Proteomic-based discovery of biomarkers for disease has recently come under scrutiny for a variety of issues; one prominent issue is the lack of orthogonal validation for biomarkers following discovery. Validation by ELISA or Western blot requires the use of antibodies, which for many potential biomarkers are under-characterized and may lead to misleading or inconclusive results. Gelsolin is one such biomarker candidate in HIV-associated neurocognitive disorders.
Samples from human (plasma and CSF), monkey (plasma), monocyte-derived macrophage (supernatants), and commercial gelsolin (recombinant and purified) were quantitated using Western blot assay and a variety of anti-gelsolin antibodies. Plasma and CSF was used for immunoaffinity purification of gelsolin which was identified in eight bands by tandem mass spectrometry.
Immunoreactivity of gelsolin within samples and between antibodies varied greatly. In several instances, multiple bands were identified (corresponding to different gelsolin forms) by one antibody, but not identified by another. Moreover, in some instances immunoreactivity depended on the source of gelsolin, e.g. plasma or CSF. Additionally, some smaller forms of gelsolin were identified by mass spectrometry but not by any antibody. Recombinant gelsolin was used as reference sample.
Orthogonal validation using specific monoclonal or polyclonal antibodies may reject biomarker candidates from further studies based on misleading or even false quantitation of those proteins, which circulate in various forms in body fluids.
The development of global proteomic profiling in the mid-1990 s raised the expectations for quick discovery of new biomarkers . More importantly, it was expected that profiling of body fluids using high throughput, sensitive and specific methods would result in bringing new and approved diagnostic and therapeutic biomarkers from bench to bedside in a fast track manner . However, soon after the first large profiling experiments were performed, researchers observed several major problems: (i) very high dynamic range of the expression of proteins in the body fluids can reach 1012 orders of magnitude, thereby excluding the possibility to quantitate both low and high abundance proteins without additional sample fractionation(s) ; (ii) range of concentration for any given protein varies from individual to individual in general population as well as in cohorts of patients; (iii) standard operating procedures - including sample preparation, mass spectrometers used, and bioinformatic database searching - varied between proteomic labs, resulting in variability and only partial overlap of results ; and (iv) orthogonal validation of biomarkers in body fluids is essential following discovery phase, however these methods often fail to confirm initial results .
Of all the issues listed above, several are beyond our control and others require more technological development; validation of quantitative proteomics data is one such issue requiring advancement [6, 7]. Examples of orthogonal validation techniques for MS-based proteomics include Enzyme Linked ImmunoSorbent Assay (ELISA) [8–10] and Western blot [11, 12]. In comparison, examples of parallel validation techniques include Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) [13, 14] and Multiple Reaction Monitoring (MRM) [15, 16]. Each technique has advantages and drawbacks for the validation of potential biomarkers. For example, orthogonal validation using Western blot or ELISA requires the use of antibodies; some of which are not well characterized and when used, may result in misleading or skewed data.
Proteomic studies from our laboratory have shown that gelsolin is differentially expressed in the plasma and Cerebrospinal Fluid (CSF) of Human Immunodeficiency Virus (HIV)-infected individuals with and without dementia [17–19]. Likewise, gelsolin circulating in the plasma of monkeys infected with simian immunodeficiency virus (SIV) is also differentially expressed between pre-infection, acute and chronic infection . We have also found that monocyte derived macrophage (MDM) activated by HIV infection in vitro produce and secrete gelsolin (Ciborowski, P.; Kraft-Terry, S. both unpublished). Taking this together, we postulated that if gelsolin is validated, it may become a candidate as a diagnostic biomarker and be justified to move to experiments using larger cohorts of patients. However, validation of the differential expression of gelsolin in body fluids occurred to be a challenging task, as quantitative Western blot did not confirm differential expression unambiguously. As further studies indicated this was caused by two major reasons. First, high variability in the immunoreactivity of commercially available antibodies and the variability in recognition of gelsolin originating from CSF or plasma resulted in ambiguity. Second, immunoaffinity purification of gelsolin followed by MS/MS revealed that although the gelsolin circulating in the plasma and CSF was the secreted form of gelsolin (plasma gelsolin; pGSN), several other forms in addition to the full-length molecule (86kDa) were also in circulation ranging in molecular weight from 10 kDa to 188 kDa . Based on these prior studies and observations, this study focused on problems with validation of gelsolin using antibody based orthogonal assays.
Four sets of human and non-human primate samples were used throughout this project: two sets of human plasma, one set of human CSF, and one set of non-human primate plasma. Human plasma and CSF samples were previously obtained from the National NeuroAIDS Tissue Consortium (NNTC, http://www.nntc.org) under request #R101. The samples were classified based only on neurocognitive status (non-demented [ND], sub-symptomatic, or HIV-associated dementia [HAD]); no other criteria (age, race, gender, T-cell count, viral load, etc.) were applied for sample selection. Two additional sample sets were obtained: human plasma from the California NeuroAIDS Tissue Network (CNTN) at the University of California San Diego, CA (from Drs. I. Grant, R. Ellis, S. Letendre) and Rhesus macaques plasma samples were obtained from Dr. Robert M Donahoe, University of Utah, UT . The UNMC Institutional Review Board approved the use of the human clinical samples (#196-05-EX).
Prior to any type of sample processing, proteases and virus were neutralized using a solution of 10 μL - 10% Triton X-100 and 50 μL - 20X cocktail of protease inhibitors (Sigma-Aldrich; St. Louis, MO) per mL of sample as described previously in Wiederin et al. .
Each CSF sample was split into separate parts: one was used for immunodepletion and another for immunoaffinity purification. Immunodepletion was performed as described in Rozek et al.  using the Multiple Affinity Removal Spin Cartridges Hu-6 (Agilent; Santa Clara, CA). Plasma samples from CNTN, San Diego were immunodepleted as described in Pottiez et al.  using the Seppro® IgY 14 LC10 Column (Sigma-Aldrich). Rhesus macaques plasma samples were immunodepleted as described in Wiederin et al.  using the ProteomeLab IgY-12 High Capacity Proteome Partitioning Kit (Beckman Coulter; Fullerton, CA). Following immunodepletion, all samples were stored at -80°C.
Non-immunodepleted plasma and CSF samples from NNTC were pooled based on source and neurocognitive status immediately before immunoaffinity purification of gelsolin. A 1 mL capacity HiTrap NHS-activated HP affinity column (GE Life Sciences; Pittsburg, PA) was used for immunoaffinity purification and was performed as described in Pottiez et al. . Protein quantity for each fraction was analyzed using a NanoDrop 2000 (ThermoScientific, Inc., Waltham, MA) and fractions containing protein were pooled, dialyzed in MilliQ water and stored at -80°C.
In addition to plasma and CSF samples, human monocytes were isolated and cultured for this project. Monocytes were isolated by leukophoresis from donors whom were HIV-1, -2, and hepatitis seronegative as described in Gendelman et al. . These monocytes were cultured and differentiated as described in Ciborowski et al.  and infection with HIV-1ADA (multiplicity of infection: 0.1) occurred 7 days post-plating. Cell supernatants were collected from both HIV-infected and non-infected control cells at day 3 post-infection.
Human recombinant plasma gelsolin protein was obtained from Cytoskeleton, Inc. (Denver, CO). Human plasma gelsolin protein was obtained from Sigma-Aldrich.
Antibodies used throughout this study included mouse anti-human gelsolin (hGSN) monoclonal antibody (mAb) (BD Biosciences; San Jose, CA), goat anti-hGSN C-20 polyclonal antibody (pAb) (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA), and rabbit anti-hGSN pAb (Abcam; Cambridge, MA). The appropriate horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmnoResearch Laboratories, Inc; West Grove, PA) were used for Western blot. A total of 1 mg goat anti-hGSN pAb antibody (Santa Cruz Biotechnology) was purified by protein-G affinity chromatography (Pierce; Rockford, IL) following manufacturer's protocol and used for immunoaffinity purification of samples.
Samples were desiccated using a SpeedVac (ThermoScientific) and resolubilized in 20 μL NuPAGE (Invitrogen; Carlsbad, CA) sample buffer with reducing agent prior to heating and gel loading. 1DE was performed using NuPAGE® Novex® precast 4-12% Bis-Tris Gels (Invitrogen) under reducing conditions. The gel was run for 90 minutes at 100 V. Human immunoaffinity purified plasma and CSF derived gelsolin gels were fixed and stained with brilliant-blue G-colloidal concentrate (Sigma-Aldrich). Remaining samples were used for Western blot.
Bands from plasma immunoaffinity purified gelsolin samples were excised using a razor blade, destained, and digested in-gel using modified trypsin. Destaining consisted of two 30-minute washes using first 200 μL of 20 mM NH4 HCO3/50% acetonitrile (ACN), then 200 μL of 100% ACN. After destaining, the gel slices were desiccated and treated with 0.1 μg/μL sequencing grade modified trypsin (Promega; Madison, WI) overnight at 37°C. Next, the peptides were extracted using 60% ACN, 0.1% TFA solution, desiccated and resuspended in 0.1% TFA. Reverse-phase C18 Zip-Tips® (Millipore; Billerica, MA) were used to purify extracted peptides following manufacturer's protocol.
Mass spectrometric analysis was carried out using an LC/ESI-MS/MS system in a nanospray configuration using a microcapillary reverse phase RP-C18 column (New Objectives; Woburn, MA). An LCQ-Deca XP Plus ion trap mass spectrometer (ThermoScientific, Inc.) was used to perform tandem mass spectrometry. Spectra were searched and proteins were identified following procedures stated in Pottiez et al. .
Following electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad; Hercules, CA) for immunodetection as previously described in Ciborowski et al. . Manufacturer's recommendations were followed for each antibodies used in the detection of gelsolin in samples; each primary (anti-hGSN) antibody was used at a 1:1000 dilution in phosphate buffered saline with 0.02% Tween-20 (PBST) and 10% (w/v) skim milk; each secondary antibody was used at a 1:20,000 dilution.
Based on the sequences identified using LC/ESI-MS/MS, the theoretical molecular weight was calculated using the ExPASy Compute pI/Mw tool http://www.Expasy.org. For each band, the peptides from the most N-terminal and C-terminal regions were selected as the form endpoints. Using the FASTA sequence for secreted pGSN, all amino acids between those N-terminal and C-terminal amino acids were identified and this shortened sequence was used to generate a theoretical molecular weight.
Previously published MS-based proteomic studies have shown that plasma gelsolin (pGSN) is differentially expressed in HIV infected humans, SIV infected monkeys and in vitro HIV infected MDM [18–20, 23]. Prior to conducting further studies using larger cohorts of samples from patients, we attempted to validate its expression using a smaller number of samples. Our validation effort using quantitative Western blot analysis gave ambiguous results and indicated that differences in validation strongly depend on which antibody was used. Therefore, the initial goal of our study was to select an anti-gelsolin antibody that when used for quantitative Western blot analysis would most closely reflect the results of proteomic profiling.
Subsequent experiments brought to light new information concerning our previous results of Western blot validations  in which we observed a single band corresponding to the full-length gelsolin molecule. Concurrent experiments of immunoaffinity purification from the same samples showed multiple forms of gelsolin. This discrepancy prompted us to further explore the specificity of anti-hGSN antibodies to explain if Western blot validation of proteomic profiling might be biased and do not reflect real levels of intact and processed forms of gelsolin. From more than 20 commercially available anti-hGSN antibodies, we selected three: mouse monoclonal, goat polyclonal and rabbit polyclonal; all were raised against an epitope from the C-terminal portion of gelsolin. A sheep polyclonal anti-hGSN antibody was also tested, but results could not be obtained due to high background (data not shown). Figure 1A is a schematic diagram of the gelsolin molecule along with the location of the epitope corresponding to each antibody. Figure 1B summarizes the specificity of those three anti-hGSN antibodies used for Western blot assay against the gelsolin found various samples. It was unexpected that that monoclonal antibody raised to a synthetic peptide (located in the C-terminal end of gelsolin) reacted only with a single band of approximately 86 kDa, corresponding to the full length and intact gelsolin molecule. This indicated the conformational dependence of the antibody; moreover, it suggested that the conformation of the entire molecule - not only its C-terminal portion - is necessary for its immunoreactivity. Furthermore, Western blot analysis using either goat or rabbit pAb recognized additional forms of gelsolin with both larger and smaller molecular weights. It is likely that the smaller molecular weight forms are truncated forms of the full-length molecule based on trends observed by LC/ESI-MS/MS (Table 1). Further analysis using LC/ESI-MS/MS revealed that the larger molecular weight form (Figure 2A, asterisked band) contains fibronectin, which co-immunopurified with gelsolin; this comes as no surprise as it is well known that fibronectin binds gelsolin .
Included is the band identification (corresponding to extracted bands in Figure 1, column A), molecular weight based on electrophoretic mobility, theoretical minimum molecular weight as calculated using ExPASy Compute pI/Mw tool, identified peptides, and peptide location in secreted pGSN. The peptide -DSQEEKTEALTSAK- was the most commonly identified peptide (in 7 of 8 bands). * - Molecular weight (M.W.) is approximate based on electrophoretic mobility in 1DE SDS-PAGE. ** - Theoretical Molecular weight was approximated using the ExPASy Compute pI/Mw tool and was calculated using the first peptide position through the last peptide position as determined using MS/MS.
Immunorecognition of hGSN by three antibodies in Western blot assay. (A) The location of epitope specific to the mouse, goat and rabbit anti-hGSN antibodies are provided in reference to the full-length pGSN. Numbers above each epitope correspond to the amino acid sequence from the full-length (with signal sequence intact) pGSN containing peptides used as antigens. (B) Summary of Western blot analyses revealing that immunoreactivity of pGSN depends on not only antibodies but also source of antigen. Total protein loaded per source per lane: 25 μg of human plasma from HIV-infected individuals, 10 μg of human CSF from HIV-infected individuals, 20 μg of cell supernate from both HIV-infected and non-infected cells, 25 μg of monkey plasma from pre- and 10 days post-infection of rhesus macaques with SIV, and 2 μg each of commercially available gelsolin. Membranes from each source were probed with mouse anti-hGSN, goat anti-hGSN, and rabbit anti-hGSN (all 1:1000) and corresponding HRP-conjugated secondary antibodies (1:20,000) diluted in PBS supplemented with 10% Tween-20 and 10% (w/v) skim milk.
Forms of immunoaffinity purified gelsolin. A and B shows 1DE analysis of gelsolin immunoaffinity purified from plasma and CSF respectively. A total of 15 μg immunoaffinity purified gelsolin was loaded per lane and gels were stained with Coommasie Brilliant Blue. Eight bands (labeled in lane A) were selected for mass spectrometric identification of proteins. A total of 2 μg recombinant gelsolin was used for analysis via Western blot; banding pattern differences were seen between goat anti-hGSN (lane C) and mouse anti-hGSN (lane D). The high molecular weight band - which is identified by an asterisk - was found to contain fibronectin, a protein known to bind gelsolin. All other bands contained gelsolin, which is further discussed in Table 1.
It was most unexpected that goat and rabbit pAb showed such great differences in immunoreactivity within samples. For example, goat pAb reacted weakly with only one form of gelsolin in the CSF whereas rabbit pAb recognized strongly two forms of gelsolin in that same sample. None of these antibodies recognized a form of gelsolin in any sample with a molecular weight lower than 45 kDa with the exception of goat pAb against commercially available human pGSN (Sigma Aldrich), which was able to recognize an additional band with a molecular weight of less than 38 kDa. In addition to the lower molecular weight forms, there were several samples in which higher molecular weight forms were detected; goat anti-hGSN pAb was able to detect these higher molecular weight forms in both human plasma and commercial gelsolin samples. Protein purification and concentration can often cause proteins to aggregate, which is a potential explanation for these higher molecular weight bands. These higher molecular weight bands warranted further investigation and using LC/ESI-MS/MS on bands excised from recombinant gelsolin, we were able to positively identify only gelsolin.
1DE of immunoaffinity purified CSF and plasma derived gelsolin revealed several bands with a wide range of molecular weights: approximately 17 kDa to >188 kDa (Figure 2 columns A and B). Although the relative concentration for each band varied between immunoaffinity purified gelsolin from plasma and CSF, the banding pattern remained consistent suggesting processing of gelsolin in the plasma and CSF is similar. Western blot analysis of recombinant plasma gelsolin using mouse anti-hGSN showed a single band at 86 kDa, which corresponds to the full-length gelsolin molecule (Figure 2 column D). In comparison, Western blot of this same sample using goat anti-hGSN revealed multiple bands at 166 kDa, 86 kDa, 64 kDa, 60 kDa, 54 kDa, and 45 kDa (Figure 2 column C).
Eight bands were selected for tryptic digestion and identification by LC-ESI-MS/MS from the immunoaffinity purified plasma sample (Figure 2 column A). Gelsolin was found in each band; the peptides that were identified and their exact location in secreted pGSN are included in Table 1. The approximate molecular weight for each band as estimated by electrophoretic mobility is included. Furthermore, a theoretical minimum molecular weight for each band based on the peptides identified in that band and calculated using the ExPASy Compute pI/Mw tool was also included in Table 1. Some peptides were identified in almost every band, whereas several were identified in only one or two bands. Based on the peptides identified, it was determined that the forms present in immunoaffinity purified samples were mainly truncated from the N-terminal end.
Due to the immunodetection pattern observed in the Western blots (Figure 1 and Figure 2: columns C and D), it was further postulated that the pGSN forms in bands 7 and 8 (Figure 2 column A) were likely either (i) at a concentration below the detectable threshold via Western blot, (ii) truncated at the C-terminal end and thereby lacking the immunogen required to be recognized by the antibody, and/or (iii) in an alternative conformation due to post-translational modifications or from changes occurring during 1DE and transfer.
Figure 3 includes a Western blot using goat anti-hGSN antibodies against a linear dilution of recombinant gelsolin. In the most concentrated sample (2 μg) of GSN, a total of 3 clear and distinguishable bands were detected: >188, 60 and 54 kDa both representing different forms of pGSN. Also detected were two bands at 166 kDa and 86 kDa; these bands however were not clear and distinguishable, but instead were oversaturated and unquantifiable. A 7-fold dilution (0.016 μg) of GSN resulted in only one clear, distinguishable and quantifiable band at 86 kDa. It was determined that immunodetection using goat anti-hGSN is dependent on the concentration of each form present in the sample.
Western blot titration of hGSN. Titration of hGSN using Western-blot assay shows limitations of this assay in quantitation of this protein. At higher concentrations other forms than full-length molecule are detected, however, 86 kDa band representing full-length molecule is oversaturated. Conversely, at low concentration, full-length molecule can be quantitated, but the presence of all other forms is missed. Goat anti-hGSN antibody was used in this experiment.
Based on the banding pattern observed, peptides recognized and their location, molecular weight observed for each band and the calculated theoretical minimum molecular weight, a schematic for each band was created (Figure 4).
Schematic model of full-length hGSN and proposed forms of gelsolin isolated from serum/plasma and CSF. Band 1 represents the full-length hGSN molecule and includes its functional and structural features. This form shows electrophoretic mobility corresponding to approximately 86 kDa. hGSN was identified by tandem mass spectrometry analysis in bands 2 to 8. Based on their electrophoretic mobility and identified peptides resulting from trypsin digestion (see Table 1 for details) we estimated their approximate molecular weight and amino acid sequence coverage. Gelsolin peptides identified in each band by LC/ESI-MS/MS are colored.
Biomarker discovery and validation - or even the complete characterization - of the plasma and/or proximal fluids (including CSF) has been a daunting task. New biomarkers have not emerged as expected, despite the effort put forth experimentally by both small, single laboratories with limited clinical samples [3, 13, 27] as well as large, research organizations like the Human Plasma Proteome Project (HPPP) . A conundrum has emerged with respect to validation of biomarkers following the discovery phase; is a lack of validation due to the assumptions that have been made about how a particular disease progresses or is it that the tools and reagents used are not adequate to the task? Accordingly, we postulate that a better understanding of the molecular mechanisms underlying diseases will help us to understand observed changes at the protein level and will also result in the validation of already discovered as well as new biomarker candidates.
For the majority of studies validation is based entirely or in part on immunoreactivity of specific antibodies. In terms of ELISA, this approach has been proven as very useful and accurate in many instances, e.g. measurement of bacterial proteins/toxins etc. However, when the dynamic changes of human proteins are measured or taken into consideration (including alternative splicing, post translational modification, regulated processing or degradation), immunoreactivity based assays become quite inaccurate. There has been and will continue to be attempts to improve these assays and thereby promote the acceptance of scientifically sound biomarkers. For example, Rifai et al. proposed a biomarker "pipeline" including discovery, qualification, verification, assay optimization, validation and commercialization to help aid in the discovery of better protein biomarkers . Several of steps in this pipeline require the use of antibodies - from immunoaffinity purification in the qualification and verification stages to immunoassays like Western blot and ELISA in validation.
Experimental data that we present in this study helps to understand why in many cases validation based on immunoreactivity may lead to inconclusive or even misleading results. Moreover, we also conclude that other methods such as MRM may provide inaccurate results in the validation of biomarkers. For example, quantitative methods requiring the use of antibodies such as ELISA and quantitative Western blot will vary depending on which antibody is used (Figure 1 and Figure 2). Additionally, it was shown that the concentration of the biomarker in question might also adversely affect the results of quantitation using Western blot (Figure 3). With respect to quantitation, the different immunoaffinity purification methods used in sample preparation must also be considered. To date, our laboratory has yet to witness non-specific removal of gelsolin forms, however it has been realized that using different methods of immunoaffinity purification may adversely affect the quantitation of protein. Therefore using different methods of immunoaffinity purification should not be used in quantitative studies, but may be included in qualitative based studies. Furthermore, based on the results of this study, we reaffirmed that the conformation of the antigen does plays a key role for immunodetection; this was seen with the Western blots using mouse anti-hGSN antibody in Figures 1 and 2 being only able to detect a single gelsolin form. Additionally, goat anti-hGSN antibody was able to pull down various forms of gelsolin in human plasma during immunoaffinity purification; however it was unable to detect all the forms following 1DE, transfer and Western blot.
Our results reported here and those reported in previous papers [30–32] have a much broader implication to which method should be used for validation and eventually which potential biomarker candidate will be used or rejected from testing on larger cohorts of clinical samples.
Although ELISA has long been considered both reliable and high throughput and it is a technique that utilizes conjugated antibodies to quantify the targeted proteins, it also has limitations in its ability to differentiate between protein forms. In comparison, the Western blot - which is able to detect expression changes in the various forms of any given protein addressing the limitation of ELISA - is not a high-throughput technique and its reliability is often questioned because of saturation of chemiluminescent signal measured with X-ray films. Standardization of Western blot is much more difficult even if a fluorescently tagged secondary antibody is used. Protein microarrays, which address the issue of high-throughput, is also based on antigen-antibody interaction and must be performed using very well characterized antibodies. If an antibody used for microarrays recognizes only one or two forms, only a fragment of information about the differential expression of any protein will be received, similar to Western blot assay. Therefore, the 2D-differential in-gel electrophoresis (DIGE) profiling method - which separates full-length forms from fragments (resulting from processing or degradation) - appears to be an attractive alternative method. In our previous profiling studies using 2D-DIGE, we were able to show that the best indicator of changes of complement C3 in CSF, which is processed by multi-step well-defined mechanism, is a "residual" α-40 chain . However, lack of good antibody to this fragment of C3 made orthogonal Western blot validation impossible at that time.
A novel approach known as Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) was developed to allow for the enrichment of targeted proteins in complex samples  and thereby could facilitate biomarker validation. This method is based on peptide quantitation in complex mixtures such as the total tryptic digest of plasma samples. The SISCAPA method first combines immunoaffinity purified native peptide using anti-peptide antibodies immobilized on 100-nanoliter column and spiked stable-isotope-labeled internal standard peptide of the same sequence. Next, both peptides are measured by ESI-MS/MS and quantity is calculated based on the ratio of heavy (standard) to light (native) peptide, much like multiple reaction monitoring (MRM, below). Although the SISCAPA method may lead to increased sensitivity, it is a technology utilizing antibodies and therefore the same concerns with respect to ELISA and Western blot are applicable.
The MRM approach for validation is based on the comparison of abundance of selected peptides originating from a sample and spiked standard [16, 34]; usually the peptides that are well ionized are selected for MRM quantitation. However, ambiguous results may occur depending on the peptide(s) chosen. For example, if one peptide is selected from N-terminal end of gelsolin and one from C-terminal region for MRM quantitation (or SISCAPA), the results of quantitative comparisons can be very different, as shown in Figure 5. More importantly, each peptide reflects a different situation; the N-terminal peptide will indicate quantity of full-length molecule and multimers whereas the C-terminal peptide will reflect the degree of gelsolin processing and/or degradation. Such an ambiguous result, if not further explained, may result in the rejection of a putative biomarker from further studies. Additionally, if a third peptide from the mid-region is selected and the quantity is averaged, the end result may not be different than the control sample and potential biomarker will also be rejected from further studies.
Peptide candidates for MRM based quantitation. Two peptides: (1) DSQEEKTEALTSAK and (2) EVQGFESATFLGYFK, derived from pGSN by trypsin digestion and representing N-terminal and C-terminal regions respectively, are well ionized and fragmented by ESI-MS/MS. As such they are excellent potential candidates for MRM based quantitation. Note that peptide (1) was identified in only 2 out of 8 bands and peptide (2) was identified in 7 out of 8 bands of pGSN circulating in plasma/CSF. Therefore, neither of these two peptides will reflect accurately levels of pGSN; additionally, use of these peptides in MRM may not validate pGSN as potential biomarker.
Gelsolin is a candidate biomarker for several neurocognitive diseases but before it can be integrated into the "biomarker pipeline" , further steps must be made to improve the immunoreactivity of anti-gelsolin antibodies. Without further antibody development, characterization and optimization, candidate biomarkers such as gelsolin will lack quantitative validation and thereby be unable to enter clinical assay development.
Validation is one of the critical steps in bringing new biomarkers from bench to bedside in translational research. Our data presented here using gelsolin as an example, highlights a set of specific problems associated with antibody based validation methods. We also briefly describe how each of the current widely accepted methods of validation has inherent weaknesses yet each are strong enough that if used alone may lead to ambiguous or even false results. Hence, conclusions based on our experimental data have a broad application as to how we should approach validation methodologically and partially explains lack of real progress in the translation of biomarkers from bench to bedside.
The authors would like to thank Dr. Robert M. Donahoe from University of Utah for kind gift of monkey plasma (supported by NIDA grant DA 04498) and Drs. Igor Grant, Ronald Ellis and Scott Letendre from the California NeuroAIDS Tissue Network (CNTN) at the University of California San Diego, CA for kind gift of human plasma samples. This work was partially supported by the National Institutes of Health (1 P20DA026146-01 and 2 P01 NS043985-05).
NH has performed the majority of the experimental work, wrote drafts of the manuscript and incorporated all suggested changes, has made drafts and final forms of all figures, and has made important contributions to the intellectual content. GP has made contributions to concept and design of the project, has been involved in revising the manuscript critically for important intellectual content, and has contributed to the designing of figures. JW has made contributions to data analysis, has been involved in revising the manuscript critically for important intellectual content, and contributed to designing of figures. PC has made substantial contributions to conception and design of the project, data analysis and interpretation, drafting of the manuscript, and has been involved in revising the manuscript critically for important intellectual content. All authors have read and approved the final manuscript.


