text:
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|Lippuner et al.
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This application claims priority to U.S. Provisional Patent application Ser. No. 60/701,460 filed Jul. 21, 2005 and entitled "Assay For ALS and ALS-Like Disorders" by inventors Ira L. Goldknopf, et. al.
The invention relates to a method for discriminating between amyotrophic lateral sclerosis (ALS) patients and patients with ALS-like disorders that express symptoms like ALS. The method is based on the use of 2-dimensional (2D) gel electrophoresis to separate the complex mixture of proteins found in blood serum and the quantitation of a group of identified biomarkers to differentiate patients having ALS from patients having other ALS-like disorders.
ALS is a devastating, fatal neurodegenerative disease that causes the progressive loss of the cells in the brain, spinal cord, and motor nerves that control muscle function. It is the third most common neurodegenerative disease in adults, after Alzheimer's disease and Parkinson's disease. Early symptoms of ALS may include arm and leg weakness, stiffness, and slurred speech. The majority of patients die within 3-5 years from the appearance of the first symptom, usually from respiratory muscle failure.
Presently, the diagnosis of ALS is a clinical one. There is no single test that can provide diagnostic certainty. The usual diagnostic process consists of a full medical history, as well as a comprehensive physical and neurological examination. The revised El Escorial Criteria, developed at a Consensus Conference in Spain in 1990, is widely accepted for the diagnosis of ALS (Chaudhuri, K. R., et al. 1995. J. Neurol. Sci. 129 Suppl.: 11-12). This set of criteria combines clinical features and laboratory test results to classify the level of diagnostic certainty into Definite, Probable, Possible, and Suspected.
Since no definitive diagnostic test for ALS is currently available, numerous studies are typically performed to rule out other medical conditions that can mimic the appearance of ALS. This is important because many of the ALS-like conditions have a much more favorable prognosis. A complete evaluation may include an electromyogram (EMG) with nerve conduction studies (NCV), magnetic resonance imaging (MRI) of the brain and spinal cord, lumbar puncture (LP) with analysis of cerebrospinal fluid (CSF), a panel of blood tests, and muscle biopsy. Because the El Escorial criteria set was originally designed for research purposes, some clinicians find them to be somewhat cumbersome (Brooks, B. R. 2000. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. Suppl 1:S79-S81).
From a clinical standpoint, familial ALS and sporadic ALS are indistinguishable (Mulder, D. W., et al. 1986. Neurology 36: 511-517; Juneja, T., et al. 1997. Neurology 48:55-57; Cudkowicz, M. E., et al. 1997. Ann. Neurol. 41: 210-221; Li, T. M., et al. 1988. J. Neurol. Neurosurg. Psychiatry 51: 778-784). In the United States, 90-95% of ALS cases are sporadic (i.e., no family history of ALS). Only 5-10% of ALS cases are familial. In 10-20% of these familial cases, a mutation can be identified in the gene for superoxide dismutase 1 (SOD 1), a ubiquitously expressed antioxidant protein (Siddique, T., et al. 1991. N. Engl. J. Med. 324:1381-1384). Over 90 different SODI mutations have been reported in different persons with familial ALS. Although tests are available that can detect SOD1 mutations, less than 20% of familial cases will have a SOD1 mutation (Orrell, R. W., et al. 1997. Neurology 48: 746751; Shaw, C. E., et al. 1998. Ann. Neurol. 43: 390-394). Thus, SOD1 mutations account for less than 2% of all ALS cases. Mutations in other genes have also been linked to small subsets of familial ALS. Clearly, genetic testing will not detect the majority of ALS cases.
In addition, the etiology of ALS remains undefined. It is even unclear what places a person at-risk of getting ALS. It is currently proposed that a combination of genetic susceptibility factors and environmental factors is involved in an increased risk for ALS. Researchers have repeatedly searched for genetic susceptibility factors that affect cellular processes that influence the survival of motor neurons, including excitotoxicity (Rothstein, J. D., et al. 1995. Ann. Neurol. 38(1): 73-84; Rothstein, J. D., et al. 1992. N. Engl. J. Med. 326: 1464-1468), oxidative stress (Comi, G. P., et al. 1998. Ann. Neurol. 43(1): 110-116), neurofilament abnormalities (Al-Chalabi A., et al. 1999. Hum. Mol. Genet. 8(2): 157-164; Vechio, J. D., et al. 1996. Ann. Neurol. 40: 603-610), inflammation, growth factors, axonal transport, and other processes (Olkowski, Z. L. 1998. Ann. Neurol. 40: 603-610; Hayward, C., et al. 1999. Neurology 52(9): 1899-1901; Drory, V. E., et al. 2001. J. Neurol. Sci. 190(1-2): 17-20). However, to date, no susceptibility factors have emerged to account for the majority of ALS cases.
There is a tremendous need for a definitive diagnostic test to confirm the diagnosis of Lou Gehrig's disease (ALS) and distinguish it from other ALS-like disorders that display similar symptoms but have different treatment options and prognosis. Clinicians have long sought such a diagnostic test in hopes of providing earlier treatment decisions and improved patient outcomes.
Proteomics is a new field of medical research wherein proteins are identified and linked to biological functions, including roles in a variety of disease states. With the completion of the mapping of the human genome, the identification of unique gene products, or proteins, has increased exponentially. In addition, molecular diagnostic testing for the presence of certain proteins already known to be involved in certain biological functions has progressed from research applications alone to use in disease screening and diagnosis for clinicians. However, proteomic testing for diagnostic purposes remains in its infancy. There is, however, a great deal of interest in using proteomics for the elucidation of potential disease biomarkers.
Detection of abnormalities in the genome of an individual can reveal the risk or potential risk for individuals to develop a disease. The transition from risk to emergence of disease can be characterized as an expression of genomic abnormalities in the proteome. Thus, the appearance of abnormalities in the proteome signals the beginning of the process of cascading effects that can result in the deterioration of the health of the patient. Therefore, detection of proteomic abnormalities at an early stage is desired in order to allow for detection of disease either before it is established or in its earliest stages where treatment may be effective.
Recent progress using a novel form of mass spectrometry called surface enhanced laser desorption and ionization time of flight (SELDI-TOF) for the testing of ovarian cancer and Alzheimer's disease has led to an increased interest in proteomics as a diagnostic tool (Petrocoin, E. F. et al. 2002. Lancet 359:572-577; Lewczuk, P. et al. 2004. Biol. Psychiatry 55:524-530). Furthermore, proteomics has been applied to the study of breast cancer through use of 2D gel electrophoresis and image analysis to study the development and progression of breast carcinoma in patients and in plasma from Alzheimer's disease patients (Kuerer, H. M. et al. 2002. Cancer 95:2276-2282; Ueno, I. et al. 2000. Electrophoresis 21:1832-1845). In the case of breast cancer, breast ductal fluid specimens were used to identify distinct protein expression patterns in bilateral matched pair ductal fluid samples of women with unilateral invasive breast carcinoma.
Detection of biomarkers is an active field of research. For example, U.S. Pat. No. 5,958,785 discloses a biomarker for detecting long-term or chronic alcohol consumption. The biomarker disclosed is a single biomarker and is identified as an alcohol-specific ethanol glycoconjugate. U.S. Pat. No. 6,124,108 discloses a biomarker for mustard chemical injury. The biomarker is a specific protein band detected through gel electrophoresis and the patent describes use of the biomarker to raise protective antibodies or in a kit to identify the presence or absence of the biomarker in individuals who may have been exposed to mustard poisoning. U.S. Pat. No. 6,326,209 B1 discloses measurement of total urinary 17 ketosteroid-sulfates as biomarkers of biological age. U.S. Pat. No. 6,693,177 B1 discloses a process for preparation of a single biomarker specific for 0-acetylated sialic acid and useful for diagnosis and outcome monitoring in patients with lymphoblastic leukemia.
Neurodegenerative diseases are difficult to diagnose, particularly in their early stages, as currently there are no biomarkers available for either the early diagnosis or treatment of neuromuscular diseases such as amyotrophic lateral sclerosis (ALS) or ALS-like disorders. There are a number of ALS-like disorders that exhibit similar clinical symptoms as ALS, but have a much better prognosis. Yet the distinction between ALS and ALS-like disorders can be difficult for the physician using current standards of care including medical history, comprehensive physical and neurological examination, MRI, electromyogram, nerve conduction studies, spinal tap for analysis of CSF, a blood test panel, and muscle biopsy.
Thus, there is a continuing need for better ways to detect and distinguish ALS patients from patients having ALS-like disorders.
The present invention is a diagnostic assay for differentiating amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, and ALS-like disorders. The method comprises collecting a biological sample from a patient having symptoms consistent with ALS, quantitating up to 34 protein biomarkers identified as related to ALS or ALS-like disorders, and determining whether or not the patient has ALS or an ALS-like disorder based on the statistical analysis of the quantity of the selected protein biomarkers.
One aspect of the present invention is a method for screening a patient for ALS or ALS-like disorders. The method includes: collecting a serum sample from a patient having symptoms consistent with ALS, separating the proteins in the serum sample by 2D gel electrophoresis, quantitating a panel of protein biomarkers, and determining whether or not the patient has a ALS or an ALS-like disorder based on the quantity of those biomarkers in the patient's serum.
The foregoing has outlined rather broadly several aspects of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and the specific embodiment disclosed might be readily utilized as a basis for modifying or redesigning the methods for carrying out the same purposes as the invention. It should be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims.
For a more complete understanding of the present invention, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:
FIG. 1 a 2D gel electrophoretic image of human serum proteins with 34 biomarkers marked and numbered.
FIG. 2 shows a linear discriminant function analysis of human serum samples from ALS patients and patients with ALS-like disorders.
FIG. 3 shows the performance of the quadratic discriminant function analysis of training and test sets of samples from ALS patients and patients with ALS-like disorders.
The present invention is a diagnostic test for differentiating individuals with amyotrophic lateral sclerosis (ALS) patients and patients with ALS-like disorders that express symptoms like ALS. The method is based on the use of 2-dimensional (2D) gel electrophoresis to separate the complex mixture of proteins found in blood serum and the quantitation of a group of identified biomarkers to differentiate patients having ALS from patients having other ALS-like disorders.
In the context of the present invention a "neuromuscular disease" is a condition wherein an individual or patient exhibits a known set of symptoms such as limb weakness, slurred speech, muscle twitching or cramping, and/or swallowing difficulty. Neuromuscular diseases include, but not be limited to amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), ALS-like diseases, Parkinson's disease (PD), and PD-like diseases.
