, 1988
|Molecular Diagnostics, Inc.
|Testing dna samples for particular nucleotide sequences
|May 28, 1985
|Dec 11, 1985
|Cetus Corporation
|Method for detection of polymorphic restriction sites and nucleic acid sequences
|May 3, 1985
|Dec 18, 1985
|Allied Corporation
|Displacement polynucleotide assay employing recombination protein and diagnostic kit
|May 3, 1985
|Jan 8, 1986
|Allied Corporation
|Displacement polynucleotide assay method and polynucleotide complex reagent therefor
|Mar 14, 1986
|Apr 1, 1987
|James Summerton
|Polynucleotide assay reagent and method.
|Mar 14, 1986
|Aug 23, 1988
|James Summerton
|Polynucleotide assay reagent and method.
|Dec 23, 1986
|Jul 8, 1987
|Molecular Diagnostics, Inc.
|Eucaryotic genomic dna dot-blot hybridization method
|Dec 23, 1986
|Dec 9, 1987
|Molecular Diagnostics, Inc.
|Eucaryotic genomic dna dot-blot hybridization method
|Feb 24, 1987
|Sep 9, 1987
|Miles Inc.
|Rapid detection of nucleic acid sequences in a sample by labeling the sample
|Feb 24, 1987
|May 10, 1989
|Molecular Diagnostics, Inc.
|Rapid detection of nucleic acid sequences in a sample by labeling the sample
|Sep 29, 1983
|Apr 12, 1984
|Massachusetts Inst Technology
|Characterization of oncogenes and assays based thereon
|Apr 18, 1985
|Nov 7, 1985
|Yale University
|Junction-fragment dna probes and probe clusters
|May 31, 1985
|Dec 19, 1985
|National Biomedical Research Foundation
|Catalyzed nucleic acid hybridization using enzymatic reagent
|Dec 19, 1985
|Jul 3, 1986
|Blair Malcolm A D
|Improved sandwich hybridisation technique for the detection of nucleotide sequences
|Mar 14, 1986
|Sep 25, 1986
|James Summerton
|Polynucleotide assay reagent and method
|Aug 18, 1998
|Feb 25, 1999
|Transgenomic, Inc.
|Chromatographic method for mutation detection using mutation site specifically acting enzymes
|U.S. Classification
|435/6.12, 435/7.5, 436/508, 436/501, 435/91.53, 536/24.3, 435/7.8, 436/504
|International Classification
|Cooperative Classification
|European Classification
|Sep 24, 1981
Owner name: MEDICAL COLLEGE OF GEORGIA RESEARCH INSTITUTE, INC
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:WILSON, LOIS B.;WILSON, JOHN T.;GEEVER, ROBERT F.;REEL/FRAME:003912/0355
Effective date: 19810917
|Feb 13, 1987
|Surcharge for late payment
|Feb 13, 1987
|Fee payment
Year of fee payment: 4
|Feb 26, 1991
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|Jul 28, 1991
|Lapse for failure to pay maintenance fees
|Oct 8, 1991
|Expired due to failure to pay maintenance fee
Effective date: 19910728