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WHAT IS THE PROPER MARKETING MIX WHEN THE RECIPE IS TAIWANESE
WHAT IS THE PROPER MARKETING MIX WHEN THE RECIPE IS TAIWANESE Lee, Monle 1998-02-01 00:00:00 While the consumer movement was popular in economically advanced countries between 1965 and 1975, developing countries were not yet ready for such issues. The consumers in these countries, due to their economic, social, and political environments, were not aware of their rights. Taiwan was a typical case. Three main reasons contributed to the ignorance of the Taiwanese consumers at the advent of the worldwide consumer movement. First, in its early years, Taiwan was basically an agricultural society. The primary industry and its commodities made product differentiation impossible. It was nature, and not human beings that decided the quality andor quantity of the products produced for the market. Everyday products were simple and few, and the choice between products, easy. Secondly, it was not until the 1960's that Taiwan turned slowly from an importoriented economy to that of an exportoriented economy. Raising tariffs and controlling the importation of products were necessary to protect local businesses from foreign competition. Thus, the rights of consumers were sacrificed. Lastly, the very nature of the Chinese e.g., psychological profile, led them to endure the suffering of any mistreatment. They would avoid a direct confrontation on any dispute. Therefore, organized activities against businesses was not possible. All of these reasons made the consumer movement unheard of in Taiwan during the 1960s. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Competitiveness Review: An International Business Journal incorporating Journal of Global Competitiveness Emerald Publishing http://www.deepdyve.com/lp/emerald-publishing/what-is-the-proper-marketing-mix-when-the-recipe-is-taiwanese-5BEdcjk00F
Lee, Monle
Competitiveness Review: An International Business Journal incorporating Journal of Global Competitiveness
, Volume 8 (2): 12 – Feb 1, 1998
/lp/emerald-publishing/what-is-the-proper-marketing-mix-when-the-recipe-is-taiwanese-5BEdcjk00F
Competitiveness Review: An International... /
Copyright © Emerald Group Publishing Limited
10.1108/eb046366
While the consumer movement was popular in economically advanced countries between 1965 and 1975, developing countries were not yet ready for such issues. The consumers in these countries, due to their economic, social, and political environments, were not aware of their rights. Taiwan was a typical case. Three main reasons contributed to the ignorance of the Taiwanese consumers at the advent of the worldwide consumer movement. First, in its early years, Taiwan was basically an agricultural society. The primary industry and its commodities made product differentiation impossible. It was nature, and not human beings that decided the quality andor quantity of the products produced for the market. Everyday products were simple and few, and the choice between products, easy. Secondly, it was not until the 1960's that Taiwan turned slowly from an importoriented economy to that of an exportoriented economy. Raising tariffs and controlling the importation of products were necessary to protect local businesses from foreign competition. Thus, the rights of consumers were sacrificed. Lastly, the very nature of the Chinese e.g., psychological profile, led them to endure the suffering of any mistreatment. They would avoid a direct confrontation on any dispute. Therefore, organized activities against businesses was not possible. All of these reasons made the consumer movement unheard of in Taiwan during the 1960s.
Competitiveness Review: An International Business Journal incorporating Journal of Global Competitiveness – Emerald Publishing
Published: Feb 1, 1998
Lee, M. (1998). WHAT IS THE PROPER MARKETING MIX WHEN THE RECIPE IS TAIWANESE. Competitiveness Review: An International Business Journal incorporating Journal of Global Competitiveness, 8(2), 34-45.
Lee, Monle. "WHAT IS THE PROPER MARKETING MIX WHEN THE RECIPE IS TAIWANESE." Competitiveness Review: An International Business Journal incorporating Journal of Global Competitiveness 8.2 (1998): 34-45.


