text:
So I just brought a nissan spec v and it doesn't wanna start when I brought it from the owner he said that it was sitting for a long time what do you think it could be why it doesn't want to start
6 Answers
why was it parked? Did the previous owner say? I'd start with him.
He said that he never had time to drive it or work on it
Hmmmm. Well, start with the basics. Are you getting spark? Fuel? Is it even turning over?
Yeah it turns on but it turns right off when you dont give it gas
So it runs but will NOT idle?
If you are this confused, you might not be able to fix this yourself- my guess, with cars that have been sitting- is that the gas has gone bad, the fuel tank is corroded, and bad juju is filling your gas lines, injectors, fuel pump, filter, etc. HEY! how's that for guessin'?!
Your Answer
Looking for a Used Sentra in your area?
CarGurus has 53,116 nationwide Sentra listings starting at $1,799.
Search Nissan Sentra Questions
Nissan Sentra Experts
Reputation 1,250
Doran Harney
Reputation 1,150
Tom Demyan
Reputation 950

subset: commoncrawl


text: Hey Miss (whyintellectual) wrote in cooking, 2014-03-17 19:22:00 Hey Miss whyintellectual cooking 2014-03-17 19:22:00 Previous Share Next Shortcuts and Substitutions Today I was making spaghetti and meatballs and realized I only had 1/3 of a jar of sauce left.This was problematic because I brown my meatballs on two sides and then let them simmer in the sauce to finish. In a flash of brilliance, I decided to drain a can of diced tomatoes and added my leftover sauce and managed to make a pretty tasty concoction for my meatballs to complete cooking in. No complaints from the boyfriend. He loved it. So now I am wondering...What are your favorite shortcuts and substitutions in the kitchen? Tags: help: substitutions, help: tips and tricks, meat: beef, pasta and noodles Post a new comment Error Anonymous comments are disabled in this journal We will log you in after post We will log you in after post We will log you in after post We will log you in after post We will log you in after post Anonymously switch LiveJournal Facebook Twitter OpenId Google MailRu VKontakte Anonymously default userpic Your reply will be screened Your IP address will be recorded Post a new comment Preview comment Help 21 comments Post a new comment 21 comments