text:
Metabolism and Caloric Intake
We all know by now that a lack of physical exertion equals a lower metabolism. At night, when the sun goes down, our metabolism goes down with it.
Before we get to the magical steps to boosting metabolism during shut eye, let's talk a little about caloric intake, gender, and the things that affect your calorie needs over the day. This will help you put it all together in the end. It's not difficult to understand – especially if you are learning for your own personal needs and if you desire to apply it to your life.
Naturally, the human body burns some calories just to stay alive, whether you are moving or not. This is why woman need between at least 1200-1600 calories per day and men need anywhere from 1800-3000 per day.
What Are Your Calorie Needs?
You can calculate your necessary caloric intake by using the following formula:
Female: 65 + (4.3 x weight in lbs.) + (4.7 x height in inches) – (4.7 x age)
Male: 66 + (6.3 x weight in lbs.) + (12.9 x height in inches) – (6.8 x age)
Males need more calories than females because they have more testosterone, and are more muscular. However, a physically active female may need the same amount or even more calories than a sedentary male. And, if you weigh more, you burn more calories when you exercise. That's why you should punch in your actual weight when jumping on the treadmill or elliptical at the gym!
For example, if you participate in low impact aerobics and weigh 155 lbs, you will burn around 350 calories in an hour. But if you weigh 205 lbs, you will burn 465 calories in the same amount of time. This is the reason why you may lose weight fast at the beginning, and then eventually hit a plateau.
It's recommended to start off your workouts slowly, because once you lose a lot of weight, you can then start increasing the time you spend at the gym or in your normal exercise routine, and still burn calories and fat.
If you weigh 300 lbs and get on the elliptical for only 20 minutes, you will burn 200 calories. Once you get to 200 lbs, if you want to keep losing weight, you must workout for 45 minutes to an hour because you will burn less than 200 calories in 20 minutes at 200 lbs than you would at 300 lbs. Does that make sense?
All of this will help you burn more calories while you sleep. But there's more.
The Magical Steps to Boosting Your Metabolism While You Sleep
Now that you understand how your body works when you are awake and active, let's talk about how you can boost your overall metabolism while hitting the hay. Just remember that physical activity will always trump whatever steps you take during the evening hours. But everything you do counts towards overall weight loss.
- Meditate – Before bed, shut off all your electronics, including your cell phone. Sit up comfortably and close your eyes. Breathe in deeply and hold it. Let it out and keep breathing like this for 15 minutes. When we exercise, we breathe in and out more deeply, which helps to burn more calories and increase metabolism. Exercising at night will keep you awake, but meditating will relax you completely and help you fall asleep while increasing metabolism at the same time.
- No Food After 7 pm – If you eat around the time the sun begins going down, you can easily pack on the pounds. Your metabolic rate starts to lower in the evening, and especially when it's dark out. If you stop eating by 7 pm you could easily lose weight, and that double chin might disappear within a week or so!
- Keep It Cool – Putting your temperature below 66 degrees was proven by an experiment to help you burn more calories. 31 people were tested in either a room at 75 degrees or 66 degrees. The colder the room the greater the calorie burn – about 7% more. But there is a catch – you can't wear heavy pj's or use warm blankets.
Now that you know how to rev up your metabolism all day long, you have no more excuses. Start out slow, and don't give up. Continue to add ways to become more active, even by getting a new hobby in addition to your regular exercise routine. Dancing, swimming, and playing golf are not only fun, but help you to get moving.
It may take some time to lose weight, but remember, it took time for you to gain it as well. The more steps you decide to follow, the faster you will see results.

subset: commoncrawl


text:
You do not see the same types of injuries during a basketball game that you would watching football. It is sad, but seeing players lie motionless after a big hit or get carted off the field has become almost routine during football games. In basketball, there are not any pads, so if someone takes a blow to the head and gets knocked unconscious, it's a scary moment.
Charlotte Bobcats rookie Michael Kidd-Gilchrist collided with teammate Jeffrey Taylor and took a pretty big shot to the back of the head during the fourth quarter of Saturday's game against the Houston Rockets. He had to be carted off on a stretcher and spent the night in a Houston area hospital.
Kidd-Gilchrist suffered a concussion but the X-rays and CT scan came back negative, so there were not any severe head or neck injuries. He was reportedly moving all his extremities and talking fine even before leaving the arena.
It looks like Kidd-Gilchrist will be fine, but he is still going to miss a few games for the Bobcats. He did not make the trip with the team to Miami for Monday night's game against the Heat and he will not be cleared to return to action until he passes the requirements of the NBA's new concussion protocol.
The Bobcats have a pretty condensed schedule going forward. They have the last two games of their current road trip Monday and Wednesday, and then they play four games in six nights before the All-Star break. With all the travelling and the frequency of games, I would not be surprised if we did not see Kidd-Gilchrist back in action until after the break.
The Bobcats do not have much to play for (aside from avoiding the title of "NBA's worst team"), so there is no sense in rushing back this year's second overall pick and risking further injury.
Image: MSN.com


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With India facing traffic jams in every city, there needs to be a new alternative to reach destinations. The roads and rails are seeing great numbers of users and takes some time to reach your destination.
Airbus is on an ambitious project on the self-flying taxi, the company is working actively to get this to reality within the next decade. The project has been named 'Vahana' (Sanskrit word for "that which carries, that which pulls").
Users of the self-flying taxi arriving at an airport would book a seat on the so-called zenHop "CityAirbus" drone, you will then proceed to zenHub helipad based on the concept by Airbus.
You will be flown to the destination at the same cost as a taxi since the ride will be shared by other passengers as well. Even the luggage will be delivered by another service called zenLuggage and the whole thing will be safeguarded by zenCyber from hackers.
Airbus has kept the CityAirbus multi-rotor, electric aircraft design under wraps; however, there is an artist impression of the self-flying taxi revealed.
The company is also working on a drone delivery service and plans are to start testing it at Singapore university by 2017.