In the context of the present invention an "ALS-like disorder" would include six main anatomical categories, as follows:
1. Cervical Spinal Compromise (Myelopathy)
2. Multiple Sclerosis
3. Lower Motor Neuron Compromise
4. Nerve Disease
5. Neuromuscular junction compromise
6. Muscle Disease
In the context of the present invention, the "protein expression profile" corresponds to the steady state level of the various proteins in biological samples that can be expressed quantitatively. These steady state levels are the result of the combination of all the factors that control protein concentration in a biological sample. These factors include but are not limited to: the rates of transcription of the genes encoding the hnRNAs; the rates of processing of the hnRNAs into mRNAs; the splicing variations during the processing of the hnRNAs into mRNAs which govern the relative amounts of the protein isoforms; the rates of processing of the various mRNAs by 3′-polyadenylation and 5′-capping; the rates of transport of the mRNAs to the sites of protein synthesis; the rate of translation of the mRNA's into the corresponding proteins; the rates of protein post-translational modifications, including but not limited to phosphorylation, nitrosylation, methylation, acetylation, glycosylation, poly-ADP-ribosylation, ubiquitinylation, and conjugation with ubiquitin like proteins; the rates of protein turnover via the ubiquitin-proteosome system and via proteolytic processing of the parent protein into various active and inactive subcomponents; the rates of intracellular transport of the proteins among compartments such as but not limited to the nucleus, the lysosomes, golgi, the membrane, and the mitochondrion; the rates of secretion of the proteins into the interstitial space; the rates of secretion related protein processing; and the stability and rates of proteolytic processing and degradation of he proteins in the biological sample before and after the sample is taken from the patient.
In the context of the present invention, a "biomarker" corresponds to a protein present in a biological sample from a patient, wherein the quantity of the biomarker in the biological sample provides information about whether the patient exhibits an altered biological state such as ALS or an ALS-like disorder.
A "control" or "normal" sample is a sample, preferably a serum sample, taken from an individual with no known disease, particularly without a neuromuscular disease.
The method of the present invention is based on the quantification of specified proteins. Preferably the proteins are separated and identified by 2D gel electrophoresis. 2D gel electrophoresis has been used in research laboratories for biomarker discovery since the 1970's (Orrick, L. R. et al. 1973. Proc. Natl. Sci. U.S.A. 70:1316-1320; Goldknopf, I. L. et al. 1975. J. Biol Chem. 250:71282-7187; O'Farrell, P. et al. 1975. 1. Biol. Chem. May 250:4007-4021; Anderson, L. and Anderson, N. G. 1977. Proc. Natl. Acad. Sci. U.S.A. 74:5421-5425; Goldknopt I. L. and Busch, H. 1977. Proc. Natl. Acad. Sci. USA 74:864-868). In the past, this method has been considered highly specialized, labor intensive and non-reproducible.
Only recently with the advent of integrated supplies, robotics, and software combined with bioinformatics has progression of this proteomics technique in the direction of diagnostics become feasible. The promise and utility of 2D gel electrophoresis is based on its ability to detect changes in protein expression and to discriminate protein isoforms that arise due to variations in amino acid sequence and/or post-synthetic protein modifications such as phosphorylation, nitrosylation, ubiquitination, conjugation with ubiquitin-like proteins, acetylation, and glycosylation. These are important variables in cell regulatory processes involved in disease states.
There are few comparable alternatives to 2D gels for tracking changes in protein expression patterns related to disease progression. The introduction of high sensitivity fluorescent staining, digital image processing and computerized image analysis has greatly amplified and simplified the detection of unique species and the quantification of proteins. By using known protein standards as landmarks within each gel run, computerized analysis can detect unique differences in protein expression and modifications between two samples from the same individual or between several individuals.
Sample Collection and Preparation
Serum samples were prepared from blood acquired by venipuncture. The blood was centrifuged at 500×g for 10 minutes, and the separated serum was divided into aliquots, and frozen at −40° C. or below until shipment. Samples were shipped on dry ice and were delivered within 24 hours of shipping.
Once the serum samples were received, logged in, and assigned a sample number, they were further processed in preparation for 2D gel electrophoresis. All samples were stored at −40° C. or below. When the serum samples were removed from storage, they were placed on ice for thawing and kept on ice for further processing.
To each 100 μl of sample, 1OO μl of LB-2 buffer (5M urea, 2M Thiourea, 0.5% ASB-14, 0.25% CHAPS, 0.25% Tween-20, 5% glycerol, 100 mM DTT, 1× Protease inhibitors, and 1× Ampholyte pH 3-10) was added and the mixture vortexed. The sample was incubated at room temperature for about 5 minutes.
Separation of Proteins in Patient Samples
The proteins in the patient and control samples were separated using various techniques known in the art for separating proteins, techniques that include but are not limited to gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, or any of the various centrifugation techniques well known in the art. In some cases, a combination of one or more chromatography or centrifugation steps may be combined via electrospray or nanospray with mass spectroscopy or tandem mass spectroscopy, or any protein separation technique that determines the pattern of proteins in a mixture either as a one-dimensional, two-dimensional, three-dimensional or multi-dimensional pattern or list of proteins present.
Two Dimensional-Electrophoresis of Samples
Preferably the protein profiles of the present invention are obtained by subjecting biological samples to two-dimensional (2D) gel electrophoresis to separate the proteins in the biological sample into a two-dimensional array of protein spots.
Two-dimensional gel electrophoresis is a useful technique for separating complex mixtures of proteins and can be performed using a variety of methods known in the art (see, e.g., U.S. Pat. Nos. 5,534,121; 6,398,933; and 6,855,554).
Preferably, the first dimensional gel is an isoelectric focusing gel and the second dimension gel is a denaturing polyacrylamide gradient gel.
Proteins are amphoteric, containing both positive and negative charges and like all ampholytes exhibit the property that their charge depends on pH. At low pH (acidic conditions), proteins are positively charged while at high pH (basic conditions) they are negatively charged. For every protein there is a pH at which the protein is uncharged, the protein's isoelectric point. When a charged molecule is placed in an electric field it will migrate towards the opposite charge.
In a pH gradient such as those used in the present invention, a protein will migrate to the point at which it reaches its isoelectric point and becomes uncharged. The uncharged protein will not migrate further and stops. Each protein will stop at its isoelectric point and the proteins can thus be separated according to charge. In order to achieve optimal separation of proteins, various pH gradients may be used. For example, a very broad range of pH, from about 3 to 11 or 3 to 10 can be used, or a more narrow range, such as from pH 4 to 7 or 7 to 10 or 6 to 11 can be used. The choice of pH range is determined empirically and such determinations are within the skill of the ordinary practitioner and can be accomplished without undue experimentation.
In the second dimension, proteins are separated according to molecular weight by measuring mobility through a polyacrylamide gradient in the detergent sodium dodecyl sulfate (SDS). In the presence of SDS and a reducing agent such as dithiothreitol (DTT), the proteins act as though they are of uniform shape with the same charge to mass ratio. When the proteins are placed in an electric field, they migrate into and through the gel from one edge to the other. As the proteins migrate though the gel, individual proteins move at different speeds with the smaller ones moving faster than the larger ones. This process is stopped when the fastest moving components reach the other side of the gel. At this point, the proteins are distributed across the gel with the higher molecular weight proteins near the origin and the low molecular weight proteins near the other side of the gel.
It is well known in the art that various concentration gradients of acrylamide may be used for such protein separations. For example, a gradient of from about 5% to 20% may be used in certain embodiments or any other gradient that achieves a satisfactory separation of proteins in the sample may be used. Other gradients would include but not be limited to from about 5 to 18%, 6 to 20%, 8 to 20%, 8 to 18%, 8 to 16%, 10 to 16%, or any range as determined by one of skill.
The end result of the 2D gel procedure is the separation of a complex mixture of proteins into a two dimensional array based on their unique characteristics of isoelectric point and molecular weight
2D SDS-PAGE Standards
Purified proteins having known characteristics are used as internal and external standards and as a calibrator for 2D gel electrophoresis. The standards consist of seven reduced, denatured proteins that can be run either as spiked internal standards or as external standards to test the ampholyte mixture and the reproducibility of the gels. A set mixture of proteins (the "standard mixture") is used to determine pH gradients and molecular weights for the two dimensions of the electrophoresis operation. Table 1 lists the isoelectric point (p1) values and molecular weights for the proteins included in this standard mixture.
|Molecular Weight (Da)
|Hen egg white conalbumin
|6.0, 6.3, 6.6
|Bovine serum albumin
|5.4, 5.5, 5.6
|Bovine muscle actin
|5.0, 5.1
|Rabbit muscle GAPDH
|8.3, 8.5
|Bovine carbonic anhydrase
|5.9, 6.0
|Soybean trypsin inhibitor
|Equine myoglobin conalbumin
In addition, Precision Plus Protein Standards (Bio-Rad Laboratories), a mixture of 10 recombinant proteins ranging from 10-250 kD, are typically added as external molecular weight standards for the second dimension, or the SDS-PAGE portion of the system. The Precision Plus Protein Standards have an r2 value of the Rf vs. log molecular weight plot of>0.99.
Separation of Proteins in Serum Samples
An appropriate amount of isoelectric focusing (IEF) loading buffer (LB-2), was added to the diluted serum sample, incubated at room temperature and vortexed periodically until the pellet was dissolved to visual clarity. The samples were centrifuged briefly before a protein assay was performed on the sample.
Approximately 100 μg of the solubilized protein pellet was suspended in a total volume of 184 μl of IEF loading buffer and 1 μl Bromophenol Blue. Each sample was loaded onto an 11 cm IEF strip (Bio-Rad Laboratories), pH 5-8, and overlaid with 1.5-3.0 ml of mineral oil to minimize the sample buffer evaporation. Using the PROTEAN® IEF Cell, an active rehydration was performed at 50V and 20° C. for 12-18 hours.
IEF strips were then transferred to a new tray and focused for 20 min at 250V followed by a linear voltage increase to 8000V over 2.5 hours. A final rapid focusing was performed at 8000V until 20,000 volt-hours were achieved. Running the IEF strip at 500V until the strips were removed finished the isoelectric focusing process.