text:
|Publication number
|Publication type
|Application number
|Publication date
|Apr 30, 2002
|Filing date
|Apr 30, 1999
|Priority date
|Apr 30, 1999
|Fee status
|Also published as
|Publication number
|Daniel C. Liebler, Thomas D. McClure, Garth Powis
|Original Assignee
|The Arizona Board Of Regents On Behalf Of The University Of Arizona
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (10), Referenced by (67), Classifications (28), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
1. Field of the Invention
The present invention relates to a method of identifying proteins which are differentially expressed in a plurality of biological samples. An important component of the inventive method is digesting the proteins contained in the samples to produce peptides, followed by labeling of the peptides. The labeled peptides are then separated, and those peptides that are differentially present in the samples are identified. The identity of the peptides that are differentially present in the samples may be used to determine which protein(s) in the original biological samples were differentially expressed.
2. Background of the Invention
Of the many genes in the genome, some are expressed in virtually all cells, whereas others are expressed in cell- and tissue-specific patterns. Cancer cells express different genes as compared to normal cells. These genes encode proteins that, in turn, regulate all aspects of cell function, including those that give rise to neoplastic characteristics. In principle, analyses of expressed genes in tumor cells versus normal cells should indicate which genes and gene products are characteristic of the neoplastic phenotype. DNA microarrays and related techniques have revolutionized the approach to this problem. Indeed, this approach is expected to yield profound insights into not only into the induction of neoplasia, but also the responses of neoplastic cells to therapeutic agents. Nevertheless, the correspondence between what genes are expressed and what proteins are produced is uncertain. Recent studies indicate that the correlation between mRNA induction and elevation in protein content for several enzymes is relatively poor. Variations in protein expression may be due not only to variations in gene expression, but also to variability in mRNA stability, translational efficiency, protein stability and turnover. Thus, analyzes of genomic expression patterns will not necessarily provide an accurate picture of the status of the truly functional cellular machinery—the proteins.
The term proteome was introduced by Wilkins and colleagues to describe the protein complement of the genome. It is estimated that human cells contain between 50,000 and 100,000 expressed proteins. Proteomics has emerged as a buzzword complement to genomics. Proteomics describes the study of the proteome and changes in its status. In its simplest form, proteomics is simply an exercise in "mining" samples to identify the proteins present. However, the main attraction of applied proteomics in cancer research is that it can reveal key differences between the proteomes of normal and neoplastic cells. In addition, applied proteomics will reveal unique proteins or protein expression patterns of neoplastic cells, both of which can serve the task of molecular diagnosis of cancer.
Previous work describing changes in the expression of single genes or changes in the status of single proteins provided very specific information that could be interpreted in a limited biochemical context. Although the impact of many specific changes in gene and protein regulation is understood, we now realize that the factors regulating cell growth and differentiation act in complex, interlocking pathways. Accordingly, changes in biochemical signaling pathways, networks, and regulatory cascades, rather than in single enzymes describe how cells grow, differentiate and die. By collectively describing complex, multicomponent systems, both genomics and proteomics promise a quantum leap in our level of understanding of the biology of cancer. A crucial task of the new biology is to make mechanistic sense of these changes. First, however, it is necessary to reliably detect and describe them. DNA microarray techniques now make this possible in the context of gene expression. However, no equivalent methodology yet exists to reliably compare, characterize and define patterns of protein expression between cells and tissues.
Investigators studying proteomics are at an enormous disadvantage compared to their genome scientist colleagues. First, unlike nucleic acids, proteins do not hybridize to complementary sequences. Second, there is no protein equivalent of the polymerase chain reaction (PCR). Proteomics thus requires other means of separating proteins in complex mixtures and identifying both low-and high-abundance species. The most powerful method currently available to resolve complex protein mixtures is 2D gel electrophoresis. In this technique, proteins are resolved on the basis of some physical property (e.g., isoelectric point) in a first dimension separation, and then by molecular weight in the second dimension. Many individual proteins from complex cell extracts can be resolved on 2D gels. Although 2D gels are currently the most widely used separation tool in proteomics, it is worth noting that reverse phase HPLC, capillary electrophoresis, isoelectric focusing and related hybrid techniques also provide powerful means of resolving complex protein mixtures.
Regardless of the means by which they are resolved, proteins must next be identified, primarily on the basis of sequence information. N-terminal Edman sequencing provides useful information in many cases, except where N-terminal modifications block analysis. The state-of-the-art approach to protein identification is mass spectrometry (MS). Spots containing the proteins of interest typically are excised from gels and subjected to proteolytic digestion. The resulting peptides may be analyzed by electrospray (ESI) or matrix-assisted laser desorption ionization (MALDI) MS. Sequence information is obtained with triple quadruple or ion trap mass analyzers by collision induced dissociation (CID) or on time of flight (TOF) mass analyzers by post-source decay. In either case, the ability of MS instruments to perform MS-MS experiments allows unambiguous assignment of peptide sequence. This information then may be used with sophisticated database search programs, such as SEQUEST, to identify proteins in World Wide Web protein and nucleic acid databases from the MS-MS spectra of their peptides. This combination of separation technology, MS analysis, and database searching makes the high-throughput identification of proteins in complex mixtures possible and has been the driving force behind the recent explosive growth of the proteomics field. With the continued growth of databases, it will possible to identify virtually all proteins from any 2D gel with these approaches. Indeed, investigators studying proteins of S. cerviseae, in which the entire genome has been sequenced, have made excellent progress in characterizing the yeast proteome.
With few exceptions, 2D gel approaches dominate the proteomics field today. Not surprisingly, a great deal of effort has been directed at overcoming the major technical limitations of 2D gel electrophoresis. Briefly, these limitations are:
(1) difficulties in solubilizing and achieving isoelectric focusing (i.e. 1st dimension) separations with proteins of a wide range of isoelectric points and solubility,
(2) difficulties in achieving run-to-run and laboratory-to-laboratory reproducibility in 2D gel profiles,
(3) problems in resolving the many proteins typically present in the 30-100 kDa MW range, and
(4) difficulty in detecting low abundance proteins.
While progress has been made in addressing all of these problems, 2D gel technology is ultimately limited by the diversity of proteins to be analyzed, both in physical properties and abundance. Continued improvements certainly can be expected, but the 2D gel approach for proteins will ultimately prove inadequate to the demands of proteomics.
Accordingly, there remains a need for improved methods of assaying for differential expression of proteins in biological samples.
It is an object of the present invention to provide a method to identify proteins that are differentially expressed in different biological samples, e.g., cell or tissue samples.
It is another object of the invention to provide a method to identify peptides derived from proteins which are differentially expressed in different biological samples.
The present invention exploits the fact that relatively short peptide sequences (e.g., 6-mer or larger; equivalent to an 18-mer oligo) are largely unique in proteins. This means that sequence identification of one or more such peptides in a protein digest is sufficient to establish the presence of the precursor protein in a sample with a high degree of confidence. Thus, if one identifies, for example, an 8-mer peptide, a search of a protein or nucleotide sequence database will permit the sequence to be localized to a specific protein.
The present invention is based, in part, on detecting the differential expression of the same protein in two examples, or the presence of protein(s) in some, but not all, samples by analysis of peptide fragments from each sample. To that end, the method of the present invention includes digesting the proteins in two samples to a mixture of peptides and then comparing the abundances of specific peptides. A protein that is abundantly expressed in one sample will give rise to greater amounts of product peptides upon digestion than the same protein expressed in another sample at trace amounts. Thus, the task of identifying differentially expressed proteins between two samples involves 1) digestion of two samples, 2) detection and selection of peptides that are present in different amounts in the two samples, and 3) sequence analysis of the selected peptides and identification of the protein precursors.
The objects of the present invention, and others, may be accomplished with a method of detecting peptide fragments of protein(s) that are differentially present in biological samples, by
digesting the proteins in a plurality of biological samples to produce peptides in each sample;
separating the peptides in the samples; and
identifying the peptides that are differentially present in the samples.
The objects of the present invention, and others, may also be accomplished with a method of identifying protein(s) that are differentially present in biological samples, by
detecting peptide fragments of protein(s) that are differentially present in biological samples as described above;
determining the amino acid sequence of at least a portion of the peptide fragments; and
correlating the amino acid sequences of the peptide fragments with the identity of the protein(s) that are differentially present in the samples.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
FIG. 1 is a schematic representation of method for selective detection of peptides from differentially expressed proteins; and
FIG. 2 is a schematic representation of method for identification of differentially expressed proteins.
The present invention provides a method of identifying protein(s), i.e., one protein or more than one protein, that are differentially present in samples. When the sample is a biological specimen, the information provided by the inventive method may be used to determine which protein(s) were differentially expressed in the original sample. As used herein, the term "differentially present" means that one or more proteins is present at a higher relative amount in a portion of the samples as compared to the remainder of the samples. The term also means that protein(s) are present in a portion of the samples that are not present in the remainder of the samples. Of course, it may be the case that protein(s) are present at a higher relative amount in a portion of the samples as compared to the remainder of the samples and protein(s) are present in a portion of the samples that are not present in the remainder of the samples.
One non-limiting, embodiment of the present invention involves the analysis of two peptide mixtures together in one analytical run. Use of differential labeling for the two samples yields one sample peptide mixture with a characteristic (e.g., red) label, whereas the peptides in the other sample mixture bear a different characteristic label (e.g., green). Once the mixtures are combined and then subjected to some analytical separation, variations in the ratio of signals from the two labels (i.e., a change in the red:green ratio) indicates different amounts of that particular peptide, and, thus, differential expression of the precursor protein. These peptide pairs are then selected for further analysis by LC-MS.
Biological Samples
The nature of the biological samples is not limited, provided that the samples contain proteins. The sample may contain whole cells or ruptured cells. The biological sample may be a blood sample, a biopsy sample. Samples containing whole cells may be processed to isolate the proteins, i.e., to separate the cellular proteins from the other cellular components, using any of the techniques that are well-known in the field of biological and diagnostic assays.
Samples may comprise cultured cells or cultured organ specimens, tissues taken from biopsy or dissection procedures, or blood or other biological fluids. One of the most important aspects of applying the inventive method to cellular systems is to reproducibly extract cellular proteins from the cell samples being compared. A failure to reproducibly extract proteins in the two samples could either mask real differences in expression or suggest false differences. Extractions are based on the methods outlined in a recent review, which entails 1) the use of DNase and RNase to degrade nucleic acids, 2) an anionic detergent (e.g., CHAPS), and 3) a reductant (e.g., DTT or b-mercaptoethanol), see Walsh et al, ABRFnews 9, pp. 11-21, 1998, incorporated herein by reference. Other adjuncts to improve protein solubility or isoelectric focusing separation in 2D SDS-PAGE include SDS and ampholytes. Adaptation of extraction methods to individual samples is largely empirical, but can be based on known methods to prepare protein samples for 2D SDS-PAGE, see Link et al, Electrophoresis 18, pp. 1314-1334, 1998; and Ducret et al, Protein Sci. 7, pp. 706-719, 1998, both of which are incorporated herein by reference.
In principle, there is no limit on the number of samples that may be analyzed. However, the number of samples is preferably 2 to 100, more preferably 2 to 25, even more preferably 2 to 10. Analysis of 2 samples is a particularly preferred embodiment of the present invention.
Protein Digestion
The proteins contained in the biological samples may be digested with any of the well-known protein digestion reagents. Such reagents may be chemical or enzymatic. Preferably, the N-termini of the peptide fragments are free, i.e, the N-terminal end of each peptide is a free amino group. In this case, the free amino groups serve as a convenient location at which to label the peptides, as discussed below. A particularly preferred reagent for the protein digestion is cyanogen bromide (CNBr). As will be recognized by one skilled in the art, the conditions of the digest are adjusted such that peptides are produced which are amenable for separation, detection and identification.
For CNBr cleavage, the peptides may range in size from 1 amino acid to 20 or more, depending on the protein sequence. Thus, the molecular weight for such peptides is from 50 to 20,000 daltons. Unique sequences may lack methionine residues (where CNBr cleaves) for stretches of more than 20 amino acids, but this circumstance is rare. For a detailed description of protein digestion see Protein Structure: Practical Approach, T. E. Creighton, ed., ILR Press, 1989, pp 117-144, incorporated herein by reference.
Peptide Labeling
The peptides from the digests may be labeled by any of the well-known techniques available in the art. The label is preferably a chemical group that facilitates facile detection of the peptides.
The peptides may be labeled at any position in the amino acid sequence, such as at the N-termini, C-termini, or at an amino acid side chain (e.g., Lys, Arg, Ser, Cys, Tyr, Glu, Asp, etc.). However, since not all side chains will be present in all of the peptides produced in the digest, labeling at the N-, or C-termini is preferred. N-terminal peptide labeling is particularly preferred.
Preferred labeling groups are fluorescent chromophores that are conventionally used as reporter groups. For example, the structurally related cyanine (Cy™) fluorescent labeling reagents, Cy3 and Cy5, may be used to produce N-terminally-tagged peptides (FIG. 2). These dyes have very similar structures, the only difference being the presence of an additional methylene group in the polyene linker chain in Cy5. Both dyes are intensely fluorescent and water soluble, which will facilitate their use in labeling peptide mixtures. Incubation of the Cy3 or Cy5 monofunctional succinimide esters with the peptide will result in N-terminal labeling of the peptides. These dyes are commercially available from Amersham Pharmacia Biotech.
Alexa™ dyes marketed by Molecular Probes, Inc. may also be used. These dyes comprise a series of fluorophores with emission maxima throughout the visible spectrum. Of these, two dyes, Alexa 532 and Alexa 568 would be especially suitable (FIG. 5). Both share a similar fluorophore and bear the same polar sulfonate and quaternary nitrogen functional groups in similar spatial orientation in the molecule. Their emission maxima are at 554 nm and 603 nm, respectively. Although these compounds are structurally related, they are not as closely matched in structure as are the Cy dyes discussed above. Therefore, the Cy dyes are preferred over the Alexa dyes.
In a preferred embodiment, the peptides in each biological sample are labeled with a different moiety that permits specific detection of the peptides from that sample. For example, in the case where two samples are analyzed, the peptides from the first sample are labeled with a red label and the peptides from the first sample are modified with a green label. In a most particularly preferred embodiment, the labels do not alter the detection of a particular peptide sequence that may be present in multiple samples. For example, in the case where two samples are analyzed, a peptide sequence modified with label (1) in sample 1 has the same detection signature (e.g., HPLC retention time, relative gel mobility) as the peptide modified with label (2) in sample 2.
Peptide Separation
A variety of techniques well-known for separating peptides may be used to separate and detect the labeled peptides. For example, peptides may be separated by a variety of techniques. Such techniques include 2D gel electrophoresis, capillary electrophoresis, isoelectric focusing and liquid chromatography. A preferred analytical method for separating the labeled peptides is high-performance liquid chromatography (HPLC). Reverse-phase HPLC is a routine analytical procedure in the field of protein and peptide analysis, and may be successfully used in the inventive method.
In reverse phase HPLC C-18 columns typically are used, although shorter-chain stationary phases provide improved resolution for larger polypeptides. Three column formats are most widely used. Analytical columns (4.6 mm I.D.) typically are eluted at flow rates of 0.5-2 mL min−1. Narrow bore columns (1 mm I.D.) are run at approximately 0.1 mL min−1. Fused silica capillary columns (0.1-0.3 mm I.D.) are eluted a flow rates of 4 μL min−1 and below. There are three advantages conferred by microscale HPLC separations. First, resolution of individual peptide components is often improved relative to standard analytical columns. Second, narrow bore and capillary columns expose the sample to less total surface area in the column and result in lower loss of sample via nonspecific adherence. Finally, low flow rates lead to highest sensitivity in ESI-LC-MS analyses, which are used for in-line sequence analysis of peptides. Indeed, capillary LC-MS has become the method of choice for high sensitivity/high throughput sequence analysis of peptides in complex mixtures.
In a preferred embodiment, the samples containing the labeled peptides are combined prior to separation. In this embodiment, a single analytical step, e.g., a single HPLC separation, produces the data necessary to identify the differentially expressed proteins in the original samples.
Identification of Peptides
An further aspect of the inventive method is identifying the peptides which are produced from proteins in the original samples that are differentially expressed. This portion of the method involves identifying peptides that are present in some, but not all, of the original samples, i.e., identifying the peptides that are present in only a portion of the original samples. For example, when a protein is present in one sample, sample 1, but not any of the other samples, separation of all of the peptides in each sample as described above will reveal that the sample 1 contains peptides that are not present in any of the other samples. These peptides are referred to as the "signature peptides". The signature peptides are derived from the protein or proteins that were present in sample 1 but not sample 2.
In an embodiment where the labeled peptides from each sample are not combined, the signature peptides can be detected by multichannel detection. For example, in an embodiment where two original biological samples are analyzed, each label may have a different wavelength where it can be specifically detected, e.g., the label in the first sample can be detected in the red region and the label in the first sample can be detected in the green region. The labels may simultaneously be monitored by a dual wavelength detector. This embodiment is shown in FIG. 1. In the Figure, the peptide separation reveals the presence of signature peptides in sample A (red label) at 8 and 13 and 24 minutes.
In order to ensure that differences in amounts of peptides reflects actual differences in protein expression, it is preferable to normalize the samples based on total protein content. In other words, the sample volumes are adjusted so that each sample has the same concentration of total protein.