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North Korean Believer's Tale of Persecution Survival
N. Korean Believer's Tale of Persecution Survival
By Gary Lane
CBN News Sr. International Correspondent
Millions of Christians around the world will unite this weekend to mark the International Day of Prayer for the persecuted church.
The annual event is a time for standing with those who suffer for following Christ.
Attacks against Christians in countries like Nigeria, Pakistan, and Egypt are increasing, yet the worst place to be a Christian is in North Korea's Hermit Kingdom.
Christians are under intense pressure in Muslim countries, but North Korea tops the Open Doors list each year as the world's worst persecutor of Christians.
Foley talked more about Bae's story and the plight of North Korea's Christians, on CBN's Newswatch, Nov. 8. Stay with CBNNews for that interview after 5pm ET.
Nearly one year after the death of Kim Jong Il, little has changed. His son and successor, Kim Jong Un, is continuing efforts to rid the nation of Christianity.
Christians are arrested, imprisoned, and even executed simply for believing in a faith other than the state religion known as Juche, or self-reliance.
CBN News protected one North Korean Christian by hiding his face and calling him by a different name: "Mr. Bae."
Bae was imprisoned for evangelizing a friend.
"There were interrogations, tortures," he told CBN News. "But the most difficult thing was sit still on the floor without moving my neck, arms, legs for more than a year."
Bae said his faith grew deeper as time passed.
"I felt as if God was with me. I would not die," he recalled. "I came to have faith and at the end I was released without any charges, so that I knew that it is not the work of man, but it is the work of God that I am alive."
Bae told his story to Rev. Eric Foley, CEO of the Colorado-based ministry Seoul USA. His family experience of Christian suffering is detailed in the new book, These Are the Generations.
The Prison Rape Elimination Act of 2003
The Prison Rape Elimination Act of 2003 (PREA) is the first United States federal law passed dealing with the sexual assault of prisoners. The bill was signed into law on September 4, 2003.
New Archibishop of Canterbury Chosen to Lead Anglicans
Anglicans to be Led by a New Archbishop of Canterbury
LONDON – The next spiritual leader of the world's 77 million Anglican Christians has been chosen.
Archbishop of Canterbury Rowan Williams is retiring after a turbulent decade dealing with the Anglican Communion's deep divisions about gay bishops and homosexuality.
British media say the new choice is Durham Bishop Justin Welby, who made a mid-career shift from the oil industry to the clergy and has made business ethics part of his work.
The 56-year-old Welby is said to be an opponent of same-sex marriage and the appointment of gay bishops, issues which have divided the Episcopal Church, the U.S. Branch of the Anglican Communion.
Queen Elizabeth is supreme governor of the Church of England and had to approve church leaders' pick for archbishop of Canterbury.
James Abram Garfield (November 19, 1831 – September 19, 1881) served as the 20th President of the United States, after completing nine consecutive terms in the U.S. House of Representatives. Garfield's accomplishments as President included a controversial resurgence of Presidential authority above Senatorial courtesy in executive appointments; energizing U.S. naval power; and purging corruption in the Post Office Department. Garfield made notable diplomatic and judiciary appointments, including a U.S. Supreme Court justice. Garfield appointed several African-Americans to prominent federal positions.
Garfield was raised in humble circumstances on an Ohio farm by his widowed mother and elder brother, next door to their cousins, the Boyntons, with whom he remained very close. He worked at many jobs to finance his higher education at Williams College, Massachusetts, from where he graduated in 1856.
A year later, Garfield entered politics as a Republican, after campaigning for the party's antislavery platform in Ohio. He married Lucretia Rudolph in 1858, and in 1860 was admitted to practice law while serving as an Ohio State Senator (1859–1861). Garfield opposed Confederate secession, served as a Major General in the Union Army during the American Civil War, and fought in the battles of Middle Creek, Shiloh and Chickamauga. He was first elected to Congress in 1862 as Representative of the 19th District of Ohio.
Throughout Garfield's extended Congressional service after the Civil War, he fervently opposed the Greenback, and gained a reputation as a skilled orator. He was Chairman of the Military Affairs Committee and the Appropriations Committee and a member of the Ways and Means Committee. Garfield initially agreed with Radical Republican views regarding Reconstruction, then favored a moderate approach for civil rights enforcement for Freedmen. In 1880, the Ohio legislature elected him to the U.S. Senate; in that same year, the leading Republican presidential contenders – Ulysses S. Grant, James G. Blaine and John Sherman – failed to garner the requisite support at their convention. Garfield became the party's compromise nominee for the 1880 Presidential Election and successfully campaigned to defeat Democrat Winfield Hancock in the election. He is thus far the only sitting Representative to have been elected to the presidency.
Garfield's presidency lasted just 200 days—from March 4, 1881, until his death on September 19, 1881, as a result of being shot by assassin Charles J. Guiteau on July 2, 1881. Only William Henry Harrison's presidency, of 32 days, was shorter. Garfield was the second of four United States Presidents who were assassinated. President Garfield advocated a bi-metal monetary system, agricultural technology, an educated electorate, and civil rights for African-Americans. He proposed substantial civil service reform, eventually passed by Congress in 1883 and signed into law by his successor, Chester A. Arthur, as the Pendleton Civil Service Reform Act.
A Poet's Death is His Life
A Poet's Death is His Life IV
by Khalil Gibran
The dark wings of night enfolded the city upon which Nature had spread a pure white garment of snow; and men deserted the streets for their houses in search of warmth, while the north wind probed in contemplation of laying waste the gardens. There in the suburb stood an old hut heavily laden with snow and on the verge of falling. In a dark recess of that hovel was a poor bed in which a dying youth was lying, staring at the dim light of his oil lamp, made to flicker by the entering winds. He a man in the spring of life who foresaw fully that the peaceful hour of freeing himself from the clutches of life was fast nearing. He was awaiting Death's visit gratefully, and upon his pale face appeared the dawn of hope; and on his lips a sorrowful smile; and in his eyes forgiveness.
He was poet perishing from hunger in the city of living rich. He was placed in the earthly world to enliven the heart of man with his beautiful and profound sayings. He as noble soul, sent by the Goddess of Understanding to soothe and make gentle the human spirit. But alas! He gladly bade the cold earth farewell without receiving a smile from its strange occupants.
He was breathing his last and had no one at his bedside save the oil lamp, his only companion, and some parchments upon which he had inscribed his heart's feeling. As he salvaged the remnants of his withering strength he lifted his hands heavenward; he moved his eyes hopelessly, as if wanting to penetrate the ceiling in order to see the stars from behind the veil clouds.
And he said, "Come, oh beautiful Death; my soul is longing for you. Come close to me and unfasten the irons life, for I am weary of dragging them. Come, oh sweet Death, and deliver me from my neighbors who looked upon me as a stranger because I interpret to them the language of the angels. Hurry, oh peaceful Death, and carry me from these multitudes who left me in the dark corner of oblivion because I do not bleed the weak as they do. Come, oh gentle Death, and enfold me under your white wings, for my fellowmen are not in want of me. Embrace me, oh Death, full of love and mercy; let your lips touch my lips which never tasted a mother's kiss, not touched a sister's cheeks, not caresses a sweetheart's fingertips. Come and take me, by beloved Death."
Then, at the bedside of the dying poet appeared an angel who possessed a supernatural and divine beauty, holding in her hand a wreath of lilies. She embraced him and closed his eyes so he could see no more, except with the eye of his spirit. She impressed a deep and long and gently withdrawn kiss that left and eternal smile of fulfillment upon his lips. Then the hovel became empty and nothing was lest save parchments and papers which the poet had strewn with bitter futility.
Hundreds of years later, when the people of the city arose from the diseased slumber of ignorance and saw the dawn of knowledge, they erected a monument in the most beautiful garden of the city and celebrated a feast every year in honor of that poet, whose writings had freed them. Oh, how cruel is man's ignorance!
"Spanish Village" by W. Eugene Smith
Tomb of the Unknown Soldier by Day Williams
Lady Justice, Virginia City, Nevada by Day Williams


