text:
Publication number | US7087896 B2 |
Publication type | Grant |
Application number | US 11/023,234 |
Publication date | Aug 8, 2006 |
Filing date | Dec 27, 2004 |
Priority date | Oct 15, 2001 |
Fee status | Paid |
Also published as | US6835927, US20030111596, US20050116159 |
Publication number | 023234, 11023234, US 7087896 B2, US 7087896B2, US-B2-7087896, US7087896 B2, US7087896B2 |
Inventors | Christopher H. Becker, Curtis A. Hastings, Scott M. Norton, Sushmita Mimi Roy, Weixun Wang, Haihong Zhou, Thomas Andrew Shaler, Praveen Kumar, Markus Anderle, Hua Lin |
Original Assignee | Ppd Biomarker Discovery Sciences, Llc |
Export Citation | BiBTeX, EndNote, RefMan |
Patent Citations (29), Non-Patent Citations (29), Referenced by (4), Classifications (7), Legal Events (10) | |
External Links: USPTO, USPTO Assignment, Espacenet | |
This application is a continuation of U.S. application Ser. No. 10/272,425, "Mass Spectrometric Quantification of Chemical Mixture Components," filed Oct. 15, 2002, now U.S. Pat. No. 6,835,927, issued Dec. 28, 2004, which claims the benefit of U.S. Provisional Application No. 60/329,631, "Mass Spectrometric Quantification of Chemical Mixture Components," filed Oct. 15, 2001, both incorporated herein by reference.
The present invention relates generally to spectroscopic analysis of chemical and biological mixtures. More particularly, it relates to a method for relative quantification of proteins or other components in mixtures analyzed by mass spectrometry without using an internal standard, isotope label, or other chemical calibrant.
With the completion of the sequencing of the human genome, it has become apparent that genetic information is incapable of providing a comprehensive characterization of the biochemical and cellular functioning of complex biological systems. As a result, the focus of much molecular biological research is shifting toward proteomics and metabolomics, the systematic analysis of proteins and small molecules (metabolites) in a cell, tissue, or organism. Because proteins and metabolites are far more numerous, diverse, and fragile than genes, new tools must be developed for their discovery, identification, and quantification.
One important aspect of proteomics is the identification of proteins with altered expression levels. Differences in protein and metabolite levels over time or among populations can be associated with diseased states, drug treatments, or changes in metabolism. Identified molecular species may serve as biological markers for the disease or condition in question, allowing for new methods of diagnosis and treatment to be developed. In order to discover such biological markers, it is helpful to obtain accurate measurements of relative differences in protein and metabolite levels between different sample types, a process referred to as differential phenotyping.
Conventional methods of protein analysis combine two-dimensional (2D) gel electrophoresis, for separation and quantification, with mass spectrometric identification of proteins. Typically, separation is by isoelectric focusing followed by SDS-PAGE, which separates proteins by molecular weight. After staining and separation, the mixture appears as a two-dimensional array of spots of separated proteins. Spots are excised from the gel, enzymatically digested, and subjected to mass spectrometry for identification. Quantification of the identified proteins can be performed by observing the relative intensities of the spots via image analysis of the stained gel. Alternatively, peptides can be labeled isotopically before gel separation and expression levels quantified by mass spectrometry or radiographic methods.
While 2D gels combined with mass spectrometry (MS) has been the predominant tool of proteomics research, 2D gels have a number of key drawbacks that have led to the development of alternative methods. Most importantly, they cannot be used to identify certain classes of proteins. In particular, very acidic or basic proteins, very large or small proteins, and membrane proteins are either excluded or underrepresented in 2D gel patterns. Low abundance proteins, including regulatory proteins, are rarely detected when entire cell lysates are analyzed, reflecting a limited dynamic range. These deficiencies are detrimental for quantitative proteomics, which aims to detect any protein whose expression level changes.
In applications that do not require large-scale protein analysis, protein quantification can be performed by fluorescent, chemiluminescent, or other labeling of target proteins. Labeled antibodies are combined with a sample containing the desired protein, and the resulting protein-antibody complexes are counted using the appropriate technique. Such approaches are suitable only for known proteins with available antibodies, a fraction of the total number of proteins, and are not typically used for high-throughput applications. In addition, unlike mass spectrometric analysis, antibody-protein interactions are not fully molecularly specific and can yield inaccurate counts that include similarly structured and post-translationally modified proteins.
Because it can provide detailed structural information, mass spectrometry is currently believed to be a valuable analytical tool for biochemical mixture analysis and protein identification. For example, capillary liquid chromatography combined with electrospray ionization tandem mass spectrometry has been used for large-scale protein identification without gel electrophoresis. Qualitative differences between spectra can be identified, and proteins corresponding to peaks occurring in only some of the spectra serve as candidate biological markers. These studies are not quantitative, however. In most cases, quantification in mass spectrometry requires an internal standard, a compound introduced into a sample at known concentration. Spectral peaks corresponding to sample components are compared with the internal standard peak height or area for quantification. Ideal internal standards have elution and ionization characteristics similar to those of the target compound but generate ions with different mass-to-charge ratios. For example, a common internal standard is a stable isotopically-labeled version of the target compound.
Using internal standards for complex biological mixtures is problematic. In many cases, the compounds of interest are unknown a priori, preventing appropriate internal standards from being devised. The problem is more difficult when there are many compounds of interest. In addition, biological samples are often available in very low volumes, and addition of an internal standard can dilute mixture components significantly. Low-abundance components, often the most relevant or significant ones, may be diluted to below noise levels and hence undetectable. Also, it can be difficult to judge the proper amount of internal standard to use. Thus internal standards are not widespread solutions to the problem of protein quantification.
Recently, Gygi et al. introduced a method for quantitative differential protein profiling based on isotope-coded affinity tags (ICAT™) [S. P. Gygi et al., "Quantitative analysis of complex protein mixtures using isotope-coded affinity tags," Nat. Biotechnol. 1999, 17: 994–999]. In this method, two samples containing (presumably) the same proteins at different concentrations are compared by incorporating a tag with a different isotope into each sample. In particular, cysteines are alkylated with either a heavy (deuterated) or light (undeuterated) reagent. The two samples, each containing a different isotope tag, are combined and proteolytically digested, and the combined mixture is subjected to mass spectrometric analysis. The ratio of intensities of the lower and upper mass components for identical peptides provides an accurate measure of the relative abundance of the proteins in the original samples. The initial study reported mean differences between observed and expected ratios of proteins in the two samples of between 2 and 12%.
The ICAT™ technique has proven useful for many applications but has a number of drawbacks. First, the isotope tag is a relatively high-molecular-weight addition to the sample peptides, possibly complicating database searches for structural identification. The added chemical reaction and purification steps lead to sample loss and sometimes degraded tandem mass spectral fragmentation spectra. Additionally, proteins that do not contain cysteine cannot be tagged and identified. In order to obtain accurate relative quantification using ICAT, different samples must be processed identically and then combined prior to mass spectrometric analysis, and it is therefore impractical to compare samples acquired and processed at different times, or to compare unique samples. Furthermore, the method is not applicable to other molecular classes such as metabolites.
Existing protein and metabolite quantification techniques, therefore, require some type of chemical calibrant, increasing the sample handling steps and limiting the nature and number of samples to be compared. It would be beneficial to provide a method for quantification of proteins and low molecular weight components of chemical and biological mixtures that did not require an internal standard or other chemical calibrant.
Various embodiments of the present invention provide methods for estimation of relative concentrations of chemical sample components by mass spectrometry without the use of an internal standard.
In one embodiment, the present invention provides a method for processing spectral data containing peaks having peak intensities. A set of spectra is obtained from a plurality of chemical samples such as biological samples containing metabolites, proteins or peptides. The spectra can be mass spectra obtained by, for example, electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), or electron-impact ionization (EI). Peak intensities in each spectrum are scaled by a normalization factor to yield peak intensities that are proportional to the concentration of the responsible component. Based on scaled peak intensities, relative concentrations of a particular sample component can be estimated. The normalization factor is computed in dependence on chemical sample components whose concentrations are substantially constant in the chemical samples. In one embodiment, these components are not predetermined and are inherent components of the chemical samples. In another embodiment, the normalization factor is computed from ratios of peak intensities between two (e.g., first and second) spectra of the set and is a non-parametric measure of peak intensities such as a median.
In an alternative embodiment, the present invention provides a method for estimating relative concentrations of a particular component in at least two chemical samples, such as biological samples containing proteins or peptides. Mass spectra are acquired, e.g., by electrospray ionization, matrix-assisted laser desorption ionization, or electron-impact ionization of the samples, and peak intensities of peaks in the spectra are scaled by a normalization factor. The normalization factor is computed in dependence on chemical sample components whose concentrations are substantially constant in the chemical samples. In one embodiment, it is computed from ratios of peak intensities in two (e.g., first and second) of the spectra and is a non-parametric measure (e.g., median) of peak intensities. Based on scaled peak intensities of a peak corresponding to the particular component, relative concentrations of the particular component can be estimated.