A further embodiment of the present invention uses an HPLC analytical system that permits resolution of the peptide mixture while permitting co-elution of peptides differing only in their N-terminal labeling. The co-elution of differently labeled peptides is important for selecting peptides present at different levels based on the ratios of the two fluorescence signals. Reverse phase HPLC is the dominant approach to characterizing peptide mixtures. Separation of complex peptide mixtures may be conducted on standard, narrow bore, or capillary columns with a C-8 or C-18 stationary phase and a water/acetonitrile/trifluoroacetic acid mobile phase.
After identifying which peptides are signature peptides, the sequence thereof can be determined. Any of the well-known methods for determining the sequence of a peptide may be used. A particularly preferred method of peptide sequence analysis is MS. A variety of MS techniques are routinely used to determine peptide sequence. For example, MALDI-TOF MS or ESI MS may be used.
Two MS ionization methods used in the field of protein analysis are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Both methods are effective means of producing gas phase ions of proteins, peptides and other biomolecules for MS analysis. ESI sources typically are used on quadrupole or ion trap mass analyzers, whereas MALDI sources are typically used with time-of-flight (TOF) mass analyzers. Although a variety of hybrid instruments have been produced. Both ESI and MALDI are capable of sub-femtomole sensitivity for peptide analysis. ESI-triple quadrupole or ESI-ion trap instruments can be used for MS-MS analyses that yield peptide sequence information. MALDI instruments equipped with post source decay capability also can generate peptide sequence information although ESI-triple quadrupole and ESI-ion trap instruments are considered the best for true MS-MS sequencing.
ESI and MALDI differ in the types of ions produced. ESI essentially samples peptide ions present in solution; thus acid-base equilibria generate singly and multiply charged ions. For larger peptides (>1000 MW) multiply charged ions usually predominate. A "multicharge envelope" is commonly seen with polypeptides>10,000 MW. Available algorithms permit deconvolution of multicharge envelopes to yield a derived spectrum indicating the molecular mass of the peptide. Algorithms are described in the following, each of which is incorporated herein by reference: Convey et al, Rapid Commun. Mass Spectrom. 2, p. 249; Loo et al, Anal. Biochem. 179, p.404; Mann et al, Anal. Chem. 61, p. 1702.; U.S. Pat. Nos. 5,844,237; 5,689,111; 5,652,427; 5,432,343.
An advantage of multicharging is that the multicharged ions fall within the mass range of quadrupole and ion trap mass analyzers (typically up to 2000-4000 Da). However, overlapping multicharge envelopes of protein/peptide mixtures generate a very complicated spectrum and can make this task very difficult. In contrast, MALDI produces primarily singly charged ions and no multicharge envelopes. TOF mass analyzers have greater mass ramps than quadrupoles and are capable of reasonably accurate mass measurements of singly charged ions up to 50 kDa or higher.
Advantages of the MALDI-TOF over the ESI triple quadrupole or ion trap instruments thus are:
(1) MALDI produces a simpler spectrum (i.e., single charging vs. multicharging) from complex samples. This facilitates rapid MS identification of the peptides of interest.
(2) TOF analyzers display a greater mass range than quadrupoles or ion traps, particularly for peptides that do not undergo significant multicharging. This may be important for some peptides with few basis sites for protonation, as the Cy dye labels occupy a protonation site and confer a net charge of minus one.
(3) MALDI-TOF: instruments offer a considerably greater speed of analysis and automation than HPLC-ESI-MS systems. The interface of the instrument with a robotic workstation automates proteolytic cleavage of the collected CNBr peptides and MS analysis of the peptide fragments.
A convenient way to perform the dual- (or multi-) channel detection is to monitor the ratio of the absorbances at the specified detection wavelengths. A deviation of the ratio from unity indicates that a signature peptide is eluting from the detector. At this point, the eluate can be transferred to a suitable storage container, e.g., a vial or a 96 well assay plate, for further analysis.
Accordingly, the signature peptides may be isolated after the separation. The isolated peptides may be further purified, if desired.
Correlation of Differentially Produced Peptides with Differentially Expressed Proteins
Using the amino acid sequences of the signature peptides, determined as described above, the identity of the protein(s) that are differentially expressed in the original samples may be determined. To do so, the amino acid sequences of the signature peptides may be used to search a database of protein sequences. This search would reveal the amino acid sequences of known protein(s) which contain the peptide sequences as subsequences therein. This analysis may reveal that one, or more than one, protein was differentially expressed in one of the original samples.
The sequence database may be a protein or a nucleic acid sequence database. As will be recognized by those skilled in the art, a nucleic acid sequence database may be searched by using the standard genetic code to determine the possible nucleic acid sequences which encode the signature peptides.
Several databases in FASTA (ASCII text) format with protein sequence information can be accessed with standard web-browsing software over the world wide web (WWW). These include, for example, the SWISS-PROT database (http:/www.expasy.ch/sprot/) and OWL database (http;/www.biochem.ucl.ac.uk/bsm/dbbrowser/OWL/OWL.html). Nucleotide sequence databases contain sequences for expressed sequence tags (ESTs), which correspond to expressed genes and gene fragments. EST sequence databases, such as the ESTdb at the National Center for Biotechnology Information (http:/www.ncbi.nlm.nih.gov/dbEST/index.html) can be accessed in the same manner as protein sequence databases.
Isolation of Genes Coding for Differentially Expressed Proteins
The amino acid sequences of the signature peptides may be used to isolate nucleic acids which encode proteins having the peptide sequences as subsequences therein. This aspect of the present invention is particularly useful when the database search of protein sequences, described above, fails to identify a protein from which the peptide was derived by digestion. Such a result may indicate that an unknown, differentially expressed protein is present in the sample.
Using amino acid sequence of the signature peptides and the genetic code, if oligonucleotide primers may be constructed that are capable of hybridizing to a nucleic acid sequence encoding the peptide sequence. The probes may be based on a sub-fragment of the sequence of a signature peptide. Hybridizing is preferably conducted under stringent conditions, which are well-known in the art. The primers can be contacted with nucleic acid obtained from the original biological samples in order to identify which nucleic acids encode the peptide, or sub-fragment thereof. Using standard techniques, such nucleic acids can then be isolated and sequenced in order to reveal the amino acid sequence of the protein. The isolated nucleic acid can then be used in routine cloning methods to produce the protein recombinantly. For example, the DNA encoding the protein may be inserted into a standard cloning vector. The vector can then be used to transform a suitable host, such as bacteria, e.g., E. coli. The host could then produce the protein, which can then be isolated. Techniques for primer synthesis, cloning, stringent conditions, and recombinant expression are described in B. Perbal, A Practical Guide to Molecular Cloning, Second Edition, John Wiley and Sons, 1988 and Current Protocols in Molecular Biology, F. M. Ausubel et al, Eds, Volumes 1-3, John Wiley and Sons, 119-1998, both of which are incorporated herein by reference.
A standard protein mixture is used as the background matrix for this Example. The object of this Example is to detect mixtures that differ by the presence or absence of one or two individual proteins. Accordingly, a commercially produced mixture of protein molecular weight standards containing the proteins listed in Table 1 below is used.
Matrix proteins
Molecular Weight
Number of Peptides
Lysozyme, hen egg white
Trypsin inhibitor, soybean
Carbonic anhydrase, bovine
Ovalbumin, hen egg white
Serum albumin, bovine
Phosphorylase B, rabbit
Apomyoglobin, horse
β-Galactosidase, E. coli
The proteins listed above are representative examples, and several similar mixtures are commercially available. If the mixture described above in the Table is used, the background matrix of CNBr peptides contains 45 peptides.
This mixture is divided into aliquots, to which known amounts of a specific protein is added. Thus, in one test experiment, sample A contains the matrix proteins, whereas sample B contains the matrix proteins plus apomyoglobin. The samples contain approximately equimolar amounts of both the matrix proteins and apomyoglobin. E. coli β-galactosidase is also used as a test protein. This protein is approximately 10-fold greater molecular weight than apomyoglobin and would thus test the ability to observe two proteins of dramatically different molecular weight. In subsequent experiments, a series of A/B samples, in which the A sample had the matrix proteins and a fixed amount of either apomyoglobin or β-galactosidase and the B sample had different amounts of the same protein, is prepared. The ratios would be varied from 1:0.1 to 10:1 in approximately 4-5 increments.
To confirm the identities of the differentially expressed CNBr peptides as apomyoglobin and β-galactosidase, HPLC peaks corresponding to the CNBr peptides are isolated, subjected to additional tryptic digestion, followed by ESI-MS-MS analysis. Peptide sequences are matched to protein sequence databases with SEQUEST and "hits' are confirmed by manual inspection of the MS-MS spectra for the tryptic peptides.
A human keratinocyte cell line (HaCaT) stably transfected with a neomycin resistance gene (pSV2 neo) and an AP-1-luciferase reporter gene is compared with untransfected HaCaT cells. The objective of this analysis is detecting the presence of the aminoglycoside phosphotransferase (APH(3′)II) that confers neomycin resistance. Cell material is isolated from culture plates by scraping with a rubber policeman and then cellular proteins are extracted as described above.
Protein Digestion
CNBr cleavage of proteins is accomplished by dissolving the protein samples in 70% formic acid containing a 50-fold molar excess of CNB and incubating the samples in the dark at 25� C. for 12 to 24 hr. BSA cleavage is used for quality control. A similar digestion protocol involving the use of 50% trifluoroacetic acid can also be used.
The efficiency of cleavage by CNBr is assessed by two methods. First, SDS-PAGE analysis of the protein mixtures is performed to assess progress the cleavage reactions. Gels will be silver stained to detect both precursor proteins and CNBr peptides. Second, HPLC-ESI-MS analyzes of the protein mixtures and the CNBr digests are conducted to confirm cleavage of the proteins to peptides and to confirm that the peptides generated are those expected based on the cleavage specificity of CNBr.
Peptide Labeling and Assessment of Labeling Chemistry
Labeling reactions with the Cy dyes or Alexa dyes are conducted in aqueous sodium bicarbonate buffer at pH 9. Amine-containing buffers (e.g., Tris) are avoided. A gel filtration step may optionally be performed to remove unreacted dye. If a separation step is needed, a gel filtration with a low molecular weight cutoff gel (e.g., Biogel P10). Solvent extraction (e.g., with ethyl acetate) or solid phase batch cleanup may also be used.
Labeling efficiency is assessed by ESI-MS flow injection analysis of dye-treated peptide mixtures.
Detection of Labeled Peptides
A Thermo Separations Products quaternary HPLC system equipped with a variable wavelength detector and dual fluorescence detectors, computer, software, is used. The solvent delivery system is compatible with standard flow capabilities (0.1-2.0 mL min−1) and can be adapted with the aid of a flow splitter to low-flow HPLC (1-50 μL min−1). PEEK tubing is used for the entire system to minimize sample loss by nonspecific adherence to stainless steel. The system is under complete computer control and signals from the two fluorescence detectors can be processed simultaneously by the data system. A Vydac 238 TP-51 monomeric, 5 μm, 250�250�1 mm C-18 column is used. Alternatively, a C-8 or C-4 column may be used for these separations. N-Terminally labeled peptides are eluted with a linear gradient of 0.1% trifluoroacetic acid/acetonitrile.
Numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
|Cited Patent
|Filing date
|Publication date
|Sep 19, 1985
|Feb 16, 1988
|Genetics Institute, Inc.
|Reagent polynucleotide complex with multiple target binding regions, and kit and methods
|May 11, 1994
|Jul 1, 1997
|Trustees Of Boston University
|Methods for the detection and isolation of proteins
|Apr 15, 1992
|May 5, 1998
|Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V.
|Determination of peptide motifs on MHC molecules
|Aug 30, 1996
|Jan 12, 1999
|Matritech, Inc.
|Methods and compositions for the detection of cervical cancer
|Sep 11, 1996
|Mar 23, 1999
|Eli Lilly And Company
|System and methods for qualitatively and quantitatively comparing complex admixtures using single ion chromatograms derived from spectroscopic analysis of such admixtures
|Jun 30, 1997
|Oct 26, 1999
|Kabushiki Kaisha Toshiba
|Gene detection method
|Jul 22, 1997
|Feb 22, 2000
|Rapigene, Inc.
|Methods and compositions for enhancing sensitivity in the analysis of biological-based assays
|Nov 23, 1988
|Apr 25, 2000
|Applied Biosystems, Inc.
|Nucleic acid detection with separation
|Dec 1, 1997
|May 16, 2000
|Oxford Glycosciences (Uk) Ltd.
|Computer-assisted methods and apparatus for identification and characterization of biomolecules in a biological sample
|Nov 30, 1998
|Jan 23, 2001
|The Regents Of The University Of California
|Detection methods using probes labeled with energy transfer coupled dyes for DNA fragment analysis
|Citing Patent
|Filing date
|Publication date
|Oct 20, 2003
|Sep 26, 2006
|Promega Corporation
|Compositions for separating molecules
|Apr 28, 2004
|Feb 13, 2007
|Proteomyx Inc.
|Methods and reagents for differential proteomic analysis
|Mar 1, 2002
|Feb 20, 2007
|Activx Biosciences, Inc.
|Protein profiling platform
|Jun 19, 2002
|Apr 3, 2007
|Cell Signaling Technology, Inc.
|Immunoaffinity isolation of modified peptides from complex mixtures
|Feb 12, 2004
|Nov 27, 2007
|John Rush
|Immunoaffinity isolation of modified peptides from complex mixtures
|Aug 11, 2005
|Dec 4, 2007
|Promega Corporation
|Compositions for separating molecules
|May 19, 2006
|Dec 4, 2007
|Promega Corporation
|Compositions for separating molecules
|Mar 28, 2002
|Mar 25, 2008
|Akins Jr Robert E
|Multi-dimensional proteomic analysis method
|Oct 20, 2003
|Apr 8, 2008
|Promega Corporation
|Methods for separating molecules
|Oct 3, 2003
|Jul 22, 2008
|Biotempt B.V.
|Methods of treatment for septic shock with urine extract
|Apr 8, 2003
|Jul 14, 2009
|Biotempt B.V.
|Treatment of multiple sclerosis (MS)
|Feb 16, 2005
|Sep 22, 2009
|Sandia National Laboratories
|High temperature flow-through device for rapid solubilization and analysis
|Jul 5, 2006
|Feb 16, 2010
|Biotempt B.V.
|Treatment of tumors using short peptides from human chorionic gonadotropin (HCG)
|Oct 30, 2007
|Aug 31, 2010
|Biotempt B.V.
|Treatment of burns
|Mar 7, 2007
|Sep 14, 2010
|Biotempt B.V.
|Control of radiation injury
|Jan 18, 2005
|Oct 26, 2010
|Biotempt B.V.
|Methods of selecting immunoregulator peptides obtained from gonadotropins
|Apr 12, 2003
|Dec 7, 2010
|The University Of North Carolina At Chapel Hill
|Methods of quantitation and identification of peptides and proteins
|May 26, 2009
|Jul 5, 2011
|1St Detect Corporation
|Driving a mass spectrometer ion trap or mass filter
|Nov 6, 2006
|Jul 10, 2012
|Biotempt B.V.
|Treatment of ischemic events
|Jul 15, 2009
|Oct 16, 2012
|Biotempt B.V.
|Control of radiation injury
|Dec 8, 2008
|Dec 18, 2012
|1St Detect Corporation
|End cap voltage control of ion traps
|Nov 30, 2011
|Apr 23, 2013
|Sandia National Laboratories
|High temperature flow-through device for rapid solubilization and analysis
|Nov 15, 2006
|Mar 25, 2014
|Biotempt B.V.
|Oligopeptide acetate and formulations thereof
|Dec 17, 2012
|Apr 22, 2014
|1St Detect Corporation
|End cap voltage control of ion traps
|Apr 4, 2013
|Aug 11, 2015
|Cell Signaling Technology, Inc.
|Analysis of ubiquitinated polypeptides
|Apr 4, 2013
|Nov 10, 2015
|Cell Signaling Technology, Inc.
|Analysis of ubiquitinated polypeptides
|Jul 29, 2010
|May 24, 2016
|Siemens Aktiengesellschaft
|Method for filtering a chromatogram
|Mar 28, 2002
|Oct 24, 2002
|Akins Robert E.
|Multi-dimensional proteomic analysis method
|May 8, 2002
|Feb 20, 2003
|Ian Melhado
|Distributed computing environment for recognition of proteomics spectra
|Jun 19, 2002
|Mar 6, 2003
|Cell Signaling Technology, Inc.
|Immunoaffinity isolation of modified peptides from complex mixtures
|Jul 22, 2002
|Mar 6, 2003
|Diversa Corporation
|Cellular engineering, protein expression profiling, differential labeling of peptides, and novel reagents therefor
|Apr 18, 2002
|Apr 24, 2003
|Ciphergen Biosystems, Inc.
|Biomolecule characterization using mass spectrometry and affinity tags
|Apr 8, 2003
|Nov 27, 2003
|Robbert Benner
|Treatment of trauma
|Oct 20, 2003
|Jul 1, 2004
|Promega Corporation
|Compositions for separating molecules
|Oct 3, 2003
|Jul 15, 2004
|Erasmus Universiteit Rotterdam
|Oct 20, 2003
|Sep 23, 2004
|Promega Corporation
|Methods for separating molecules
|Apr 12, 2003
|Oct 28, 2004
|Borchers Christoph H.
|Methods of quantitation and identification of peptides and proteins
|Feb 12, 2004
|Jan 6, 2005
|John Rush
|Immunoaffinity isolation of modified peptides from complex mixtures
|Apr 28, 2004
|Apr 7, 2005
|Tran Nathaniel Tue
|Methods and reagents for differential proteomic analysis
|Apr 8, 2004
|Oct 13, 2005
|Robbert Benner
|Compositions capable of reducing elevated blood urea concentration
|Aug 11, 2005
|Jan 12, 2006
|Promega Corporation
|Compositions for separating molecules
|Oct 13, 2005
|Jun 29, 2006
|Robbert Benner
|Compositions capable of reducing elevated blood urea concentration
|May 19, 2006
|Sep 7, 2006
|Promega Corporation
|Compositions for separating molecules
|Jul 5, 2006
|Jan 25, 2007
|Biotempt B.V.
|Treatment for tumors
|Nov 15, 2006
|Aug 23, 2007
|Khan Nisar A
|Oligopeptide acetate and formulations thereof
|Oct 31, 2007
|Mar 27, 2008
|Biotempt B.V.
|Administration of gene-regulatory peptides
|Sep 21, 2007
|Mar 27, 2008
|Khan Nisar A
|Immunoregulatory compositions
|Oct 30, 2007
|Jul 17, 2008
|Robbert Benner
|Treatment of burns
|Jan 25, 2008
|Aug 14, 2008
|Promega Corporation
|Methods for separating molecules
|Mar 7, 2007
|Sep 11, 2008
|Floyd Randolph Simonds
|Sand control screen having a micro-perforated filtration layer
|Oct 30, 2007
|Oct 2, 2008
|Khan Nisar A
|Peptide compositions
|Feb 12, 2008
|Oct 30, 2008
|Biotempt B.V.
|Treatment of trauma-hemorrhage with short oligopeptides
|Oct 31, 2007
|Dec 25, 2008
|Khan Nisar A
|Treatment of neurological disorders
|Feb 16, 2005
|Aug 27, 2009
|West Jason A A
|High temperature flow-through device for rapid solubilization and analysis
|Apr 9, 2009
|Sep 10, 2009
|Biotempt B.V.
|Methods and uses for protein breakdown products
|Jul 15, 2009
|Nov 26, 2009
|Biotempt B.V.
|Control of radiation injury
|May 16, 2007
|Aug 26, 2010
|Arcxis Biotechnologies, Inc.
|Pcr-free sample preparation and detection systems for high speed biologic analysis and identification
|Jul 1, 2010
|Jan 13, 2011
|Biotempt B.V.
|Control of radiation injury
|Apr 14, 2009
|May 5, 2011
|Biotempt B.V.
|Gene regulator
|Dec 14, 2010
|May 12, 2011
|Cell Signaling Technology, Inc.
|Analysis of ubiquitinated polypeptides
|Jul 29, 2010
|Jul 26, 2012
|Siemens Aktiengesellschaft
|Method for Filtering a Chromatogram
|Mar 1, 2002
|Sep 12, 2002
|Activx Biosciences, Inc.
|Protein profiling platform
|Apr 18, 2002
|Oct 31, 2002
|Ciphergen Biosystems, Inc.
|Biomolecule characterization using mass spectrometry and affinity tags
|Jul 22, 2002
|Apr 17, 2003
|Diversa Corporation
|Cellular engineering, protein expression profiling, differential labeling of peptides, and novel reagents therefor
|Jul 22, 2002
|Nov 13, 2003
|Diversa Corp
|Cellular engineering, protein expression profiling, differential labeling of peptides, and novel reagents therefor
|Aug 24, 2009
|Feb 25, 2010
|The United States Of America, As Represented By The Secretary Of Agriculture
|Rapid identification of proteins and their corresponding source organisms by gas phase fragmentation and identification of protein biomarkers
|Aug 24, 2009
|Jul 8, 2010
|The United States Of America, As Represented By The Secretary Of Agriculture
|Rapid identification of proteins and their corresponding source organisms by gas phase fragmentation and identification of protein biomarkers
|U.S. Classification
|436/86, 536/25.32, 435/267, 536/18.7, 536/25.41, 536/24.32, 436/89, 536/26.6, 204/451, 204/601, 536/25.4, 536/22.1, 536/23.1, 204/610, 436/16, 435/804, 435/272, 536/24.31, 536/24.3, 204/459, 435/6.14
|International Classification
|Cooperative Classification
|European Classification
|Mar 4, 2002
Owner name: ARIZONA BOARD OF REGENTS OF BEHALF OF THE UNIVERSI
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LIEBLER, DANIEL C.;MCCLURE, THOMAS D.;POWIS, GARTH;REEL/FRAME:012628/0934
Effective date: 20020228
|Sep 26, 2002
Owner name: ARIZONA BOARD OF REGENTS OF BEHALF OF THE UNIVERSI
Free format text: CORRECTIVE TO CORRECT ASSIGNEE S NAME AND ADDRESS PREVIOUSLY RECORDED ON REEL 012628/FRAME 0934.;ASSIGNORS:LIEBLER, DANIEL C.;MCCLURE, THOMAS D.;POWIS, GARTH;REEL/FRAME:013153/0920
Effective date: 20020228
|Jul 18, 2005
|Fee payment
Year of fee payment: 4
|Dec 7, 2009
|Maintenance fee reminder mailed
|Apr 30, 2010
|Lapse for failure to pay maintenance fees
|Jun 22, 2010
|Expired due to failure to pay maintenance fee
Effective date: 20100430