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Home / Area News / Trovinger, Hopson lead Mona Shores past Midland Dow 35-12 in D2 district finals
Trovinger, Hopson lead Mona Shores past Midland Dow 35-12 in D2 district finals
By Mitchell Boatman
NORTON SHORES – Something about playoff football brings out unsung heroes.
For Mona Shores on Friday night, it was seniors Kolbe Trovinger and Jaylen Hopson who stepped into the spotlight in a 35-12 win over Midland Dow in a Division 2 district title game at Sailor Stadium.
Both players are regular contributors for the Sailors, but with a slew of offensive playmakers on the team, they don't always get the attention.
But Trovinger and Hopson both made some very big plays to spark the Sailors at the end of the first half, before the team pulled away in the second.
Trovinger scored the game's first touchdown and put the Sailors up for good, while Hopson scored the second TD with an exciting pick-six.
"The first score was huge, and I think even bigger when Hopson got the pick-6," said Mona Shores Coach Matt Koziak. "That was kind of a back-breaker right before the half."
Kolbe Trovinger breaks tackles on the outside and takes it in for the Sailor's first touchdown. Photo/Eric Sturr
The win gives Mona Shores its third district title in program history, and second in a row for the very first time.
"It feels great," Koziak said. "I don't think it's really set in yet. It's a special group, and to be able to win it at home for our seniors, our last game on this field for these seniors, we're going to enjoy it. The more time that passes, I'll enjoy it even more."
The Sailors are two wins away from making another trip to Detroit's Ford Field, where they fell in the Division 2 state finals last season.
"It motivates us a lot," Trovinger said about last year's state final. "We honestly were so close that game and it slipped away at the end. We want to get back to Ford Field and win it this time. We're not just trying to get there, we're trying to win it."
The Sailors will travel to play Portage Northern in the regional round of the playoffs next week.
Neither team was able to find the end zone on their first two drives. The Sailors finally broke through on their third possession with a 15-yard touchdown run by Trovinger. The senior took a jet sweep to the left end, turned upfield and dove to the pylon, just staying in bounds. The extra point put the Sailors up 7-0.
On the next possession, the Chargers attempted a trick play that backfired. Quarterback Shane Juday handed off to Kaleb Studebaker on a reverse. Studebaker ran right before firing back across the field to Juday.
Mona Shores' Brady Rose finds the opening for a big gain. Photo/Eric Sturr
Hopson read the play perfectly, jumped the route, intercepted the pass and returned it 55 yards for a Sailor touchdown.
"I waited for a bit and saw the quarterback come out for the pass," Hopson said. "I just came up and got it right on time. I saw nothing but green grass. I just kept going, I knew I had (the touchdown)."
The Sailors led 14-0 at halftime.
The Chargers got the ball to start the third, but punted after three plays. On the ensuing drive, the Sailors extended the lead to 21-0 on a 60-yard drive that ended with a one-yard touchdown plunge by quarterback Caden Broersma.
On the following kickoff, Mona Shores was flagged for an illegal block. The penalty was awarded as a spot foul, which moved Dow from its own 25-yard-line to the Sailor 40. Four plays later, Juday found Carter Kohtz for a 27-yard touchdown pass. A missed extra point left it 21-6 with 3:47 to play in the third.
The Chargers then secured an onside kick and moved deep into Sailor territory on a pass interference penalty two plays later. But the Mona Shores defense held strong and Brady Rose intercepted a pass in the end zone on third down to end the threat.
The Sailors scored on the second play of the fourth quarter on a 46-yard scoring burst by Rose, who eluded several defenders on his way to the end zone. The extra point made it 28-6.
After the Sailor defense forced a turnover on downs, Broersma connected with a wide open Keondre Pierce for a 38-yard touchdown pass, making the score 35-6 lead with 3:39 to play.
The Chargers scored the final points of the contest on a 10-yard run by Juday with 2:52 remaining.
Trovinger led the Sailors with 103 yards rushing. Broersma rushed for 72 yards, threw for 94 and totaled two scores. Rose totaled 70 yards rushing with a touchdown. Pierce was the leading receiver with 70 yards and a score.
Mona Shores' Tre Word battles on the defensive line with his Midland counterpart. Photo/Eric Sturr
Mona Shores' Brady Rose hauls down a 47-yard touchdown pass from Caden Broersma. Photo/Eric Sturr
The Mona Shores defense swallows Midland's quarterback for a loss. Photo/Eric Sturr
Mona Shores' Jaylen Hopson breaks up a pass to stall Midland's drive. Photo/Eric Sturr
Mona Shores – District Champions
← Previous Story WMC volleyball team beats North Muskegon, wins long-awaited district championship
Next Story → Grand Haven's playoff run ends with a 35-7 loss to Hudsonville in district finals