Additionally, the present invention provides a method for detecting a component present in substantially different concentrations in at least two chemical samples, such as biological samples containing proteins or peptides. Mass spectra of the samples are obtained, e.g., using electrospray ionization, matrix-assisted laser desorption ionization, or electron-impact ionization. Peak intensities in each spectrum are scaled by a normalization factor computed in dependence on chemical sample components whose concentrations are substantially constant in the chemical samples. In one embodiment, the normalization factor is computed from ratios of peak intensities in two (e.g., first and second) of the spectra and is a non-parametric measure (e.g., median) of peak intensities. A peak is then identified that has substantially different scaled peak intensities in at least two of the mass spectra. In an additional embodiment, the component corresponding to the peak is identified. A relative concentration of the component in the samples can be computed based on the scaled peak intensities of the corresponding peak.
Another embodiment of the present invention is a program storage device accessible by a processor and tangibly embodying a program of instructions executable by the processor to perform method steps for the above-described methods. An additional embodiment is a computer readable medium storing a plurality of normalized peak intensities obtained by any of the methods described above.
Various embodiments of the present invention provide methods for relative quantification of a substance present at different concentrations in different chemical samples using mass spectrometry. Unlike many prior art mass spectrometric quantification methods, which require internal standards or detectable tags to be added to each sample, or which require multiple samples to be combined for analysis, embodiments of the present invention allow relative quantification to be performed directly from acquired mass spectra. In some embodiments, no additional sample processing steps are required, and quantification can be performed on previously acquired data that were not intended to be compared. The methods can be useful for small sample volumes that would be overwhelmingly diluted by an internal standard. They are also useful for samples that contain multiple components of interest or of which the components of interest can be determined only after measurements are performed (unanticipated components).
Although embodiments of different methods will be described primarily in the context of mass spectrometry, it is to be understood that the methods are applicable to any type of spectroscopy or spectrometry yielding spectra containing signals (or peaks) whose intensities or areas are proportional to component concentrations. Mass spectrometry is believed to be an important tool for proteomics and metabolomics research, because it provides for sensitive detection and identification of all types of proteins and metabolites over a large dynamic range. However, the detected ion intensity may depend upon many factors in addition to sample component concentration, such as ionization efficiency, detector efficiency, sample size, and sample flow rate. For this reason, additional methods are traditionally employed to provide for quantification of detected components. While protein and peptide ionization for mass spectrometry conventionally employ MALDI (matrix-assisted laser desorption ionization) or ESI (electrospray ionization), the invention is applicable to any suitable current or future ionization method, as well as any suitable detection method, such as ion trap, time-of-flight, or quadrupole analyzers. In addition, the method can be applied to data obtained from gas chromatography-mass spectrometry (GC-MS), particularly using electron-impact ionization (EI), a highly reproducible ionization method. One application of embodiments of the invention is analysis of mixtures of metabolites and proteins that are enzymatically digested prior to analysis; other embodiments are used for relative quantification of any type of chemical or biological sample.
Some embodiments of the invention rely on the assumption that biological samples, particularly those of interest in proteomics and metabolomics research, consist of complex mixtures of multiple biological components, of which only a minority are relevant or important. The large majority of components are at relatively constant concentrations across samples and subject populations. For the purposes of discovering biological markers of disease, these constant components provide little useful information. Rather, it is the difference in protein expression between, for example, healthy and diseased subjects, that is important. Differentially expressed proteins (or other organic molecules) may serve as biological markers that can be measured for diagnostic or therapeutic purposes. In embodiments of the present invention, the majority of components whose concentrations do not vary across samples are used to normalize the concentrations of components that do vary. Thus this background level of substantially unchanging proteins serves as an intrinsic internal standard by which the relative concentrations of varying proteins can be measured. This intrinsic internal standard can be used to correct for both drift in instrument response and also overall differences in sample concentrations (e.g., dilute versus concentrated urine). Note that high accuracy of relative quantification depends in part on consistent sample processing techniques.
One embodiment of the invention is a method illustrated by the schematic mass spectra 10 and 12 of
The spectra 10 and 12 shown correspond to two different samples, both of which yield component peaks at particular values of mass-to-charge ratio (m/z), labeled as A, B, C, and D. As used herein, a peak is a local maximum in signal intensity, with respect to one or more of m/z, chromatographic retention time, or any other suitable variable. Peaks are characterized by the value of the variables at which they occur. The intensity value (height, area under the curve, or other suitable intensity measure) of the peak is referred to as its peak intensity. Note that the two spectra have completely different intensity scales. In the spectrum 10 of
Although the absolute intensity values vary widely between the two spectra, the relative abundances of components represented by peaks A, B, and D are essentially the same in the two spectra. Thus it is assumed that these three components have substantially equal or constant concentrations in the two samples. The substantial constancy of concentrations is represented as the substantial constancy of intensity ratios. That is, the ratio of intensities of peaks A and B, A and D, and B and D are substantially constant. Equivalently, the ratio between each component in the two spectra is substantially constant. That is, the ratio of peak A intensity in the second spectrum 12 to peak A intensity in the first spectrum 10 is approximately equal to the ratio of peak B intensity in the second spectrum 12 to peak B intensity in the first spectrum 10. These ratios are approximately 70:1. As used herein, a substantially constant concentration or substantially constant ratio refers to one that fluctuates by no more than a value approximately equal to the coefficient of variation (CV) for peak intensities in spectra of similar types of samples. For serum sample spectra obtained using currently optimal sample preparation techniques and current instruments, a current value is approximately 25%. As will be appreciated by those of skill in the art, numerous error sources exist for LC-MS and GC-MS data, including the sample preparation techniques, chromatographic method, and ionization method. While lower coefficients of variation may be achieved when measuring limited numbers of molecules in relatively simple samples, it is not expected that similar numbers can be obtained for simultaneous measurement of thousands of molecules in complex biological samples. This value may decrease with future improvements in sample preparation methods and instrumentation.
In contrast, the component represented by peak C varies in relation to the other peaks. Any of the ratios between C and A, C and B, and C and D are substantially non-constant between the two spectra, changing by more than the approximate CV, preferably more than about 25%. The ratio of peak C intensity in the second spectrum 12 to peak C intensity in the first spectrum 10 is approximately 70:3, substantially different from the 70:1 ratio for all other peaks. Since this ratio changes by a factor of three, it can be assumed that the concentration of a chemical component associated with peak C is three times greater in the sample of the first spectrum 10 than in the sample of the second spectrum 12.
The structure of the component associated with peak C can be determined subsequently. In some cases, the peptide or other molecule corresponding to the mass-to-charge ratio of peak C is known. In other cases, tandem mass spectrometry can be performed to fragment the ion of peak C and obtain its mass spectrum, from which the structure of the ion can be determined. Typically, a protein-containing sample is enzymatically digested before mass spectral analysis, and there are multiple peptide peaks varying according to the same ratio. In many cases, the peak list can be compared with spectral libraries to determine the identity of the varying component. Other analysis can be included to account for multiply charged ions or modifications, such as oxidation, to a portion of the peptides. Also, accurate mass measurements can be employed to aid in molecular identification.
A flow diagram outlining general steps of a method 20 of one embodiment of the present invention is shown in
In a second step 24, a normalization factor is computed for each spectrum (or a subset of the spectra) in the set. The normalization factor is computed in dependence on chemical sample components whose concentrations are substantially constant among the analyzed chemical samples. The constant components are represented by peaks whose intensity ratios remain substantially constant across spectra, as described above. Typically, it is not known a priori which components will be at constant concentration; that is, the constant components are not predetermined. In fact, it is often the object of the study to determine which components do vary among samples. The constant components are not added to the samples for quantification purposes; rather, they are inherent components of the samples being analyzed.
In one embodiment, one of the spectra is selected as a reference spectrum, and ratios are computed between peaks in the spectrum to be normalized (the test spectrum) and the reference spectrum. Ratios can be computed for all peaks or for some fraction of the total number of peaks. The reference spectrum can be of the same general type of sample (e.g., same biological fluid such as serum) but is not otherwise closely matched. Peak ratios are computed for peaks at the same value of m/z (and retention time or other position variable, for hyphenated methods), within predefined tolerances, resulting in a list of ratios. The majority of values in the list are substantially equal, representing components whose concentrations do not vary between the test and reference spectra. In one embodiment, the normalization factor is computed from the list of ratios using a non-parametric measure. Most preferably, the normalization factor is the median of the list of intensity ratios. Alternatively, the normalization factor can be the mode of the list of intensity ratios. Non-parametric measures such as a median or mode are insensitive to outliers and therefore minimize the effect of non-constant components on the normalization factor. An example of a normalization factor obtained from the median of the ratios of peaks in two peptide samples derived from human serum is shown in
In an alternative embodiment, if constant components are known a priori, then intensities of peaks corresponding to these components can be used as the normalization factor, or can be used to compute the normalization factor.