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Active member
All though I have set an image of size 300x250 px as OG image in my installation, I always get someother image found in a thread as OG image. So I thought of debugging the OG in fb tools but I always get a 404 error in the debugging. Dont know whats causing it. Attached is a screenshot of the Open Graph debugger result. Dont know whats causing this. Any help? Thanks
143.8 KB Views: 11


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Tony G calls out Team Full Tilt to insure player deposits
On a recent episode of the Hardcore Poker Show, Party Poker spokesman and all-around poker magnate Tony G called on the members of Team Full Tilt Poker to guarantee player deposits which he estimated at $100 million.
Tony G first spoke on the podcast about the April 15th crackdown, saying that it was inevitable, and had to happen. He went on to say, "There was no legal standing, no one knew for sure, and now they do," when discussing the legality of online poker in the US, and what laws the sites still operating in the US market were possibly violating.
Tony G then went on to call out Team Full Tilt members –Howard Lederer and Phil Ivey in particular and by name—to insure players at the site that they would receive their money:
"If the whole team Full Tilt could guarantee some of that money back, I think that would be a big help. From what I understand about $100 million of Full Tilt's money has been frozen," he said, stressing that he sees the Team Full Tilt as having a big part of the responsibility in the process.
"I think people like Howard (Lederer) and Phil Ivey should come forward and guarantee that money, and ensure that people are being paid back. They could make a very firm position, and they could come forward. The Team Full Tilt needs to step up."
You can watch the entire episode here
Tags: 100 Million, Crackdown, Full tilt Poker, Guarantee, Howard Lederer, Magnate, Members, Money, Party Poker, Phil Ivey, Podcast, Spokesman
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text:
What should i get?
I don't know very much about weapons. I'm starting to learn a little bit here and there. I would like to know what pistol I should purchase. Any suggestions?
My extremely limited opinion is to get a 9mm. It is a useful caliber and inexpensive to learn to shoot. I will probably get a Glock 17 or Smith & Wesson M&P 9. There are a lot of great options out there.
all will tell you - go to a range and see what you like. I agree!
Many here would recommend a .22 pistol to learn the basics with, and I tend to agree got the most part.
What would be a great idea is to find a range that rents handguns, try as many as you can, with as much ammo as you can, and get what feels best to you.
Some folks want the latest and greatest, or the prettiest, or the one they hear about the most in online forums like this one. That is not the way to go.
You need something that you are comfortable with and enjoy shooting. If you don't like it, you won't shoot it, if you don't shoot it, your skills with it will suffer.
Shooting is a perishable skill, just like riding a motorcycle, driving a car, and many other things.
To ask what gun to buy is similar to asking what work boots to buy. Everyone's feet are different, as well as the job they work. What suits one, may not suit anyone else.
You also need to know for sure what the primary use will be. Carry gun? Plinker? Wall hanger? Collectible? Let us know so we can give you better advice.
Pretty open ended question. What do you intend to do with it? Self protection? Target? Hunting? Will you be carrying it on your person, leaving it home for home protection? Lots of stuff to consider.
If you just want a good gun to learn with, IMHO you'd be best off with a .22 revolver. Easy and simple to load and shoot. If you really want a semi-auto, I'd suggest a Ruger MKIII. Great pistol for not too much money. I can't really suggest a particular revolver as I don't own a .22 revolver and it's been a while since I've shot one.
Hopefully you can get someone you know who knows about guns to help you pick one out and bring you to a range to learn to load and shoot it properly. If not, find out if there's a range near you that rents guns so you can try some out with their help.
Good luck and welcome to the wonderful world of guns! Kiss your $$ goodby! :)
If you want a pistol to learn the basics. I would recommend you look into the Ruger 22 Semi-Auto Target Pistols, Browning Bench Mark or similar pistols. They are great pistols to learn basic fundamentals and handling techniques and are not that expensive. I addition, 22 ammunition is very reasonable to shoot! These pistols will rapidly develop confidence and good techniques and will be a good choice! I would recommend also that you check around and find a good instructor to get you started on the correct fundamentals!
1. Go take a class on handguns. Here is a NRA site that will help find something... http://www.nrahq.org/education/training/index.asp
2. Go try lots of different guns. Much of purchasing a gun is how it feels to you.
3. Buy two guns: a .22lr and a major caliber (9mm, 357, 40, 45).
If there is one thing everyone on here can agree upon, it is everyone needs a 22. It will always be the most economical way to shoot and therefore means you will shoot more often and become more proficient.
Honestly, if the 22 isn't your first gun, it should be your second.
Don't get hung up on brands. I own Springfield, Smith & Wesson, Ruger, Winchester, Remington... Any of the major manufacturers make fine guns... even Glock (ugh).
Glock 19 (9mm) IMO ;)
Trip286 and Scott have it right just follow their post. Good Luck.:)
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"Thank you for your support"
About: Pilgrim Hospital / Paediatrics Pilgrim Hospital Paediatrics PE21 9QS
Posted by Alaarahman (as ),
My daughter has been admitted with abdominal pain. I am not going to talk about the surgical part because it was expected from my colleagues. My observation today about the nurses and senior nurses in the ward, very organised, friendly and reassured my daughter throughout her stay. Thank you for your support especially Laura and Ms C Harris.