Members who did not renew their membership by July 31 were dropped from the Member Directory and membership roster, unless that member made prior arrangements. Former members in good standing can reinstate their membership by submitting the membership fee. Former members are not required to submit a new membership application, unless their information has substantially changed.
IF are joining as a new member, please be sure to submit a Membership Form after submitting your membership fee.
Former members who missed the deadline ... don't despair. We want you to come back. We know that some members may have simply forgotten to pay. Former members in good standing can rejoin at any time without submitting a new Membership Application. However, if you have moved, built a new website, changed your phone number, etc. You may want to submit a new application so that our records are current.
We have opened the FALDP Certification Exams year round. We plan to keep the exams open all the time to allow members who are seeking certification to have easier access. The exam links remain inside the Members Only area. If you are a member and did not receive the email with the new login for the Members Only area, call 800-515-0496 or email staff@faldp.org and request it.
We have invited a videographer to come to the conference and take footage of our attendees and the conference. We'll be posting video of the conference on our YouTube Channel, our Facebook Page, and on our site. Take a look at our Conference Archives to see pictures and clips from past conferences.
Registration for our 8th Annual Conference is open. Go to our Annual Conference Page to register. The conference fee by itself is $95. During August/ September the conference fee plus membership is $170.
We invite attendees to bring along a guest at no additional fee. So bring a friend, a colleague, a staff member, a business partner, or prospective member. We may cancel this invitation to bring a guest as registrations come in, so please register early. At this time (8/14) we still have space for attendees to bring a guest. But, we expect that in the next week or so, we won't have the extra space left for the guest seats. If you plan to attend and bring a guest, please check in advance to make sure we still have seating.
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HMCS Donnacona members have memorable visit with veteran Wren
Navy News / December 30, 2020
by Sub-Lieutenant Simon Bullock, HMCS Donnacona
Two days before Remembrance Day, I received the most remarkable call.
"What the heck is going on for Remembrance Day this year?" said the woman on the line.
"Well, ma'am, due to the pandemic and public health measures, there will be no grand ceremonies, and citizens are encouraged to participate in the act of remembrance online," I said.
"Well, that's a shame!"
"I completely agree and understand."
We talked for a bit longer and it became clear I wasn't speaking to just anybody, but a woman of distinct character and passion. I later found out her name was Vicky Lockwood-Drummond, a Second World War veteran of the Women's Royal Canadian Naval Service (WRCNS).
"You must not talk to a lot of 97-year-old ladies, do you?" she continued.
"No, ma'am."
"Well, I'm old and you're going to need to speak up and clearly for me."
"Aye, ma'am."
I collected her personal details, phone number and home address.
Immediately after the call, I burst into my colleague's office at Her Majesty's Canadian Ship Donnacona, the Naval Reserve Division in Montreal, to tell them about the remarkable lady I had just spoken with.
"We have to do something for this lady, a card, a coin, something!"
I set to work coordinating with the Naval Association of Canada (NAC) to validate her credentials, and reached out to Lieutenant (Navy) Anne-Marie Belisle for public affairs oversight and consultation, Non-Public Funds to procure a ship's coin, and my Command team to review the copy of the letter to be drafted.
A flurry of activity later, everything was nearly ready to go.
On Remembrance Day, I arrived at my office early in the morning to draft a letter on behalf of the Commanding Officer in Copperplate script.
A few hours later, having just finished the letter, Acting Sub Lieutenant (A/SLt) Cole Henry Forster and I traveled to Valleyfield, Que., in order to assist the Chief of Staff (Eastern Region) Commander Mathieu Leroux and our ship's Coxswain, Chief Petty Officer 2nd Class Pascal Rioux. A somber wreath laying followed, in which we paid our respects to fallen comrades, but also the memory of His Majesty's Canadian Ship Valleyfield, a River-class frigate that was torpedoed and sunk in May 1944 during the Battle of the Atlantic.
Back at the Naval Reserve unit, I pursued my frenzied activity, making calls to A/SLt Forest to serve as the supporting Navy personnel, and Charles O'Leary from the NAC. Mr. O'Leary informed me that he would be joined by Anthony Colucci, also from the NAC. Some grand plans were hatched; however, I cautioned them lightly about turning this into anything too big as we didn't want to startle Ms. Lockwood-Drummond!
We arrived at Ms. Drummond's address with time to spare in a tentative formation outside her door and paused for a moment. I knocked gently at first, then more firmly.
"Deborah, hold on, I have to get the door," I heard Ms. Lockwood-Drummond say.
The door creaked open.
"Deborah, I have to go! THE NAVY IS HERE!"
"I'm Sub-Lieutenant Simon Bullock, we spoke earlier this week on the phone. How are you?" I asked.
"Oh my goodness, this is just wonderful! The Navy came to visit me! This means so much to me, you don't even know."
We were cautious to ensure we kept our physical distance due to the pandemic and were careful to keep our masks on at all times.
It didn't take her long before she launched into stories from her days in service. Her anecdotes were as full of life as she was, even in her advanced age.
Energy and vitality springing from every word, her eyes smiled and lit up as youthfully as the uniformed young lady framed on the shelf over her shoulder.
During our visit, we sang a tune or two popular during the war, and she recounted her favourite memories of her time in the WRCNS, in her own words below.
"Violet? You're not a violet, Violets are too nice; you're feisty."
"The first woman joined the Women's Royal Canadian Naval Service in October 1942; I joined on February 8, 1943. I tried to join on February 2, but due to me having lost an eye at eight years old, the recruiter deemed me unfit.
I said, what do you mean, I can't see!? I'm seeing you just fine right now.
Well, I was able to get the Admiral's number and called him to explain the absurdity of me not being able to serve due to missing one eye. I see just fine!
Well, six days later I got a call back and the Admiral had told them to let me in. That's how I became known as Vicky; I was victorious."
On burial and ceremony
Vicky went on to tell us about her burial plot in the Field of Heroes and how she wishes to have her family contact us when she dies to have us attend.
"Well I hope that isn't for quite some time," replied A/SLt Forest.
"Yes, I'm sure you can make it to 100," added Mr. Colucci.
"Oh God I hope not. Let's not drag this out; I've had a great run," replied Vicky.
"I really, really hope that the Navy could be present at my funeral, it would mean so much to me."
"As sad a day as that would be, it would be an honour. HMCS Donnacona will be there," I promised.
"You can't see it under this mask, but I'm smiling ear to ear," said Vicky.
"Your eyes are smiling and say enough," chimed in Mr. O'Leary.
"When Irish Eyes are smiling sure it's like a morn in spring," he sang in a light playful tone.
Without pausing, Vicky had caught on and echoed back: "In the lilt of Irish laughter, you can hear the angels sing."
Our faces all lifted as Mr. O'Leary and Vicky harmonized the rest of the verse.
"When Irish hearts are happy all the world seems bright and gay.But when Irish eyes are smiling sure they'll steal your heart away."
All of our eyes were a-glow peaking over our masks, and I thought I saw a tear fill her eyes.
Throughout all of her stories, she always would end on the note of how incredibly important the Navy was to her and her family. How much she loved it.
We said our heartfelt goodbyes and left her with a handwritten card and ship's coin.
"Oh, I am just so pleased! This is the most wonderful day," she said.
"I can't wait to call all my girlfriends and tell them that the Navy came to visit me! This is just the best. I have to call all of my family now and tell them. Thank you so, so much!"