In the next step 26, normalized spectra are computed by scaling each peak, or each desired peak, by the normalization factor. If the normalization factor is the median of intensity ratios of the reference to test spectra, then the peaks are multiplied by this factor.
Any desired quantitative analysis can be performed on the normalized spectra. For example, in step 28, peaks are located whose intensity varies substantially between at least two spectra. Substantially varying peaks differ by at least the approximate CV, e.g., by at least 25%. The intensity ratio of two such peaks occurring within a specified m/z and position tolerance indicates the relative concentrations of the component responsible for the peak in the two samples. Subsequent analysis may be performed using conventional methods to determine the identity of the compound or compounds responsible for the peak differences. In proteomic analysis, a single protein is digested into multiple peptide fragments, yielding multiple peaks. Conventional algorithms and public databases can be employed to identify the responsible protein.
While it may be possible to determine manually or using a simple automated algorithm which peaks of the normalized spectra vary, more complex methods may also be used. For example, in one embodiment of the invention, an analysis algorithm can be applied to the normalized spectra to determine which peaks are most responsible for the variance among spectra. One possible algorithm is principal component analysis (PCA), but other techniques including, but not limited to, ordinary least squares, principal component regression, and partial least squares can also be used. PCA is known in the art and will not be described in detail herein. Briefly, PCA reduces the dimensionality of the spectral data by introducing new variables, termed principal components, that are linear combinations of the original variables. Originally, each spectrum is represented as a vector of normalized intensity values at each relevant mass-to-charge (m/z) ratio or m/z and retention time pair. The first principal component accounts for as much of the variance in the data as possible, and each succeeding component accounts for as much of the remaining variance as possible. In many cases, enough information is contained in the first two or three principal components for the Euclidean distances between points in principal component space to indicate the similarity between spectra.
To determine which peaks differ most in intensity among samples, it is useful to determine which peaks contribute most to each principal component. This can be accomplished by examining the coefficients in the linear combinations that make up the principal components to locate peaks with the highest absolute value of coefficient. Once the set of relevant peaks is known, ratios (between spectra) of their normalized intensities can be obtained to determine the relative quantity of the corresponding ion (and peptide or protein) in the different samples. If it is known that multiple peaks correspond to peptides obtained from the same protein, an average is computed of their ratios to determine the protein's relative quantity in the different samples. Note that when the ratio is computed from all peptide peaks originating from the same protein, each peak is an independent measure of the protein concentration, effectively lowering the measurement standard deviation.
The intensities used in obtaining the quantification ratios and performing the analyses can be computed in a number of different ways. The most suitable intensity measure typically depends upon the type of data acquired. A simple measure is the maximum intensity value of the identified peak. Alternatively, the intensity can be the peak area (or volume for three-dimensional data). It is to be understood that the term "intensity," as used herein, refers to intensity measures computed in any desired manner. The selected measure typically depends on the particular data. In many cases, equivalent results are obtained using a variety of different measures.
Note that in some embodiments of the invention, it is sufficient to know which peaks are varying among samples, and it is not necessary to quantify the relative concentrations. Normalization is useful in this case to allow accurate identification of the varying peaks.
In one embodiment, it may be desirable to add one or more spiked molecules to aid in quantification. These molecules may be matched to a known sample component (e.g., a deuterated or other isotopically-labeled version) or not matched to any components. The spiked molecules can be added to the samples at a known concentration and their signal intensities used to normalize spectral signals and computed sample component concentrations.
Although not limited to any particular hardware configuration, the present invention can be implemented in software by a system 30 shown in
The computer implementing the invention can contain a processor 42, memory 44, data storage medium 46, display 48, and input device 50 (e.g., keyboard and mouse). Methods of various embodiments of the invention are executed by the processor 42 under the direction of computer program code stored in the computer 32. Using techniques well known in the computer arts, such code is tangibly embodied within a computer program storage device accessible by the processor, e.g., within system memory 44 or on a computer readable storage medium 46 such as a hard disk or CD-ROM. The methods may be implemented by any means known in the art. For example, any number of computer programming languages, such as Java, C++, or LISP may be used. Furthermore, various programming approaches such as procedural or object oriented may be employed.
In an alternative embodiment, normalized peak intensities, e.g., computed according to any of the embodiments described above, are stored on a computer readable medium. In another embodiment, the normalized peak intensities are stored in a database.
It is to be understood that the steps described above are highly simplified versions of the actual processing performed by the computer, and that methods containing additional steps or rearrangement of the steps described are within the scope of the present invention.
The following working examples illustrate embodiments of the invention without limiting the embodiments to the particular details described.
5-Component Protein Mixtures
A method of one embodiment of the invention was implemented using three five-component protein mixtures in which two of the components varied in concentration,
while the remaining three were constant. Relative mass concentrations within the samples were as follows:
Bovine Bovine Bovine Sample Horse ribonuclease serum cytochrome Human number myoglobin A albumin C hemoglobin 1 1 1 1 1 1 2 1 1 1 5 0.2 3 1 1 1 0.2 5
All three samples were denatured by 6 M guanidine hydrochloride, reduced by 10 mM dithiothreitol at 37� C. for 4 hours, and alkylated with 25 mM iodoacetic acid/NaOH at room temperature for 30 minutes in the dark. The denaturant and reduction-alkylation reagents were removed from the mixtures by buffer exchange against 50 mM (NH4)2CO3 at pH 8.3 three times using 5-kDa molecular weight cut-off spin filters. Modified trypsin at 1% weight equivalence of the proteins was added to the mixtures for incubation at 37� C. for 14 hours. The same amount of trypsin was again added, and the mixtures were incubated at 37� C. for another 6 hours. Each resulting sample was divided into four aliquots.
Electrospray ionization liquid chromatography-mass spectrometry was performed on the twelve aliquots using a binary HP 110 series HPLC directly coupled to a ThermoFinnigan LCQ DECA™ ion trap mass spectrometer or MicroMass LCT™ ESI-TOF mass spectrometer equipped with a nanospray source. Fused-silica capillary columns (5 μm C18 resin, 75 μm internal diameter �10 cm) were run at a flow rate of 300 nL/min after flow splitting. An on-line trapping cartridge allowed fast loading onto the capillary column. Gradient elution was achieved using 100% solvent A (0.1% formic acid in H2O) to 40% solvent B (0.1% formic acid in acetonitrile) over 100 minutes.
The resulting spectra were normalized using an embodiment of the normalization method in which the normalization factor was the median of intensity ratios, yielding an average coefficient of variation of 17% for the four replicates, an improvement of 5% over the non-normalized results. Principal component analysis (PCA) was performed on extracted normalized peaks, and the first and second principal components are plotted in
Average Ratio of Integrated Peak Areas (Theoretical value 5.0) Peak m/z Sample 1:Sample 2 Sample 3:Sample 1 537.01 5.20 3.85 564.60 3.88 5.36 818.74 5.00 3.45 932.77 5.77 5.51 1150.85 2.49 5.87 Average ratio 4.47 4.81 Coefficient of variation 29% 23% Error 11% 3.8%
Differences in signal values substantially exceeding the coefficients of variation represent components occurring in different concentrations.
Normalized Peak Intensities of Human Serum Sample Spectra
Human serum samples were analyzed to determine measurement variability after normalization using one embodiment of the present invention. Pooled human serum was purchased from Sigma-Aldrich (for proteome studies) and obtained from four anonymous healthy donors at the Stanford Blood Center (for metabolome studies). The serum was fractionated into serum proteome and serum metabolome using a 5-kDa molecular weight cut-off spin filter. Twenty-five μL of the serum proteome was diluted with 475 μL of 25 mM PBS buffer (pH 6.0) before being applied to affinity beads from ProMetic Life Sciences for removal of human serum albumin and IgG. The albumin- and IgG-depleted serum proteome was denatured, reduced, alkylated, and trypsin digested following the procedures described in Working Example 1 to yield 200 μg proteome. The serum metabolome was desalted using a C18 solid-phase extraction cartridge. The proteome fraction was divided into 10 samples and the metabolome fraction into 90 samples.
Mass spectra were obtained of the proteome samples using the LC-MS instruments and procedures described in Working Example 1. The metabolome procedure differed in that the chromatographic separation was performed with a gradient of 10% to 25% of solvent B in 40 minutes, followed by 25–90% solvent B in 30 minutes. 2000 peaks were selected from each spectrum and normalized using the median intensity ratio as described above in one embodiment of the invention.
Human Serum Spiked With Non-Human Proteins and Small Molecules
Human blood serum proteome spiked with horse myoglobin and bovine carbonic anhydrase II, as well as human blood serum metabolome spiked with low-molecular weight species, were analyzed using methods of embodiments of the invention. The spiking is not part of the quantification method, but was rather used to test the method.