Isoelectric focused strips were incubated on an orbital shaker for 15 min with equilibration buffer (2.5 ml buffer/strip). The equilibration buffer contained 6M urea, 2% SDS, 0.375M HCl, and 20% glycerol, as well as freshly added DTT to a final concentration of 3O mg/ml. An additional 15 min incubation of the IEF strips in the equilibration buffer was performed as before, except freshly added iodoacetamide (C2H4INO) was added to a final concentration of 40 mg/ml. The IPG strips were then removed from the tray using clean forceps and washed five times in a graduated cylinder containing the Bio Rad Laboratories running buffer 1× Tris-Glycine-SDS.
The washed IEF strips were then laid on the surface of Bio Rad pre-cast CRITERION SDS-gels 8-16%. The IEF strips were fixed in place on the gels by applying a low melting agarose. A second dimensional separation was applied at 200V for about one hour. After running, the gels were carefully removed and placed in a clean tray and washed twice for 20 minutes in 100 ml of pre-staining solution containing 10% methanol and 7% acetic acid.
Staining and Analysis of the 2D Gels
Once the 2D gel patterns of the serum samples were obtained, the gels were visualized with either a fluorescent or colored stain. SyproRuby™ (Bio-Rad Laboratories) was the preferred stain. Once the protein spots had been stained, the gel was scanned and a digital image of the protein expression profile of the sample was obtained.
The digital image of the scanned gel was processed using PDQuest™ (Bio-Rad Laboratories) image analysis software to first locate the selected biomarkers and then to quantitate the protein in each of the selected spots. The scanned image was cropped and filtered to eliminate artifacts using the image editing control. Individual cropped and filtered images were then placed in a matched set for comparison to other images and controls.
This process allowed quantitative and qualitative spot comparisons across gels and the determination of protein biomarker molecular weight and isoelectric point values. Multiple gel images were normalized to allow an accurate and reproducible comparison of spot quantities across two or more gels. The gels were normalized using the "total of all valid spots method" which assumes that few protein spots change between serum samples, and that changes average out across the whole gel. The quantitative amount of the selected biomarkers present in each sample was then exported for further analysis using statistical programs.
Initial Biomarker Selection
The 2D gel patterns of 92 serum samples collected from normal control subjects were compared with each other. The 92 normal samples all gave similar 2D gel protein patterns. The normal protein expression pattern was then compared to the gel patterns obtained in serum samples of 183 patients diagnosed with a neuromuscular disease. The comparison of the protein expression pattern of normals and neuromuscular patients identified at least 34 protein spots seen on 2D gels that differed in protein concentration.
Once the 92 normal serum samples and the 183 neuromuscular disease serum samples had been run on 2D gels and the initial 34 identified protein spots were quantitated in each serum sample, the results were analyzed using statistical programs to determine which biomarkers to include in the assay for ALS in order for the assay to have a sensitivity, specificity, and positive and negative predictive values to be of clinical use to physicians.
Initially, the mean and standard deviations of the biomarkers were used to select the biomarkers and to assess the statistical significance of concentration differences in the biomarkers between the control sera and the neuromuscular disease sera. However because of the number of biomarkers studied, subsequent studies used multi-variant statistical programs to select the biomarkers. A linear discriminate functional analysis was initially employed to determine the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each biomarker and a number of combinations of biomarkers in determining the difference between normal serum and serum taken from patients diagnosed with neuromuscular disease. However, the analysis of the training set and test sets of ALS patient samples have shown the quadratic discriminant analysis of the data sets to be superior to the use of linear discriminant analysis. Therefore, even though a linear discriminant analysis would be included as an embodiment of the present invention, the preferred embodiment of the present invention uses a quadratic discriminant analysis of the data. Both linear and quadratic discriminant analyses are further described below.
Biostatistical Discriminant Function
The quantitative amount of the selected biomarkers present in each sample was then analyzed using a biostatistical discriminant function. The concentrations for the set of selected biomarkers were entered into a biostatistical algorithm and the sample was classified as either ALS or as an ALS-like disorder based on a comparison to a database of values collected from the individuals in the training set from which the discriminant function was derived,
The output of discriminant analysis is a classification table that permits the calculation of clinical sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). These terms are defined herein as follows: (1) the clinical sensitivity measured how often the test yielded positive results in diseased patients, in the case of the present invention, patients with ALS; (2) the clinical specificity measured how often the test gave negative results in non-diseased individuals, in this case patients with ALS-like disorders; (3) the negative predictive value (NPA) measured the probability that the patient would not have the disease and therefore have an ALS-like disorder when values were restricted to all individuals who tested negative; and (4) the positive predictive value (PPV) measured the probability that the patient had the disease (i.e., ALS) when values were restricted to those individuals who tested positive.
A standard discriminant function analysis was performed to determine the subset of biomarkers that would be most useful in differentiating individuals with ALS from those individuals with ALS-like disorders. Discriminant analysis has been well-validated as a multivariate analysis procedure. Discriminant analysis identified sets of linearly independent functions that successfully classify individuals into a well-defined collection of groups. The statistical model used assumed a multivariate normal distribution for the set of biomarkers identified from each disease group.
Where xij represented the p-tuple vector of biomarkers from the ith patient in the jth group, j=1, and {overscore (x)}. . . j represented the p-tuple centroid of the jth group, made up of the mean biomarker values from the jth disease group, then S represented the estimate of the within group variance-covariance matrix. The discriminant function was then that set of linear functions determined by the vector α that maximizes the quantity:
The outcome of the discriminant analysis was a collection of m-1 linear functions of the biomarkers (m) that maximized the ability to separate individuals into disease groups. The vector α is the p-tuple vector which contained the coefficients that, when multiplied by an individual's biomarkers, produced the linear discriminant function, or index that was used to classify that individual. In general, if m biomarkers are used, a maximum of (m-1, g-1) discriminant functions are determined where g represented the number of groups.
Where aj(k) represented the kth p-tuple discriminant function. Then the value of that discriminator for the ith patient is aj(k)′xi. Thus for each patient there are k such values computed, which are used in a classification analysis. The discriminant functions themselves are linearly independent (i.e., for each pair of the m discriminant functions) aj(k) and aj(l), then aj(k)′aj(l)=0. Thus, the m-1 discriminant functions provide incremental and non-redundant discriminant ability.
Identifying the discriminant function involved identifying the coefficients λ from the linear algebraic system of equations |H−λi(H+E)|=0 where H and E were the one way analysis of variance hypotheses and error matrices respectively. It is this computation that was provided by SAS™ statistical software. The SAS software program identified the collection of best discriminators using a forward entry procedure where the p-value to enter and the p-value to stay in the model are each 0.15.
While the discrimination procedure was fairly robust in the presence of mild departures from the normality assumption, it was very sensitive to the assumption of homogeneity of variance. This means that the variance-covariance matrices of the groups between which discrimination was sought must be equal. In this circumstance, these variance-covariance matrices can be pooled. However, in the situation where the variance-covariance matrices are not equal (multivariate heteroscedasticity), this pooling procedure is suboptimal. In this circumstance, the individual variance-covariance matrices have been used.
The use of the two within-group variance-covariance matrices is an important complication in the computation of discriminant functions. When the homoscedasticity assumption is appropriate, the within group variance-covariance matrices can be pooled, producing a linear discriminant function. The use of the within-group variance-covariance matrices produced a quadratic discriminant function (i.e., where the discriminant function is a function of the squares of the proteomic measures).
Classification Analysis
Individuals with either a prior diagnosis of ALS or ALS-like disorders were randomly allocated using a 4:1 ratio into either a training set or a test set. Disease classifications were based on clinical symptoms and family history.
Discriminant analysis was applied to the training set, from which the contribution of each individual biomarker was determined. The SAS™ statistical software program was then used to determine the linear combinations of biomarkers that provided an optimum classification of individuals into disease groups. Alternatively, the programmer can manually select different combinations of biomarkers to be incorporated into a quadratic discriminant function to optimize the classification of individuals into disease groups. Once an individual discriminant model was "trained" by optimizing performance using a representative set of samples (the "training set"), the same set of discriminators were then used to classify an independent set of individuals (the "test set"). Thus, the test set was used to examine the validity of identifying individuals with ALS from those with ALS-like disorders using the selected biomarkers. The test set consisted of ALS patient samples only, as there were an insufficient number of ALS-like patient samples to adequately construct independent training and test sets of the ALS-like disorders.
Thirty-four protein biomarkers were identified in the training set that both individually and/or jointly discriminated ALS patient samples from samples taken from patients with ALS-like disorders. Various sets of biomarkers (representing one, multiple or all thirty-four biomarkers) were then used to analyze the training set and then the test data set, using the same discriminant functions built against the training data set to determine the ability of each set of biomarkers to predict ALS. Individuals were classified as ALS or having ALS-like disorders based on clinical symptoms and family history. Each of the 34 protein biomarkers were assessed individually through discriminant analysis to determine its ability to predict ALS.
2D Gel Electrophoretic Controls
Representative samples from individuals with known cases of ALS and ALS-like disorders were run as positive and negative reference controls. Serum containing all of the selected biomarkers was also provided as a reference standard. A reference control was periodically run as an external standard and for tracking overall performance and reproducibility. In addition, 2D gel images from samples classified as ALS and ALS-like disorders were used for reference. The spot locations were noted for the selected biomarkers, as well as for landmark proteins commonly found in human serum (see FIG. 1).