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Added: Wednesday, June 10, 2009
In this clip, a tanned young tranny named Saigon lays back in bed while fondling with her boobs. She reached between her legs and took out a succulent looking meat and began stroking it. Someone handed her a dildo and began cramming it deep into her tight rump.


text:
what i wore to the gym and i had army cargos on, with really tatty batterd up vans trainers
ok seen this girl about a couple times at the gym very very attractive my type!!
but today first time ever she kept looking at me i clocked her loads of times from across the gym then she would come near me and look at me when she was picking up weights and in the car park she went to gym with a friend and her mate was driving so she was passenger as i was walking to my car and they was backing out she looked then looked down then full on watched me walk. why is she looking at me? im ugly as shit and very doubt she was attracted to me so that can't be an answer, was it the way i was dressed? is she looking because she was discusted with how i looked?
im asking because i dont know when it comes to women so i can't tell IF she likes me, if she hates me IF she does like me then she needs her eyes testing lol
Most Helpful Girl
its probably what you were wearing haha!!! cargos to the gym? really? ha!!! cute. probably just clownin what u were wearing. or maybe she was checking u out.00|00|0
so if she was checking me out which i highly doubt , im very shy how would i go about doing so? plus i dont wanna look like a twit either lol and if things go tits up we go to the same gym so it could be awkward or shall i just leave it? end of the day im at the gym to work out not pull
just wait and see if she does it again. but this time wear something practical. then you'll know she's looking at you and not your funky outfit. then go in for the kill. even if you just start with a hello :)
Most Helpful Guy
You look ok to me maybe she was checking you out.10|10|0
see im very shy how would i go about doing so? plus i dont wanna look like a rwit either lol and if things go tits up we go to the same gym so it could be awkward
go about asking her? or shall i just leave it and add it to the pile of what ifs?
Just be friendly with her and see how it feels when you are talking to her. Start off with a "Hi" and see how she responds.