Human serum was obtained and fractionated into serum proteome and serum metabolome as described in Working Example 2. The two non-human proteins were spiked into 20 μg of unprocessed human serum proteome at amounts ranging from 100 fmol to 100 pmol. The spiked proteome samples were denatured, reduced, alkylated, and trypsin digested following the procedures described in Working Example 1. Varying amounts of an equimolar test compound mixture were added to 100 μL of the metabolome prior to sample clean-up using the solid-phase extraction C18 cartridge. The components added were des-asp1-angiotensin II, [val4]-angiotensin II, vitamin B12, and α-endorphine. Spiked mixture amounts varied from 50 fmol to 100 pmol per component. Resulting samples were analyzed by LC-MS as described in Working Example 1 and peaks identified and normalized using one embodiment of the invention.
Similar results are shown for the serum metabolome in
It should be noted that the foregoing description is only illustrative of the invention. Various alternatives and modifications can be devised by those skilled in the art without departing from the invention. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variances which fall within the scope of the disclosed invention.
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11 | Fiehn et al. (2000) Nat. Biotechnol. 18:1157-1161. | |
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13 | Gygi et al. (1999) Nat. Biotechnol. 17:994-999. | |
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15 | Ji et al. (2000) J. Chromat. B. 745:197. | |
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17 | Koradi et al. (1998) J.Mag. Res. 135:288-297. | |
18 | Nelson et al. (1995) Annal. Chem. 67:1153. | |
19 | Nielsen et al. (1998) J. of Chromatography A 805:17-35. | |
20 | Oda et al. (1999) Proc. Natl. Acad. Sci. USA 96:6591. | |
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22 | Pravdova et al. (2002) Analyitca Chimica Acta 456:7792. | |
23 | Prazen et al. (1998) Anal. Chem. 70:218-225. | |
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26 | Stein (1999) J. Am. Soc. Mass Spectrom. 10:770-81. | |
27 | Wang et al. (1987) Analytical Chemistry 59:649. | |
28 | Wang et al. (2003) Anal. Chem. 75:4818. | |
29 | Wingdig et al. (1996) Anal. Chem. 68:3602-3606. |
Citing Patent | Filing date | Publication date | Applicant | Title |
US7462819 * | May 16, 2006 | Dec 9, 2008 | The University Of Western Ontario | Statistical methods applied to surface chemistry in minerals flotation |
US8543625 | Oct 16, 2009 | Sep 24, 2013 | Intelliscience Corporation | Methods and systems for analysis of multi-sample, two-dimensional data |
US20060289740 * | May 16, 2006 | Dec 28, 2006 | Smart Roger S C | Statistical methods applied to surface chemistry in minerals flotation |
US20100100577 * | Oct 16, 2009 | Apr 22, 2010 | Intelliscience Corporation | Methods and systems for analysis of multi-sample, two-dimensional data |
U.S. Classification | 250/282, 250/281 |
International Classification | H01J49/04, B01D59/44, H01J49/00 |
Cooperative Classification | H01J49/0036 |
European Classification | H01J49/00S3 |
Date | Code | Event | Description |
May 3, 2005 | AS | Assignment | Owner name: SM PURCHASE COMPANY, LLC, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SURROMED, INC.;REEL/FRAME:015972/0122 Effective date: 20050131 |
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Register | FAQ | PM | Events | Groups | Blogs | Calendar | Mark Forums Read |
Skookum Robotics Skookum Robotics SK-360 SK-540 & SK-720 Digital Flybar |
Thread Tools | Display Modes |
Registered Users
Be aware of this tail issue (when using DX8 and/or satellites)
Hi guys,
Its the issue that your tailservo just moves a few hundred steps between the end points rather then (theoretically) 2048 steps... Maybe it just happens with my config, but I'm not sure:
How to check if you have the problem: Power-up your Tx and your heli so the servos move (not just the SK720). Connect the SK720 to the PC so it enters setup mode. Now slowly move the rudder on your Tx and hold the tail-rod slightly between your fingers. If you can feel a ratchet movement (i.e. feel every step the servo does) you have the same problem as me. How to make sure - How to fix The only fix that worked for me every time is to rebind the SK720 to the Tx. If you're not sure if you have the problem just rebind and feel the movements afterwards. If its more smooth than you had the problem and fixed it with the rebind. Background story Today I ran again in the problem with my tail servo ratchet movement (see here). It seems that the problem still exists. Art (Skookum support) agreed to the problem as he saw it in the logs I've sent him a couple of month ago. The signal the SK720 sends to the tail-servo are just a few hundred steps. We couldn't find out though when this happens. Yesterday I had a really loud tail noise during flying (sounded like a turbine). First I thought its just the wind blowing the sound directly to me. But now I think its this issue again. By accident I checked the tail-rod during setup again and I felt the ratchet movement again... Only rebinds resolved the problem. It seems to me, that changing values in the setup software (maybe just tail-servo relating values) causes the problem. Because after my session with Art a view month ago I did the rebind and everything was fine. Anyway... I just wanted to share and let you know about that Soko
The future of setup... now! Soko Heli Toolbox app for a next generation setup experience. ONE tool for ALL setups of ALL sizes and ALL brands of helis www.soko-heli-tools.com |
Skookum Robotics Skookum Robotics SK-360 SK-540 & SK-720 Digital Flybar |
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subset: commoncrawl


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Zigolini's Pizza – Wood-Fired Pizza in Midtown NYC
That should be a whole new category – "wine bar pizza." I've had too many "pizzas" lately that were nothing more than pita bread with a few toppings on it. And all of them were at wine bars. Not that I mind a snack like that but can we stop calling them pizzas?
Yes! bar pizza!
http://slice.seriouseats.com/archives/2008/01/a-list-of-regional-pizza-styles-slideshow.html#show-85728 -
@JustinM-so true! Actually, that is just how I felt about this pizza!!
@A-Tooch-And THERE ya go! Something else I NEVER knew even existed! -
Have you been to Motorino? My fav thin crust pizza! Let's go on a #soulmate date there xo
I'm not a soulmate but I do live directly above Motorino so maybe I can joinnn? 🙂
[…] their indigenous foods. In one day, you can travel through 3 or 4 states and try everything from New York pizza to Connecticut hot dogs to Rhode Island […]


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Thread: Can We Call Them Commies Now?
Results 21 to 30 of 34
- Join Date
- Nov 2009
- Posts
- 1,800
here's some Minute Men protesting along side the American Third Position
They are a White Nationalist group and their homepage features videos of speakers from the Tea Party rallies: http://american3p.org/category/american-voice/
The party Director of the American Third Position has written several anti-Semitic articles,a few of which are titled:Originally Posted by Kevin MacDonald
They were at an immigration protest with the Minute Men and they were at the Tax Day Tea Party rally and happily display those tea party videos on their front page.
That doesn't mean the Minute Men are a White Nationalist organization, does it?
That doesn't mean the Tea Party is a White Nationalist organization, does it?
They actually seem very active with Tea Party rallies: http://american3p.org/american-voice...ss-the-nation/
A3P activist Josh Bates and his wife joined thousands of Georgians and South Carolinians at the Augusta Tea Party, where they spoke with scores of attendees and passed out hundreds of party fliers.
Recounting the successful nature of his outreach effort, Josh said, "One of the last people I spoke to was a man from South Carolina who, after hearing what I had to say about the A3P, stated he would like to get something started in South Carolina. We exchanged information, and I hope to be in touch with him soon to help him get started."
He continued, "After all was said and done, I had given out all of my literature and still had swarms of people asking for more information on the party. I ended up having to write our website and contact information on sheets of paper from my notebook to give out, due to the demand. All in all, it was a great evening. My wife and I got to meet a lot of people who are enthusiastic about the A3P cause, and I have already received numerous emails from some of the people I spoke with."Last edited by Wei Wu Wei; 06-10-2011 at 02:42 AM.
Originally Posted by Adam Smith - Wealth of Nations
- Join Date
- May 2008
- Posts
- 11,970
I'm kind of relieved that my union isn't involved (UAW).
How can you tell that they are Third Position members? I heard the voice over of the camera claiming that they were, but I didn't hear anyone admit to it or see anything that identified them as such. Could you cite the point in the video where someone who isn't the narrater could confirm this, or is it just more BS?
Sounds like they are trying to make it seem that way. But if they had shown up at a Tea Party event, where are the photos? It's not like the left hasn't tried to tie the Tea Party in with fringe groups before. They've even sent agent provocateurs into rallies with signs that were meant to present a false impression. This looks like more of the same to me.--Odysseus
Sic Hacer Pace, Para Bellum.
Before you can do things for people, you must be the kind of man who can get things done. But to get things done, you must love the doing, not the people!
lol you are trying so hard.
There is Tea Party stuff all over the Third Position website, including photos:
In fact here is a video of the Chairman of the American Third Position, William D. Johnson, speaking at a Tea Party rally (notice the giant banner behind him).