text:
"Spectacular gorge" Top 5 Page for this destination Kolasin Transportation Tip by rayzepp
Kolasin Transportation: 3 reviews and 0 photos
The train to Kolasin is an engineering marvel. Perhaps as much as half of the route is inside longs tunnels through the high mountain.The views, when you emerge from a tunnel, are out of this world.
The train from Podgorica is cheap, fast, and comfortable. The train station in Podgorica is right at the bus station.
The train is quite an alternative to road transport, because the road stays at the bottom of the gorge and you look up at the mountains, while the railroad is far, far above the streambed and road.
This train journey is one of the best train trips in the world. It should be the subject of documentaries and television series.
Mode: TO
Type: Train
More Reviews (2)
Hotels (2)See All Up in the mountains
Been to Kolasin?Share your travels with the world!
rayzepp's Related Pages
"The Cambodia Less Traveled"
- Member Rank:
- 0 9 2 2 7
- 14 Reviews
- 0 Photos
- Add Friend
- Follow
- Send Message
Have you been to Kolasin?Share Your Travels


text:
Watermelon Is Vitamin-Filled And Yummy
Whenever I eat watermelon, I feel like I'm not really eating anything — watermelon is the fruit equivalent of celery. But nutrition experts say that the juicy fruit has tons of Vitamin A and C, as well as lycopene, an antioxidant that might help prevent cancer and cardiovascular disease. Oh, and spitting seeds helps develop muscles in your lips. (I made up the last one, but it seems like it could be true, doesn't it?) [Newswise]
5 Signs You Belong Together
We've all heard the old saying that there's someone out there for everyone. Here are a few signs you can look for to figure out just how well you and your partner mesh together.
3 Reasons to Stay Single
There are plenty of benefits to being in a relationship, but there are just as many reasons for staying single.
9 Sexual Communication Tips
Talking about sex can be stressful, Here are nine helpful tips to make it easier.
Top 5 Gifts for Couples
Are you looking for something you can share with your partner? Check out these great recommendations to find something that you'll both love.
Top 5 Gifts for Him
Not sure what to get your man? Check out some of the top gift ideas from the guys at Adam & Eve.
Indulge in a little "me time"
Adam & Eve Adult Toys. Always Discreet, Free Shipping and Bonus Gifts.