text:
Biona Organic Orange Juice Pressed (1 litre)
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Dietary Summary:
Organic smooth orange juice, simply pressed, not from concentrate.
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Product description
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BHP Billiton's petroleum boss Tim Cutt has assured investors that its $US20 billion ($21.6 billion) shale business in the US is set to turn a profit this year for the first time but has played down the near-term prospect of developing the group's LNG prospects off Western Australia.
Mr Cutt told a conference in New York that the US onshore business should be a strong contributor to pre-tax profits this financial year, following on from a profitable second half of 2013-14.
The business, which is swallowing up about $US4 billion a year of capital expenditure, should then be cash-flow-positive by 2015-16, and be throwing off towards $US3 billion a year in cash by the end of the decade.
BHP's shale business, built from $US20 billion of acquisitions in 2011, suffered a writedown in some of its gas assets the following year after US gas prices slumped. However, the division has driven significant growth for the petroleum business, mostly due to the lucrative Eagle Ford shale activities in southern Texas, and promises to deliver more from the emerging Permian shale play in the west of the state.
The emphasis of the growth in the shale business comes after BHP last month said it would review its smaller petroleum assets for sale as part of a comprehensive overhaul of its portfolio centred around a demerger. The high-returning oil and gas business will remain with BHP, becoming a bigger part of the pared-down portfolio.
In his speech in New York, Mr Cutt revealed that BHP, which sold its British venture in Liverpool Bay last year, was actively marketing its petroleum assets in Pakistan while other smaller assets around the world were under review and could be divested.
Mr Cutt said BHP would prioritise the highest-return opportunities in petroleum, with a near-term focus on deep-water in the Americas, and the shale liquids business. However, its "greenfield LNG" business, which includes the huge undeveloped Scarborough gas field operated by ExxonMobil off Western Australia, appears to be lower priority.
"The takeaway for you guys today is that we will not move forward with these projects unless they are economically attractive and, more importantly, they are competitive within the overall portfolio," Mr Cutt said.
Similarly, the expansion of the Mad Dog oil venture in the US Gulf of Mexico, operated by BP, is still under scrutiny, after last year's decision to rework the development plan to cut costs that were set to reach towards $US20 billion, on some estimates.
BHP has earmarked about $US1.5 billion a year on capital expenditure in its conventional oil and gas assets, primarily in the Gulf of Mexico and Australia, as it seeks to maintain stable production from that part of the business.
Mr Cutt was very enthusiastic about the potential for BHP's new deep-water play in Trinidad, where it has identified a giant oil prospect, Pegleg, which is bigger than the large Shenzi, Mad Dog and Atlantis fields in the Gulf of Mexico. Pegleg lies in the Orinoco River Basin, which Mr Cutt said was "one of the last large deltaic basins" to be explored in deep water.
A major seismic survey in the Trinidad acreage is due to be completed by the end of this year, with about 12 months of studies to follow before drilling.
"We are very excited about Trinidad," Mr Cutt said. "We have a large position, and we hope it will be another heartland for us."
Deutsche Bank energy analyst Paul Young said it looked as if BHP was "hanging its hat" on Pegleg to some extent, and probably needed to add more acreage, though he noted the company had recently outbid some majors including BP for several new exploration permits in the western part of the Gulf of Mexico.
"It clearly looks like BHP is stepping up in a big way" in its exploration in the Gulf, he said. "It certainly tells you they are focusing more on offshore."
In the US shale business, BHP is continuing to focus on liquids-rich areas, which were prioritised for drilling after the slump in gas prices. Mr Cutt said that while the Haynesville shale activities could reap 30 per cent returns, BHP was working to improve its fracking technology before moving into full development.
The prized Black Hawk venture in the Eagle Ford shale in southern Texas was set to become BHP's largest single producing petroleum asset this year and was generating margins of some 75 per cent at current prices, Mr Cutt said.
However, he signalled that the earlier-stage Permian play in western Texas could become even more significant for BHP, noting the company's 74,000 acres in its core area, larger than the 57,000 acres in the Black Hawk.
"We know what kinds of returns the Black Hawk is delivering so we feel good about this," Mr Cutt said, describing the Permian as "fantastic".
He made public for the first time that BHP is focusing its work in the Permian in the Wolfcamp shale, which Mr Young said contrasted with some other companies targeting different rocks.
BHP reiterated its target of building a 100,000 barrels of oil equivalent per day business in the Permian by the end of the 2018 financial year.
"The well results to date are positive and repeatable and there is additional upside to the development program given our longer-term outlook for the field," he said.
However, Mr Young said he expected BHP would have to deploy more drilling rigs in the Permian in 2015-16 and potentially form some joint ventures or add acreage to reach its production target.
Mr Cutt said BHP's efforts to drive productivity in drilling were paying off, with some wells in the Black Hawk now costing less than $US2 million each, down from an average of $US5.5 million in 2012-13. Wells in the Permian were more difficult but costs had fallen from $US7 million two years ago to $US4 million in some cases.