But I guess this all must just be secret leftists infiltrating everything and working behind the scenes really hard to make people look badOriginally Posted by Adam Smith - Wealth of Nations
- Join Date
- Jun 2011
- Location
- Indiana
- Posts
- 3
06-10-2011, 01:27 PMWhenever somebody says that these guys are communists, they are very quick to throw around charges like McCarthyism and Red-Baiting, but is it okay to call someone a communist if they co-sponsor and march in a communist parade behind a communist banner, surrounded by people who openly call themselves communists? Just asking.
Not as hard as you are. You've found one obscure extremist group that managed to infiltrate a single Tea Party event. I guarantee that they won't be welcome at future ones. The equivalence of this on Mayday would have been if SEIU held a labor rally, and a group of communists who had hidden the worst parts of their agenda had shown up and then raised their flags when the cameras were on. But that isn't what happened. SEIU knew that they were co-sponsoring the Mayday event, which is a communist version of the Thanksgiving Day Parade, except that instead of giving thanks, they try to drum up resentment. They knowingly associated with communists, planned it out in advance and allowed themselves to be led behind a red banner. There's nothing to indicate that the Tea Partiers knew who Johnson was, since he was simply introduced as a local attorney, and his entire speech was about fiscal issues.
No, it's just a small fringe group that is trying to piggyback on the tea party's successes. They are helped by the fact that their name doesn't actually imply anything about their racism, and that they present a fake front of moderation at the event that they attended. I'd never even heard of them until this thread. OTOH, ANSWER has been around since the beginning of the war, and is not shy about its communism. Now, apparently, SEIU isn't, either.--Odysseus
Sic Hacer Pace, Para Bellum.
Before you can do things for people, you must be the kind of man who can get things done. But to get things done, you must love the doing, not the people!
« Previous Thread | Next Thread » |


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Credits & Info
- Uploaded
- Aug 6, 2003 | 5:50 PM EDT
If you liked this, check these out!
X and Zero: The Fight P.1 by Supersonic89
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Author Comments
sorry guys for the wait i got hella lazy
Rated 5 / 5 stars
dood,this movie is the best!when u make more plz,by all that is good and game-related tell me!!!!! im a big fan of these, plz make different ones with zero in them...
Rated 5 / 5 stars
lol "Is that my grandma flashing her tits"
That's funny what megaman said LMAO
-To all those that hate this and/or don't like sprites........THEN DON'T WATCH IT U FUCKING MORONS IT'S AS SIMPLE AS THAT....and if u hate it bcuz it's sprites...well your just an ass if you have made a SINGLE sprite movie or if ones in your favorites and you hate all sprites you a hypocrite.
-Great flash man I'm gonna go watch the other series now CYA
Rated 4 / 5 stars
Pretty good, youve come a long ass way since your first flash :D And im sick of people talking about bad grammar and shit, WHO GIVES A FUCK!? Its a mistake! Get over it! But yeah nice one and fuck Im seriously thinking of putting this series in my Favorites :D:D:D OH YEAH!
Rated 4 / 5 stars
funny......sum1 shulda killed zero once it started zero is a basterd
Rated 4 / 5 stars
Pretty good.
Yay.....Guardian-Dragon.......read what i put for #3. it goes for this 2 you stupid baby. Anyways...again....pretty good movie yet again. good finish.
thanks :D


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MyChart Login
Home > Health & Wellness > Health Library > Circumcision: Gomco Clamp
The Gomco clamp is a metal device with a bell-shaped end. During circumcision using a Gomco clamp, the baby's foreskin is stretched over the bell, and the clamp is tightened over his foreskin.
If the foreskin can't be pulled back (is not retractable), it usually needs to be cut before the clamp is put in place. The foreskin is cut away and the clamp removed.
Applying the Gomco clamp is:
ByHealthwise StaffPrimary Medical ReviewerAdam Husney, MD - Family MedicineSpecialist Medical ReviewerMartin J. Gabica, MD - Family Medicine
Current as ofNovember 20, 2015
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Item | Clutch Plate, Heavy Duty |
For Use With | Hild Model Machines with Plate Drive |
Package Quantity | 1 |
Weight | 0.650000 |
This item is restricted for international sale. This item is no longer available and cannot be ordered online. Some item(s) may be available at a DrillSpot branch. For assistance, call your local branch.


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WikiProject Somalia
WikiProject Somalia | Main page | Data sources |
Somalia, Africa |
latitude: 5, longitude: 48 |
Browse map of Somalia 5°00′00″ N, 48°00′00″ E |
Edit map |
External links:
Use this template for your city |
Somalia is a country in Africa at latitude 5°00′00″ North, longitude 48°00′00″ East.
2011 droughts
More information about Droughts in the horn of Africa on the HOT blog |
Somalia and the whole of the Horn of Africa have been struck by droughts. Currently the only area to do some mapping specifically in response to this crisis, is around the refugee camps in Dadaab (Kenya) However general improvements to the map of Somalia may help aid organisations at this time.
General mapping
/Roads import of data from MadMappers requires (JOSM expert) volunteers help out with checking and merging in data. If you know how to do this kind of thing, join in! (See /Roads) Some other datasets are described at /VectorAndMapData.
There's also a certain amount of remote mapping to be done using low-res Landsat.
As ever, we can always benefit from people doing on-the-ground mapping. If you are in a position to do this, we'd love to hear from you, and help you get started. Please contact HOT to say hello. You can also read more here: Map Making Overview
Muqdisho - Bing imagery available
Kismaayo has been mapped with a 1992 map from the National Imagery and Mapping Agency.
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Office Space to Rent in Dombey Street, WC1
We have a variety of serviced offices available to rent in Dombey Street, WC1. Each office space is offered at the best rates and suited to your individual needs and requirements.
We have a variety of serviced offices available to rent in Dombey Street, WC1. Each office space is offered at the best rates and suited to your individual needs and requirements.
A virtual office could be up and running in a matter of minutes with: Prestige address, live receptionist, direct dial number, message taking service, mail forwarding, main line number, company voicemail and access to meeting rooms.
Self-contained building, approx. 3,000sqft over 6 floors. 40-50 workstations with 2 executive offices and lower ground boardroom benefiting from natural light. There is also a kitchen area and roof terrace. Fully managed inclusive of: rent, business rates and service charge, utilities, cleaning and maintenance, furniture (if required), buildings insurance, fully network cabled, bandwidth (can bring own lines), key phone entry, facilities management, decoration and upkeep and fire detection. Flexible terms available from 12 months.
Meeting Rooms Only
Offering flexible licenses in non-branded period style office buildings conveniently located in and around Soho and Central London. Offices are fully furnished with complete telecomms and IT infrastructure pre-installed. Prime locations and the latest IT and Telecoms infrastructure are the focus, with the target market being Start-ups, Small and Medium Enterprises.
The serviced offices at this centre occupy a prime location in the heart of Clerkenwell between Russell Square, Farringdon and Chancery Lane. The business centre is situated within an impressive 18th Century Georgian building that has been recently refurbished to provide high quality office space, with excellent natural light and period features. The centre maintains its heritage but creates superb unbranded offices that come fully furnished, with a wide range of state-of-the-art IT and Telecoms and 24/7 access. You will also be able to benefit from the onsite team who will act as your company receptionist and assist you with anything you may need. Meeting rooms are available to book upon request from 2-10 people and offer a full catering service and audio visual equipment. Great building features include; fire places, high ceilings, staircases, renovated vaults, a large courtyard and a roof terrace.
This business centre operates over 3 floors. The entrance and reception area have just finished a recent full refurbishment complete with modern design Amourcoated walls providing a very welcoming first impression to your visitors. Offices are newly refurbished ranging in size from 2 workstations to whole floor plates, offering views over to Red Lion Square. Full air conditioning, lifts to all floors, 24 hour manned security, fully refurbished, modern British made desks and modern working environment with latest technology telecoms and internet provision.
This business centre is located in London's central business district; these offices offer clients an elegant corporate environment and a prestigious business address. With excellent access to transport links, these offices are fully managed and fully furnished to help you save time and money. With a receptionist, stylish meeting and conference rooms, high spec IT infrastructure and support services available, the aim is to help you focus on your own business, while the centre takes care of all your office requirements.
At the centre you'll benefit from the following: instant occupancy with no start-up costs; simple, flexible agreements; fully equipped conferencing and meeting rooms, video conferencing; wide-ranging business support services; dedicated comms rooms with Cat 5e cabling; State of the art VOIP telecoms, bandwidth provision and network access; 24/7 access; 24-hour remote security monitoring. In addition, there's a highly trained, Institute of Customer Service accredited centre team on hand to support you and your business. This centre offers all-inclusive pricing, so you can move in with peace of mind that there will be no unexpected costs each month.