As a pharmacist, I always understood the importance of proper drug disposal, but it wasn't until I watched my 2-year-old craft a staircase out of a shoebox, a club-sized package of paper towels and a box of cereal in his determination to retrieve an out-of-reach cookie that I witnessed its importance. What if he climbed high enough to get into the medicine cabinet? So many others have realized this, too. One of the most frequent questions I was asked in my career as a retail pharmacist was "How do you get rid of old medications?" Whether it was expired bottles of over-the-counter medications, unused antibiotics or unwanted narcotics from a recent surgery, there was a constant murmur about what to do with these medications. Clearly you don't want them just sitting around, but what are you supposed to do with them?
The easiest way to dispose of medications is in the trash. Simply mix the medicine with an undesirable substance like cat litter, coffee grounds or dirt. Place it into a container that you can seal, like an empty jar of peanut butter or a plastic bag with a zip top, and throw away in your regular household trash. I still hear of folks flushing their medications down the toilet, but this practice is only recommended for a select few flushable medications.
If throwing medicines in the trash worries you, another very popular option is through the National Drug Take Back Days. This is a day where you can drop off any medication with no questions asked to your local participating law enforcement or fire station. There are two National Drug Take Back Days each year (typically in April and October). The U.S. Food and Drug Administration cites that, since 2010, well over 5 million pounds of unused, unwanted medications have been disposed of. The next National Drug Take Back Day is April 28. To find a location nearest to you, check the Drug Enforcement Agency's (DEA) website.
A third option is to find a DEA-authorized collector in your area. These are public sites that have registered with the DEA to maintain a collection receptacle to take back and dispose of medications. You'll most commonly find one of these receptacles in a community pharmacy or a hospital with an on-site pharmacy.
Keep in mind, there may be some restrictions on these take back programs, and for those who are still looking for options, there are mail-in options, or your local waste management agency can direct you to other local programs.