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From Hellgate Global Wiki / Hellgate Resurrection Wiki
Gamblers are found in occasional stations and are easily identified by the glowing red dice above their heads. They sell a range of Armor and Weapons all of which are unidentified and show as white items until they have been purchased and identified.
The first gambler is Ko who is situated in Covent Garden Station and is the first NPC you see on exiting Covent Garden Approach


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Battery, Sealed Lead Acid, 6V, 10Ah, Faston
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Zoro #: G2028853Mfr. Part #: 2UKH2
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Battery, Battery Chemistry Sealed Lead Acid, Voltage 6, Battery Capacity 10Ah, Housing Material ABS, Terminal Type Faston, Terminal Connection 0.187 In. Male Tab, Height 3-7/8 In., Width 5-15/16 In., Depth 2 In., Black, For Use With All Purpose
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- *3: Hazardous material


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V for Vermuth Makeup Artist
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About MeAfter an extended hiatus due to a foot injury, I am back in action! A note about limitations: my left heel was shattered in May, 2010 and as a result I have limited balance and range of motion on that side. I can only wear a 4 inch heel (excluding platforms) and have a little trouble negotiating uneven ground for outdoor shoots. Other than that, I'm practically back to my old self.
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MUAH is not required. No make up artists are credited in my portfolio; I typically do my own work, but would be glad to work with anyone.
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Ridgeline Hybrid Down Insulator W je popolna novost v seriji Rigeline. Midlayer predstavlja izjemno uporaben kos opreme, saj ga lahko uporabimo za zgornji sloj v toplih dneh in odlično izolacijo v mrzlih. Ridgeline Hybrid Down Insulator sestavljata Pertex Endurance, ki zagotavlja odlično vodoodpornost in odpornost na veter ter PrimaLoft Down notranjost, ki omogoča hitro sušenje, in ohranjanje toplote, hkrati pa ohranja izjemno lahkotnost midlayerja.


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2013 Mercury Dime 1oz Silver Comemorative Bullion Coin
- Sold for: Start Free Trial or Sign In to see what it's worth.
- Item Category: Coins & Currency
- Source: eBay
- Sold Date: May 17, 2013
- Channel: Auction House
.999 1oz Silver Bullion. Makes the Mercury dime look nice and big. Minted this year. It is commemorate the Mercury Dime series.===========================================================Welcome to Pirate's Cove Coins! Shop till you drop, we love to hear those bags rumbling. Few things to remember: We do not give refunds on graded coins, you get exactly what is in the photo. On ungraded coins or other items, we give you 7 days to return the items after you receive them unless otherwise stated in the description. We do not pay for return postage, and please return item exactly as you received it unopened (if in a container) to get a refund. All winners must pay in 48hrs, the item will be shipped the next business day after payment is received. We accept paypal. We do report winners that do not pay.We will give you a 5 star rating and we hope you will return the favor. If you have an issue please contact us, we will try and help if it is possible. Cheers matie, and


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Okay ladies.. how do I make tummy time not so terrible?
So, Charlie is 14 weeks old. I let him go at his own pace for everything and only pay attention to articles and webstuffs as a general guide. Charlie HATES tummy time. His legs are just going crazy, he kicks a lot and even stands a little on them when Dad holds him up, but when he goes down for tummy time those arms just don't move! I'm not concerned about it yet, he'll get to things when he gets to them. But I don't like him being so upset! I've tried getting down to his level, ratting a little toy in front of him, I've tried standing behind him and just letting him do his thing without me in the way, it's good for maybe 30 seconds, they he goes into meltdown mode. I am not a cry it out mom so that is not an option.
I know tummy time is very important, how do I make it less awful for him?