High Holborn is centrally located, with Holborn tube station on the door step providing you with the Central & Piccadilly line, it also has an array of restaurants and coffee shops surrounding it making High Holborn a fantastic working environment. After having a complete new makeover this building provides 6 office floors as well as an amazing, generous break out area in the basement. Cycle in and utilise the brand new showers and drying room with your bike securely locked for safe keeping.
This centre provides professional office and studio space in a modern business centre in Holborn. The building has recently undergone a refurbishment and now offers a stylish open plan reception area and high-spec meeting room. Just minutes from Chancery Lane tube station, this centre is well placed for access to both the financial centre in the City and the business, entertainment and media heartland of the West End.
Below you can see the nearest areas to Dombey Street, WC1 where you can find serviced offices:


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Tuesday, July 24, 2012
Auckland Art & Craft Fair
This weekend is the Auckland Art & Craft Fair and I'm more than a little excited. If you can make it up to Auckland for the event I highly recommend it- it's a great chance to support the New Zealand handmade community and a perfect opportunity for a bit of shopping.
It's extra special for me this year with my good friend Dane setting up his own stall to sell goodies from his store The Lost Boys Club. If you see him make sure you say hello, and for that matter if you see me floating around I'd love to meet you too!
If you're planning to join us there it's being held at the Aotea Centre in the CBD from 11 - 3 for only a gold coin entry :)
(Collage images used from: Hunter Gatherer, Bird in a Bunny Suit, Bron Alexander, Bonjour Sweetie, Baka, The Lost Boys Club)
7 comments:
Sounds like a lot of fun.
Christie x
Dark Blue Stripes


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DIY Powerhouse: Elekit's TU-8340VK Amplifier Is Not Your Grandfather's Dynaco Elekit TU-8340VK Amplifier, Part 1 of 2 by Steve Graham In fact, this new amplifier isn't even your [...] 2017/03/13 /// 0 Comments
Domestic Harmony, Chapter 6 (The Conclusion) Domestic Harmony: The 6th and concluding part of the Jamie and Paul series By Steve Graham As followers of [...] 2017/03/10 /// 1 Comment
Photos from the Coherent Speakers audio meet in Hamilton Well, many thanks to Frank Fazzalari; his audio meet was a resounding success! There was a great turnout of [...] 2017/03/08 /// 0 Comments
Coherent Audio to host local meet in Hamilton on Mar.6th Our friend Frank Fazzalari of Coherent Speakers is organizing an Audio Meet in Hamilton, that will showcase [...] 2017/02/27 /// 0 Comments
My Audio Life: Part 6 My Altec Adventures (continued) By Steve Graham The media room renovation is almost complete. The [...] 2017/02/08 /// 0 Comments
Good News / Bad News: Final update on the Linestage and 6DJ8 articles by Steve Graham Since the last update I've been corresponding with Hari S. (I hope I've spelled [...] 2017/02/06 /// 2 Comments
Mundorf XO Part 3, aka The Law of Diminishing Returns This will be the last part of my writeup on Frank Fazzalari's amazing crossovers for the Radian [...] 2017/01/11 /// 0 Comments
Domestic Harmony, Chapter 5: The Truth About Anna Domestic Harmony: Part 5 of the 'Jamie and Paul' series By Steve Graham The Truth about [...] 2017/01/10 /// 0 Comments
The Hana EH: Redefining the budget phono cartridge Many have long regarded Denon's DL103 as the benchmark in "budget moving coil" cartridges. [...] 2017/01/10 /// 8 Comments
Sun Valley 211/845 SET Tube Amp Kit from Japan Some cool news from Victor Kung of vkmusic.ca: VK Music will soon carry the SunValley 211/845 amp kit from [...] 2017/01/06 /// 0 Comments


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UNLOCKING THE CANINE ANCESTRAL DIET - HEALTHIER DOG FOOD THE ABC WAY by Steve Brown (See other books by author)
Click here to get the Ebook version
Summary: In Unlocking the Canine Ancestral Diet, dog nutrition expert Steve Brown shows how you can bring the benefits of the canine ancestral diet to your dog by feeding him differently as little as just one day a week. And no, you won't need to lead a pack of dogs on a hunting expedition! Just follow Steve's well-researched and easy to follow ABCs to make improvements to whatever your dog currently eats.
Ancestors and canine cousins of our dogs didn't eat "krunchy kibble" or "meat 'n gravy in a can." They ate what they found or caught... and it wasn't cooked or "enriched" either! It was high in protein, with balanced fats, and usually included a few fruits, vegetables and grasses. Steve Brown, an expert on canine nutrition, shows how you can bring the benefits of the canine ancestral diet to your dog by feeding him differently as little as just one day a week. And no, you won't need to lead a pack of dogs on a hunting expedition! Just follow Steve's well-researched and easy to follow ABCs to make improvements to whatever your dog currently eats. BONUS! Raw food or home prepared feeders will learn how to balance nutrients more precisely, especially fats, for optimum health. A dog diet to get wild about! • Learn about the latest research on the importance of protein and healthy fats in your dog's diet. • Find out why commercial foods can't include these fragile-but-crucial nutrients, and how you can make sure your dog gets them. • Just one day a week, or more frequently if you choose, follow the simple recipes that balance the nutrition in the commercial food you are feeding—wet or dry! What experts are saying about Unlocking the Canine Ancestral Diet Brown has done his homework and is able to present his case for feeding a nutrient mix that more closely matches what has been health food for hundreds of generations of canines. Unlocking the Canine Ancestral Diet is chock full of information that supports his premise that the ancestral diet is good stuff for today's dogs. It's a good read, packed with scientifically-backed information, and it presents a practical approach for achieving the goal of helping us to make a healthy diet for our dogs. As easy as ABC. Randy Kidd, author Dr. Kidd's Guide to Herbal Dog Care This book is enlightening and invaluable. I especially like the fact that his guidelines can help balance canine diets no matter what the caregiver's level of commitment is to the ancestral diet. Dry food feeders can greatly enhance their pet's nutrition with just one homemade meal a week. Raw fed dogs can benefit from a balancing meal as well. Doug Knueven, DVM, CVA, CAC, author The Holistic Health Guide: Natural Care for the Whole Dog To prevent chronic disease in dogs you must get a critical balance of species appropriate foods and fats in your companion's diet. Steve Brown understands this and you can too by reading his book.Karen Becker, DVM Steve Brown turned his passion for canine health into developing leading-edge products and educational programs to improve canine nutrition. He developed Charlee Bear Dog Treats®, one of the best selling dog treats in the US and, in 1998, he developed Steve's Real Food® for Pets, the first AAFCO-compliant, frozen, raw meat-based diet. Brown is the co-author of See Spot Live Longer and has written numerous articles on canine nutrition published in leading pet health and veterinary journals. Steve lives outside of Eugene, Oregon. Click here to view an excerpt. What reviewers are saying... THE VITAL CIRCLE "We just discovered a great new book about the role of omega-3s in canine health. Canine nutrition expert Steve Brown presents what must be the most exhaustively researched resource on the diets dogs have eaten over many millennia. And in Unlocking the Canine Ancestral Diet: Healthier Dog Food the ABC Way, he's used that research to show how people can enhance their pets' health by approximating the ancient canine diet as closely as possible. He finds evidence that most dogs ate diets that were high in protein, with balanced fats – including omega-3s, sometimes in large amounts (as with salmon-loving wolves) and usually included a few fruits, vegetables and grasses. Brown presents a compelling analysis of dogs' nutritional needs, and provides practical ways to meet them in his "ABC" diet plan, which owners only need to provide one day per week. Here's what Brown means by an "ABC" diet: • Add high-quality protein with hearts, eggs, and sardines • Balance the fats with sardines • Complete the nutrition with hearts, eggs, sardines, and vegetables. He also custom-tailors the plan to each animal's weight and to an owner's preferences for various kinds of commercial dog food (dry, wet, etc.). Unlocking the Canine Ancestral Diet represents a landmark in canine nutrition research … and in practical feeding advice for owners." Craig Weatherby MIDWEST BOOK REVIEW "Dogs weren't meant to eat dried animal byproducts. "Unlocking the Canine Ancestral Diet: Healthier Dog Food the ABC Way" is a guide for pet owners who want a more healthy and vitamin packed diet for their dog, drawing back on their instinctual diet of real meat, uncooked and raw, along with the scattered fruits and vegetables they may have ate. Stating that treating your dog to this special diet if only once a week will have numerous long term positive effects, "Unlocking the Canine Ancestral Diet" is a fine read that should not be ignored for those who care about the health of their dog." James A. Cox, Editor DOG WORLD "Steve Brown is not a veterinarian or a nutritionist, but he has extensively researched canine nutrition since 1998. Unlocking the Canine Ancestral Diet presents his recommendations on enhancing the average canine diet. Brown admits that we don't yet fully understand canine nutritional requirements and that much information is extrapolated from human nutritional studies. He writes, for instance, "Cooking tomatoes increases the amount of lycopene that humans can absorb. This is probably true with dogs, as well." …Essentially, Brown's program advocates feeding his ABC diet once a week, which entails adding high-quality protein to commercial food, balancing fats, and completing nutrition with fresh foods. He includes recipes for homemade diets, and provides nutritional analysis which are often missing from most dog food recipes. However, these recipes might not be practical for anyone lacking time to prepare complicated meals containing costly ingredients." Amy Fernandez DOGS IN CANADA "ABC stands for amount, balance and complete, and the ABC philosophy recommends feeding fats, proteins and carbohydrates in amounts approximating what your dog's ancestors ate. Brown points out what he believes to be weaknesses in modern dog food, explains the chemistry of fats, and describes how to complete your dog's diet with fresh foods, including recipes for replacing, altering or augmenting whatever you're currently feeding. For those reluctant to invest the time to cook for their dog on a daily basis, Brown provides a plan for practicing ABC just one day a week ." Natalie Ann Comeau
Dig In


text:
Valerie Singleton, John Noakes and Peter Purves team up to present a series of programmes celebrating the 40th anniversary of the show that led to phrases such as "Here's one I made earlier" and "Get down, Shep" becoming ingrained on the nation's psyche.