text:
The lack of a strong engine on the 2006 model is a serious drawback for the comfortable but aged Volvo S80.
- Supportive front seats, spacious backseat, soothing ride, optional all-wheel drive.
- Weak engine for a luxury sedan, suspension has little inclination for back-roads driving, stability control isn't standard.


text:
"Empire state building" Empire State Building Tip by kentishgirl
Empire State Building, New York City: 371 reviews and 605 photos
You must must must go!!!!!!!!
Get the picture??!!!
The lift is faster than anything ever!
If you can pull yourself away from the views
when you are at the top, there is a coffee bar and a shop.
We stood and watched the planes from the airports, the cars which looked like toys they were so small.
It really is indescribable - you just have to go!
Address: 350 5th Avenue
Directions: 1, 2, 3 or 9 (Seventh Avenue Lines), A, C or E (Eighth Avenue Subway) to 34th Street/Penn Station.
Phone: (212) 947-1360
Website: http://www.esbnyc.com/
More Things to Do in New York City (2)
More Reviews (4)
kentishgirl's Related Pages
"Spread Your Wings Before They Fall Apart......"
- Member Rank:
- 0 0 1 9 6
- 1,390 Reviews
- 2,202 Photos
- Add Friend
- Follow
- Send Message
Have you been to New York City?Share Your Travels


text:
Tata Aria price in Osmanabad start at Rs. 11.85 Lakh. The lowest price model is Tata Aria Pure LX 4x2 and the most priced model of Tata Aria Pride 4x4 priced at Rs. 17.05 Lakh.
* includes only fuel cost
It will help us serve you better content and best deals
One of our representatives will soon contact you.


text:
Diamond Plus
By: HoaPV
Welcome to the walkthrough for the iOS game Diamond Plus, if you are looking for ways to make it to the next level, and to improve gaming experience as a master player, you've come to the right place. Here you will find Diamond Plus tips, tricks and guides, as well as the opportunity to share your own discoveries to help others move on and beat the game.
If you can't find the answer to your question you are welcome to post it in the comments section below. This way others who know the answer can help you by sharing their own tips and advices.
Diamond Plus game app is developed by HoaPV and available on iTunes and Google Play. For more details on the app, check out our review.
- App name: Diamond Plus
- Seller: HoaPV
- Category: Board
- Updated: Sep 24, 2014
Diamond Plus iOS/Android Gameplay
Check out these tutorial for all levels of Diamond Plus. We'll continue to update this list with the latest answers as new levels are released.
Diamond Plus Answers
- Basic Game Guide Part One - my first commentary plus diamond smg gameplay
Hit like if this helped you!
Until the Walkthrough is completed, feel free to ask for help in the comments section.


text:
Download presentation
Presentation is loading. Please wait.
Published byJulissa Ranger Modified about 1 year ago
The Profile of a Fraudster Who is most likely to commit occupational fraud?
International Fraud Awareness Week This presentation was created by the Association of Certified Fraud Examiners (ACFE) for International Fraud Awareness Week. Visit www.FraudWeek.com for more information.www.FraudWeek.com
The Cost of Occupational Fraud According to the ACFE's 2012 Report to the Nations on Occupational Fraud & Abuse, organizations lose an estimated five percent of their annual revenues to fraud. Applied to the estimated 2011 Gross World Product, this figure translates to a potential global fraud loss of more than $3.5 trillion. The median loss caused by the occupational fraud cases in our study was $140,000. More than one-fifth of the frauds involved losses of at least $1 million. The frauds lasted a median of 18 months before being detected.
The Profile of a Fraudster A fraud suspect might not be easy to pick out of a crowd - or from a rap sheet. However, ACFE research helps identify certain common traits and red flags.
Profile of a Fraudster The Age Factor More than half of all cases in the study were committed by individuals between the ages of 31 and 45. Generally speaking, median losses tended to rise with the age of the perpetrator. This trend is also reflected in tenure at an organization.
Profile of a Fraudster The Perpetrator's Department Fraud offenders were most likely to be found in one of six departments: Accounting (22%) Operations (17%) Sales (13%) Executive/upper management (12%) Customer service (7%) Purchasing (6%)
Profile of a Fraudster A Clean Record Most of the fraudsters in the study had never been previously charged or convicted for a fraud-related offense. Only six percent of the perpetrators had been previously convicted of a fraud offense. This finding is consistent with prior ACFE studies.
Profile of a Fraudster Warning Signs and Red Flags The most common behavioral red flags displayed by perpetrators: Living beyond one's means (36 percent of cases) Experiencing financial difficulties (27 percent of cases)
Profile of a Fraudster More Behavioral Red Flags Unusually close association with vendor/customer (19%) Control issues; unwillingness to share duties (18%) "Wheeler-dealer" attitude (15%) Divorce/family problems (15%) Irritability, suspiciousness or defensiveness (13%) Addiction problems (8%) Refusal to take vacations (7%)
Profile of a Fraudster Tone at the Top High-level perpetrators cause the greatest damage to their organizations. Frauds committed by owners/executives were more than three times as costly as frauds committed by managers, and more than nine times as costly as employee frauds. Executive-level frauds also took much longer to detect.
Profile of a Fraudster The most common crimes committed in the executive suite: Corruption (54%) Billing schemes (33%) Expense reimbursement fraud (21%) Fraudulent statements (21%) Non-cash (16%) Other schemes include skimming, cash on hand, payroll fraud, cash larceny, check tampering and register disbursements
Additional Resources The ACFE's 2012 Report to the Nations on Occupational Fraud & Abuse is drawn from a survey of 1,388 Certified Fraud Examiners (CFEs) who investigated fraud cases between January 2010 and December 2011. In addition to studying the traits of perpetrators, the Report includes data on how occupational fraud is committed and detected, as well as the characteristics of the victim organization. The Report to the Nations is available for download online at the ACFE's web site: www.ACFE.com/RTTN. The Report is in PDF format.
Additional Resources Find more information about preventing and detecting fraud at the following web sites: The Association of Certified Fraud Examiners (www.ACFE.com)www.ACFE.com The Federal Bureau of Investigation (www.FBI.gov)www.FBI.gov The National White Collar Crime Center (www.nwc3.org)www.nwc3.org U.S. Government Accountability Office (www.GAO.gov)www.GAO.gov Internal Revenue Service (www.IRS.gov)www.IRS.gov
About the ACFE The ACFE is the world's largest anti-fraud organization and premier provider of anti-fraud training and education. Together with nearly 65,000 members, the ACFE is reducing business fraud world-wide and inspiring public confidence in the integrity and objectivity within the profession. Visit ACFE.com for more information.
Similar presentations
© 2017 SlidePlayer.com Inc.
All rights reserved.


text:
Topeka Symphony plans concert at Washburn
TOPEKA — The Topeka Symphony will present music by Brahms and Menotti on Saturday in White Concert Hall on the Washburn University campus.
The 7:30 p.m. program will be conducted by conductor candidate Christopher Kelts.
The concert will open with a Concerto for Violin by Menotti. Guest violin soloist Brian Lewis is a native of Ottawa, Kan. Brian was a Topeka Symphony youth talent winner when he was in high school and has performed with the Topeka Symphony and Sunflower Music Festival. The second half of the program will be Symphony No. 2 by Brahms.
Kelts has been music director of the Kansas City Civic Orchestra since 2008. He is also director of orchestras at Washburn University and first conductor of the newly established Kinnor Philharmonic in Kansas City. Kelts, a violist, holds a doctorate in orchestral conducting from UMKC. He grew up in St. Louis.
Season ticket packages are still available and start at $59. New subscribers who purchased the last three concerts receive 50% off the regular subscription price. A Flex Pass, good for any combination of four admissions, is also available for $115. Single tickets for this concert are $32, $27 and $22, with full-time students at half price.
At 6:30 p.m., Kelly Huff, Washburn professor of music history, will present an informal talk about the evening's program in the Bradbury Thompson Alumni Center. The concert preview is free to all ticket holders and reservations are not required. Beverages will be available for purchase.


text:
Continuing Education
The ATC is the joint meeting of the American Society of Transplant Surgeons (ASTS) and the American Society of Transplantation (AST). ATC provides a forum for exchange of new scientific and clinical information relevant to solid organ and tissue transplantation and brings together transplant physicians, scientists, nurses, organ procurement personnel, pharmacists, allied health professionals and other transplant professionals. The educational offerings provide attendees the opportunity to learn cutting-edge advances in research and exchange of ideas and practice in the field of solid organ and tissue transplantation.
As the accredited provider for this activity, ASTS remains strongly committed to providing the best available evidence-based clinical information to participants of this educational activity and requires an open disclosure of any relevant financial relationships with commercial interests that create a conflict of interest. It is not the intent of ASTS to disqualify anyone from participating in this educational activity but to resolve any conflicts of interest that may arise from financial relationships with commercial interests. All conflicts of interest are reviewed by the ASTS CME Program to ensure that such situations are properly evaluated and, if necessary, resolved. ASTS educational standards pertaining to conflict of interest are intended to maintain the professional autonomy of the clinical experts inherent in promoting a balanced presentation of science. Through our review process, all ASTS CME activities are ensured of independent, objective, scientifically balanced presentations of information. Disclosure of any or no relevant financial relationships will be made available prior to educational activities.
This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society of Transplant Surgeons and the American Society of Transplantation. The American Society of Transplant Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
The American Society of Transplant Surgeons designates this live activity for a maximum of 38.25 AMA PRA Category 1 CreditTM. Final credit hours are subject to change and will be provided on-site. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
This activity is eligible for a maximum of 24.25 ACPE credits; Final credit hours are subject to change and will be provided on-site. See final CPE activity announcement for specific details.
This activity is eligible for continuing nursing education credit available.
Transplant Coordinators
This program has been submitted to the American Board of Transplant Certification for review.
CME/CE Instructions
Please take a brief moment to complete the evaluation for ATC 2017, and claim your continuing education credits. Your feedback is very important to us in our ongoing effort to help meet your educational and professional practice needs and to deliver excellence at future educational activities.
You will be emailed instructions on claiming CME/CE. To begin use link that is applicable to your credentials and access your evaluation survey, claim your credits, and print your CE/CME Certificate and/or obtain an ATC Certificate of Attendance. The date to claim CME credit will be announced at a later date.
For questions related to nurse contact hours, pharmacist ACPE credits or transplant coordinators CEPTCs, email email@example.com
For questions related to CME or a certificate of attendance, please email email@example.com.
To claim CME:
You will receive an email with a link to claim CME on the last day of the conference. This email will include detailed instructions on claiming CME/CE.