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Value of a Preferred Stock
Unlike common equity, preferred stocks pay a fixed dividend. As such, the value of a preferred stock can be calculated using the dividend discount model. The value of the preferred stock is essentially the present value of the dividend in perpetuity, where k is the required return.
Formula 13.1
|Value of preferred stock
Example: Calculating the value of a preferred stock
Assume that Newco's preferred stock pays out to an investor an annual dividend of $8 per share. Given a rate of return of 10%, what is the value of Newco's preferred stock?
Value of Newco's preferred stock = ($8/0.10) = $80
Value of a Common Stock
Much like a preferred stock, holders of common stock can also receive dividends. However, dividends on common stock are not guaranteed, nor are they a fixed amount from year-to-year. As such, we can value common stock using dividends over various time horizons.
1. One-year Holding period
Consider a one-year holding period, in this time frame, an investor will receive a dividend and the price of the common stock at the end of the one year, both discounted back by the rate of return on the common equity.
Keeping this in mind, we can calculate the value of the common stock as follows:
Formula 13.2
Example: Calculate the value of a stock with a 1-year holding period
Newco expects to pay its shareholders common equity, $0.25 per share this year. The investor anticipates Newco's stock will close the year at $30 per share. Given a rate of return of 10%, what is the value of Newco's common stock?
2. Multiple-year holding period
Given investors can hold a common stock for over a year, it is useful to value a stock over the investor's expected holding period. In this case, the DDM model can be used.
Formula 13.3
|Value of common equity
Example: Calculate the value of a stock with a multiple-year holdingperiod
An investor plans to hold Newco's stock for 2 years. Newco expects to pay its shareholders common equity, $0.25 per share over the next two years. The investor anticipates Newco's stock will close the end of that time period at $40 per share. Given a rate of return of 10%, what is the value of Newco's common stock at the end of the two-year time period?
3. Infinite Period DDM
To value a common stock using the infinite period DDM, the calculation is simplified much like it is when valuing a preferred stock with infinite dividends.
Formula 13.4
Where k = rate of return, g = growth rate
Example: Calculate the value of common stock with infinite dividends
Newco paid a $0.25 annual dividend in 2004 and expects to pay $0.265 in 2005. Assuming a 6% growth rate and a required rate of return of 10%, calculate the value of Newco's stock.
Valuing the Common Stock of Companies with Supernormal Growth
When valuing a common stock of a company which is experiencing significant growth, we take a similar approach to valuing the common stock with a multi-year holding period. The difference in the approaches is related to the dividend. A multi-year holding period approach assumes a stable dividend, whereas the dividend changes given the supernormal growth in this approach.
Formula 13.5
Example: Calculate the value of common stock with temporary supernormal growth
An investor plans to hold Newco's stock for 3 years. In that time period, Newco plans to grow at a rate of 6% in the first two years and 3% thereafter. Newco's last dividend was $0.25. Given a rate of return of 10%, what is the value of Newco's common stock at the end of the three-year time period?
To begin, the dividend in each time period must be calculated [D = D0(1+g)]
Since we expect the dividend to grow indefinitely in year 3 and on, the present value of the stock price in year 3 is calculated as follows:
The value of Newco's common stock is as follows:
Using the DDM to Develop an Earnings Multiplier Model
Developing the earnings multiplier using the DDM begins with the basic determinant of price. Recall, to determine the price of a stock using the DDM, the infinite dividend is divided by the required return minus the growth rate as follows:
To determine the price to earnings multiple, the price of the stock is simply divided by the earnings per share of the stock as follows:
Formula 13.6
Example: Determining a company's price-to-earnings ratio using the DDM
With Newco's $0.25 dividend payout, an EPS of $1.00, calculate the stock's P/E ratio assuming 10% required return and 5% growth.
In terms of changes in the dividend, the required return and the growth rate affect the DDM. Changes in these variables will also affect the stock's P/E ratio.
Valuation Of A Preferred StockDetermining the value of a preferred stock is important for your portfolio. Learn how it's done.
Digging Into The Dividend Discount ModelThe DDM is one of the most foundational of financial theories, but it's only as good as its assumptions.
Managing Wealth
The Advantages of Preferred DividendsPreferred dividends are cash distributions a company pays on its preferred shares.
Why Dividends MatterSeven words that are music to investors' ears? "The dividend check is in the mail."
The 3 Biggest Misconceptions of Dividend StocksTo find the best dividend stocks, focus on total return, not yield.
Managing Wealth
An Example of Dividends in ArrearsLearn about the concept of dividends in arrears and which shares of stock guarantee payment of accrued dividends even if the company doesn't turn a profit.
How Dividends Affect Stock PricesFind out how dividends affect the price of the underlying stock, the role of market psychology and how to predict price changes after dividend declaration.
Financial Advisor
4 Reasons a Company Might Suspend Its DividendLearn about the four most common reasons a company may choose to suspends its dividends, including financial trouble, funding growth and unexpected expenses.