The Reproducibility of Biomarker Identification and Quantification
The consistency and reproducibility of quantifying biomarkers using 2D-gel electrophoresis was characterized. To optimize reproducibility, each sample was preferably run in triplicate and each set of replicate samples was analyzed as a group. This maximized the overall accuracy of spot identification and biomarker quantification. The average percent Co-efficient of Variation (% CV) is 11±7% for 10 biomarkers quantified from a single image scanned 10 times. The average % CV is 23% for a set of 25 biomarkers quantified from 12 separate processed aliquots of the same sample. The range in biomarker concentrations for this group of biomarkers ranged from a low of 248 ppm to a high of 15,548 ppm normalized concentration of spot per total detected spots in the 2D gel.
The protein concentrations employed in the discriminant function were relative values obtained by normalizing the intensity of each spot to all detected spots in the image. The linear range in protein concentrations was 0.5 to 1,000 ng per spot. The concentration of any given spot was the absolute amount of protein in that spot divided by the total protein loaded onto the gel. The total amount of protein loaded onto a gel was typically about 100 μg.
Serum is primarily comprised of a highly conserved distribution of the most abundant proteins, such as albumin and immunoglobulin, which enhance efforts to ensure the reproducibility and consistency of biomarker detection and quantitation. The selected biomarkers represented a minor fraction of the total serum protein. Therefore the concentration of the selected biomarkers varied significantly as a function of disease state without significantly shifting the overall distribution and concentration of the major serum proteins. Discriminant biostatistics were employed to establish the dynamic concentration range of the selected biomarkers useful in differentiating ALS patients.
Biomarker Stability
The effect of multiple freeze/thaw cycles on protein stability and sample integrity was investigated. A serum sample was collected and aliquoted. One aliquot was processed without freezing, while other aliquots were frozen at −80° C. and thawed repetitively. A second set of serum samples was diluted into loading buffer and aliquoted. The second set of samples, similar to the first set, had one aliquot processed without freezing and other aliquots frozen at −80° C. and thawed repetitively.
Triplicate samples were processed as described. The scanned images of the 2D gels were analyzed, and the quantities of each of the 34 neurodegenerative biomarkers of interest were determined. The results illustrated that freezing and thawing either undiluted or diluted serum samples up to 10 times had no significant effect on the serum protein profile or on the abundance of the selected biomarkers.
In addition, sample deterioration was investigated over a one-year period. Twenty-one selected biomarkers were quantitated in control samples stored at −80° C. An aliquot of each control sample was processed several times each quarter, or each 3 month time period. The results demonstrated that there was no significant increase or decrease in the quantity of biomarker detected over a one-year time frame for samples stored at −80° C., beyond that which is typically observed for processing replicate samples.
Samples Analyzed
Serum samples were obtained from 136 ALS patients and 31 patients having ALS-like disorders. All individuals with symptoms of a neuromuscular disorder were evaluated and diagnosed by a neurologist. All individuals diagnosed with ALS were classified as Probable or Definite ALS by the revised El Escorial criteria (Brooks, B. R., et al. 2000. Amyotroph. Lateral Scier. Other Motor Neuron Disord.1(5):293-9).
The ALS-like disorder controls included individuals with the following conditions: Benign Fasciculations, Brachial Amyotrophic Diplegia, Brachial Plexopathy, Cervical Myelopathy, Lumbosacral Radiculopathy, Cervical Radiculopathy, Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP), Corticalbasal Ganglionic Degeneration (CBGD), Diabetic Neuropathy, Cervical and Lumbar Stenosis, Guillain Bane Syndrome—Axonal type, Inclusion Body Myositis (IBM), Idiopathic Sensory Ataxia, Inflammatory Peripheral Neuropathy, Lewy Body Dementia, Inflammatory Myelopathy with Polyneuropathy, Monomelic Amyotrophy, Multiple Sclerosis, Muscle Spasms, Muscular Dystrophy, Myasthenia Gravis, Myotonic Dystrophy, Progressive Bulbar Palsy, Multiple System Atrophy, Multiple System Atrophy with Subdural Hematoma, Progressive Muscular Atrophy, Spinal Bulbar Muscular Atrophy (Keimedy's disease), Spinal Muscular Atrophy (SMA), Spinal Cord Syrinx with history of Spinal Meningitis, and Vascular Parkinsonism.
Ninety of the 136 ALS samples were randomly selected for use in the training set for constructing the discriminant function. All 31 of the ALS-like disorder samples were used in the training set due to an insufficient number of patients in this group. Thus, the training set contained 90 ALS patient samples and 31 samples from patients having ALS-like disorders. Once the discriminant function was developed, the remaining ALS samples were used in a validation set.
Differentiating ALS and ALS-like Disorders
The preferred embodiment used all 34 biomarkers of interest. To assay patient samples based on all 34 biomarkers the training set used in training the discriminant function included all 34 biomarkers. Although a variety of different combinations of biomarkers were also tested that gave comparable statistical performance, they are not specifically described herein but would be performed in a similar fashion.
As shown in FIG. 1, the 34 biomarkers were resolved by 2D gel electrophoresis of human serum proteins. The proteins were visualized by the sensitive (≦1 ng protein/spot out of 100 μg serum proteins per gel) and linearly staining (linearity and dynamic range of from ≦1 ng to ≧1000 ng) SyproRuby™ fluorescent stain. The stained gels were scanned and the digital image of the 2D gel was analyzed using PDQuest™ quantitative digital image analysis software.
The quantitative results were then subjected to linear and quadratic discriminant analysis using the SAS™ statistical software. The results, shown in FIGS. 2 and 3, indicated that the quadratic discriminant analysis was superior to the linear discriminant analysis. The linear discriminant analysis only correctly classified 23 of the 31 patient samples from ALS-like disorders (74% specificity) and only 102 of the 114 ALS patient samples (89% sensitivity). Although these results could be clinically useful, use of the quadratic discriminant analysis properly classified all 31 of the patient samples from ALS-like disorders (100% specificity) and all 114 ALS patient samples correctly (100% sensitivity). Thus, the quadratic discriminant analysis was selected as the preferred embodiment.
When a randomized 80% training set (ALS+ALS like) and a test set (independent samples of ALS+duplicate and replicate ALS sample data, not included in the training set) validation was performed using quadratic discriminant analysis (see FIG. 3), the performance was perfect for these samples (i.e., 100% specificity and 100% sensitivity).
When samples from ALS-like disorders that were not included in the training set were used in a test set, a few of those samples were misidentified as ALS. Inasmuch as the ALS-like patients represent a large group of diseases with similar symptoms but somewhat different anatomical and biological features, it was postulated that the 31 samples of ALS-like serum did not provide a sufficiently representative and robust model for the ALS-like classification. Thus, a larger number of ALS-like disorder samples in both the training and validation sets will be acquired, analyzed and added to the training and test datasets.
Assay for ALS vs. ALS-Like Disorders
Definitive diagnostic tests to confirm the diagnosis of Lou Gehrig's disease (ALS) and distinguish it from the ALS-like disorders that display similar symptoms but have different treatment options and prognosis have long been sought by clinicians in hopes of providing earlier treatment decisions and improved patient outcomes. ALS is a devastating, fatal neurodegenerative disease that causes the progressive loss of the cells in the brain, spinal cord, and motor nerves that control muscle function. It is the third most common neurodegenerative disease in adults, after Alzheimer's disease and Parkinson's disease. Early symptoms of ALS may include arm and leg weakness, stiffness, and slurred speech. The majority of patients die within 3-5 years from first symptom, usually from respiratory muscle failure.
Presently, the diagnosis of ALS is a clinical one. There is no test that provides diagnostic certainty. The usual diagnostic process consists of a full medical history, and comprehensive physical and neurological examinations. The revised El Escorial Criteria, developed at a Consensus Conference in Spain in 1990, is widely accepted for the diagnosis of ALS (Chaudhuri, K. R., et al. 1995. J. Neurol. Sci. 129 Suppl.: 11-12). This set of criteria combines clinical features and laboratory test findings to classify the level of diagnostic certainty into Definite, Probable, Possible, and Suspected.
As mentioned previously, no definitive diagnostic test for ALS is currently available. Numerous studies are generally performed to rule out other medical conditions that can mimic the appearance of ALS. This is important because many of the ALS-like conditions have a much more favorable prognosis. A complete evaluation may include an electromyogram (EMG) with nerve conduction studies (NCV), magnetic resonance imaging (MRI) of the brain and spinal cord, lumbar puncture (LP) with analysis of cerebrospinal fluid (CSF), a panel of blood tests, and muscle biopsy. Because the El Escorial criteria set was originally designed for research purposes, some clinicians find them to be somewhat cumbersome (Brooks, B. R. 2000. Amyotroph. Lateral. Scler. Other Motor Neuron Disord. Suppl 1:S79-S81).
The etiology of ALS is undefined and it is unclear what places a person at-risk of getting ALS. A combination of genetic susceptibility factors and environmental factors is thought to be involved. Researchers have searched for genetic susceptibility factors that affect cellular processes that influence the survival of motor neurons; however, to date, no susceptibility factor has emerged to account for the majority of ALS cases.
In summary, the diagnosis of ALS is currently based on clinical criteria and the results of electrodiagnostic studies. Numerous neuroimaging and blood studies are generally performed, mostly to rule out the presence of other medical conditions that may mimic the clinical appearance of ALS. To date no definite biochemical or genetic test is available to definitively diagnose ALS, or to differentiate ALS from ALS-like conditions.
The present invention provides an assay for differentiating ALS from ALS-like disorders. The assay is comprised of the following steps: (1) collecting a serum sample from a patient; (2) running triplicate 2D gel electrophoreses of the patient sample; (3) staining the 2D gel; (4) creating a digital image of the 2D gel; (5) quantifying the protein concentration in selected protein spots on the 2D gel; and (6) performing a statistical analysis on the quantity of the selected proteins to determine the likelihood of the patient having ALS or an ALS-like disorder.
While the methods have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods including the sequence of steps in the methods. Certain agents may be substituted by one of skill and similar results may be achieved, as will be appreciated by one of skill in the art. Such modifications or substitutions to the methods of the present invention are deemed to be within the spirit, scope and concept of the invention as defined by the disclosure and its claims.