How or why it became such a success is unclear, but it clearly grabbed children's imagination in a way no other programme did - or has since.
For the presenters, the joy of Blue Peter was the chance to do the things most people dream of. Valerie Singleton, who presented the programme between 1962 and 1972, said: "I always liked the part where we got to touch the exhibits, where most people could only look."
Saturday's specials will include Carry on Blue Peter, with bloopers and out-takes, It's a Dog's Life, about the Blue Peter dogs, and A Right Royal Reunion, with Valerie Singleton talking to the Princess Royal about their 1971 Kenya trip.
The following Friday, on the anniversary of the programme's first showing, a special 40th birthday edition with past presenters will be aired.
Peter Purves, who joined in 1967, said: "At the time I wasn't aware of its immense appeal. I remember the renaming ceremony for the restored Blue Peter locomotive where 50,000 people turned up. The police were unprepared for it."
For 40 years, the appeal has never really waned. Blue Peter has become a cornerstone of the BBC. The regular features such as the "makes" (with parents being pestered for washing-up bottles), appeals (raising pounds 25m over the past 15 years) and summer expeditions played their part in creating a continuity that helped to keep children watching.
Valerie Singleton said she has seen changes but the show still keeps the bare bones that make Blue Peter special: "All the things you now associate with it came about when I was on the programme."
Stuart Miles, the longest serving current presenter, believes there are two main reasons why the show remains as popular as ever: "One is that there's something comfortable about it, with the presenters and pets, and the other is that there's always an edge to what goes on. You never know what's going to happen this week."
The show has had 25 presenters, 26 if you count Anita West who was on for six months before being replaced by Valerie Singleton. She is never mentioned in BBC archives, although one of the programmes on Blue Peter night promises to set the record straight.
- More about:
- 1990s TV
- Children's TV
- Dogs
- Pets


text:
Spectramast LC (Canada)
This page contains information on Spectramast LC for veterinary use.The information provided typically includes the following:
- Spectramast LC Indications
- Warnings and cautions for Spectramast LC
- Direction and dosage information for Spectramast LC
Spectramast LC
This treatment applies to the following species:ceftiofur hydrochloride sterile suspension
Veterinary Use Only
Lactating Cow Mastitis Treatment
For Intramammary Infusion Only
SPECTRAMAST® LC sterile suspension is an oil-based sterile suspension containing 125 mg of ceftiofur (as ceftiofur hydrochloride) per 10 mL disposable syringe. Ceftiofur is a broad-spectrum cephalosporin antibiotic that exerts its effect by inhibiting bacterial cell wall synthesis. Like other β-lactam antimicrobial agents, the cephalosporins inhibit cell wall synthesis by interfering with the enzymes essential for peptidoglycan synthesis. This effect results in lysis of the bacterial cell and accounts for the bactericidal nature of these agents. The chemical name of ceftiofur hydrochloride is 5-Thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[[2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetyl]amino]-3-[[(2-furanylcarbonyl)thio]methyl]-8-oxo, hydrochloride.
Chemical Structure:
INDICATIONS: SPECTRAMAST LC sterile suspension is indicated for the treatment of clinical mastitis in lactating dairy cows caused by Escherichia coli, Streptococcus dysgalactiae and coagulase negative staphylococci. Cows with systemic clinical signs caused by mastitis should receive other appropriate therapy under direction of a licensed veterinarian.
Dosage and Administration
Infuse one (1) syringe (10 mL) into each infected quarter. Repeat this treatment once in 24 hours.Directions For Use
Wash teats thoroughly with warm water containing a suitable dairy antiseptic. Dry the teats thoroughly. Milk out the udder completely. Using an alcohol swab provided, wipe off the end of the affected teat using a separate swab for each teat. Choose the desired insertion length (full or partial) and insert tip into the teat canal; push plunger to dispense the entire contents, massage the quarter to distribute the solution into the milk cistern. Following infusion it is advisable to dip all teats in an approved teat dip.
The syringe is designed to provide the choice of either insertion of the full cannula as has traditionally been practiced, or insertion of no more than 1/8 inch of the cannula, as reported by Eberhart, R.J. et. al. 1987. Current Concepts of Bovine Mastitis, 3rd Edition, National Mastitis Council, Arlington, VA.
a. Full insertion: Remove the white end cap by pulling straight up as shown. Gently insert the full cannula into the teat canal; carefully infuse the product.
b. Partial insertion: Remove the white end cap by pulling straight up as shown. Gently insert the exposed white tip into the teat canal; carefully infuse the product.
CLINICAL EFFICACY: In 1999-2000, the efficacy of ceftiofur was demonstrated in a pivotal field dose confirmation efficacy study in lactating dairy cattle with clinical mastitis in one quarter. Ceftiofur was formulated in stable cotton seed oil sterile suspension. Cows with mastitis were enrolled in the study if characterized with visually abnormal milk (clots, flakes, or watery secretion) or if udder swelling, heat, pain or redness were present and the milk was not yet visually abnormal but California Mastitis Test (CMT) gave results of 2 or greater. A total of 13 trial sites enrolled 352 cows in the study. Cows were assigned to one of three treatment groups: non-treated control, 62.5 mg ceftiofur, and 125 mg ceftiofur (SPECTRAMAST LC sterile suspension). Each treatment group received an intramammary infusion twice at a 24-hour interval in the affected quarter. The non-treated controls received no therapy. Three different definitions for cure were used for analysis purposes: 1) a clinical cure was defined as the milk and udder returning to normal 14 days after the last treatment and remaining normal at the 21 day time point; 2) a bacterial cure was defined as the absence of the pre-treatment pathogen at 14 and 21 days post-treatment; and 3) a protocol cure was defined as the absence of the pre-treatment pathogen at 14 and 21 days post-treatment and return to normal 14 days after the last treatment and remaining normal at the 21 day time point. Of the 337 cows analyzed, the clinical cure rate for the non-treated control group was 54.7% (64/117) compared to 69.4% (75/108) for the 62.5 mg treatment group and 78.6% (88/112) for the 125 mg treatment group. The 125 mg treatment group's clinical cure rate was significantly greater than the non-treated control (P=0.002). Of the 146 cows analyzed, the bacterial cure rates were 41.3% (19/46) for the non-treated control group, 45.6% (21/46) for the 62.5 mg treatment group and 70.4% (38/54) for the 125 mg treatment group. The 125 mg treatment group was significantly better than the non-treated control group (P=0.006). For the 146 cows analyzed, the protocol cure rate was 63.0% (34/54) for the 125 mg treatment group, 41.3% (19/46) for the 62.5 mg treatment group and 23.9% (11/46) for the non-treated control group. The 125 mg treatment group was significantly better than the non-treated control (P<0.001) for treatment of clinical mastitis.
Based on this pivotal study, 125 mg of ceftiofur administered via intramammary infusion twice at a 24-hour interval was effective in the treatment of clinical mastitis in lactating dairy cows caused by Streptococcus dysgalactiae, Escherichia coli, and coagulase negative staphylococci.
ANIMAL SAFETY: A pivotal udder irritation study was conducted in which udder irritation was assessed in 17 cows, following the intramammary infusion of SPECTRAMAST LC sterile suspension twice at a 24-hour interval. A slight increase in Somatic Cell Count (SCC) to levels <150,000 cell/mL was observed following infusion in normal cows with very low pre-infusion SCC (<41,000 cell/mL). This elevation is not unexpected with oil-based suspensions. No changes in body temperature were noted during this study. Mild increases in udder palpation scores were noted in 8/17 cows; moderate increases in udder palpation scores were noted in 3/17 cows; moderate increases in strip cup scores were noted in 3/17 cows; a marked increase in strip cup score was noted in 1/17 cows. These increases were transient, and could be related to microbial infections. This pivotal safety study demonstrated that SPECTRAMAST LC sterile suspension is clinically safe for use in lactating dairy cows. In two clinical field efficacy studies involving 971 lactating dairy cows, no reports of udder irritation or adverse events were noted following infusion. Collectively, these three studies demonstrate that the intramammary infusion of SPECTRAMAST LC sterile suspension twice at a 24-hour interval is clinically safe for use in lactating dairy cows.