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Faculty in the News
Martha Chamallas Media Hits
The following is a list of selected media coverage for Martha Chamallas. The links below will direct you to sites that are not affiliated with the Moritz College of Law. They are subject to change, and some may expire or require registration as time passes.
In one corner of the law, minorities and women are often valued lessOctober 25, 2016
Professor Martha Chamallas was quoted in The Washington Post about how demographic averages—like earnings and employment data based on race and gender—oftentimes determine how much compensation victims or their families receive after injuries and accidents. Women and people of color tend to receive less than white or male victims.
Chamallas described the practice as "something Ruth Bader Ginsburg and civil rights advocates [fought] in the 1960s."
Sex saga at firehouse a turnoff to women recruits?October 12, 2014
Professor Martha Chamallas was quoted in The Columbus Dispatch regarding a recent scandal at a city fire station and the possibility it could alienate women in the fire and police divisions.
"A highly sexualized culture sends a coercive message that, unless you play along and become part of this scene, you will not be accepted into this group and receive proper recognition," she said. "It's not that women don't like sex or have affairs, but this type of environment often produces sexual harassment."
In court, color still mattersApril 3, 2012
Professor Martha Chamallas's work about race and gender bias in the legal system was referenced in a column published in the Tampa Bay Times.
According to Chamallas, race- and gender-based tables result in significantly lower awards for minority men and women.
Judge eyes race question in ferry crash payoutSeptember 8, 2008
Professor Martha Chamallas was quoted in Newsday about whether race should contribute to determining a person's life expectancy.
Chamallas said race has cropped up routinely in studies she has done from the 1990s through 2005 of personal injury cases. While longevity and medical care are different issues than economic loss caused by an inability to work, Chamallas said the result sought is usually the same: determining a proper monetary award.
E-mails show close friendship gone sourNovember 29, 2006
In an article in The Columbus Dispatch about sexual harassment suits between a school board member and the district's superintendent, Professor Martha Chamallas says that earlier welcomed behavior is relevant when a relationship turns sour. However, she notes that later forms of behavior could be harassment.


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Condition and Calculation Not Working TogetherAsked by dlasson on August 19, 2015 at 12:43 PM
Please see the section entitled "HealthCare FSA." For the field "Annual Healthcare Election," users will enter a numerical value (currency). If the value is LESS than $2550, I want the "Per Pay" field to perform its calculation. If the value is GREATER than $2550, I want the message (listed in the condition) to appear either in the "Annual Healthcare Election" field or the "Per Pay" field.
Also, the default "$" is not showing in the "Annual HealthCare Election" field.
Duplicate issue in the next field, "Dependent Care FSA," the only change being that the maximum is $5000.
JotForm Support
This is because you have a conditional logic for the rule greater than then you setup a formula inside the form calculation widget (Per Pay Healthcare). This is causing a conflict to what action to be done. You need to set a stricter and more straight forward action.
I cloned your form for testing purposes, you can check how it works here: http://form.jotformpro.com/form/52305862245958
1. First, removed the formula in your form calculation widget for the field "Per Pay Healthcare...".
2. Now use conditional logic to perform a calculation, we will use the same options to where you inserted the text for the maximum value, but in this case we will perform a calculation when the value is less than 2550. Below is the condition I setup, you'll notice I set a "Is equal to", this is because greater than and less than means it doesn't include the actual value. In this case, if the value IS 2550 and below then perform the calculation.
3. Now here are the conditions for that specific field:
Apply the same method in the other fields and the calculation should work.
Regarding the follow up question on "$" not showing, I believe this is because you used the "Number" field, unfortunately, it doesn't accept non numerical values. You need to update that field to used a text box instead and set the validation to "Currency" then add a default value "$".
However, this means you need to update the conditions you have already set.
Do let us know if you need more assistance on this.
Regarding the "$": If I change the field to "text box" as you suggested, I don't get numerical condition options (i.e. "greater than, less than") when I go to set up the conditions that I need.
For some reason, I cannot duplicate your solution. Specifically, the "Per pay Healthcare..." field is not computing.
In that case I would recommend simply cloning the form of my colleague: http://form.jotformpro.com/form/52305862245958
That way you just do few clicks and you have the same form in your account - no extra steps ;)
You can see how to clone it to your account by following the steps here: How to Clone an Existing Form from a URL
Do let us know of course if you happen to have any further questions or issues and we would be happy to assist.
A condition is not working in one of my sections. Under "Voluntary Life"/ "Employee Coverage," I would like the "Max Coverage Amount Exceeded" field to appear if "Desired Coverage Amount" exceeds 500000. See the condition working for "Flexible Spending Accounts/ Healthcare" (though a different amount there).
JotForm Support
Hi, it seems like this is a different concern with the original thread, I see it's a different form. For us to better assist you, I went ahead and opened a separate thread for it, please refer to this link instead: http://www.jotform.com/answers/658188. We will address it shortly.
Thank you.