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"The team at Carver did a great job for CRESS Systems - from finding the suitable property to handling the finer points that can complicate a completion. Would work with them again. "
"All staff were very helpful every time we called, Jack, Alison and Julie were most helpful. and would like to thank them for all their help in selling our shop"
"Highly recommended very professional & extremely helpful staff this is the second property I have rented through carver commercial & will definitely use them in the future "
"All the staff were most helpful and did a very good job in what turned out to be difficult circumstances. very good team work led by Julie Wallin "
"We appointed Carver Commercial to dispose of a vacant office in Darlington and were delighted with the professionalism and service we received during the transaction completing in a very timely and orderly manner. "
Jenny Haunch Managing Director Brooksbank Industries Limited Skipton Yorks.
"Carver Commercial completed the sale of our investment property on Gallowfields Trading Estate Richmond, a site of 18 business units plus compounds. Whilst on the market Carvers also let the vacant units for us as they became available, to maintain a good level of income. We have been extremely satisfied with the efforts of the team who have been very professional, responsive to our needs and helpful throughout the process. We would have no hesitation in recommending their agency and professional services in the future."
"We are extremely pleased with the service we received from Carver Commercial. They were very professional,but at the same time friendly and helpful, keeping us informed every step of the way as to what was happening with the sale of our property we highly recommend them. "
"On behalf of the Directors of Georgetown Victoria to express our thanks to Julie, Chris, Jack, Alison and Helen for all your hard work in securing tenants for all the units at Henson Road. I look forward to working with you and your team on this property and other opportunities in the future. Many thanks once again."
Danny Richards, Managing Director, IndustraCare Limited.
"" Nothing was ever an issue for your team or too much trouble and to summarise, the sale of this property was only made possible by the very hard work of Jack, Chris and Alison. I wouldn't hesitate to recommend Carver Commercial and i would use your company again without a second thought.""
""Very good service from Chris, Jack and the Follow up from Alison all of whom showed true professionalism through out""
""It has been a pleasure to deal with Carvers. I have found the team - Alison, Jack and Chris very proactive and quick to respond throughout our negotiations.""
"In 2014 I had several vacant shop properties in the centre of Darlington. I approached Carver Commercial for help in finding tenants for these properties, and in a relatively short period of time they secured tenants for them all. I can thoroughly recommend Carvers to anyone who has commercial property to let, they go the extra mile for their clients and are my first choice for help with commercial property management."
" Carver Commercial managed to dispose of a number of our assets in a challenging market, they kept me informed of the progress every step of the way. I would not hesitate to recommend them to others. "
" Carver Commercial has acted on our behalf for many years as letting agents and commercial property advisors within the Tees Valley area. Their local knowledge is unsurpassed and with a dedicated commercial property team in Darlington we have always found their finger to be on the pulse when tracking local, regional and national property requirements. Julie Wallin and her team are second to none and if there's a property requirement for the area Carver Commercial will know about it! "


text:
Ex-minister's son 'admitted drinking before crash'
Hirschson was driving a motorcycle with his girlfriend when he crashed, critically injuring her; released to house arrest.
By YAAKOV LAPPIN
APRIL 5, 2012 16:59
Police in Hod Hasharon 311
(photo credit: Zaka / Anshil Za'ak)
Police indicted the son of a former finance minister on Thursday with driving a motorcycle under the influence of alcohol and negligent conduct that led to a serious crash which injured his girlfriend.
Police prosecutors said at the Tel Aviv Magistrate's Court that Barak Hirschson, son of former finance minister Avraham Hirschson, "confessed during questioning to drinking alcohol an hour before the accident, and driving anyway."
Hirschson, 28, has 22 prior traffic violations, police said, including a 2006 conviction for drunk driving.
According to the indictment, Hirschson's blood-alcohol level at the time of the accident was 1.5 times higher than the legal limit.
Hirschson's girlfriend was critically injured in the crash this week, suffering a head injury that led to brain damage and spinal fracutures. She remains hospitalized at the Ichilov Hospital in Tel Aviv.
Judge Shmuel Melamed rejected the police's request to keep the defendant in custody until the end of the trial, and sent Hirschson to house arrest.
Hirschson's attorneys said he had never been involved in any criminal incident and described his prior traffic violations as minor.
Former minister Avraham Hirschson is serving a five-anda- half year prison sentence for theft of funds.


Arbit Scratchpad: Chaos & Fishes Cannibalizing their Offsprings!
The second iterate results in a 2-cycle periodic solution and then later this branches out and a 4-cycle periodic solution appears. Thus as R passes through a series of bifurcation values the character of the solution passes through a series of bifurcations, as period doubling of the periodic solutions.
As R increases through successive bifurcations, every even periodic solution branches into a double-periodic solution and this happens when R is such that the eigenvalue of the periodic solution passes through −1. The distance between bifurcations in R-space also gets smaller and smaller.
Bifurcation to higher periodic solutions eventually leads to chaotic behavior: Initially observe the solution oscillates between two points then it exhibits a 4-period solution then the solutions become chaotic. Again around a small window of R = 23-25 we see that the solutions exhibit regular periodic solutions after which they again become aperiodic/chaotic. So the same sequence of bifurcations get repeated and it is akin to a fractal, and the periodicity-aperiodicity-periodicity sequence is also repeated.


Within your hosting account create a database and a user with access to that database. It is important that you assign the proper database rights for that user.
CHMOD the temp directory to 777.
CHMOD the FCKeditoruserimages directory to 777.
CHMOD 755 the config_write. php file.
DELETE the install folder and all files in it.
Acc Autos 5.0 scripting tags: database, car, add, vehicle listing, acc autos, car dealership, search, car selling, cars. What is new in Acc Autos 5.0 software script? - Unable to find Acc Autos 5.0 news. What is improvements are expecting? Newly-made Acc Autos 5.1 will be downloaded from here. You may download directly. Please write the reviews of the Acc Autos. License limitations are unspecified.


Active Nation Scottish Cup holders Rangers were forced to battle for a 3-3 draw after almost being dumped at the fourth round by Hamilton.
Walter Smith's men appeared to be cruising after goals from Steven Whittaker and Kenny Miller, before Simon Mensing pulled a goal back from the penalty spot.
Accies then stunned their visitors when Marco Paixao and Mickael Antoine-Curier both netted in injury time of the first half, with Rangers suffering the added blow of Steven Davis and Kris Boyd limping off injured. However, Miller spared Rangers' blushes when he grabbed his second of the day from the penalty spot to ensure both teams have to do it all again.
Rangers needed just four minutes to take the lead. A Davis free-kick into the box was met by the head of Lee McCulloch who tried to pick out Boyd. Richie Hastings attempted to clear but the ball fell kindly for Whittaker to rifle high into the net.
Accies then squandered a great chance to level when a long ball from Trent McClenahan found Hastings in front of goal but he somehow managed to blast over from five yards when it looked easier to score.
Billy Reid's men were left rueing that miss when Rangers doubled their advantage after 30 minutes. A Davis ball into the box was touched on by Boyd for Miller and his finishing was clinical as he drilled past Tomas Cerny into the bottom corner.
Just when Rangers thought they were cruising, Accies pulled a goal back when Kirk Broadfoot was judged to have fouled Mark McLaughlin in the box and Mensing stepped up to convert with a powerful strike from the spot after 38 minutes.
The home side then stunned the visitors when they netted twice in stoppage time to head into the break with the lead. Paixao rifled home the rebound when Antoine-Curier's effort was blocked after a McArthur free-kick into the box caused all sorts of problems for McGregor.
Boyd then limped out of the action before Rangers were dealt another blow when Hamilton netted a third. David Weir slipped attempting to intercept Mensing's through ball and Antoine-Curier showed great composure to slot into the back of the net, with Allan McGregor stranded.
The Glasgow giants were back on level terms after 62 minutes when Elliott was harshly deemed to have deliberately handled in the box. Miller took responsibility for the pressure kick and slotted low past Accies goalkeeper Cerny. Accies where then dealt a blow when Hastings was dismissed with 10 minutes to go after picking up his second yellow card of the day for a foul on substitute Andrew Little.