text:
Ronald Epstein
Senior HTF Member
- Joined
- Jul 3, 1997
- Messages
- 56,806
- Real Name
- Ronald Epstein
NEW 2020 1080p master from 4K scan of best surviving nitrate elements!
Run Time 134:00
Subtitles English SDH
Audio Specs DTS HD-Master Audio 2.0 - English, MONO - English
Aspect Ratio 1.37:1, 4 X 3 FULL FRAME
Product Color BLACK & WHITE
Disc Configuration BD 50
Special Features: Commentary by Film Historian Jeanine Basinger; Making of Featurette "Sergeant York: Of God and Country"; Classic Cartoon "Porky's Preview"; Vintage Short "Lions for Sale"; Theatrical re-issue trailer (HD).
Thank you for supporting HTF when you preorder using the link below. If you are using an adblocker you will not see link. As an Amazon Associate HTF earns from qualifying purchases

subset: commoncrawl


text:
Iceland Opens Europe's Biggest NP
Iceland may not have many trees, but it makes up for it by overwhelming visitors with a vast network of volcanoes, glaciers, canyons, calderas, rivers, lakes, and the most powerful waterfall in Europe. Starting June 7, you can visit all these features in one place: Vatnajokull National Park. Covering 13,000 square kilometers and 13 percent of Iceland, it's Europe's biggest national park.
Right now, the park is mostly pure backcountry, but extensive plans have been put in motion to open several ranger stations, visitor centers, and other services by 2012. From the spectacularly-named Olof Yrr Atladottir, director general of the Icelandic Tourist Board:
"Visitors are a vital part of Iceland's rural economy and contribute positively to the protection and sustainability of our magnificent landscape."
"Personally, I see a myriad of possibilities for all sorts of activities opening up such as action tours, eco-recreation, study visits and learning experiences as well as the chance to experience the nurturing silence of Iceland's highlands."
Travel officials expect the national park to boost visitation to Iceland from 30,000 to 42,000 per year — that's still far less than most major U.S. parks get in a year, and we're talking about the entire country. You have to figure half of those visitors just want to get loaded in Reykjavik and visit Bjork's birthplace.
— Ted Alvarez
Iceland to open Europe's largest national park (Opodo Travel News)


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It's 11:20 PM and I still haven't managed to get a post up today. As a result I'm turning to the lamest of devices. Even lamer than last night's haiku. A Facebook meme. Forgive me David Foster Wallace for I have sinned.
So my understanding is that you're supposed to pick 12 music albums that are essential to you, however you want to define that. You're not supposed to think about it too much, which won't be a problem for me because the clock is ticking. I can say that these 12 albums are ones I'd hate to be without. If they were the only 12 I had I could probably get by pretty well.
With no further ado and in no particular order:
- David Byrne Uh Oh
- Lyle Lovett The Road to Ensanada
- Nanci Griffith Other Voices, Other Rooms
- Supertramp Breakfast in America
- Dire Straits Brothers in Arms
- OK Go Of the Blue Colour of the Sky
- Orchéstre Baka de Gbiné Gati Bongo
- Jonathan Coulton A Thing a Week Three
- "Weird Al" Yankovic Straight out of Lynwood
- Various Artists Pearls in the Snow: The Songs of Kinky Friedman
- Willie Nelson Greatest Hits (and Some That Will Be)
- The Judys Washarama
I am really going to be glad when this week is over. Tomorrow's post might be even more lame.


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28 of the 35 animals that we received from Florida last Friday went up for adoption on September 13 at our Sharonville facility. A lottery drawing was held for animals with more than one interested party.
The other 7 animals are currently undergoing further medical treatment and will become available for adoption in the near future.
Please note that these are not displaced animals with owners. These animals were being housed in shelters in Ft. Lauderdale, Florida for adoption. The animals were then evacuated to make room for the displaced animals of Hurricane Irma.


text:
Why can't I see digital images of all portraits?
The Gallery is actively engaged in digitising its collection, and brings approaching 10,000 new images on to the website each year.
We cannot guarantee a date when any unillustrated portrait will be digitised, but we aim to get recent acquisitions illustrated on the website within months of their acquisition.
If you wish to purchase a print of any unillustrated portrait, please contact Rights and Images.
- Rights and Images on +44 (0)20 7312 2473/4/5
- [email protected]


text: Ryan Mauskopf is an illustrator, graphic designer, and musician living and working in New York City. His musical pseudonym Professor Soap has a new animated interstellar music video to accompany his latest release 'Spacetime Fabric Softener'. All of Professor Soap's music can be downloaded for free via his BandCamp page or head over to RyanMauskopf.com to download 2 bonus tracks, a HD version of the music video and storyboard artwork! You can aslo head over to Ryan's website to see his other animated projects.


text:
OEDA would not exist with out the help of dedicated volunteers! Our volunteers are community members with a variety of backgrounds who give their time and energy to support our vision of promoting healthy body images and positive relationships with food.
We need volunteers to help in all aspects of our organization including administration, development, programming, event planning, and education.


text:
"Truck Parking Space"
Truck Parking Space is a cool truck game, it's almost as if you were a driver in the trucking industry. Your parking spot has been outlined for you in each stage and all you have to do is use your ARROW KEYS to drive your large truck around each level and park it in the designated spot! Do your best to be fast and accurate as touching walls will destroy your truck and taking too long will end your turn!
ARROW KEYS LEFT and RIGHT to steer. UP and DOWN to accelerate and brake.
More Bus, Caravan, Tank, Plane and Truck Parking Games:
Other Parking Games:
More to Play:
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text:
Storm2's unique piezo-actuated touchscreen was acting up in any statistically significant way, but apparently, there'd been enough with dodgy lower left corners to prompt RIM to do something about it. BerryScoop and BlackBerry Leaks are reporting that Verizon stores have been ordered to return their existing stock in favor of new Storm2s with "silicone actuators" added to "to improve touchpad performance and tactile response," and don't worry, current customers -- it seems you're more than welcome to exchange your old unit for an upgraded one provided you're "experiencing touchpad performance issues" (wink, wink). Unfixed handsets need to be out of stores by March 11, so it looks like you can definitely find the new ones hanging around this week -- let us know if you can tell the difference.
RIM tweaking Storm2's SurePress underpinnings, Verizon units getting swapped out
Sponsored Links
Verizon owns Engadget's parent company, Verizon Media. Rest assured, Verizon has no control over our coverage. Engadget remains editorially independent.


text:
Read the full article
This article was originally published in issue #15
To read the article in its entirety, view the digital magazine
When it comes to the big brand names in bass there aren't many better known than this one. You've seen Ampeg many times in the hands of the very best in bass guitarists - usually plugged into the company's massive 8x10 sealed 'fridge' cabinets with SVT valve amplifiers atop. They're the dream of many a budding bass player - often regarded as the ultimate Rock rigs. I myself owned an SVT 2 Pro for quite some time - a wonderful sounding amp with lots of volume available. It was a total beast in terms of tone but... sadly, the weight! These things are built for the road and come with near bullet-proof construction and of course a price point that is out of reach of many a weekend warrior and semi-professional alike.
But Ampeg has a wide range of products to suit every pocket, back and volume requirement, all taking a slice of that famous 'Ampeg tone'. So if small and quirky is your bag, then this is going to be a favourite! After the success of Ampeg's Micro VR stack range, based on the original VR heads and cabinets, comes this diminutive rig that takes its tone and stylings from the classic CL range.
Don't get me wrong, this isn't just a toy, the Micro CL 'mini fridge' is very much a working man's rig despite looking like SVT CL's 'Mini-Me' clone, which means dreams of owning a CL stack can be realised even if your living space is a bit of a tight squeeze!
The Micro CL really is cool and includes all the familiar Ampeg looks and front panel lay out. The amplifier head has a solid-state derived 100W output into the 8 Ohm cabinet and features a truly 'plug and play' layout. Simply plug in to one of the inputs; you can select the padded input if your bass (like mine) has a very high output and you need to stop the input from distorting.
Next to the master volume (there is no gain control) there is a simple and musical three band EQ. By 'musical' I mean that the wide frequency range that each control covers means that there are no harsh tone peaks when boosted. Not that much boosting is needed. Even at 12 0'clock on each setting, it sounds full and fat. In the video, for my particularly bright sounding bass (in comparison to that of a passive P for example) I set up a little boost on the treble and a little boost on the bass control. I found that as the cabinet had no tweeter, the top end was naturally softened a little, but the resulting tone was nicely rounded. Once you have your tone dialled in, then it's over to the connections on the right hand side. Usefully, you can use the head on its own without the cabinet connected, as a private practice amplifier. You can plug an MP3 player in to the AUX socket that features its own level control, and monitor both your bass sound and your favourite tracks through headphones.
If you do want to use the ban, however, you'll find it's a vertically aligned (which will please the geeky types) unit having two 10 inch Ampeg speakers. The cabinet is also rated at 100W and has a load of 8 Ohms. The stack includes a 1/4" jack speaker lead - there are no Speakons on this rig. A power lead is also provided in the box to get you up and running quickly. Oh, and there's an effects loop for when you're feeling adventurous.
In use, the Ampeg is a little peach, but don't expect it to contend with its bigger brothers. Yes it will keep up with a conservative drummer and happily do acoustic type gigs with moderate volumes, but if you have a guitarist who cranks even a 50W valve combo amplifier, or a drummer who likes to leather the kit a bit, then this stack will leave you wanting for more booty. That said, the tone is just brilliant! Whilst we were in the studio we plugged a bass distortion pedal in to the Micro CL to see if we could get that famous Ampeg grind - and boy did we! This leads me to believe that you could use this set up to great affect recording a filthy distorted bass track, without the need for a big bass stack. Mix in a little clean DI to the master mix too and I think it'd sound sweet! In the studio we joked that if this head was coupled to a 500W power stage and uprated cones in the cabinet, it'd be a wolf in lambs clothing!
We all liked this rig. I think it's a real eye catcher, not because of flashing lights and crazy design, but just because we're so used to seeing this proportioned rig some three times larger. It makes me smile as does its ease of use. It scores well for being quirky and cute and having great tone but won't win the dB war.
This article was originally published in issue #15
To read the article in its entirety, view the digital magazine