text:
5217 Roseberry Drive,Doylestown PA, 18902
Sold Price
3 beds , 2 Full baths
Status: Sold
Berkshire Hathaway HomeServices Fox & Roach, REALTORS
(866) 486-5636
Last Updated: 2/23/2017 7:27 AM
Description for 5217 Roseberry Drive Doylestown, PA 18902
Fabulous opportunity for a well cared for home. Three bedrooms with a beautiful master bathroom with soaking tub. Open floor plan and an eat in kitchen add to the appeal. Nice size deck, ample shed, and driveway add to the home's value. There are two separate entrances, enter through the living room or the mud room/laundry room. Snow removal, trash pick up, water and sewer are all included in your monthly lot rent. Located in the Central Bucks School District. Schedule your appoint for a private tour.
Listing Information
Valley View
LP Gas Water Heater, Public Water Service
Public Sewer
School Information
Room Information
First Floor Laundry
Interior Features
Central Air
No Basement
Exterior / Lot Features
2 Parking Spaces,
Financial Considerations
Inside AccessTM
Current market conditions for 5217 Roseberry Drive
Active Market Trends
Source:MLS DATA
Median list price$559,000
Days on market131
Properties for sale216
Sold Market Trends
Source:MLS DATA
Median sold price$380,000
Sales to list price9,642.0%
Properties sold152
Median sq ft price$172
Arrows indicate 90 day trend
Listing Price History for 5217 Roseberry Drive
Doylestown Real Estate
Doylestown is located in Pennsylvania. Doylestown, Pennsylvania 18902 has a population of 8,381.
The median household income in Doylestown, Pennsylvania 18902 is $64,659. The median household income for the surrounding county is $76,859 compared to the national median of $53,046. The median age of people living in Doylestown 18902 is 48.5 years.
Loading nearby sales and history ...
Ask a Question?
Berkshire Hathaway HomeServices Fox & Roach, REALTORS
(866) 486-5636
Start a no spam email conversation
My Drive Time
Enter your work address to view actual drive times with traffic for this property
Here is the weekday INRIX Drive Time from this property to .
When you register, you will receive ...
Weekday drive times
Plus all these great features
Register or login today to take advantage of Drive Time
Nearby homes for sale
Inside AccessTM
Recent sales nearby
Doylestown Real Estate
Nearby Communities


text:
Tampa Pro Live WebcastWatch Tampa Pro Live! SPoTlight Productions is hosting a live webcast of the contest, which kicks off this weekend, March 21st-23rd. The webcast starts March 23rd @ 12pm EST. Check it out!!
Posted March 21st, 2008 by Emily
Fucken awsome!!!!
Posted By: Thomas aka Bigfoot on March 21st, 2008 at 07:34PM PST
sik ass
Posted By: drew Fatality on March 24th, 2008 at 06:34AM PST
yea - chili kon karne
Posted By: filip on March 13th, 2011 at 11:47AM PST
diclofenac topical amoxil mobic 7.5 tab albuterol effexor bupropion without prescription ventolin hfa erythromycin 250 mg where to buy motilium cheap vardenafil online
Posted By: JudithVot on January 4th, 2017 at 11:29PM PST


text:
Download presentation
Presentation is loading. Please wait.
Published byKendall Woolridge Modified over 2 years ago
CLASS II MALOCCLUSION Class II malocclusion can be divided into two types: Class II Div I – the lower incisor edges lie posterior to the cingulum plateau of the upper incisors – Increased overjet – upper central incisors are proclined Class II Div II – the lower incisor edges lie posterior to the cingulum plateau of the upper incisors – Overjet is minimal but may be increased – upper central incisors are retroclined
AETIOLOGY OF MALOCCLUSION There are four major factors which contribute to the aetiology of any malocclusion : Skeletal factors Soft Tissues Dental factors Local factors Usually, a malocclusion is a result of the combination of all four of the above factors.
AETIOLOGY OF CLASS II DIVISION I MALOCCLUSION Skeletal pattern A class II skeletal pattern is usually present in patients with a class II division I malocclusion. This is most commonly due to a retrognathic mandible. It is also possible for patients to present with a protruded maxilla but this is less common. Size discrepancies between the mandible and maxilla may also be the cause of the class II malocclusion and would be due to decreased mandibular size and increased maxillary size. Vertical skeletal discrepancies are also common.
AETIOLOGY OF CLASS II DIVISION I MALOCCLUSION Soft tissues The soft tissues exert forces on the teeth and can contribute to the malocclusion Patients with class II division I malocclusion usually have incompetent lips due to the prominence of the upper incisors. This can encourage upper incisor proclination and lower incisor retroclination as the lower lip is drawn behind the upper incisors and therefore worsening the incisor relationship. However, dentoalveolar compensation can occur to mask the skeletal problem if the patient postures the mandible forwards or uses their circumoral muscles to achieve an oral seal. A high lip line can cause protrusion of the upper incisors as it means the lip is no longer placing force over the upper incisors to prevent them splaying outwards
AETIOLOGY OF CLASS II DIVISION I MALOCCLUSION Dental factors Crowding in the upper arch can result in a lack of space for the upper incisors, pushing them labially out of the arch and cause an increase in overjet.
AETIOLOGY OF CLASS II DIVISION I MALOCCLUSION Local factors Habits such as digit sucking can have a significant effect on a patients malocclusion. Digit sucking causes proclination of the upper incisors and retroclination of the lower incisors. This will cause an increase in overjet and can exacerbate an existing class II malocclusion.
AETIOLOGY OF CLASS II DIVISION II MALOCCLUSION Skeletal pattern Class II division II malocclusion is usually associated with a class II skeletal pattern. The vertical skeletal pattern is also typically reduced which results in the over eruption of the lower incisors and an increased overbite, due to an absence of an occlusal stop.
AETIOLOGY OF CLASS II DIVISION II MALOCCLUSION Soft tissues A high lower lip line is common in class II division II and this causes retroclination of the upper incisors. A high lip line can also cause retroclination of the upper incisors due to forces being applied to the teeth.
AETIOLOGY OF CLASS II DIVISION II MALOCCLUSION Dental factors Crowding is common in patients with a class II div II relationship. The retroclination of the upper incisors leads to a smaller arc circumference creating a lack of space. Lower arch crowding can also be seen as the retroclination of the upper incisors and increased overbite can cause the lower incisors to tilt lingually and thus cause crowding. In a few cases, the increased overbite can be traumatic and has been seen to cause ulceration of the palatal tissues and stripping of the lower labial gingival tissues.
Aetiology of Class III
Definition Angles Classification: mandibular first molar is anteriorly placed in relation to the maxillary first molar. British Standards Incisor classification: lower incisor edge occludes anterior to the cingulum plateau of the upper incisors Angles classification is a more useful definition due to incisal camouflage
The 4 Factors Skeletal pattern Dental factors The soft tissues Environmental issues
Skeletal pattern Increased mandibular length A more anteriorly placed glenoid fossa – leading to mandibular prognathism Reduced maxillary length A more retruded position of the maxilla leading to maxillary retrusion A reduced cranial base angle (ANB <2o) Often a mixture of two or more of the above Growth patterns are important in vertical discrepancies
Dental Maxillary incisors lie in a similar plane to a normal occlusion, however can be proclined due to soft tissue influences Mandibular incisors are often seen to be more anteriorly placed than in normal occlusions A narrow maxillary arch coupled with a broad, anteriorly placed mandibular arch can cause a class III malocclusion
Soft Tissues Tend to mask the malocclusion, not cause e.g. dento-alveolar compensation
Environmental Enlarged tonsil Difficulty in nasal breathing Congenital anatomic defects Disease of the pituitary gland Hormonal disturbances Habit of protruding the mandible Posture Trauma and disease Premature loss of 6 Irregular eruption of permanent incisors or loss of deciduous incisors.
Twin Studies Concluded that genetics are not solely responsible for a class III malocclusion – it is highly multi-factoral
Aetiology of Anterior Openbite Definition; the anterior teeth in the maxilla do not occlude with the opposing teeth in the mandible in any mandibular position.
Subcategories of AOB DENTAL OPEN BITE; Cases of AOB in which the vertical skeletal pattern is not contributory. SKELETAL OPEN BITE; The open bite is at least partly due to the vertical facial form, usually the AOB develops due to excessive vertical growth. These are usually more severe than dental open bites, often with only the most distal molars in contact.
Dental Open Bite Digit Sucking; Open bite caused by digit sucking is characterised by an asymmetrical open bite, the greater extent being on the side the digit is most commonly placed, and upper incisal proclination (effects on the lower incisors are more variable), and often over eruption of posterior teeth as a result of the digit acting as a barrier to eruption of the anteriors.
Dental Open Bite (continued) Abnormal Tongue Function; abnormal tongue thrusts are often noted in patients with AOB however it is believed that these are often a result of the AOB or other factors, and Proffit suggests tongue thrusts are not likely to be the causative factors in an AOB, more likely abnormal tongue resting positions may have an effect as the durations involved in thrusts, whether endogenous or adaptive is too short to cause any displacement but the durations involved when the tongue is at rest are much greater. Often an abnormally large tongue can result in unusual tongue resting positions resulting in AOB, such cases will require tongue reduction as part of their treatment.
Skeletal Open Bite Neurological Disturbances Muscular Dystrophy Muscle Weakness Syndromes Cerebral Palsy (certain forms) Lengthening of the lower face and accompanying anterior open bite in a 15 year old patient resulting from muscle weakness due to muscular dystrophy.
Other Causes of Open Bite IATROGENIC OPEN BITE; Failure to prevent over eruption of posterior teeth when biteplanes and functional appliances are used may also give rise to AOB, as may extrusion of the molar teeth during fixed appliance treatment. PATHOLOGICAL OPEN BITE; Trauma to the facial skeleton, acromegaly and cleft palate are all associated with localised AOB.
Aetiology of crossbites Crossbite: one or more teeth have a more buccal or lingual position in relation to its opposing tooth or teeth. Figure 1: Anterior Crossbite Figure 2: Posterior Crossbite Local causes: crowding, retention, trauma. Skeletal causes: mismatch in the widths of the dental arches and/or an anteroposterior skeletal discrepancy. Soft tissues/habits: digit-sucking. Rarer causes: TMJ disorders, cleft palate.
Canine Displacement Displacement of canines can be classed into palatal and buccal displacements. Displacement of the crypts – this is thought to be the cause of the rarer forms of canine displacement e.g. Horizontal displacement. Long path of eruption Short-rooted or absent upper lateral incisors Crowding Retention of primary deciduous canine Genetic factors- Evidence has been gathered supporting the genetic theory of canine displacement on the basis of 5 : 1)The occurrence with other dental anomalies. 2)Bilateral occurrence of palatally displaced canines. 3)Sex differences with palatally displaced canines- affects females more often than males. 4)Familial occurrence. 5)Variations in different populations.
Summary Aetiology of malocclusions: Class II Class III AOB Crossbites Canine Displacement Further information can be found on uSpace: Group name: Ortho Wiki 09 Aetiology of Malocclusion Link: 09-aetiology-of-malocclusionhttp://uspace.shef.ac.uk/clearspace/groups/ortho-wiki- 09-aetiology-of-malocclusion
Similar presentations
© 2017 SlidePlayer.com Inc.
All rights reserved.