text:
Category Archives: Media
Colin Emerson & Norman Bonney present the ESS petition to The Scottish Parliament
January 17, 2014 5:55 pm , Admin
Posted in: Campaigns, General update, Media , Tagged: colin emerson, norman bonney, petition, religious representatives, scottish parliament, secularism
Edinburgh Secular Society comment on the 2011 census religion statistics
October 8, 2013 4:11 pm , Admin
News release – For immediate release – 8th Oct 2013
Edinburgh Secular Society comment on the 2011 census religion statistics.
• "No religion" polls at 37 per cent…higher than The Church Of Scotland.
• Church of Scotland polls at 32 per cent…a 10 per cent fall since the 2001 census.
• Another 7.5 per cent of the total Scottish public turn their back on the Kirk since the last count.
• ESS questions the mandate of the Church of Scotland to have privileged access to our education system.
The religious statistics produced after Scotland's 2011 census show, as many had expected, that religion is an ever decreasing choice for many Scots. The 2011 census figures for Scotland reveal such plunging numbers declaring religious beliefs that it is predicted in a few years the total number of people declaring any religious belief will be a minority. The most striking figure is that "no religion" has increased dramatically to 37 per cent. This is higher than The Church of Scotland at 32 per cent which is a reduction of well over 400,000 from its numbers in the 2001 census.
An ESS spokesperson said, "We respect individual adult religious choice but the Church of Scotland cannot presume to speak for anyone other than its followers. It continues to impose its minority beliefs in our non-denominational schools and together with other religions has unelected representatives on all Scottish education committees. Almost two million Scots now declare that they have no religion. So what does that mean for the age-old religious assumption of church involvement in our education system? Religious Observance is still compulsory and evangelising missionaries use this to infiltrate the school system."
ESS continued:
"The right to evangelise is an important religious freedom but it does not extend to recruiting from our schoolchildren."
ESS calls on the Scottish Government to begin the process that will :
Repeal the legislation that requires Religious Observance is all Scottish schools
Repeal the legislation that imposes unelected religious representatives on all Local Authority Education Committees.
***Ends***
Speakers available for TV/Radio and other interviews. For further information contact Gary McLelland – Chairman on 07813060713 or Neil Barber – Press and Communications Officer on 07986183977.
ESS is a leading secular campaign group in Scotland. We are based in Edinburgh and cover a range of national issues relating to Secularism. ESS believes in freedom of religion and freedom from religion.
www.edinburghsecularsociety.com
Posted in: General update, Media, News Release , Tagged: census, church of scotland, education, non-denominational, nones, press, religion, religious observance, religious representatives
Leading constitutional academic criticizes the treatment of former Archbishop
October 6, 2013 12:01 am , Admin
News release – For immediate release – Sunday 6th October 2013
Leading constitutional academic criticises the treatment of former Archbishop.
Honorary President of Edinburgh Secular Society has issued a call for additional information about the safety and security of the former Cardinal, Keith O'Brien.
Edinburgh Secular Society speaks out against the treatment of the former Archbishop of Edinburgh and St Andrews by the hierarchy of the Catholic Church.
Professor Norman Bonney (pictured left – picture available here) is Honorary President of Edinburgh Secular Society, a position dating back to the mid-19th Century (see here). Prof. Bonney is a social science researcher, specialising in UK constitutional matters (http://www.normanbonney.blogspot.com/).
Speaking on behalf of the Society, Prof. Bonney asked: "Is the man at liberty or is he being held under constraint? Does he know that he is entitled as UK citizen to live wherever he would choose in the UK and the EU."
Prof. Bonney goes on: "As well as being excluded from the conclave of Cardinals that elected the current Pope he attempted to set up home in Dunbar (Scotland) but was apparently coerced by the Church to go into seclusion and exile.
"This case bears all the hallmarks of the 'kidnapped by a sect' story that happens from time to time in relation to much less significant denominations. The public of Edinburgh and St Andrews that the cleric attempted to serve over a long period deserve assurance that the Cardinal is a free man who is fully advised of his options by independent lawyers and is not held unwillingly under constraint."
Prof. Bonney continued:
"There are numerous disturbing aspects of the treatment of this individual that deserve investigation by journalists, and perhaps by the police. Probably he is entitled to an old age pension and as a homeless person the state and the local authority would have certain obligations to assist him to re-establish himself independently. There might even be a charitable congregation or organisation that might support him to live independently. Clearly, however, he will be under constraint. If he wishes to have a secure future with decent living conditions he may feel constrained to accept the authority of the Church and continue in his current circumstances.
"But there are alternatives. He could write a candid biography, like the former Scottish Episcopal Bishop of Edinburgh, Richard Holloway, which might be a best seller and provide for a more comfortable and freer retirement. He could certainly perform a valuable public function by elaborating further on the idea that Roman Catholic priests might be allowed to marry that he floated in his farewell interview while still in office."
Prof. Bonney is available for TV/Radio and other interviews. For further information contact Gary McLelland – Chairman on 07813060713 or Neil Barber – Media Officer on 07986183977.
Prof. Bonney speaks on behalf of Edinburgh Secular Society.
ESS is a leading secular campaign group in Scotland, based in Edinburgh we cover a range of National issues relating to secularism. ESS believes in freedom of religion and freedom from religion.
Posted in: General update, Media, News Release , Tagged: archbishop, catholic church, keith o'brien, norman bonney, president
Edinburgh Secular Society member Neil is featured in Herald Scotland
September 22, 2013 2:15 pm , Admin
Posted in: General update, Media , Tagged: neil gregory, parents, prayer spaces, prayers, religious education, religious observance, secular education


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redpajama_set_name: RedPajamaGithub


Gordon Huether Studio is by implication committed to enhance and beautify a space through the placement of artistically conceived and architecturally integrated artwork by drawing its cues from two essential design elements: light and space. In an ever so rapidly moving, disposable world, the Studio is committed to a much sought after precision, pristine craftsmanship and material longevity. It is a crucial part of the artist's philosophy to personally work with the client from inception of each project until the installation of the finished piece. A holistic understanding of the project needs, specifications, the client's expectations and visions, are elements, which provide the foundation of Huether's conceptual phase.
All of the work Huether designs is fabricated at the studio in Napa, CA. Our team of skilled professionals is internationally recognized for their innovative approach and creative craftsmanship in a wide range of glass work, composite fabrication, welding, wood and metal working. Each project receives detailed attention from its initial stages of planning and collaborating on design, throughout the entire fabrication process and the final installation of the work.
Private commission work by Gordon Huether Studio, as the term suggests, is a personal matter for the artist. A vital part of the design-development phase is getting to know our clients – their creative vision and the project intent. The production and final realization of the project is guided by an initial concept. Naturally, this concept morphs, changes and is shaped by factors such as space, creative vision of the client and the artist, materials used and the possibilities they offer.
From the inception of each project up to its completion and beyond, Gordon Huether is dedicated to providing high quality work, including a smooth production process, integrative, professional and prompt communication as well as a state-of-the-art design concept. Drawing from his extensive experience in collaborating with architects, landscape architects and interior designers, the artist is able to manage a diverse set of variables relating to project scope, materials and location.


text:
var sendMessage = require('../lib/sendMessage');
sendMessage('3 ' + process.argv.slice(2).join(' '));


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There are two sounds in the Hilton Head area. Calibogue Sound is located on the South end of the island and Port Royal Sound is on the North End. Homes that front on Calibogue Sound are in Sea Pines Plantation, the Point Comfort area, and the Spanish Wells area. Some property is located directly on the sound, but many homes are situated on rivers and creeks that are accessible by boat from the sound. Homes on the Port Royal Sound are located in Port Royal Plantation and Hilton Head Plantation. Homes that have docks and also have sound views are located in the Skull Creek area of Hilton Head Plantation.