text:
BIO 12N: Sensory Ecology of Marine Animals
Animals living in the oceans experience a highly varied range of environmental stimuli. An aquatic lifestyle requires an equally rich range of sensory adaptations, including some that are totally foreign to us. In this course we will examine sensory system in marine animals from both an environmental and behavioral perspective and from the point of view of neuroscience and information systems engineering.
Terms: Aut | Units: 3 | UG Reqs: WAY-SMA
Instructors: Thompson, S. (PI)
2020-2021 Autumn
3 units |
UG Reqs: WAY-SMA |
Class #
Section 01 |
Grading: Letter or Credit/No Credit Exception |
| Remote: Synchronous
| Students enrolled: 14 / 16
09/14/2020 - 11/20/2020 Mon, Wed 11:30 AM - 12:50 PM at Remote with Thompson, S. (PI)
Instructors: Thompson, S. (PI)
Notes: Introductory Seminar. Preference to first-year students; sophomores admitted if space available. Advance sign-up process and deadlines at http://introsems.stanford.edu.
Additional Resources: (Login to view additional resources)
Filter Results:


text: Would you like to plan a custom trip? Please fill out the form below and we will contact you soon. Name* First Last Email* Phone*Mountain Bike or Moto Enduro?Mountain BikeMoto EnduroWhat level of difficulty am I comfortable with?Entry LevelIntermediateAdvancedHighly AdvancedWhat time of year do I want to take my trip?SpringSummerFallWinterWhere do I want to go?ArizonaCaliforniaUtahNevadaUT/NVUT/AZWhere do I want to go?IdahoCaliforniaUtahWyomingUT/IDID/WYWhere do I want to go?IdahoCaliforniaUtahArizonaNevadaUT/IDUT/AZUT/NVWhere do I want to go?CaliforniaUtahArizonaNevadaUT/AZUT/NVHow many days do I want my trip to be?1234567How many total people will be riding on this trip?12345678910Am I bringing my family?*YesNoMy family consists of:*My significant otherMy significant other & 1 dependantMy significant other and childrenWIll my family members be riding on this trip?*Yes my significant other will be riding on the tripNo my family is pretty self sufficientWIll my family members be riding on this trip?*Yes all of themYes my children who are over 13 years of ageNo my family wants to find other activities in the areaDoes my family want help from Advenduro Tours finding activities in the area?*Yes my family would like help from Advenduro Tours finding activities in the areaNo my family is pretty self sufficientAm I bringing, shipping, or renting a bike?*I need to rent at least one bikeYes I need a shipping addressNo I will bring the bikes I needPlease ship your bike to: Advenduro Tours 3388 Paiute Rd St. George, UT 84790 so that it arrives at least one week prior to your tripHow many bikes do I need to rent?*12345678910What is my preferred Rental Package?*Green PackageBlue PackageBlack Diamond PackageAm I renting any gear?*Yes I need to rent gearNo I have what I needWhat gear do I need to rent?*BootsChest ProtectionGlovesHelmet & Eye ProtectionKnee & Elbow ProtectionPersonal Hydration BackpackIf gear is not listed below please add your request to the additional details below.Do I want Avenduro to handle my hotel and dinners?*Yes - please handle everythingNo - I will take care of my hotel and dinner on my ownAdditional Details


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Disney's Frozen character costumes are going to be one of the hottest items for Halloween this year. The most popular character costumes are no doubt Princess Elsa and Princess Anna's costumes. The more common Princess Anna costume is her blue/magenta one but she also wears a very nice black/olive gown in the movie.
If you are enamored by Princess Anna's gown that she wore for her sister, Princess Elsa's coronation, you can get your very own copy of that gown for Halloween or for cosplay. This is a high quality adult version of the costume and it comes in various sizes from XXS all the way to XXXL. If you want a better fit, you can provide your body measurements to the manufacturer and they will custom tailor the dress for you at no extra charge.
The Frozen Princess Anna Coronation Gown Cosplay Costume is made from satin and it even comes with the black satin-laced necklace with the bronze symbol of Arendelle as shown in the picture.Buy Now


text:
This software installation guide is designed to help users successfully install the BlueStorm/LP serial adapter under the QNX 4.2x platform.
Before beginning the software installation, please be sure to read the product manual for hardware installation/configuration instructions.
To make the instructions easy to follow, all commands that have to be issued on the command line are written in bold text.
NOTE: You must be logged in as root to perform this installation
Preamble: Getting Your System Ready
Before you begin the installation process, you will need to have two applications running so you will be able to read from the DOS diskette. To run these applications, enter the following two commands from the command line:
# Fsys.floppy &
# Dosfsys &
These particular applications may be running by default (these are QNX applications), and therefore you may not have to issue the previous commands. Please see the QNX documentation for more details about Dosfsys and Fsys.floppy
Installing the Driver
- Go to the root directory by issuing the command:
# cd /
- If you downloaded the *.tar file from the website, you will need to untar it first.
The *.tar file contains qnx4.F and readme.txt files.
- tar xvf BL-QNX4_XXXX.tar
Where XXXX is the version number
- tar xvf BL-QNX4_XXXX.tar
- To install the driver:
# install -u /..PATH../qnx4
Where PATH is the location of the qnx4.F file
- If you are using the driver diskette, issue the following command:
# install -u /dos/a/qnx4/qnx4
- Once the installation script starts, follow instructions to finish the installation. You can also see where the files are being copied to
Command Line Arguments
The BlueStorm/LP driver, Dev.ctiser, can be controlled with the following command line arguments:
# Dev.bluestorm &
The command above will start Dev.ctiser, the default BlueStorm/LP serial driver (32-bit version).
# Dev.bluestorm -zd Dev16.ctiser &
This command starts Dev16.ctiser instead of Dev.ctiser by using the -zd option.
# Dev.bluestorm -N bl &
This command gives the BlueStorm/LP serial ports unique names (bl) instead of the default "ser".
If you look in the /dev directory, you would see 8 bl entries (bl1 – bl8), representing your Titan serial ports.
For more command line options, issue the following commands:
# use Dev.bluestorm
# use Dev.ctiser
Dev.bluestorm operates with the following command line parameters:
- ID=addr,write,read Check card for ID
- -b number Define initial baud rate (default 9600)
- -C number Size of canonical input buffer (default 256)
- -d Set DSR on initially (default)
- -D Set DSR off initially
- -e Set options to "edit" mode
- -E Set options to "raw" mode (default)
- -f Enable and lock hardware flow control (default)
- -F Disable hardware flow control
- -h number Define high-water mark for input flow control
- (default 3/4 point of input buffer)
- -I number Size of raw input buffer (default 2048)
- -l number Define low-water mark for input flow control
- (default 1/4 point of input buffer)
- -m Disable modem carrier processing (HUP)
- -M Enable modem processing (HUP) (default)
- -N name Prefix to register (default "ser")
- -O number Size of output buffer (default 2048)
- -p Enable packet mode. (Modem status change causes "packet" event).
- -P Disable packet mode (default).
- -r Set RTS on initially (default)
- -R Set RTS off initially
- -s Enable ohflow split (CTS only required) (default)
- -S Disable ohflow split (CTS+DSR required)
- -t number UART FIFO threshold (1, 4, 8 or 14). 0 disables. (default -t4 or 16)
- -V Enable verbose mode, great for debugging
- -w Enable hardware shutdown on SIGPWR.
- -W Disable hardware shutdown on SIGPWR. (default)
- -x Enable and lock software flow control
- -X Disable software flow control (default)
- Dev.bluestorm &
- starts driver for BlueStorm/LP, device names will start at /dev/ser1.
- Dev.bluestorm -N bl &
- starts the driver naming the devices /dev/bl1, /dev/bl2 etc
- NOTE: Be sure to include the space after the -N argument.
RS485 Mode Support
The BlueStorm/LP includes support for Full Duplex, Half Duplex and Multi-Drop Slave modes. Connect Tech provides the "ctty" utility to enable and disable these modes.
The settings that are enabled/disabled by the ctty utility are volatile, meaning that if the system needs to be rebooted, the settings will be lost. Because of this, it is recommended that you place the ctty command in the sysinit startup file. This way if you do need to reboot your computer, your settings will be re-enabled at boot time. If you choose to do this, you will also have to start the driver in the sysinit file as well.
NOTE: the ctty command won't work unless the driver is running.
The default mode is Full Duplex, so you will only need to invoke the ctty command if you need to run in Half Duplex or Multi-Drop Slave modes.
Here are some command line examples. We assume here that the driver is already running:
# ctty +rts < /dev/ser3
This will enable Multi-Drop Slave mode on port ser3 *Note* Jumpers on Bluestorm must also be properly set.
# ctty +rts < /dev/ser3
This will enable Half Duplex mode on port ser3 *Note* Jumpers on Bluestorm must also be properly set.
# ctty -rts < /dev/ser3
This will enable Full Duplex mode on port ser3 *Note* Jumpers on Bluestorm must also be properly set.
NOTE: When running in Half-Duplex or Multi-Drop Slave mode, the driver must be started with Hardware Flow Control OFF.
# Dev.bluestorm -F &
Hardware Flow Control is set to ON by default, so this argument is necessary.
Editing the sysinit File
Before you begin editing the sysinit file, you will have to determine exactly which sysinit file to edit. To do so, you will have to find the current node number of your system. This can be easily done issuing the following command:
# sin info
This will give you the current node number of your system, as well as other information. Once you know the node number of your system, you can determine which sysinit file you need to edit. The node number will actually be the extension of the sysinit file to be edited.
For example, if you found that the current node number of your system is 6, you will have to edit the file sysinit.6. All sysinit files will be located in the /etc/config directory. If you are going to edit the sysinit file to load the driver at boot time, make sure that you add this entry at the end of the sysinit file. It is also recommended that you add a sleep command of 1 as well.
For example, to start the default BlueStorm/LP driver, add these lines to the sysinit file:
Dev.bluestorm &
Sleep 1
You can also add the ctty commands in the sysinit file, but make sure to place them after the line where you start the serial driver
Testing the Newly Installed Ports
You can perform a simple loopback test with a terminal program (i.e. qtalk) and a loopback connector to ensure that the newly installed ports are functioning.
Connect the loopback connector to the port you want to test and run qtalk. When you type a character, you should see it on the screen, which means that the port is working. If you remove the loopback connector, the characters that you type should not appear on the screen.
Assuming that you want to test ser1 with qtalk, run:
qtalk -m /dev/ser1