(1) Milk taken from treated cows during treatment and within 96 hours after the latest treatment must not be used as food. (2) No meat withdrawal period is required when this product is used according to label directions and dosage. (3) To limit the development of antimicrobial resistance: SPECTRAMAST LC should only be used to treat individual cases of clinical mastitis. - The choice of this product as the most appropriate treatment should be confirmed by clinical experience supported where possible by pathogen culture and drug susceptibility testing. The extra label use of this product is not recommended. (4) Penicillins and cephalosporins can cause allergic reactions in sensitized individuals. Topical exposure to such antimicrobials, including ceftiofur, may elicit mild to severe allergic reactions in some individuals. Repeated or prolonged exposure may lead to sensitization. Avoid direct contact of the product with the skin, eyes, mouth and clothing. Sensitization of the skin may be avoided by wearing latex gloves. (5) Persons with a known hypersensitivity to penicillin or cephalosporins should avoid exposure to this product. (6) In case of accidental eye exposure, flush with water for 15 minutes. In case of accidental skin exposure, wash with soap and water. Remove contaminated clothing. If allergic reaction occurs (e.g., skin rash, hives, difficult breathing), seek medical attention. KEEP OUT OF THE REACH OF CHILDREN.The material safety data sheet contains more detailed occupational safety information. To report adverse effects in users, to obtain more information or to obtain a material safety data sheet, call 1-800-461-0917.
As with all drugs, the use of SPECTRAMAST LC sterile suspension is contraindicated in animals previously found to be hypersensitive to the drug.
CAUTIONS: Cows with systemic clinical signs caused by mastitis should receive other appropriate therapy under the direction of a licensed veterinarian. Do not reuse. If redness, swelling and/or abnormal milk persists, discontinue use and consult a veterinarian. It is recommended that milk cultures be used to aid in decision-making for clinical mastitis therapy.
CLINICAL MICROBIOLOGY: Ceftiofur has demonstrated in vitro and clinical activity against Streptococcus dysgalactiae, Escherichia coli and coagulase negative staphylococci. The in vitro activity of ceftiofur against mastitis pathogens are summarized in Table 1 and 2.
Table 1. Ceftiofur Minimum Inhibitory Concentration (MIC) Values of isolates from Field Studies Evaluating Clinical Mastitis in Dairy Cows in the U.S. During 2000.
Pathogen |
Number of isolates |
MIC range |
Coagulase-negative staphylococci (CNS) |
33 |
1.0 |
≤0.06-2.0 |
Streptococcus dysgalactiae |
32 |
≤0.06 |
≤0.06-0.05 |
Escherichia coli |
35 |
0.5 |
≤0.06-1.0 |
* MIC for 90% of the isolates
Table 2. Ceftiofur MIC Values* for Mastitis Pathogens from Diagnostic Laboratories in the U.S. and Canada.
Organism |
Country of Isolation |
No. |
Date Tested |
Summary of MIC Data |
Range |
Streptococcus dysgalactiae |
United States |
15 |
1991-1992 |
1.0 |
≤0.06-2.0 |
United States, Canada |
64 |
2000 |
≤0.06 |
≤0.06-0.5 |
United States |
152 |
1997-1999 |
0.25 |
0.25-4.0 |
Escherichia coli |
United States |
39 |
1991-1992 |
1.0 |
0.25-1.0 |
United States, Canada |
52 |
2000 |
0.5 |
≤0.06-1.0 |
Coagulase (-) staphylococci |
United States, Canada |
139 |
2000-2001 |
1.0 |
≤0.06-2.0 |
* The clinical significance of this data is not known
** MIC for 90% of the isolates
MIC90 values not calculated for organisms with less than 10 isolates.
Based on pharmacokinetic, milk residue and clinical effectiveness studies in dairy cattle following intramammary infusion of ceftiofur and the MIC and disk (30 µg) diffusion data from mastitis pathogens, the following breakpoints are recommended by the Clinical and Laboratories Standards Institute (CLSI) (Table 3).
Table 3. Current recommended interpretative criteria established by CLSI for ceftiofur for bovine mastitis.
Bovine Mastitis Organisms |
Disk Content |
Zone Diameter |
MIC breakpoint |
Staphylococcus aureus |
30 µg |
≥21 |
18-20 |
≤17 |
≤2.0 |
4.0 |
≥8.0 |
Streptococcus dysgalactiae |
Streptococcus uberis |
Streptococcus agalactiae |
Escherichia coli |
S - Susceptible
I - Intermediate
R - Resistant
Standardized procedures require the use of laboratory control organisms for both standardized diffusion techniques and standardized dilution techniques. The 30 µg ceftiofur sodium disk should give the following zone diameters and the ceftiofur sodium standard reference powder (or disk) should provide the following MIC values for the reference strain. Ceftiofur sodium disks or powder reference standard is appropriate for ceftiofur hydrochloride and ceftiofur sodium (Table 4).
Table 4. Acceptable Quality Control Ranges for ceftiofur against CLSI recommended American Type Culture Collection (ATCC) reference strains.
Organism Name |
Zone Diameter |
MIC Range |
Escherichia coli (25922) |
26-31 |
0.25-1.0 |
Staphylococcus aureus (29213) |
- |
0.25-1.0 |
Staphylococcus aureus (25923) |
27-31 |
- |
Store between 15 and 25°C. Store PLASTETS in carton or pail until used. Protect from light.PRESENTATION: SPECTRAMAST LC is available in cartons of 12 and pails of 144 x 10 mL PLASTET disposable syringes, with one individually wrapped 70% isopropyl alcohol pad per PLASTET.
Zoetis is a trademark and Spectramast is a registered trademark of Zoetis or its licensors, used under license by Zoetis Canada Inc.
Zoetis Canada Inc., Kirkland QC H9H 4M7
NAC No.: 1198347.4
Order Desk: | 800-663-8888 | |
Technical Services Canada: | 800-461-0917 | |
Technical Services USA: | 800-366-5288 | |
Website: | www.zoetis.ca |
Every effort has been made to ensure the accuracy of the Spectramast LC information published above. However, it remains the responsibility of the readers to familiarize themselves with the product information contained on the Canadian product label or package insert. |
Copyright © 2017 North American Compendiums. Updated: 2017-02-06


text:
No matter what the outcome
of the Progressive Insurance Automotive X PRIZE, SABA Motors' team leader,
Simon Saba, will have no regrets.
Although he does not have a background in automotive, he knew that the
time was now to take advantage of an opportunity that he believes will never
come again.
Simon, a mechanical
engineer, who works in the field of lasers and fiber optics technology,
followed his entrepreneurial spirit when he heard about the Progressive
Automotive X PRIZE. Understanding
the technical and financial challenges of the competition, he nevertheless
jumped in and has not looked back.


text:
We've seen then mature into two stunning young ladies who not only have good sense but have great taste. The daughters of Run-DMC rapper, Reverend Run, Angela and Vanessa Simmons are now taking their own show on the road with a spin-off on MTV called "Daddy's Girls." The girls revealed to Blackvoices.com that their new show will follow them now that they are living together in L.A., and developing their "Pastry" shoe and clothing line. But that's not the only thing keeping the girls busy. Vanessa is still securing various acting roles and just started filming a new movie with Marques Houston. Angela is turning her eye towards the music game premiering the video for her new song "Center of Attention" on the Pastry website.
Black Celeb Couples


text:
Join Occupy Reno and Code Pink for the Protest – April 17th 4-6 pm
At the Vassar Street Post Office
tax day protest 2
Latest Tweets
- The Families Funding the 2016 Presidential Election fb.me/4A3pcu4mx 1 year ago
- Hi everyone, this page will disappear this week. Please go ahead and like Occupy Reno's new page to stay... fb.me/7udBpUu6J 1 year ago
- Organize your workplaces people! Right to Work only means Right to Union Bust. fb.me/7tUgWoxWS 1 year ago
- fb.me/3QuLvr9m9 1 year ago
- fb.me/32RvVDJSP 1 year ago


text:
By Ron Richter on Wed 03/13/2013 06:33pm
On July 1 of this year, the state tax on gasoline and diesel fuel in Wyoming will jump from 14 cents a gallon to 24 cents. The last time the fuel tax was raised in Wyoming was 1998. Representative Mike Madden explains why legislators felt that the tax hike was needed.
The increase is estimated to generate over $70 million for the 2014 fiscal year, with the majority of the funds dedicated to the state's highways. State estimates show that a typical Wyoming family can expect to pay a little over $100 extra at the pump annually.
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