text:
Consult a Product Specialist
Special Import. Usually dispatches in 20-23 Days. Conditions Apply*
Special import. Dispatches in 20-23 days from International Warehouse. Conditions Apply*
EMI starts from Rs. 777.56.
Click here for options.
T-Rex Engineering replay Box Delay Pedal
We have had many requests for a more straight sounding delay pedal that is clearer and more true to the incoming guitar signal. By using new hardware, we not only created a very studio-like sounding delay, but we were able to increase the delay time to a maximum of 3 seconds. It sounds more modern and it doesn't distort or warble - it merely sends out what you put in, only a little later and in a very pleasing way. Just like all our other delays, the dry signal is kept analog, so there is no latency or coloring from the converters to the dry signal. Tap Tempo works great for keeping the delay time in sync with the guy banging on the skins, but there is something to be said about subdivisions. A straight up quarter note delay works great for most sounds, but the rhythmic pattern that emerges from a perfect dotted eighth delay is quite nice and triplets can be just the ticket in swing or more rootsy types of music, so we put in all three options. You only have to tap the footswitch to the beat of the song, and the Replay Box will do the rest of the work for you by adjusting the delay time to 1/1, 2/3 or 3/4 of your foot taps, depending on the switch setting. Try tapping a perfect triplet with your foot while you play you will know why the switch comes in handy! The Replay Box features stereo in- and outputs, so you get the most out of those stereo modulation boxes you sometimes step on. Not only is the dry stereo image kept intact, but the DSP processes each side individually, according to the knobs settings.


text:
Part-Time Music Researcher
BMISan Francisco, CA
Part Time Job
POSITION SUMMARY: Occasional part-time work that can be 0-15 hours weekly depending on availability of assignments. Work consists of music and market research in businesses in various states. Assignments include onsite research in businesses during late evenings, including weekends, and can require possible travel and overnight stay.
LOCATIONS: CA (Northern), CO, CT, IA, ID, KS, MD, MT, NC, ND, SC, SD, TX, UT, VA, WA, WI, WY, WV
• Conduct in-person research in businesses as assigned.
• Complete detailed reports of music used in a business during in-person research.
• Recording of music performance(s).
• Photographing both exterior and interior of locations.
• Must be 21 years of age
• Able to conduct on-site research in businesses during late evenings, including weekends
• Available for possible travel and overnight stay
• Strong interpersonal skills
• Good communication skills, both written and oral
• Proficient in basic computer skills
• Good time management and organizational skills
• Detail oriented
LEARN MORE ABOUT THE BMI TEAM! SEE OUR VIDEO AT: https://youtu.be/tqocYocXNGU


text:
ecschneic4Forums Member
Content count14
Last visited
Everything posted by ecschnei
ecschnei posted a question in Question & AnswerDoes anyone know how to setup the LG OLED B6P TV so that when you are watching HDR blue-ray movies and HDR Roku that the TV will display the control4 navigator correctly? Thanks in Advance
Thanks Matt for taking the time to answer all the questions. Wish you was here in Kansas City, MO...LOL. Is fishing that celerity cable through walls difficult? I know fiber optic cables are sensitive to bending. I wasn't sure if I could just attach it to the end of the HDMI that is there and use it to pull through. There is the possibility that I might try and DIY this part of my project. The last thing I want to do is kink a $400 cable.
So what would you use for the hdmi issue? Balun? I apologize if my spelling is off.
ecschnei replied to Ajbarrister's question in Question & AnswerMine has a very subtle delay (30 sec.) but.....I have the Wink driver pack installed for other reasons and run the MyQ through it...so if you want an example of too many layers...LOL
LOL. Doesn't solve the need to reboot the TV to switch between HDR and not but it answers one question. Since I will probably end up running a different cable for the HDMI, what gauge speaker wire would you run to Klipsch floor speakers that are 8 ohm at 125 cont/500 peak watts? The original installer ran 16 gauge. Also since the speaker and receiver can run Bi-Amp would you run enough cable to do so?
but not the most cost effective due to my equipment rack is in the basement and the TV is upstairs. Could the 50 ft. HDMI cable running from the receiver to the TV not be HDR 4K compatible? would that cause it to randomly blink since I have noticed this happens as well?
Sorry, LG TV on monitor out of Marantz sr-7011 reciever EA-5 on DVD input of reciever Samsung Blue-Ray on Blue-Ray input of reciever Even thought the blue-ray has HDR turned off in the settings, when a HDR movie is put in the TV pops up a window that says the TV needs to restart. I'd like to either set the TV so it doesn't ask or fix it so the other inputs can be "scaled up" to HDR so the TV doesn't need to be restarted every time... Thank you,
ecschnei replied to wappinghigh's topic in ShowcaseI have to throw my 2 cents worth in....Dave really knows his stuff. Alan sent me his way when I had some specific driver requests. Very professional and extremely talented. Thanks Dave! Eric
ecschnei posted a question in Question & AnswerI've seen some network questions come up in here so I'll see what comes up since this is an active forum with lots of experience. I have the following hardware and am still having reliability issues. The problem is I don't have the experience to know where my issues are. I'm also too complicated for a run of the mill tech but too small for the "big boys" to mess with. TWC Arris cable modem Netgear R8500 Router Ubiquiti EdgeSwitch ES-48-500W Qnap 48TB 8 x 6TB NAS with 2 10GbE network connections 2 AP's on other floors 12 IP cameras some POE some wireless using Blue Iris HC-800 running 2.9 I'm around Kansas City, MO so if anyone knows of someone or is interested in taking on a client that is limited in funds since it's all tied up in hardware...LOL please let me know. Thank you,
I have yet to find a 48 port POE gigabit switch that is "dumb". I also transfer large files to my 48 TB NAS so I wanted to have no loss of speed. I understand that my system is probably overkill but I have limited number of outlets where my rack is so I didn't want multiple switches when one should do it all. Thank you for pointing me in a direction to look and I will see if I can correct it.
Well, update as of last night, I couldn't get my router to allow blue iris to transmit on the port i had setup. everything is green until the last step where it verifies the server pings back. It was off and on for the last month and then last night just quit. I was told that since I had a level 2 managed switch that I use none of the features of (other than the POE ports and the 2 optical connections to my NAS) that it could be filtering out and causing issues. I will provide more details as I go and if it doesn't go smoothly then I will post pictures of a server rack after 3 sticks of dynamite reboot it.....
ecschnei replied to Audio Obsessions's topic in General Control4 Discussion3 hikvision DS-2CD2145F-IS 4MP 2.8mm outdoor domes 4 dlink dcs-2630L 3 dlink dcs-936L 2 dlink dcs-5222LB and a partridge in a pear tree. lol
ecschnei replied to Audio Obsessions's topic in General Control4 DiscussionIt looks amazing in blue iris on pc and remote app with my android phone.
ecschnei replied to Audio Obsessions's topic in General Control4 DiscussionHello everyone. I'm having stability issues with my hc-800 running 2.9 with blue iris driver integration. Seems to be the camera's don't show up all the time and the resolution is grainy. I believe the drivers for the 12 camera's are loaded fine but i can only get the snapshot url to test ok some of the time. I've very green and have been teaching myself control4 as I go. Any assistance would be very appreciated. If I can send any log files or provide more information please let me know. Thank you in advance.


text:
Republicans are threatening a mass blockade of President Barack Obama's three most important judicial nominees ahead of an expected Thursday vote on the first of them.
Backed by House colleagues and GOP attorneys general from around the country, Senate Republican leaders are whipping opposition to advancing the nomination of Patricia Millett -- or anyone else -- for the D.C. Circuit Court of Appeals, which is one notch below the Supreme Court and often has the final word on matters of executive authority.
"This week, Sen. Reid has teed up President Obama's court-packing plan for the D.C. Circuit Court of Appeals, a court that some people call the second most important court in the nation," Senate Minority Whip John Cornyn (R-TX) told reporters. "The last thing we need to do when money is tight ... is throw more money at unneeded judges on this court, in an attempt to simply pack the court in order to tilt that court ideologically in a way that favors the big government agenda of the Obama administration."
"We intend to stop it," he said.
Cornyn's court-packing claim is misleading -- it is a reference to President Franklin Delano Roosevelt's attempts to expand the size of the Supreme Court; Obama merely wants to fill vacancies on the appellate level. But he repeated the talking point in multiple op-edsrecently, laying the groundwork for a mass filibuster and pressuring GOP senators not to allow Obama to fill any of the three vacant seats on the D.C. Circuit court.
"I can't think of anything more ridiculous," Senate Majority Leader Harry Reid (D-NV) said Wednesday. "Making nominations to vacant judgeships is not court-packing. It's the president's job." Talking up Millett's qualifications as a Supreme Court litigator and her integrity, he said, "It is truly a shame that Republicans would filibuster this exceedingly qualified nominee for unrelated political reasons."


text:
The Scorpion King (2002)
( Bluray )
|The Scorpion King (2002)
|Rating: 5.5/10 (92,037 votes)
Director: Chuck Russell
Writer: Stephen Sommers (story), Jonathan Hales (story), Stephen Sommers (screenplay), William Osborne (screenplay), David Hayter (screenplay)
Stars: Dwayne Johnson, Steven Brand, Michael Clarke Duncan, Kelly Hu
Runtime: 92 min
Rated: PG-13
Genre: Action, Adventure, Fantasy
Released: 19 Apr 2002
Info: http://www.imdb.com/title/tt0277296/
|Plot: In an ancient time, predating the pyramids, the evil king Memnon is using the psychic powers of his sorceress Cassandra to fortell his great victories. In a last ditch effort to stop Memnon from taking over the world, the leaders of the remaining free tribes hire the assassin Mathayus to kill the sorceress. But Mathayus ends up getting much more than he bargained for. Now with the help of the trickster Arpid, tribal leader Balthazar and an unexpected ally, it's up to Mathayus to fufill his destiny and become the great Scorpion King.
Jadwal Serial TV, Anime & K-Drama Buffering? Ada 2 Server silahkan pilih yang cepet. More Info Tekan CTRL+F5/Command+Shift+R jika Player tidak muncul Cara Download Film & Subtitle ada di F.A.Q & Fanpage
Veuillez activer / Por favor activa el Javascript![ ? ]
COMMENTS Would you like to comment?