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Here's an email that I sent to Mark yesterday when he asked me about joining this blog.
This is an execellent start on a very important and very difficult issue facing Torah Judaism today. First of all, I think that many Rishonim beginning with the Sefer Hachinuch and such Gdolei Acharonim such the Chafetz Chaim state that it is assur to remind a BT of his prior background and behavior. That being a given, I think that there are many BTs today who are integrated into the frum community on all of these issues. Many, if not all of my contemporaries in NCSY in the 1960s and 1970s are very much a part of the frum community and are rabbinic and lay leaders. The OU Board of Directors is loaded with many BTs. Their POV is accepted on a wide variety of issues.
In one of his Teshuvah Drashos, RYBS pointed out the difference between Teshuvah mAhavah and Teshuvah MYirah as being the difference between integrating one's past into one's new life or totally eradicating the past. I think that many BTs walk a fine line on this very issue.
Parenting, Relationships, Plateauing, Education and Shidduchim are issues that affect all Jews. Integration and handling the relationship with family relatives who are "not yet frum" are issues which IMO are very much related to BTs. I think that Nefesh and specifically R D A Twerski or D B Holzer could be very helpful on these issues,
I think that the above issues are present. I can't say whether they are magnified in importance as one moves across the hashkafic spectrum, but they do exist everywhere in different degrees. I once heard a joke that in Lakewood a BT is defined as someone learnig there whose parents went to RIETS, YU and Stern!
I do think that the focus has to be on what unifies all sectors of Klal Yisrael and IMO the unifying factors are Torah, Avodah and Gmilus Chasadim. All of the various hashkafic approaches ranging from the Rambam's rationalism to Chasidus, Mussar, TIDE and TuM are both wonderful in the abstract but exceedingly hard to implement . Each of these approaches has experienced intellectual "hardening of the arteries" . I have found that for myslef that all of these approaches have a lot to offer, and that aspects of all enhance my Avodas HaShem. OTOH, when the hashkfa threatens to supplant halachic norms., I can jettison those aspects which don't enhance my Avodas haShem.
IMO, too many BTs are "sold" a package that Torah and learning Torah is the "answer" to one's problems and life , as opposed to being presented with the idea that Torah is an "approach" to life that gives you a set of tools in every way to deal with the world, as opposed to a definitive answer. While halacha by its definition involves a psak, hashkafa is a much more flexible affair. IOW, as many Rishonim and Acharonim state, Daas Torah has nothing to do with Hashkafa. That's why we see such varied schools of Parshanut such as Ramban, Ibn Ezra, etc. "Answers" strike me as overly authoritative when in fact every hashkafa has its pluses and minuses. Based upon my reading of some BTs who posted on the Slifkin affair, this is an issue that is definitely present.
I also think that way too many BTs think that they can "walk the walk, talk the talk and look the part" without acquirring true textual literacy in Chumash, Tanach, Shas and Poskim.IMO, that may be one of the reasons why ArtScroll is so successful, but it also has led to too many Jews who are dependent on a crutch named ArtScroll for the rest of their lives.
These are a few of the issues that I think that I would feel comfortable blogging about.


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Sept 1st $25 FREE Credit
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text: Steel Notes Magazine October 2016 The physical CD will be available at our shows and our website plus we will be releasing them separately as singles on CDBaby, Amazon, iTunes, GooglePlay, and most popular digital music platforms. We are very excited!!! MBW: So you have a website ... How about sharing it with the whirled ?? BC: Thank you for the shameless plug!!! www.theblackcatsnyc.com MBW: Is there anything else as far as contact info you'd like to add ??? BC: Our e-mail address is firstname.lastname@example.com. We can be also found at SoundCloud, Bandsintown, FaceBook, Instagram, Twitter, Google+, ReverbNation, YouTube, Tumbler, & Pintrest, all links are on our website. MBW: Being one of the hottest bands in New York City how would you describe it??? Andrew: You're too kind Marlowe, there are a ton of great bands and talented musicians playing in NYC so that means a lot. We just play what comes natural to us and it seems to work. Don: the Black Cats NYC offers a modern spin on the Rock and Roll that we all fell in love with in a new package that is a raw, raunchy and very fun... and you can dance to it! MBW: Do you have any fun stories to tell us about doing your shows ??? ... any memorable moments ... favorite venues etc. ??? BC: February 14, 2015 we played a Valentine's Show at the Delancey. It was the first time either of us had met or played with "the Living Legend" Alan Merrill. That was a huge moment for us! We have played with a lot of great bands, important bands, in our past but Alan is the real deal and we are happy to say a great pal! As for venues they are all great! MBW: 134 Steel Notes Magazine www.steelnotesmagazine.com


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Two years ago Google began encrypting organic Search terms for anyone who was logged in to Google, preventing webmasters understanding what terms drove traffic to their site from those Visitors. At the time, Google said that this would apply to only a single digit percentage of Visitors; however it is clear that the actual figure has slowly climbed and can be 50% or more. All users will now be switched to secure search; as reported by Search Engine Watch (http://ow.ly/pbF5x) who were told by Google:
"We added SSL encryption for our signed-in search users in 2011, as well as searches from the Chrome omnibox earlier this year. We are now working to bring this extra protection to more users who are not signed in".
All the other Search Engines will still provide Search Terms and Google's Webmaster Tools will let you see the terms for Paid Search. However, that still leaves webmasters with the problem of understanding what terms are working from an SEO point of view and, whilst there are techniques available to get more information if you have a Webmaster Tools account, these involve some effort to set up and are approximate at best. N.B. The alleged reason for this is privacy, although I can see no logic for organic searching needing to be private, but not paid searching! The industry view is that it is a ploy by Google to encourage site owners to use paid search – what do you think?


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What a great company I have found for my bulk text messaging needs. In this Tatango review I am gong to emphasis that now only am I fan of their unparalleled expertise and service, but many, many industry folks agree too. When I went looking for a text message marketing company, I asked around and did some research, what I found was that Tatango is one of the leaders in the field.
If your searching for an SMS text marketing company in Canada that can deliver results and deliver your bulk text message campaigns to the Canadian mobile marketplace, Tatango should be on the top of your list.
Tatango – Humble Beginnings
When a young Derek Johnson was attending school at the University of Houston, he had a very good entrepreneur spirit and in 2007 he started what would soon become Tatango. Derek saw a chance for businesses to start mobile marketing through cell phones and has created a leading sms marketing platform used by thousands of businesses.
He saw that everybody was texting and wondered if there was an easy way to reach those customers with out having to text individual numbers. So, he set to solve the problem in the current market place of supplying an easy to use bulk SMS text messaging system that would revolutionize that way the business would be accomplished for the foreseeable future and beyond.
Tatango in the Press
As I stated before I was not the only one impressed with how easy to use the Tatango SMS text platform was to get professional looking and accepted mass text messages out to my customers, but there are many industry insiders that agree with me as well. There are several text message marketing reviews that call Tatango the Best!
The Seattle Times
"Johnson believes the product does something unique, and hopes he is one the way to creating the Yahoo or Gmail of text messaging."
"Whether you want to stay in touch with 5, 10, 15, 20, 500 or 5,000 people all at once, it's possible but it's not easy to do, unless you use a service like Tatango."
"Toss your phone tree in the trash. Through this service, you can send mass text messages from your phone or PC to large groups for easy, instant communication."
Tech Crunch
"The service is dead simple to use, as it should be. I made the jump from accountless bum to en masse messaging mogul in all of about 2 minutes."
"Perhaps the best thing going for Tatango is its completely intuitive feature set. Once you've registered, it doesn't take long to get your circle of contacts connected."
Tatango Reviews have all been glowing with its ease of use and awareness of the changing market place. Tatango also stresses best practice guidelines to make sure that not only are your marketing campaigns successes, but also respected.
Tatango Testimonials
You won't be able to look very hard to find glowing endorsements from clients that use Tatango. You can stop looking for Tatango Alternatives because the reviews are in and Tatango is leading the way. Here are a few samples of happy clients…
Adam McLane (Youth Specialties)
"If you need to send mass text messages, I only recommend Tatango."
Erik Jue (University of Houston)
"Tatango has saved us in many tight spots and made communication much easier."
You can see with Tatango the results are there for the taking you just have to get started. Currently, I know you can try Tatango for FREE with their 7 day trial.
Tatango Review Summary
My Tatango review show all the success that Tatango has to offer a small or even larger businesses to get you going on the path to successful mass text messaging campaign. Please do not hesitate to give this mobile marketing company a try and see if you can be happy as others who have experienced the Tatango difference.
Take Tatango for a FREE Text Drive (Click Below)
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Highlights / General details
One of the Brazilian Passions: Soccer! Watch the biggest teams in Rio play!
Experience the excitement of the most fanatic fans in the world. Anthems and waving flags set the mood and the electric atmosphere that surrounds the spectacle that is a Brazilian soccer match.
Seating is individual, under cover and our guide is always in attendance
Tour only offered on match days. On days with multiple games, only one time may be offered.
Roundtrip Transfer to Most Hotels in Rio de Janeiro; Guide; Tickets to the Stadium.
Food and Beverages; Gratuities.
Hours of operation
Available: Sunday, Wednesday, Thursday & Saturday; Departure times vary.
Approximately 4 hours.


text: I don't think I'm wrong when saying that all of us used to draw stuff when we were kids. I enjoyed reproducing everything that hit my eyes, including furniture, and my drawings were pretty good actually – or at least that's what I like to think. While having drawings of furniture in mind, try to imagine that those average, child made scribbles would actually project themselves into real life furniture. That's what Korean furniture designer and artist Jin il Park did. From the pictures, you'll be given the impression that you're looking at some hand-made drawings did in a hurry, without putting any effort to it. Well, you'll be wrong, as these are pieces of real furniture made from wire. Yeap, that's right, the artist managed to materialize his sketches and create a functional collection of furniture that consists in two chairs, two lamps and one table. I can't tell from the pictures if the chairs are actually comfortable, but I'm sure of one thing: this collection is awesome and I would love to have at least one of the pieces from the Drawing furniture series in my room.


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Exploring the dynamics of transport in the Dutch limes
Very little is known about local scale transport in the Roman period in the Netherlands, and therefore this paper aims to reconstruct and examine local transport networks through an integrative approach, using data and research from palaeogeography, physiology, archaeology and computational archaeological approaches. This study particularly focusses on the role of different modes of transport and the role of the natural environment. Firstly, a palaeogeographic reconstruction is presented encompassing both the natural and the cultural landscape. Transport networks for several modes are then reconstructed by calculating least cost paths that incorporate functions from physiological research. These networks are analyzed using standard network analytical procedures. Interesting interpretations can be inferred from the results of these procedures, including some relating to the different characteristics of the transport modes but notably also the relative important role of stone-built settlements in the networks and the relative lack of control that the Roman forts have over the transport network.
Full Text:PDF
Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 3.0 License
eTopoi. Journal for Ancient Studies (ISSN 2192-2608)


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It is set to music written by American gospel musician, singer, songwriter, guitar player and harmonica player, Buddy Greene. With irregular meter, the tune is usually just called MARY DID YOU KNOW. This has a 4/4 time signature. In the key of F, the first few notes are DEFG A_.
The song was first recorded by Christian recording artist Michael English in 1991, when English and Lowry were both members of the Gaither Vocal Band. Lowry has recorded it several times, the most well-known version is with the Gaither Vocal Band on their Christmas album "Still the Greatest Story Ever Told" released in 1998.
It has been recorded by may other contemporary-Christian-music artists, and reached No. 6 on the American CCM Magazine's AC Chart.
The song is regarded as a modern Christmas classic amongst American evangelicals, but has crossed denominational boundaries and is also used in many liturgical churches.
It has received varying reviews from musicians and liturgists in traditionalist churches (eg here or this one) - but at least some see it as having a place in public worship, or as a private devotional song, by people who understand that the questions in the text are literary devices, rather than expressions of doubt.
Professional recording - praise band:
Accapella duet:
Instrumental - electronic organ:
Soloist with orchestra:
Praise band - live recording:
LyricsThe lyrics are copyright so cannot be reproduced here. But they are currently available on this website. The song has three verses, and a bridge between the 2nd and 3rd verse. A rough outline is:
1 Mary did you know that your baby boy would someday walk on water? ...
2 .... Will give sight to a blind man? ... O Mary, did you know?
O Mary, did you know?
The blind will see, the deaf will hear
The dead will live a-gain
The lame will leap, the dumb will speak
The praises of the Lamb.
3 .... Is Lord of all creation?
.... This sleeping child you're holding is the great I AM! .,...


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More About WEE-SPARKLE
Visit My SparkPage Send Me SparkMail
Recipes I've Shared:
These cookies would probably be even more low carb if I had had an artificial brown sugar, but since I didn't we'll go with what we have.
I ground my own almond flour from raw almonds. I used my food processor and simply ground it up. I understand that a coffee grinder (preferably and unused one) works better.
Yummy, quick and easy Italian sauce.
This is a simple recipe from simple people created during a simple time.
This recipe is a guesstimate. I prepared the my meatloaf on a broiler pan, the potatoes cooked under them in the pan. I assuming about 1/2 a cup of juice came from the meatloaf. Meat juice estimate is included in nutritional information, but not in ingredients list.
We decided to mix it up a bit. I've made boiled cabbage for a long time, but wondered what would happen if I made it 50/50 with red cabbage. The red cabbage has a slightly more peppery flavor. The result was stupendous! Sweet and peppery, buttery and beautiful! For less calories you could probably reduce the butter by half.
Recipe from the Quaker Oats box
The goal of this recipe was to lessen the carbs, it is still a high calorie food. At 17g of carbs per cookie (at an estimated batch of 3 dozen), it's not exactly a low carb food either, but can be a nice occasional treat.
Original recipe came from Truvia website. I've made adjustments to suit my taste and hopefully to lower the carbs (I removed the oats the original recipe called for in addition to the flour).
Delicious source of protein
I like my brussel sprouts cooked softly, just not a big fan of the whole tender crisp thing.
You'll note that this recipe is slightly different from the box recipe
Yummy and delicious!
This recipe is about a simple as it gets. It's just what we eat 2-3 times a week, so it's simpler just to create it as a recipe.
This recipe is about a simple as it gets. It's just what we eat 2-3 times a week, so it's simpler just to create it as a recipe.
I use this to make chicken parmesan
Add a slice of provolone
1 Cup of Pasta
and 1 serving of my homemade italian sauce
for a meal at 690 calories
Delicious over Garlic & Herb Roasted Potatoes
Recipes I've Rated:
Recipe Collections I've Shared:
|Low Carb Offerings


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Laos – the land of smiles and striking scenery. From the mountains of Vang Vieng, to the colonial charm of Luang Prabang, this land-locked country has a lot to offer, not least the area of 4,000 Islands, an archipelago in the middle of the Mekong by the Cambodian border.
But first, that border needs negotiating – and I choose that word carefully. Continue reading Been there, Don Det, got the shi*ts


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JOS and Rainbird AI Team Up to Deliver Artificial Intelligence to the Asian Marketplace
04 Aug, 2017, 13:07 BST
LONDON, August 4, 2017 /PRNewswire/ --
JOS, one of Asia's leading providers of IT services and solutions, announced a milestone partnership with Rainbird AI as the first system integrator to bring Rainbird's unique artificial intelligence (AI) solutions to Asia. The alliance adds AI capabilities to JOS's IT solution portfolio, whilst helping the UK-based Artificial Intelligence expert to bolster its position in the Asian market.
Speaking at the AI Summit in Hong Kong, JOS's Group Managing Director Mark Lunt said: "We are delighted to announce this strengthening of our partnership with Rainbird, who bring proven skills and great, real-world experience to our customers in Asia."
Rainbird Technologies Chairman James Duez said: "We are excited to be working with JOS to bring our technology to Asia. We anticipate that JOS will become a strong agent for Rainbird in the wider Asian marketplace."
Mr Lunt added: "We are making significant progress in one project already underway for a client who will use Rainbird's AI technology in the automotive industry."
The project is the first in what is expected to be a significant number of broad ranging applications for Rainbird's technology in Asia. As early adopters of AI in the region, JOS is well positioned to capture the benefits of this important technological trend.
This collaboration further demonstrates JOS's leading edge as one of Asia's most innovative solutions providers.
About JOS
JOS is a systems integrator, solutions provider and technology consultancy with deep local and industry knowledge and an exceptional ability to execute, with 2,200 IT professionals working across Asia's major business hubs. JOS is a division of JTH Group, a member of the Fortune Global 500-listed Jardine Matheson Group.
For more information, visit JOS.com.
About Rainbird
Rainbird was created in response to demand within progressive enterprises for new, faster and more accurate knowledge for complex decision making, at scale. With Rainbird, solutions can be modelled by the people with the expertise, fully audited to satisfy compliance teams and implemented using an open architecture. Rainbird was founded in 2013 by tech entrepreneur James Duez and ex-Adobe AI Computer Scientist Ben Taylor. For more information, visit rainbird.ai.
SOURCE Rainbird Technologies


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delve a little deeper....
Oh I'm good!
Am thrilled to see I have another Field Review on the Punternet Website. Here it is if you'd like to read it!
Click here
Not only is it fabulous to get these reviews, they do wonders for my ego but it's lovely to know someone enjoyed their time with you enough to make the effort to write a report. Of course these things never hurt business and any record from a third party to prove I am who I say I am and that I do offer a good service is a good thing!
I'm thrilled because it is so lovely and from a lovely man (and no I'm not just saying that!)
In other news my campaign to spur England on to victory was futile, but at least I don't have to keep thinking of fun, sexy, relvant pictures to take!
Leave a Reply.
March 2020


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Walmart Launches Money Transfer Service, with Limits
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Walmart's new "Walmart-2-Walmart" transfer service will enable consumers to send money to each other by using the retail chain's store network.
The retailer on April 24 will introduce this service in partnership with Euronet Worldwide's subsidiary Ria Money Transfer. Walmart-2-Walmart will support domestic cash and PIN debit transfers at Walmart's 4,000 U.S. locations. Consumers can send up to $50 for a fee of $4.50, and up to $900 for a fee of $9.50.
"The service leverages our footprint and large scale to enable a low-cost service," said Daniel Eckert, senior vice president of services for Walmart U.S. on an April 17 conference call. "There is a large underbanked population in the U.S. that uses money transfer as a primary method."
The program is a potential competitor to Western Union, though the Walmart program is limited. It does not allow transfers outside the U.S and has no online component. It also prohibits transfers larger than $900.
The Walmart service will not change the retailer's relationship with MoneyGram, which enables a full range of transfer services from Walmart's stores, Eckert said. MoneyGram was told of Walmart's interest in a money transfer service during Walmart's most recent contract extension negotiation about a year ago, Eckert said. He did not elaborate on the details of those discussions and MoneyGram did not immediately return a request for comment.
"MoneyGram continues to be a very valuable partner for us. What Walmart does is provide some added choices for customers. We do best when we provide a broad assortment of products at a low price and let customers choose," Eckert said.
Walmart also offers financial services such as prepaid debit cards through partnerships with American Express and through Green Dot.
As with Walmart's other forays into financial services, the retailer is structuring the transfer service to minimize regulatory pressures. The new program's restrictions enable Walmart to avoid certain rules regarding transfers, Eckert said.
"With 'will call' money transfer services there are certain breakpoints where we are required to capture additional information and file activity reports with authorities in states and the national government," Eckert said. "That builds on added costs."
While Ria operates an international transfer service, Walmart currently plans to limit transfers to the U.S.
That may put the program at a disadvantage, says Phil Philliou, who operates Philliou Partners, a payments consultancy.
"I'm wondering what the use case is in the U.S. How valuable will this service be?" Philliou says.
The larger opportunity in money transfer is the international market, he says. "The Walmart service sounds like a good experiment, and if Walmart has aspirations to get involved in the international market, where someone could send money from the U.S. and pick it up in Mexico, that is very interesting," he says.