text:
3 accommodations in Grindavik
Northern Light InnNorthern Lights Road 1, Grindavik | 3 star hotelfrom $251
- Wanted to Stay Forever
- Clean and comfortable
- Great Hotel.
Silica HotelGrindavik | 4 star hotel
- Family of 4 - great time
- Clinical luxury
*Prices above are provided by partners for one room, double occupancy and do not include all taxes and fees. Please see our partners for full details.


text:
Explore related topics
Cheddar Gorge in Somerset, England. In 1903 the remains of a human male, since named Cheddar Man were found a short distance inside Gough's cave, he is Britain's oldest complete human skeleton having been dated to approx. 7150 BCE. Mitochondrial DNA taken from the skeleton has been found to match that of Adrian Targett, a man living in the area today indicating that Cheddar Man is a very distant ancestor, amazing!
Stone circle, Ham Hill, Somerset
Several miles north of Ashton Cottages Somerset. Glastonbury is a spot on the Mendip hills known as Deerleap. It is well known by local artists because of the far reaching views it provides over the Somerset Levels and of Glastonbury Tor nestled in amongst the hills. On misty mornings it is the perfect spot to appreciate just how flat the landscape is and how it would've looked thousands of years ago when the area was under water. On mornings like this it becomes an island once again.
Glastonbury Tor in Somerset, England. Glastonbury Tor is known as being one of the most spiritual sites in the country. Its pagan beliefs are still very much celebrated. It's a beautiful place to walk, unwind and relax. Distance from Shaftesbury to Glastonbury Tor is 30 miles
Wookey Hole caves , near Wells Somerset, England. Everyone should visit these caves, breathtaking!
Woodland Path - Longwood, Somerset, UK.
Dunster Castle. Exmoor. Somerset, UK. The castle is wonderful but the Grounds are exceptional ☆
Roman baths, Bath, UK
Glastonbury Tor, Somerset Levels - © David Noton
Lynmouth, Exmoor National Park, Somerset, England, United Kingdom,


text:
To ensure you always have a supply of Cooper Complete multivitamins and mineral supplements, join our Vitamin Delivery Subscription Program. You choose how often you would like a shipment, with 30, 60, 90 and 120 day options. If you have two or more bottles (of the same product or mix and match among products) shipped at the same time, you get free shipping!
Vitamin Subscription Enrollment Is Easy
Display the production information for the first item you would like to add to your cart, and click on the blue "Get Started" button to create a subscription. From there, choose a shipping frequency of 30 days, 60 days, 90 days or 120 days and the quantity desired, and add the item to your cart. Repeat this process for as many different products as you like, and choose a shipping frequency that best matches your use of that product. For example, you might want Advanced Omega-3 fatty acids and Joint Health supplement every 30-days, and a bottle of Basic One Multivitamin & Mineral Supplement every 60-days. Click here to start your subscription for Cooper Complete Advanced Omega-3.
Receive Advance Notice of Upcoming Vitamin Deliveries
You will receive an email a week before each shipment, reminding you the next vitamin delivery is scheduled to ship. The email includes a list of the items and quantities in the order and the address the order will ship to. Advance notice of your upcoming order gives you the opportunity to adjust the details of your vitamin auto delivery as needed.
Change or Cancel Your Vitamin Subscription
You have complete control to edit all the details of your vitamin subscription yourself, including the ability to cancel your vitamin subscription at any time.
To change or cancel your vitamin subscription, log-in to your Account and underneath your order history you will see the order listed under My Subscriptions. You have full access to:
- Add or change the items in the order
- Add a new payment method
- Add a new shipping address
- Cancel future shipments
- Change the quantity of an item
- Change the method of payment
- Change the credit or debit card number
- Change the credit or debit card expiration date
- Change the shipping frequency
- Change the next delivery month
- Change the delivery date of the recurring order to a specific renewal day within the month
- Postpone the next shipment
- Remove items from future orders
Subcribe to Vitamin Auto Delivery
To create a subscription for vitamin auto delivery, create or log-on to your Account, or enroll by calling 888.393.2221.
Cooper Complete subscription program is the easiest, most economical ways to order your multivitamins and supplements.


text:
Waukesha has a population of 64,825 and is located in Waukesha county.
The average in-state tuition for full time undergraduate students in forensic nursing schools in Waukesha during the 2009 - 2010 school year was $22,470.00. The average tuition was $22,470.00 for non-residents studying in Wisconsin.
Books and supplies for forensic nursing schools in Waukesha cost an average of $1,120.00. Students who live on campus while attending a forensic nursing school in Waukesha, can expect to pay an average of $7,023.00 for room and board. Students who live at home can expect average costs to be $3,133.00. Students have a choice of one forensic nursing college in Waukesha to attend. Forensic Nursing programs in Waukesha reportedly graduated 75 students in the 2008 - 2009 school year.
|Average Salary
|Number Employed in City
|Registered Nurses


text:
(this is his dominant personality type)
(he also shows elements of this type)
51 | Los Angeles, California
There is nothing sexier than a soulful happy smile for no reason other than just being happy in that moment.
He has a broad overview of reality and likes to tackle large, complex issues and weigh all the variables involved.
He seeks an authentic connection with just about everyone you meet, and you want to have in-depth discussions with friends and colleagues about ideas and feelings.
He likes those who can focus on their goals – individuals who complement his restless soul-searching.
A few excerpts from his personality test:
- I am more enthusiastic than most people
- It is important to respect authority
- I have a wide range of interests
His 14 interests
in his own words...
My friends would describe me as confident and sophisticated yet easy going, humble and compassionate, I am a strong confident man, My sense of humor is always present and I get greater joy in giving rather than receiving. I enjoy life greatly but dream to share all it's special moments with my future best friend and lifetime partner someone to grow old with. I will dedicate my heart and soul to her and nothing will be more important than her happiness being in my world. I have a son.he mean the world to me,looking for s woman that will accept him as I will accept hers also.well I am trying this if it will work for good I'm looking for a woman of character, loyalty, and honesty. I like to hold hands, walk on the outside and open the doors. I want someone willing to take time to build a relationship. Someone wanting to invest mentally, emotionally and physically.. I enjoy romancing a woman. There are 365 days in a year that gives me plenty of opportunities to make her feel special. I feel one of the best parts of a relationship is romancing a woman and building a friendship. I want this woman to be my equal partner, best friend and lover.. Someone that wakes up happy every day. Ok, not every day but most days! To me every morning is a new day to make the best of. Looking for someone with a positive outlook and loves to have fun! you are looking for the one night stand or bad guy type girl then you should look for someone else.. I want a long term, passionate, committed relationship with the RIGHT woman. A woman that is willing to openly communicate about everything. From how their day went to what they like best about sex My partner should like to work hard so she can play hard. Keeping herself fit. I like working out with my boy he mean the world to me,I make time to write, travel, drink wine, create music, surf, play soccer, ski or snowboard, cook, read, run,laying down the beach photography, flying. A great set of eyes will win me over easily!
About him:
Living situation:
- Live with kids
Religious affiliation:
- Christian / Catholic
Relationship history:
- Widowed
- More than $250,000
Eye color:
- Brown
Hair color:
- Salt and pepper
Ethnic background:
- White/Caucasian
What he's looking for:
Age Range:
- 38 - 55
relationship history:
- Any
- Not at all
- Every once in a while
- A few times a week
Ethnic background:
- Any
Religious affiliation:
- Any
- Any


text:
WE are using this platform to inform all the candidates who applied for admission into The Lagos State University, LASU that the management of the Lagos State University hereby informs applicants for the 2014 POST UTME examination that the examination has been postponed to Saturday, August 9th, 2014.
According to the school management of LASU, the reason for the postponement of the screening exercise is due to the declaration of Monday and Tuesday July 28 and 29, 2014 as public holidays by the Federal Government.
The Lagos State University 2014/2015 Post-UTME application form will be on sale till midnight of August 7th, 2014, while the venue AND time of the examination remain unchanged.
Candidates should take note that the payment for the Lasu post ume form as well as the filling of the application form is done fully online.
If you are yet to apply for the screening exercise, you can access the registration guideline here – Lasu 2014/2015 Post-utme registration.
Please share this info with our friends on twitter and Facebook.


text:
DVD Valves has embraced the philosophy of continuous development and new idea generation according to customer demands and needs.
For the practice of this philosophy, DVD Valves uses some of the most sophisticated technology tools in the R&D Department.
Unigraphics CAD/CAM Software
As a result of a new idea from the R&D Department, the CAD/CAM design is done in a computerized software. The design is done both in 2D and 3D drawings and it allows us to visualize the product, to clearly see the dimensions and to construct the molding with CAM systems in CNC Machines. As a result, dimensions of the molds are exactly what it needs to be.
CFdesign Computational Fluid Dynamics Software
The CFD Software allows us to analyze the characteristic of the valve without even manufacturing it. Simulations made on the 3D drawing of the valve enable us to identify the points of development. Therefore we can act quickly and revise the design on computer in case there is a development opportunity. This assures we develop the most ideal design. With CFD Software, we can analyze flow characteristics, cavitation damages, kv values, pressure loss values etc.
Computerized Hydraulic Laboratory
After the manufacturing of the product, with the fully computerized hydraulic laboratory, DVD Engineers can analyze the behavior of the valve and make calculations on pressure loss values and kv values. As a result of these tests, real results are compared with CFD results and necessary actions are taken accordingly.


text:
Find your new home in Las Cruces, NMYou are viewing homes 1 to 20 of 459 homes for sale
Learn more about homes in Las Cruces, NM
Las Cruces Home inventory breakdown by property type
Of the 459 homes for sale in Las Cruces, the breakdown of property types
is as follows:
55% Land/lot, 44% Single Family, 1% Multi Family and 0% Other.
Area details, home facts and figures
Information from in and around Las Cruces...
You are Viewing 1 to 20 of 459 homes for sale in Las Cruces. These homes are comprised of Land/lot, Single Family, Multi Family and Other. The average list price of these property results is 170,015. Prices are displayed as US Dollar. For more information on these homes for sale in Las Cruces please contact our member brokerages to schedule a showing or view an open house.
The 459 homes for sale in Las Cruces, NM are represented by 1 member brokerages serving the Las Cruces area. Member brokerages representing the 459 homes include Steinborn & Associates Real Estate.


text:
The Stone Paper Company Ltd
Waterproof Paper, Environmental Mineral Paper, Food Grade Paper,
These bags are great for Bus, train, plane or taking your shopping home. Made from Rockstock rich mineral stone paper they also have folding wheels attached to take the load. For a production enquiry with your Company branding please contact info at stonepaper.co.nz
Print this page
New Piggy Back labels now on Rocktak Rockstock rich mineral paper is used with a removeable water based acrylic food
Energy Globe National Award
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub