A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.
Glycosylation is one of the most important post-translational modifications of proteins secreted in serum and is related to protein folding (1), quality control, sorting, degradation, and secretion. The glycan helps to stabilize polypeptide folding and indirectly allows glycoproteins to interact with various lectins, glycosidases, and glycosyltransferases by acting as recognition "tags" (2, 3). The glycoform distribution and degree of glycosylation of a glycoprotein can be significantly altered by diseases such as cancer, and glycoproteins have been reported in association with a variety of abnormal phenomena in patients with cancer (4–10). Thus, quantitative analyses of the glycosylation of proteins may be useful in discovering biomarkers for cancer.
Human plasma is clinically the most important sample, although it contains an extraordinarily large and complex proteome, with its dynamic range spanning more than 10 orders of magnitude in concentration. As conventional proteomic technologies only work within a small subset of this dynamic range (11), glycoprotein analysis in low concentrations typically requires glycoprotein enrichment methods, such as reactions with lectin (12–18), hydrazide chemistry (19, 20), HILIC (21, 22), or other procedures (23, 24) to reduce sample complexity while enriching the glycoproteins. However, such complex pretreatment steps are a cause of poor reproducibility. Also, the identification of glycopeptides and characterization of glycan information from mass spectrometric analysis is difficult because the instrumental sensitivity for glycopeptides is lower than that for general peptides, and a general database for glycopeptides is lacking. These factors make the discovery of a glycoprotein biomarker, and its validation in real clinical samples, extremely difficult.
Two analytical strategies have been used with glycoproteins: glycan and glycopeptide analyses. Many current glycan analyses chemically or enzymatically cleave glycoproteins to generate a pool of glycan moieties prior to mass spectrometry-based analysis (25–27) These analyses provide both quantitative and structural information on glycan moieties. However, because the sample is a pool of glycan moieties from a glycoprotein mixture, these analyses are unsuitable for the detection of specific glycoforms released from an interesting glycoprotein and for the identification of glycosylation sites.
For glycopeptide analysis, deglycosylated peptides released by endoglycosidase are generally analyzed by MS (18, 19, 25, 28–31). This method is useful for identifying glycosylation sites; however, it usually requires glycopeptide enrichment. Enrichment methods such as lectin affinity chromatography or reaction with hydrazide beads have been widely applied for the analysis of glycoproteins at low concentrations.
However, lectin affinity methods have shown less satisfactory in enrichment efficiencies. For example, in a membrane protein study of breast tumor cells, only 25 of 88 proteins (28.4%) and 34 of 152 proteins (22.3%) were identified as glycoproteins using a concanavalin A (Con A) lectin affinity column, and only 22 of 87 proteins (26.4%) and 27 of 146 proteins (18.5%) were identified as glycoproteins using a wheat germ agglutinin lectin affinity column (31). The more general approach of the hydrazide method may isolate a higher number of glycoproteins or glycopeptides compared with lectin affinity methods designed to enrich them with specific glycan moiety. Although McDonald et al. (28) reported considerable nonspecific binding of abundant proteins to hydrazide resin in their analysis of cell surface glycoproteins, they identified 589 proteins in HeLa cell plasma membranes using the hydrazide method, and 191 (32%) corresponded to glycoproteins. Lee et al. (32) demonstrated binding of nonglycosylated proteins with both the lectin affinity and hydrazide methods of glycosylation enrichment. These low enrichment efficiencies are attributable to weak binding of glycoproteins or glycopeptides and nonspecific binding of abundant proteins, and can result in data with low reproducibility. It is not adequate for quantitatively analyzing a large number of samples. Thus, a simpler and more practical MS-based proteomics technology without the need for any complex and irreproducible glycoprotein enrichment steps or ambiguous glycan structural interpretations is desirable for the discovery of new glycoprotein biomarkers.
Tryptic digestions tend to be incomplete when applied to glycosylated proteins, because of glycan steric hindrance (25, 33) However, the ways in which post-translational modifications affect trypsin digestion can provide crucial clues for the discovery of new glycoprotein biomarkers. For example, if there are differences in the degree of glycosylation of a protein between normal and diseased states, it would result in quantitatively different peptide products in accordance with the degree of glycosylation.
In particular, because peptides adjacent to N-linked glycosylation sites are sterically affected by bulky glycan groups during proteolytic digestion, the relative abundance of nonglycosylated tryptic peptides adjacent to N-linked glycosylation sites is altered post-digestion. Many studies (18, 34, 35) and a review article (36) have reported that the degree of glycosylation can be aberrantly high in diseased samples. Thus, we hypothesized that these nonglycosylated tryptic peptides adjacent to N-linked glycosylation sites may be a valuable biomarker, revealing quantitative differences in the glycosylation states of patients with cancer. Nonglycosylated tryptic peptides are not only detected with higher sensitively by MS, but also are readily identified and quantified using existing proteomic technologies, because of the absence of heterogeneous glycans.
In the current study, targeted nonglycopeptide biomarkers were identified and validated among glycoproteins in human plasma. First, the effect of glycan steric hindrance in glycoproteins was demonstrated via a tryptic digestion of glycosylated alpha-1-acid glycoprotein (AGP-T)1 and deglycosylated AGP treated with PNGase F (AGP-PT). AGP-T and AGP-PT indirectly indicated highly glycosylated disease plasmas and less glycosylated normal plasma, according to the degree of glycosylation. Second, one pooled normal and ten hepatocellular carcinoma (HCC) human plasma samples (sample information in supplemental Table S1) were analyzed using a label-free method consisting of nano-ultrahigh-performance liquid chromatography (UPLC) followed by quadrupole time-of-flight (Q-TOF) MS to identify candidate nonglycopeptide biomarkers. Nonglycosylated tryptic peptides adjacent to N-linked glycosylation sites from glycoproteins were targeted as candidate biomarkers because of observed, quantitative differences between normal and HCC plasma samples. Third, newly discovered nonglycosylated tryptic peptides were quantitatively validated by multiple reaction monitoring (MRM) using a nano-UPLC/triple quadrupole mass spectrometer from ten normal and eighteen HCC human plasma samples (sample information in supplemental Table S2).
Nonglycosylated tryptic peptide biomarkers were discovered without the need for complex glycoprotein enrichment methods using this conventional MS-based proteomics technology, where many of the limitations inherent in currently accepted glycoprotein biomarker discovery methods were avoided.
AGP standard protein (source: human), glucose-6-phosphate dehydrogenase (G6PD) standard protein (source: yeast), [Glu1]-fibrinopeptide B (GFP), dithiothreitol, iodoacetamide, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO). Trypsin for protein digestion was obtained from Promega (Madison, WI). Peptide-N-Glycosidase F (PNGase F) for deglycosylation was supplied by New England Biolabs (NEB, Beverly, MA). Acetonitrile was of HPLC grade and obtained from J.T. Baker (Phillipsburg, NJ). Water was deionized through a Millipore system prior to use (Millipore, Eugene, OR). An HP1100LC system (Agilent, Santa Clara, CA) was used to deplete the levels of the six most abundant proteins using a multiple affinity removal system (MARS) consisting of an LC column (Agilent, 5185–5984), solutions of Buffer A for sample loading, washing, and equilibration (Agilent, 5185–5987), and Buffer B for elution (Agilent, 5185–5988). The stable isotope-labeled peptides TEDTIFL*R, TINPAV*DHCCK, ENFLFLTP*DCK and GQYCYEL*DEK (isotopically labeled [13C and 15N] at amino acid sites marked with asterisks) were obtained from Anygen Co. (Kwangju, Korea). Carbamidomethylation of cysteine residues was performed in our laboratory.
Two AGP standard solutions were prepared at concentrations of 100 μg/10 μl. AGP solutions were reduced by adding with 1 μl of 1 m dithiothreitol at 60 °C for 30 min. Proteins were alkylated by adding 5 μl of 500 mm iodoacetamide in the dark at room temperature and allowing the solution to react for 30 min. One of the 100-μg AGP solutions was deglycosylated by reacting with 1 μl of PNGase F (500,000 u/mg) at 37 °C for 6 h followed by incubation and digestion in a solution of 10:1 trypsin (total protein:trypsin, by weight) at 37 °C overnight (AGP-PT). The other AGP solution was incubated in 1 μl of deionized water, instead of PNGase F, at 37 °C for 6 h. This solution was digested as described above (AGP-T). The two AGP digests were dried in a SpeedVac. The dried samples were diluted in mobile phase A and, as needed, spiked with isotope-labeled TEDTIFL*R prior to LC/MS/MS analyses.
Plasma samples were obtained with informed consent and in accordance with IRB guidelines from Yonsei University College of Medicine (Seoul, Korea).
Plasma samples of blood from healthy donors and HCC cancer patients were divided into four equal volume bags with an appropriate concentration of K2EDTA. Each aliquot was frozen and stored at −80 °C until use.
The six most abundant proteins in human plasma: albumin, transferrin, IgG, IgA, haptoglobin, and α1-antitrypsin were depleted using a HP1100LC system (Agilent) equipped with a multiple affinity removal column (MARC; Agilent). Crude human plasma samples were diluted by a factor of five with Buffer A (for example: 80 μl of Buffer A was added to 20 μl of human plasma) containing the proper amount of protease inhibitor (Complete Protease Inhibitor Mixture tablet (Roche, Indianapolis, IN)) and filtered through 0.22-μm filters by centrifugation (16,000 × g, room temperature, 1–2 min). Diluted crude plasma (70–100 μl) was injected at a flow rate of 0.25 ml/min; flow-through fractions were collected and stored at −20 °C. Depleted plasma samples were desalted by centrifugal filtration using 5000-Da MWCO (molecular weight cutoff) VIVASPIN filters (product VS061; Sartorius, Göttingen, Germany).
Aliquots (5–10 μg) of human plasma samples that had been quantitatively analyzed by Bradford protein assays were diluted with 100 mm Tris-HCl buffer (pH 8.00). An internal standard of G6PD was prepared in 100 mm Tris-HCl buffer (pH 8.00). The plasma and internal standard solutions were digested using the same AGP digestion process without the deglycosylation step. Briefly, human plasma samples and the internal standard were reduced, alkylated, and incubated as above. Digested samples were dried using a SpeedVac. Dried samples were frozen and stored at −20 °C until use. For label-free quantification, digested internal standard solutions were spiked equally prior to liquid chromatography tandem MS (LC/MS/MS) analyses. For MRM quantification, stable isotope-labeled peptide mixtures in each plasma sample were spiked equally prior to LC/MS/MS analyses.
Digested AGP samples were dissolved in mobile phase A, and stable isotope-labeled peptide standards of AGP were spiked equally in each AGP sample prior to nano-LC/electrospray ionization (ESI)-MS/MS analyses. Digested plasma samples were also dissolved in mobile phase A, and an internal standard of digested G6PD was added equally to each plasma sample prior to nano-LC/ESI-MS/MS analyses. MS/MS experiments for identification and quantification of proteins from peptide mixtures were performed using a nano-LC/MS system consisting of a nano ACQUITY UPLC system (Waters Corp., Milford, MA) and a Q-TOF mass spectrometer (Premier; Waters Corp.) equipped with a nano-ESI source. An autosampler was used to load 5-μl aliquots of the peptide solutions onto a C18 trap column (I.D. 180 μm, length 20 mm, and particle size 5 μm; Waters Corp.). The peptides were desalted and concentrated on the trap column for 5 min at a flow rate of 20 μl/min. The peptides were then separated at a flow of 450–500 nL/min on an analytical microcapillary column (C18, I.D. 75 μm, length 150 mm, particle size 1.7 μm; Waters Corp.). Mobile phase A consisted of water with 0.1% formic acid. Mobile phase B contained acetonitrile with 0.1% formic acid. Gradient elution began with 1% B for 0.33 min, ramped to 35% B over 84.67 min, then to 50% B over 0.5 min, retained at 50% B for 6.5 min, then ramped to 95% B over 0.5 min, retaining for 7.5 min and then to 1% B for another 0.5 min. The column was equilibrated with 1% B for 19.5 min before each run. For AGP, the total run time was 75 min with ramping to 40% B over 39.67 min. All analyses were performed using positive and V mode ESI using a nano-spray source. In label-free analyses, for accurate MS measurements, a lock mass of [Glu1]-fibrinopeptide B(GFP) at 300 fmol/μl was injected once every 30 s during a single LC/MS/MS run at a flow rate of 0.5 μl/min with NanoLock spray source. The voltage applied to produce the electrospray was 2.2 kV and the cone voltage was 35 eV. Argon was introduced as a collision gas at a pressure of 20 psi. DDA mode was used for AGP samples and to focus the database of human plasma samples. The MS scan range was 400–1600 m/z and the spectral acquisition time in each mode was 1.0 s with a 0.1-s interscan delay for DDA mode. Data-dependent peak selection of the three most abundant MS ions was applied and the collision energy was used as a means of charge state recognition. Elevated energy (MSE) mode was used for label-free quantification of plasma samples and all samples were analyzed in triplicate.
MSE is used in LC/MS studies in order to obtain fragment information about small molecules in complex mixtures, based on acquisition of exact mass data at alternating high and low collision energies. In low-energy MS mode, data were collected at constant collision energy of 5 eV to yield peptide molecular weight information. In elevated MSE mode, the collision energy was increased from 20 to 35 eV for peptide fragmentation. All MS data generated during the elution of each peptide from LC were collected for the identification and quantitative analysis of the peptide. In both low energy (MS) and MSE modes of acquisition, the mass scan range was 50–2000 m/z with spectral acquisition times of 1.5 s and 1.0 s, respectively, for MS and MS/MS modes with a 0.1-s interscan delay in MSE mode.
Peak lists were generated and processed using the MassLynx software (ver. 4.1; Waters Corp.). MS spectra were smoothed once using a 5-point Savitzky-Golay method, centered on the top 50% of each peak. The resulting raw files from each analysis were automatically processed into a single *.pkl peak list file. The resulting *.pkl peak list files were processed against the International Protein Index (IPI) Human database (version 3.22; 57,846 sequences; 26,015,783 residues) using the Mascot search engine (version 2.1; Matrix Science, London, UK). Mascot was used with monoisotopic mass selected, a precursor mass tolerance of ±1.5 Da, and a fragment mass tolerance of ±0.8 Da. Trypsin was selected as the digestion enzyme, with one potential missed cleavage. ESI-Q-TOF was selected as the instrument type. Oxidized methionine, carbamidomethylated cysteine, propionamide cysteine, and pyroglutamate (N-term E, Q) were chosen as variable modifications for identifying nano-UPLC/ESI-Q-TOF results in DDA mode. Deglycosylated AGP analyses also included deamidation (N). In a Mascot search, AGP came up as the first hit with high-scoring peptides above the search threshold (expected p < 0.05, peptide score >42). All of the Mascot search results from normal human plasma, and individual HCC plasma samples were summed. The focused database was composed of sequences from all of the identified proteins in the pooled normal plasma sample and the ten HCC individual plasma samples plus the sequence of the G6PD internal standard.
A continuum of LCMSE data was generated and processed using ProteinLynx Global Server software version 2.2 (PLGS2.2; Waters Corp.). Protein identifications were performed by using the forced database composed of the Mascot search results from the previous step. The ion detection, clustering of peptide components by mass and retention time, and data normalization were performed using PLGS, as described by Silva and et al. (37, 38). Briefly, all raw data were corrected for mass accuracy by m/z 785.8426, corresponding to the GFP ([Glu1]-fibrinopeptide B) standard that was used as the lock mass. MS spectra were smoothed with a 7-point Savitzky-Golay method, centered on the top 80% of each peak. Trypsin was used as the digestion enzyme with one potential missed cleavage. ESI-QTOF was selected as the instrument type. Oxidized methionine and carbamidomethylated cysteine were chosen as modifications. For quantification, exact mass was selected with a mass tolerance of 20 ppm and the fine retention time window was 1-min. The intensity measurements for the entire data set were normalized to the internal standard, G6PD. From these criteria, the peak, which was detected at least twice, was used to quantify both normal and HCC individual plasma samples.
MRM experiments were performed with a nanoACQUITY UPLC system (Waters Corp.) and a TSQ quantum ultra EMR triple-quadrupole mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with a nanospray source. With the exception of the gradient, LC conditions were the same as those described above for the selection of candidate biomarkers. The gradient began with 5% B for 0.33 min, then ramped to 40% B over 39.67 min, to 50% over 10 min, to 95% over 0.5 min, retained at 95% B over 9.5 min, and then ramped to 5% B over 0.5 min. The column was equilibrated with 5% B for 14.5 min before the next run. Optimized conditions were used for MRM quantification: quadrupoles Q1 and Q3 were held at 0.7 m/z FWHM with a scan time of 50 ms per peptide and 2.0 kV spray voltage. Optimum transitions and collision energy parameters were determined for each peptide by infusion of 10 pmol/μl peptide solution (Table III). Three transitions for each peptide were selected and monitored. First, precursor ions with a specific mass were transmitted from Q1 to the collision cell for fragmentation. Three fragment ions were then transmitted through Q3, yielding the signals used for quantification (39).
For quantitative comparisons of the label-free plasma samples, the results of exact mass and retention time (EMRTs) from the PLGS analysis were exported to an Excel spreadsheet (Microsoft, Redmond, WA), and all data were normalized using a linear regression analysis. Validated MRM results of target non-glycopeptides from the normal and HCC cancer groups were compared statistically using MedCalc (version 10.1.8.0, demo version). The diagnostic accuracy of each peptide from the candidate biomarkers was evaluated using receiver operating characteristic (ROC) curve analyses. The area under the curve (AUC) is reported at the 95% confidence interval (CI). The related sensitivity and specificity were determined.
Successful enzyme digestion results in a solution of peptides that are completely and accurately cleaved at expected sites. This only occurs when the enzymes can efficiently access the reactive cleavage sites. Thus, steric hindrance in a protein that interferes with enzyme access affects proteolytic digestion (25, 33). This effect is more pronounced in proteins such as N-linked glycoproteins that have been highly modified post-translationally. Steric hindrance from the glycan group(s) in N-linked glycoproteins has been thought to significantly affect the efficiency of proteolytic digestion if the enzymatically active amino acid is adjacent to the N-linked glycosylation site, resulting in quantitatively different peptide products in accordance with the degree of glycosylation.
This concept is shown in Fig. 1. The illustrations depicting N-linked glycoproteins are based on an article by Bunkenborg (40). Both normal (left) and aberrant (right) glycosylated N-linked glycoproteins are shown. After tryptic digestion, most of the peptides that are not related to the N-linked glycosylation sites will be present at the same concentration, with a relative abundance close to unity. However, concentrations of peptides that were adjacent to N-linked glycosylation sites, such as peptide 7, will be lower due to steric hindrance by the glycan in aberrantly glycosylated N-linked glycoproteins. Because steric hindrance increases with the size of the glycan unit, the abundance of peptide 7, which boasts a relatively large glycan moiety, will be significantly decreased. In diseased plasma samples, aberrant glycosylation of glycoproteins, such as increased levels of fucose and sialic acid, the addition of polylactosamine units, or higher-order branching of N-linked glycans, are known (33). The peak area ratios (aberrant/normal) of specific nonglycosylated tryptic peptides that originate from sites adjacent to N-linked glycosylation sites are thus expected to be lower than those of other peptides. These peptides were the target of this study: nonglycosylated tryptic peptides from N-linked glycoproteins.
Effects of glycan steric hindrance and the degree of glycosylation on the efficiency of proteolytic digestion are illustrated. If the degree of glycosylation or size of the glycan unit(s) differs between normal and diseased samples, then these differences will be reflected in the relative abundance of nonglycosylated tryptic peptides that were adjacent to N-linked glycosylation sites following digestion. In the diseased sample shown here, nonglycosylated tryptic peptides (4 and 7) and N-linked glycosylated peptides (5 and 8) will be cleaved with a different efficiency compared with those in a normal plasma sample. Furthermore, the relative abundance of nonglycosylated tryptic peptide 7 will differ in the mass spectra of normal and diseased samples. Peptide 4 will not be affected by glycan steric hindrance because "R" (the active site for trypsin) is distant from the N-linked glycosylation site. Thus, peptide 4 will be detected at similar levels in both the normal and diseased samples. Other peptides, except nonglycosylated tryptic peptide 7 and peptide 4, will be present at similar levels. Full lines represent general peptides and dotted lines indicate nonglycosylated tryptic peptides. Glycopeptides 5 and 8 could not be identified in the MS analyses and are not displayed in the third step.
First, the effect of glycan steric hindrance on trypsin digestion was demonstrated using AGP as a model compound. Two AGP digest solutions were prepared and analyzed by LC/MS/MS. One consisted of a tryptic digest of AGP (AGP-T). The other consisted of a tryptic digest of deglycosylated AGP that had already been treated with PNGase F (AGP-PT).
Fig. 2A shows a quantitative comparison of peptides from AGP-T and AGP-PT samples. The peptide sequences are given in Table I, where the underlined sequences indicate the quantified peptides. The nearest N-linked glycosylation sites in each identified peptide are indicated as 72N and 103N (in parentheses). Methionine oxidation generally occurs when a methionine residue is exposed to oxygen during digestion, and cysteine carbamidomethylation occurs during treatment with iodoacetic acid. The peak intensity ratio was much lower for the nonglycosylated tryptic peptide TEDTIFLR than for the other peptides (peptide 2, 3, and 4), which had peak intensity ratios of approximately one.
Quantitative comparison of tryptic peptide MS intensities from glycosylated AGP (AGP-T) and deglycosylated AGP (AGP-PT) samples. AGP-T; tryptic digests of glycosylated AGP. AGP-PT; tryptic digests of deglycosylated AGP with PNGase F. A, The ratio of peptide MS intensities. Peptide 1: TEDTIFLR, nonglycosylated tryptic peptide adjacent to an N-linked glycosylation site. Peptide 2: TYMLAFDVNDEK. Peptide 3: NWGLSVYADKPETTK. Peptide 4: EQLGEFYEALDCLCIPR. Italic bold cysteine residues and methionine residues were carbamidomethylated and oxidated, respectively. B, MS spectra of TEDTIFLR of AGP-T and AGP-PT. (I) AGP-T, (II) AGP-PT. AGP-T and AGP-PT samples were spiked equally with the stable isotope-labeled peptide TEDTIFL*R (of TEDTIFLR) prior to quantitative LC/MS/MS analyses. TEDTIFLR was adjacent to an N-glycosylated site and exhibited a lower abundance in AGP-T relative to AGP-PT.
This indicates that the production of enzyme-digested peptides that had been adjacent to the N-linked glycosylation site in AGP was affected by the steric hindrance of the glycan unit. Peptide TEDTIFLR was produced by tryptic cleavage at both sites 73K and 81R. However, because of the steric hindrance by the glycan linked at 72N, trypsin's access to the 73K cleavage site was inhibited. Indeed, it caused the peptide TEDTIFLR to be present at low levels in the AGP-T sample. Notably, the same peptide exhibited a much higher abundance in the AGP-PT sample where the glycan had already been removed by PNGase F treatment prior to tryptic digestion. Furthermore, the degree of this effect would increase with the size of the glycan and decrease with the length between the enzyme active site and the N-linked glycosylation site.
Stable isotope-labeled peptide, TEDTIFL*R, was synthesized and spiked equally into the AGP-T and AGP-PT samples prior to LC/MS/MS analysis. The relative abundance of peptide TEDTIFLR (light) and peptide TEDTIFL*R (heavy) in the AGP-T and AGP-PT samples is shown in Fig. 2B. The level of TEDTIFLR peptide in the AGP-T sample was less than 10% of that in the AGP-PT sample. This experiment again demonstrates that the amount of tryptic peptide, TEDTIFLR, was quantitatively different in AGP-T and AGP-PT samples.
A label-free quantitative LC/MS/MS method was applied to identify nonglycosylated tryptic peptide biomarker from HCC human plasma. Many of the labeling approaches in quantitative proteomics have potential limitations, including complex and expensive sample preparation and incomplete labeling. Conversely, label-free quantification approaches do not have these limitations and, above all, are not limited by the number of samples. Label-free methods are limited only by the reproducibility of LC and the accuracy of mass measurements in mass spectrometry.
In this study, all 11 plasma samples (one pooled normal and ten HCC plasma samples) exhibited highly reproducible retention times and accurate mass measurements for separated peptides. The standard deviations in the retention time and in the mass accuracy of the eight tryptic peptides from the internal standards (G6PD) were 0.19–0.30 min and 0.53–3.45 ppm, respectively (supplemental Table S3).
All of the plasma samples were analyzed in DDA mode for identification prior to label-free quantification. All identified proteins from 11 analyses (one normal and ten HCC human plasma samples) were combined to form a focused database. This database consisted of 94 proteins including single peptide identifications. The small size of this database, relative to the total human database, increased the reliability of label-free MSE results by the PLGS algorithm. Each sample was analyzed in triplicate, in MSE mode by LC/MS/MS. Peptides were identified and quantified by PLGS, developed by Sliva et al. (37, 38). Quantitative results were statistically normalized by linear regression, although the retention times of the label-free results were normalized by the internal standard peptides in PLGS.
Between the two groups, 133 peptides were quantified; 17 nonglycosylated tryptic peptides related to N-linked glycosylation sites are listed in Table II. In terms of the average ratio from all quantified peptides for each protein, the normal plasmas were similar to those of the HCC plasmas (Table II). In general, it ranged within ±30%. At the peptide level, however, some peptides changed over 30% between the two groups or showed quite different ratios in comparison with the averaged ratios of the protein levels.
We selected four targets of peptides, 1, 2, 4, and 7, as nonglycosylated tryptic peptide biomarkers that were not only adjacent to N-linked glycosylation sites, but also exhibited quantitative differences between normal and HCC human plasma samples (marked with an asterisk in Table II). Despite their proximity to N-linked glycosylation sites, peptides 8, 9, 13, 14, 15, and 17 showed small quantitative differences of less than 20% and were excluded from consideration of nonglycosylated tryptic peptide biomarkers. Nonglycosylated tryptic peptides, such as 3, 5, 12, and 16, were also not selected because they were distant from the N-linked glycosylation sites. Also, peptides 10 and 11 were not selected based on their poor MS/MS spectra by MSE (supplemental Fig. S1 presents MS/MS spectra of all nonglycosylated tryptic peptides in Table II except for peptides 10 and 11; the peptide probabilities from the PLGS search algorithm were over 95 for all peptides). Isoform HWM of Kininogen 1 precursor was identified by peptides 3, 4, 5, and 6. Among these peptides, peptide 4 was selected based on its proximity to N-linked glycosylation site.
Fig. 3A shows the extracted ion chromatogram of peptide TEDTIFLR, a nonglycosylated tryptic peptide biomarker (No. 1 in Table II) from AGP. Compared with those of other peptides (Figs. 3B, 3C, 3D), TEDTIFLR from HCC plasma showed a relatively low intensity in peak abundance. Such a result is consistent with our previous experimental data, obtained as a proof of principle. As we expected, this demonstrated that the difference in a protein's glycosylation state between a normal and diseased sample can be determined by specific nonglycosylated tryptic peptides. Other MS-based label-free results showing only quantitative differences in specific nonglycosylated tryptic peptide biomarkers are presented in supplemental Fig. S2.
Chromatograms of nonglycopeptide biomarker candidates and general peptides from AGP targeted proteins are shown as acquired by the MS-based label-free method (A–D). Nonglycosylated tryptic peptides and general tryptic peptides were differentially quantified between the normal and HCC groups. Italic bold cysteine residue and methionine residue were carbamidomethylated and oxidated, respectively. A, AGP - TEDTIFLR, nonglycosylated tryptic peptide biomarker candidate. B, AGP - TYMLAFDVNDEK, general peptide. C, AGP - EQLGEFYEALDCLR, general peptide. D, AGP - TLMFGSYLDDEK, general peptide.
Although the label-free method showed quantitative differences in the nonglycosylated tryptic peptide TEDTIFLR of AGP, the Western blot analysis of AGP was very similar between the normal and HCC plasma samples (supplemental Fig. S3). Western blot analysis without specific glycoprotein enrichment did not reveal any distinguishing information between the normal and disease groups, despite differences in the glycan groups or degree of glycosylation reported previously (41–43). Western blot analysis shows total protein levels and cannot accurately detect changes in protein glycosylation.
At the peptide level, peptide TEDTIFLR from AGP, peptide TINPAVDCCK from afamin precursor, peptide ENFLFLTPDCK from isoform HMW of kininogen 1 precursor, and peptide GQYCYELDEK from vitronectin precursor protein could be identified as nonglycosylated tryptic peptide biomarkers for HCC, without glycoprotein enrichment.
Triple quadrupole mass spectrometry yielded very specific and sensitive results, because all targeted peptides were monitored by two mass filters (MS1: parent ion, and MS2: specific fragment ions of the parent ion) during the MRM. MS-based MRM assays (44–49) have been proposed as an alternative to antibody-based biomarker verification due to the high throughput, selectivity, and sensitivity of using two mass filter stages. This approach was used to verify selected nonglycosylated tryptic peptide biomarkers in ten normal and eighteen HCC human plasma samples.
All of the plasma samples were prepared as in the previous identification step. Prior to the LC/MS/MS run, all of the plasma samples were spiked equally with four stable isotope-labeled standard peptides (heavy) of selected native nonglycosylated tryptic peptides (light) and analyzed by triple quadrupole mass spectrometry combined with the same UPLC system used for the label-free analyses. The optimum transition ions and collision energy conditions for each peptide were determined through direct infusion nanoflow-ESI experiments with stable isotope labeled standards (Table III). Calibration curves for each peptide also showed strong linearity in the range of concentrations from 10 to 500 fmol for the human plasma sample (2 μg; supplemental Fig. S4). The MS and MS/MS tolerances were Δ1 Da and Δ0.7 Da, respectively.
Quantitative MRM analyses were run in triplicate for each sample. Data that resulted in detection events in two or more of the three trials, within CV (coefficient of variation) < 30%, were accepted. supplemental Table S4 shows the CV% of light/heavy ratios of targeted nonglycosylated tryptic peptides from normal and HCC plasma samples by MRM. Data that yielded results under the limit of quantification for each peptide were removed. MRM chromatograms of the targeted nonglycosylated tryptic peptide and those of stable isotope labeled standard peptides are shown in supplemental Fig. S5.
The results of the MRM analyses were analyzed statistically using MedCalc (version 10.1.8.0). ROC curves and quantitative scatter plots showing the MRM results in both the normal and HCC groups are shown in Fig. 4. The target nonglycosylated tryptic peptides were present at quantitatively lower levels in the HCC samples relative to the normal group. This was consistent with the results from the label-free quantitative analysis (Table II). This corroborates the results of the label-free analyses described above. The area under the ROC curve (AUC) values of each peptide are shown in the Table IV. Sensitivity for the four targets ranged from 55.6 to 88.9%. Specificity was between 75 and 100% at the 95% confidence interval (CI). The AUC of peptide GQYCYELDEK, from a vitronectin precursor protein, was particularly high (0.978).
ROC curves and scatter plots of target nonglycopeptides from normal and HCC plasma, as determined by MRM quantification using triple-quadrupole mass spectrometry. Italic bold cysteine residues were carbamidomethylated. A, GQYCYELDEK- Vitronectin precursor. B, TEDTIFLR – AGP. C, TINPAVDHCCK- Afamin precursor. D, ENFLFLTPDCK- Isoform HMW of kininogen 1 precursor.
In fact, the mother proteins of four nonglycosylated tryptic peptides, AGP (41–43, 50), afamin precursor (51, 52), isoform HMW of kininogen 1 precursor (53), and a vitronectin precursor (35, 54–57), are well-characterized proteins related to HCC. Most studies have suggested that changes in the glycosylation of proteins are associated with hepatic diseases.
Recently, an antibody-lectin sandwich assay that recognizes asialo-alpha-1-acid glycoprotein (AsAGP) was developed and applied to 610 serum specimens from patients with hepatic diseases, 41 healthy donors, and 155 patients with nonhepatic diseases. AsAGP was validated as a marker for hepatic disease, with an AUC value of 0.919 (42). Pei et al. reported that afamin expression was significantly reduced in HCC tumor tissue compared with expression in normal hepatocytes (51). For kininogen, the level of the fucosylated kininogen (Fc-Kin) was significantly higher in patients with HCC (53). The AUC from the combination of Fc-Kin, Golgi protein 73 (GP73), and alpha-fetoprotein (AFP) as multipanel biomarkers was 0.93, which was higher than the individual AUC values of 0.79 for Fc-Kin, 0.88 for GP73, and 0.75 for AFP. Lee et al. found higher expression of the N-linked glycopeptides from vitronectin in HCC tumor tissue and HCC patient plasma (35).
In this study, we developed a novel mass spectrometric approach for the discovery and validation of biomarkers in human plasma targeting specific nonglycosylated tryptic peptides in an N-linked glycoprotein. Based on our hypothesis, steric hindrance by glycan groups in N-linked glycoproteins would significantly affect the efficiency of tryptic digestion if enzymatically active amino acids were adjacent to an N-linked glycosylation site. This results in quantitatively different peptide products in accordance with the degree of glycosylation. We identified four nonglycosylated tryptic peptide targets using one pooled normal and ten HCC plasma samples, and verified them using ten normal and eighteen HCC plasma samples.
The current approach has significant benefits for the discovery and validation of biomarkers. In terms of sample preparation, complex, time-consuming procedures and those with low reproducibility, such as glycoprotein/glycopeptide enrichment, are not necessary, whereas they have previously been required in conventional glycoprotein research. In terms of sensitivity, because we do not target glycopeptides, but instead nonglycosylated tryptic peptides, they can readily be detected and identified by MS without PNGase F treatment. This approach also reveals differences in the degree of glycosylation in glycoproteins, which are not seen by Western blot analyses. Above all, this approach is simple and practically applicable to large numbers of complex clinical samples, such as plasma.
We thank Dr. S. Y. Cho for his helpful advice in plasma sample preparation.
↵* This work was supported by the Converging Research Center Program (2011K000884) through the Ministry of Education, Science and Technology (2009-008146) and Korea Basic Science Institute research grant (G31121).
This article contains supplemental Figs. S1 to S5 and Tables S1 to S4.


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The Impact of Poor Health on Retirement Age
Posted on September 1, 2007 March 26, 2017 By economica
by Christopher Bruce
This article first appeared in the autumn 2007 issue of the Expert Witness.
In personal injury cases, plaintiffs often argue that their disabilities will induce them to retire early and, therefore, cause a reduction in lifetime earnings. As such claims are largely based on the plaintiff's own testimony, it is often difficult for the courts to determine whether the claim is credible and, if so, to identify the number of years by which retirement will be accelerated.
The purpose of this article is to assist the court in these determinations by providing a survey of the academic literature on the effects that health limitations have on the age of retirement. As the first edition of my textbook, Assessment of Personal Injury Damages (Butterworths, 1985) contains a survey of the early literature on this topic, I concentrate in this paper on articles published since 1990.
This yields a set of eight studies. Of these, two (Disney, for Britain, and Campolieti, for Canada) reported only that a negative change in health or disability status among individuals over 50 had a "significant" negative effect on the age at which those individuals retired.
Of the remaining studies, two provided data concerning the impact of alternative levels of health status on the probability that 50-65 year-olds would be working. Au, Crossley, and Schellhorn, using Canadian data from 2000-2001, reported that even a minor change in health status, from "very good" to "good," would reduce the probability of employment by 10 percent. (See Table 1.) And a change from "excellent" to "poor/fair" could reduce employment by as much as 40 percent (among males).
Similarly, Cai and Kalb, using Australian data from 2001, found that a change in health status from "excellent" to "poor" would reduce the probability that individuals would be in the labour force by approximately 16-18 percent. (See Table 2.)
At age 55, these reductions in probabilities imply that individuals in poor health will retire between one and two years earlier than those in very good health. This is consistent with Gustman and Steinmeier's finding, for the United States, that individuals who were "limited in the kind or amount of work" in which they could engage could be expected to retire two years earlier than those not so-limited.
Berger and Pelkowski, for the United States, and Jimenez-Martin, Labeaga, and Prieto, for Spain, also found impacts that were similar to those found by Campolieti and Au, et. al., but using somewhat different measures of health status.
Jimenez-Martin et. al. reported that 55-65 year-old individuals with "severe disability" were 14.6 percent less likely to be employed than were the non-disabled, and that those with "very severe disability" were 28.5 percent less likely to be employed than were the non-disabled.
Berger and Pelkowski found that among 51-61 year-old couples in which both the husband and wife had (at the beginning of the study period) been healthy and employed, the effect of a health problem was to reduce the probability that the wife would be working by 19 percent and that the husband would be working by 35 percent.
Finally, McGarry found that a change in health status from "good" to "fair" would reduce the probability that a 62 year-old would be working from approximately 45 percent to 40 percent.
To summarise, regardless of the country that is investigated, the evidence is clear: a reduction in health, from "good" to "fair or poor" will have a significant, negative impact on the probability that 50-65 year-old individuals will be working. Although the precise effect of such a reduction varies from study to study, there appears to be fairly consistent evidence that the average effect is to reduce the age of retirement by approximately two years (for example, from age 61 to age 59).
Au, D. W., T. Crossley, and M. Schellhorn (2005) "The effect of long-term health on the work activity of older Canadians." 14 Health Economics, 999-1018.
Berger, M., and J. Pelkowski (2004) "Health and family labor force transitions." 43 Quarterly Journal of Business and Economics, 113-138.
Cai, L., and G. Kalb (2006) "Health status and labour force participation: Evidence from Australia." 15 Health Economics, 241-261.
Campolieti, M. (2002) "Disability and the labor force participation of older men in Canada." 9 Labor Economics, 405-432.
Disney, R., C. Emmerson, and M. Wakefield (2006) "Ill health and retirement in Britain: A panel data-based analysis." 25 Journal of Health Economics, 621-649.
Gustman, A., and T. Steinmeier (2000) "Retirement in dual-career families: A structural model." 18 Journal of Labor Economics, 503-545.
Jimenez-Martin, S., J. Labeaga, and C. Prieto (2006) "A sequential model of older workers' labor force transitions after a health shock." 15 Health Economics, 1033-1054.
McGarry, K. (2004) "Health and retirement: Do changes in health affect retirement expectations?" 39 Journal of Human Resources, 624-648.
Christopher Bruce is the President of Economica and a Professor of Economics at the University of Calgary. He is also the author of Assessment of Personal Injury Damages (Butterworths, 2004).
Posted in Article, Christopher Bruce, Disability and earnings, RetirementTagged Disability and Earnings, newsletter (volume 12 issue 2), Retirement
Autumn 2007 issue of the Expert Witness newsletter (volume 12, issue 2)

redpajama_set_name: RedPajamaCommonCrawl


Painter, illustrator and Belgian poster artist. Large original lithograph circa 1920. Signature in the board at the bottom right. Titled on the lower right. Dimensions of the subject 28 x 79 cm. Dimensions with margins 35 x 85 cm. Original proof very rare in the state. Thank you for your questions. The item "fernand fernel rare original automobile lithograph ed dietrich" is on sale since Monday, July 2, 2018.
It is in the category "art, antiquities \ art of the nineteenth and before \ prints, engravings, lithos". The seller is "jicky28" and is located in / in chapet. This item can be delivered anywhere in the world.


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Graduates praise value of industry experience at hired@UCB placement fair
Graduates from University College Birmingham were back on campus seeking the next generation of students to take on valuable industry placements.
More than 30 companies were in attendance at a placement fair held by the University's hired@UCB employability team.
The event, dedicated to students from The Business School and College of Food, saw employers spread across three floors of McIntyre House to talk with students about the development and training available through work placements.
And among the representatives in attendance were several former UCB students encouraging the current crop to follow in their footsteps.
BA Hospitality Business Management graduate Liz Burton, who is now General Manager at The Ivy Collection restaurant in Birmingham's Temple Row, revealed how going on placement during her course helped her gain the experience needed to start her career.
"I did my placement at a hotel in Dublin and it was my first time working away from home," said Liz. "It was an amazing experience for meeting new people, and I had to build a life in a new place which I think was really valuable.
"It gave me enough experience to get my first proper job out of university because I had actual experience on my CV and not just a degree.
"Several of our management team came through UCB and I know the calibre of the students here is high," she added. "Quite a few of our staff go to UCB now so we know we can potentially find some good people here."
Fellow ex-UCB student Simon Warren, who graduated in MA Culinary Arts Management and is now Restaurant Manager at The Ivy Temple Row, explained how a successful industry placement can open the door to a job opportunity at the same company.
"Students coming to work for you could be fantastic employees in the future," he said. "If a student had a successful placement and potential then, there could be a job available for them afterwards to go into.
Simon, who did his course placement at a hotel in Cyprus, added: "I know how beneficial placements can be for students and business because I went through it myself.
"Hospitality can be quite a testing environment and it's good to know what it's like before you get into it."
Other companies attending the event included the Eden Hotel Collection, represented by former UCB Hospitality Business Management student Ali Abdullahi.
"We've had UCB students on placement and part-time work before," said Ali, who currently works at the group's Arden Hotel in Stratford-upon-Avon. "We look for people who are driven and self-motivated – the students we have from UCB are all driven and they've got that passion they need to work hard.
"They're enthusiastic and want to learn, so hopefully again we can find more passionate students who want to come and work for us.
"Placements give you a clear indication of what you could become potentially and how hard you have to work to be there. It gives you an idea of the reality of the work."
One current student who already knows the value of placement is Joshua Topkar. The second year BA Culinary Arts Management student secured a placement at Dunboyne Castle Hotel and Spa in Ireland with the help of the hired@UCB team.
"This was my first time working as a chef so I've gained a lot of practical and theoretical skills," said Joshua. "Plus I've learned how to work in a functioning kitchen."
Also in attendance at the placement fair were the likes of Marriott, Hilton, Whitworths, Holiday Inn, Jurys Inn and Mitchells & Butlers, while the hired@UCB placement team were on hand to assist students.
Find out more about our range of courses within the College of Food and The Business School.
For more information about our hired@UCB employability service, click here.
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Our Schools Why Choose Us? Our Industry Links History of UCB Awarding of Degrees and our Partnership with The University of Birmingham Policies and Procedures OFS Transparency Return Accolades and Awards Mission Statement Enterprise Hive Latest News University League Tables Statement Job Vacancies
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San Francisco is one of California's most popular vacation destinations. Although it is only 45 minutes from Napa Valley and Sonoma, it's wine country is both unique and beautiful.
Santa Cruz is a great place to go on a wine tour. Explore four different wineries and at the end of your tour explore a sweet Santa Cruz neighborhood and feel free to shop, walk around or sit down and enjoy a nice dinner.
Most people drink alcohol because it makes them feel good.
But that's true only up to a certain point, after which alcohol makes you feel worse, then bad, then sick, then…well…dead.
Creekview Vineyards was established in 2005 and released their first vintage in 2007. The winery and vineyards are situated in the hillsides just east of the town of San Martin in the Santa Clara Valley wine region.


The poor export showings in December and January marked the first time in 27 months that shipments have fallen in consecutive months on an annual basis and are an indication of tougher times ahead for Asia's fourth-largest economy, experts here said.
Sung Tae-yoon, an economics professor at Yonsei University in Seoul, said the downturn is due to weaker global chip prices, with an unfavorable trade environment adversely affecting the country's export prospects as a whole.
"South Korea seems to be losing its competitive edge in the export sector," he claimed.
Reflecting this, most thinks tanks, trade-related organizations and brokerages warned outbound shipment growth of the world's sixth-largest exporting nation may be halved in 2019 from the year before.
Samsung Securities Co. adjusted export growth numbers from 5.5 percent to 2.5 percent, while Eugene Investment Co. downgraded the country's export growth estimate from 3.5 percent to just 1 percent.


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Q: React Navigation Custom Header rendering multiple times I have a simple React Navigation 5 stack component. I am inserting a custom Header component into the screenOptions of the navigator like so:
    <NavigationContainer>
      <Stack.Navigator screenOptions={ {headerShow:true, header:(p)=>
        <Header/> }}>
        <Stack.Screen name="Home" component={Home} />
        <Stack.Screen name="Notifications" component={AssetExample} />
      </Stack.Navigator>
    </NavigationContainer>

My header component is quite simple as well.
export default function Header({
  navigation
}) {
  console.log("WHATS HAPPENING HERE")
  return (
    <View style = {styles.container}>
        <Text style = {styles.paragraph}>
          Something text here
        </Text>

    </View>
  );
}

The console log statement in the header prints multiple times and I am unsure why that's happening.
Here is the link to the snack as well https://snack.expo.io/@mmanojr/restless-crackers
Any help is much appreciated.

redpajama_set_name: RedPajamaStackExchange


text:
Community Action for Justice in the Americas
Building a grassroots people's movement for social, economic, and environmental justice throughout the Americas.
Call for International Solidarity
The following call for international solidarity needs our attention. Please help the people of this region of Colombia. Either as an individual or as an organization copy, paste and then sign this letter and then email it directly to the International Peace Observatory at info@peaceobservatory.org
CALL FOR INTERNATIONAL SOLIDARITY TO CEASE TO STATE PERSECUTION AGAINST THE PEASANT'S ASSOCIATION OF THE CIMITARRA RIVER VALLEY (ACVC)
Peasant's Association of the Cimitarra River Valley Barrancabermeja, Colombia
We make a call out for national and international solidarity to cease the state persecution of the Peasant's Association of the Cimitarra River Valley (ACVC), an organization that carries out community, political and social initiatives in the rural municipalities of Yondó, Remedios and Segovia, in the department of Antioquia, and Cantagallo, San Pablo, Simití and Santa Rosa in the southern part of the department of Bolívar. They also have a regional office in the city of Barrancabermeja, all of which are within Colombia´s Middle Magdalena region.
The ACVC implements the goals of the community through 120 Villages Community Councils, cooperatives, fishing and mining committees, women's and youth collectives, along with other peasant worker associations, in the framework of integral defense of human rights and land, as well as the implementation of productive projects for food security, coca crop substitution, and the training and promotion of peasant- farmer organizing processes.
On Saturday, September 29th, 2007 officials of Colombia's Adminstrative Security Department (DAS) detained Andrés Gil, the ACVC's Office Coordinator in Barrancabermeja; Mario Martínez, the treasurer of the ACVC; Óscar Duque, member of the ACVC's Work Team in the Middle section of the Cimitarra River Valley; and Evaristo Mena, member of the ACVC through the Village Community Council of the La Poza, Cantagallo – all of which have been recognized community leaders for over two decades in the region of Middle Magdalena. They are currently imprisoned in Bucaramanga (department of Santander). Today, October 2nd, they will be taken to court to enter pleas.
These detentions are part of the new juducial process against the ACVC, which has also included raids of the apartment and the office of the organization in Barrancabermeja, during which three CPU's with 11 years of documentation of the association's projects, accounting, documents, photographs, and other files were taken. These actions comprise a new state attack to dismantle the peasant organizing process of Middle Magdalena region and disintegrate community development initiatives and the defense of human rights in the Cimitarra River Valley.
This offensive is not new. It is part of a series of interventions that the Colombian State has made against the ACVC over a period of many years and has manifested in acts like the government's incompliance with the agreements signed during the Peasant's Exodus of Middle Magdelana in 1998 and its indolence in applying security measures made to protect members of the ACVC, which were requested by the Inter- American Human Rights Commission.
Likewise, the legal designation of the Peasant Reserve Zone of the Cimitarra River Valley – collectively- owned land established under the Law 160 in 1994 (also known as agrarian reform) which is the equivalent to an Indigenous people's reservation or Afro- Colombian's collectively- owned land – was revoked in 2003. This designation seeks to prevent, if not eliminate, large landholdings, and facilitate processes of redistribution and prevention of division of the plots for mega-development. Additionally, it regulates the settlement on untitled land, giving preference to peasants with scarce resources. That is why the Peasant Reserve Zone is seen as a barrier to the execution of resource exploitation projects like the recently signed agreements with AngloGold Ashanti Mines, a transnational gold company, and investment in the agroindustrial monocrop African palm oil.
On top of all of this, the actions of the Colombian National Army in the region have been directed against the civilian population, by means of: murders of peasant-farmers, arbitrary detentions, use of informants (people often pressured to make up false intelligence for Army), alliances with paramilitaries, robbery, torture and other serious human rights violations.
We reiterate our call out for solidarity and invite our friends, allies, and solidarity organizations – in Colombia as well as in the rest of the world – to promote the following initiatives:
- Solicit a hearing in the Colombian Congress regarding the human rights situation in the Peasant Reserve Zone of the Cimitarra River Valley.
- Denounce, accompany and search for political support for the ACVC in Canada, United States, Europe and Latin America.
- Request a hearing of the Inter- American Human Rights Commission of the Organization of American States to review the case of persecution of the ACVC.
- Convene a meeting of the European Parliament and the European Commission to expose the violations and persecution against the ACVC.
- Expose and disseminate cases of murders, violations and persecution against the ACVC before the World Organisation Against Torture (OMCT), the Human Rights Commission of the United Nations and the International Criminal Court.
Once again we declare that the freedom, physical integrity and life of the members of the ACVC is the sole responsibility of the institutions and government officials who, in the name of the State, should work to guarantee the rights of the peasant population instead of promoting their persecution.
For these reasons, we the undersigned, support the preceding declaration of the ACVC, reaffirming their total legality and legitimacy. We request the immediate release of the imprisoned and suspension of the arrest warrants against the rest of the board of directors.
Posted by CAJA at 9:18 PM 12 comments:
Noam Chomsky: American Foreign Policy in Latin America (2)
This is Part 2 of this video. For Part 1 page down.
Posted by CAJA at 6:58 PM 8 comments:
Noam Chomsky: American Foreign Policy in Latin America (Part 1)
Professor Chomsky looks at the history of the inequitable relationship between the United States and Latin America.
Way Cool
Thanks Jay for setting this up, we can use this site for continuing discussions when we run out of meeting time, for any kind of conversation or article,book,etc.. any member thinks would be of interest, for posting pictures or for staying in touch when we are travelling, whatever. We just want to see the conversation remain civil and hopefully focused on the "project" to which we are all committed.
As a board member I just want to say thatI believe our re-organization of late ( no paid staff, no office rent, membership based funding, etc) will make us a more authentic organization. Authentic in the sense that we don't have to tailor our programs or even ideology to fit the moderate views of foundation donors, we can fight for the kind of change these large entities are uncomfortable with. This deep change is of course what the times call for, it is the historical mission of all who realize the gross inequity built into our social, political and economic systems.(this is Dave,by the way)
Update from Scott in Colombia
Photo of Alonso Campiño (and hundreds of other people!) singing the Arauca anthem at the start of the Senate hearing in Saravena. His shirt has the photos of Leonel Goyeneche, Alirio Martinez, and Jorge Prieto – three renowned community leaders that were killed by the army in Caño Seco on August 5, 2004
On November 12, 2002, the military and police rounded up more than 2,000 people in Saravena and took them to the sports arena in the first mass arrest in the state of Arauca. On September 27 of this year, more than 2,000 people again filled the arena – this time to denounce the human rights abuses committed by the military and police during the past five years. The Human Rights Commission of the Colombian Senate held a public hearing in the arena that day which lasted for more than six hours.
Bernardo Arguello was the first victim to testify. He described being taken from his home and brought to the arena on that morning of November 12. "We were tied together and forced to walk past a car with polarized windows." Two paid informants inside that car pointed out the people to be arrested. Bernardo was imprisoned for 15 months for "rebellion." It appears that his real crime was being a leader of the Saravena Community Water Company – a very inspiring company that is owned and managed by the people of Saravena. Bernardo returned to Saravena after his release from prison and, less than a month later, he was arrested again – this time for "terrorism" and he spent another 26 months in prison. "I will continue working for our communities as I have always done," said Bernardo, "but I fear for my safety, and I hold the government responsible for what could happen to me and my family."
Sandra was the second victim to testify. She described the murder of her husband by the military during the La Cabuya massacre on November 20, 1998. The army killed five people in the community that night, including a woman who was seven-months pregnant. "They pounded on the door and my husband opened it – begging them not to kill him," said Sandra. "They knocked him to the ground and then took him outside and shot him." She said that as a victim of government repression, she wants the truth to be known and justice to be achieved. Sandra concluded, "I hold the military and police responsible for what could happen to me," in reprisal for her testimony.
Alonso Campiño read the report of the Arauca Social Organizations. The military and police came to his home early in the morning of August 21, 2003 during the second mass arrest in Saravena. Alonso's three-week-old son, Marlon, was grabbed from his father's arms. His twelve-year-old son, Camilo, was forced to the ground and a soldier put a boot on his back. Alonso was arrested for "rebellion" and spent three years in prison and house arrest. It appears that his offense was being a teacher, vice president of the Arauca labor federation, and a leader in the movement to prevent Occidental Petroleum from drilling for oil in the territory of the U'wa indigenous people. Alonso said, "More than 120 community leaders have been kidnapped (arrested) by the government in Arauca, and there could be more arrests prior to the October 28 elections."
Our friends in Saravena worked really hard in preparation for the Senate hearing, and they were very glad that the arena was filled and people had the courage to denounce the abuses they had suffered. Two hundred people filed complaints, either publicly or in private, during the course of the hearing.
The organization of the public hearing was generously supported by the Lutheran World Federation which is working with four excellent social organizations in Arauca. I returned to Colombia just three days before the hearing, and I'm very fortunate to be working now as a volunteer with the Lutheran World Federation as their representative in Arauca.
In love and solidarity,
Community Action for Justice in the Americas (CAJA) challenges and seeks to fundamentally alter the unfair distribution of wealth, power, and resources. Through education, advocacy, and grassroots organizing, we strive for social, economic, and environmental justice in solidarity with marginalized people throughout the Americas.
Scott Nicholson: Scott is one of the founders of Community Action for Justice in the Americas. For the past several years, he has been working in Colombia to provide on-the-ground accompaniment to social leaders whose lives have been threatened because of their work to bring peace and basic human rights to their country.
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Mariah Carey Becomes First Artist To Score Number One Hit Singles In Four Decades
Bow to your queen.
All bow to the queen of the charts!
There are thousands of songs released each year, so to score a number one hit is not an easy task.
Most singers would be happy to score one chart topper.
But pop diva Mariah Carey just proved that she's not just an ordinary singer.
A feat that's never been done before
According to news agency AFP, the 49-year-old made histoy by becoming the first singer to achieve a number one hit in four separate decades.
Based on sales, streaming data and radio airplay, her ever-popular holiday tune "All I Want For Christmas" topped the Billboard Hot 100 for the week dated 4 January 2020.
With that, Carey has had a number one single in the 1990s, 2000s, 2010s and 2020s.
The song was first released in 1994 and gained new fame after its inclusion on the soundtrack for the 2003 film 'Love, Actually.'
But it only topped the charts for the first time in late 2019, thanks to a change in Billboard's rules as to what can be included on the top songs list.
For this year's festivities, Carey released a new music video for the song in YouTube and to date, it has over 30 million views. She also released the song as a stand-alone CD single for the first time.
According to Billboard, it is her 19th number one hit, putting her just one song behind The Beatles' overall record of 20 top songs.
And here's a fun fact: CNN reported that the song has earned Carey more than USD60 million (RM246 million) in royalties since the day it was released!
Carey celebrated the announcement with a Tweet:
Yaaaaaaaaay!!! WE. DID. IT. 🐑🍾🎊🎉💖🥳🤯
But.... what's a decade? https://t.co/OaNK23GFuN
— Mariah Carey (@MariahCarey) December 30, 2019
Congratulations, we say!
Ayumi Hamasaki Is Losing Her Hearing Completely
Bollywood Fans, Kareena Kapoor Is Coming To Malaysia On 7 July
What's Inside An Oscar Nominee's Goodie Bag? We Find Out
The World Just Lost The Man Who Played The Best James Bond


Cocoa butter is the edible vegetable fat from cocoa beans, extracted from the cocoa beans during the process of making chocolate and cocoa powder. Cocoa butter has only a mild chocolate flavor and aroma.
There were 1,040 U.S. manufacturing establishments producing chocolate and cocoa products in 2001. These establishments employed 45,913 people and shipped $12 billion worth of goods that year.
California led the nation in the number of chocolate and cocoa manufacturing establishments (with 116) followed by Pennsylvania (with 107).


Summit Brewing Co. has released the newest beer in its Unchained series of limited-edition, individual brewer creations.
The beer, created by Gabe Smoley, is a 100 percent organic ale. It's a somewhat hoppy, slightly sweet beer that should go well with almost any food.
Smoley had to create his own yeast strain to ensure the beer was 100 percent organic. Now, that's dedication.
The beer is available in most liquor stores.


In high school, students have the freedom to choose their own classes. Right?
Several Granite Bay High School students say they had little influence on their schedules, some having no involvement in the decisions of what classes they would take as a new student.
A junior boy, who wished to remain anonymous to keep his thoughts truthful and honest, transferred to GBHS in August 2017.
The student says he didn't have a choice in his electives, either.
"I was hoping to take journalism or some other elective that was listed in the catalog," the anonymous student said.
The student was also discouraged from taking any AP classes, and was placed in standard CP courses instead.
Junior Jillian Conklin was enrolled in a Spanish 3 class, but her teacher later decided she wasn't yet ready to take the course. She was instead placed in Spanish 2 again, even after passing it in the past term.
Conklin said she is very happy in Beginning Piano, and says she felt better when everything was taken care of.
Counselor Tiffani Gieck says there are many factors to consider when creating a transfer student's schedule, including the timing of transfer, availability of class options in the master schedule, and credit and graduation requirements.
Students often transfer from schools with traditional schedules with 6-7 classes to the RJUHSD block system, with 4 classes per semester. This further challenges counselors help students adjust well.
When a transfer student is cleared by the district for enrollment, the counseling department tries to place them in classes as soon as possible.
If a student's family is flexible in when to start attending GBHS, counselors try to have them start at the beginning of a grading period to make the transition easier.
Gieck says it is easier for students to adjust with a manageable course load.
The counseling department assesses a student's past transcript to choose classes that will lead the student to success.
Student Government, the Peer Resource Center, and Link Crew collaborate to involve the new student into GBHS life.


text:
module Capistrano
  module Sneakers
    VERSION = "0.2.0"
  end
end

redpajama_set_name: RedPajamaGithub


'Construction requires all our help to achieve lower carbon emissions across UK'.
47% of UK carbon emissions are from servicing buildings.
With this in mind Chambers Builders Ltd is committed to a safe, sustainable and practical construction industry. Its importance is all around us in terms of where we live and work, and how we move from one place to another. The construction sector is responsible for our homes, our hospitals, our schools, our roads, our water works, our railways, our offices and everything practical in which we live and use. Most importantly the FMB is committed to ensuring that government objectives for sustainability are practical.
Chambers Builders Ltd hopes to be able to play a fuller and more constructive role in building the new greener Britain, by establishing its new recycling centre as part of Chambers Builders Ltd Site Waste Management Plan.


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Author: Annie Bonner, WaterAid America
Annie Bonner is corporate relations manager for WaterAid America
With 768 million people living without clean drinking water around the world, many people think that we just need more money to solve the world's water crisis. Currently, about $8-9 billion is spent each year to provide clean drinking water and toilets to communities living without these most basic human needs. Last year, The World Health Organization estimated that it would take $400 billion to get water to everyone around the world, and $2 trillion to achieve universal access to water and sanitation when combined with the cost of maintaining new and existing infrastructures. These, of course, are estimates.
So will more money solve the water crisis? Of course more funding will help us reach more people with clean water, but the investment required goes far beyond a dollar amount. It takes smart partnerships and careful planning to develop projects that offer sustainable programs that empower the local community. This takes more than money; it takes time, and getting all the right people at the table.
Recently, I was in Ouagadougou, Burkina Faso to launch a new project between The Coca-Cola Africa Foundation and WaterAid that is making safe drinking water a reality for people living in one of the poorest suburbs of the nation's capital. Our extensive meetings included people from Coca-Cola and WaterAid from both the United States and Burkina Faso, a local government official, people from our local NGO partner, and representatives from the local water utility company, all together, at the same table. It's not a surprise that the word "local" comes through three times in that sentence; that's where and how projects can thrive and people feel responsible; that's where change happens. At the table in Ouagadougou, our conversations took place in both English and French to make sure that everyone understood their role, and agreed to be accountable for their responsibilities. The investment is not just on the side of the funder, it's from everyone at the table – the funders and recipients alike.
Our collaboration in Ouagadougou will bring five water standpipes to a community called Balkuy, and our WaterAid team in Burkina Faso, alongside local partner AMUS and water utility ONEA are making good progress to reach this goal. More than 6,000 people will have access to clean, safe drinking water this year as a result of the collaborative project. Given that 1.7 million people achieved access to safe water with the help of WaterAid during the last year alone, I often catch myself referring to this as a relatively small project. However big or small, I cannot begin to count the hours that we have spent on making it a successful collaboration, most especially our staffs and partners in Ouagadougou. I cannot begin to quantify the investment of the collective time, energy, passion and dedication of everyone at the table, but having met the families and children of Balkuy, I can tell you it's worth every penny.
The views expressed are solely those of the author and do not necessarily represent the views or positions of WWF.
You can also read this blog post on Coca-Cola's Unbottled.
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SEATTLE — Tim Beckham hit two of Seattle's three home runs off Boston ace Chris Sale, and the Mariners improved to 3-0 with a 12-4 win over the World Series champion Red Sox on Thursday.
Edwin Encarnacion added a solo shot off Sale as part of a four-run third inning. Ryon Healy and Domingo Santana both homered off Boston's bullpen as Seattle finished with five long balls.
Beckham's homer started Sale's problems, but it was his inability to finish off the second inning that raised his pitch count and led to two runs. Sale issued a two-out walk to David Freitas, gave up a single to Dee Gordon and hit Mitch Haniger with a pitch to load the bases.
Seattle starter Marco Gonzales (2-0) was shaky early, but pitched into the sixth and allowed three earned runs to pick up his second victory. Boston had nine hits off Gonzales, but very little solid contact until the sixth inning when doubles by Eduardo Nunez and Christian Vazquez led to a pair of runs and ended Gonzales' day.
Mariners: LHP Yusei Kikuchi (0-0) will make his anticipated home debut for Seattle. Kikuchi threw 4 2/3 innings in the team's second game in Tokyo, allowing one earned run, but failed to make it through the fifth.


text:
Q: display iterator in jsp I have the following code:
@Override
public Iterator retrieve() throws SQLException {
    List<PasalBean> pasalObject = new ArrayList<PasalBean>();
    try {
        Class.forName(dbDriver);
        con = DriverManager.getConnection(url);
        ps = con.createStatement();
        rs = ps.executeQuery("select * from T_PASAL WHERE ID_PASAL = '" + id_pasal + "' ORDER BY ID_PASAL");
        while (rs.next()) {
            pasalObject.add(new  PasalBean(rs.getInt(1), rs.getString(2), rs.getString(3), rs.getString(4)));
        }
    }catch (Exception e) {
        System.out.println("Error Data : " + e.getMessage());
    }
    return (Iterator) pasalObject;
}

How can I display it in JSP?

A: First, this code will fail. You cannot cast a List to Iterator. Make it return a List.
Then, what you need in order to display it in a servlet is:
request.setAttribute("list", retrieve());
request.getRequestDispatcher("/path/to/jsp").forward();

And then in the JSP:
<c:forEach items="${list}" var="item">
   ${item.somePropertyOfTheBean}
</c:forEach>

It would be good to place the retrieve() method in a separate, data-access class (DAO), and not directly in the servlet.


text:
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LPL Financial Research Publishes Outlook 2020
"Our strategists are committed to delivering market insights that empower LPL advisors in their efforts to make informed, risk appropriate decisions suited to their clients' unique financial goals." – John Lynch, LPL executive vice president and chief investment strategist
CHARLOTTE, N.C. – Dec. 3, 2019 – Leading retail investment advisory firm and independent broker/dealer LPL Financial LLC today announced that its annual "Outlook 2020" is now available. View the new interactive version of "Outlook 2020" or download the printable version. The report, "Bringing Markets Into Focus," contains investment insights and market guidance from LPL Research for 2020, a year that will bring a highly charged U.S. presidential election and a possible breakthrough in international trade and policy discussions. These and other critical factors outlined in the report are likely to influence the longest running bull market in U.S. history.
"We expect the U.S. economy to continue to grow in 2020, which we believe will support stock market gains, but we are increasingly mindful of the advanced age of the economic expansion and bull market. There will continue to be uncertainty in the markets, but we continue to believe there is a low likelihood of recession in the coming year," said John Lynch, executive vice president and chief investment strategist, LPL Financial. "Our strategists are committed to delivering market insights that empower LPL advisors in their efforts to make informed, risk appropriate decisions suited to their clients' unique financial goals. More Americans entrust advisors to provide financial advice than ever before. By focusing on the fundamentals and market signals, we believe investors can make these emotional decisions for their diversified long-term portfolios most effectively."
The LPL Financial Research report emphasizes the following:
Policy: Progress on trade remains central to the 2020 outlook. Progress on U.S.-China trade discussions needs to come sooner rather than later if economic growth or corporate profits are going to improve in 2020. Based on recent signals from the yield curve and economic reports, LPL Research analysts report that they suspect the Federal Reserve (Fed) is in a wait-and-see mode as it gauges the impact of its 2019 rate cuts, which can take time to flow through to consumers and businesses.
Economy: Expected GDP growth of 1.75 percent. LPL Financial Research analysts expect 1.75 percent U.S. gross domestic product (GDP) growth in 2020, reflecting the potential for continued trade and geopolitical uncertainties amid the gradual slowing of the economy at this point in the economic cycle. Their forecast reflects the U.S.-China trade dispute dragging into the first part of 2020 and increased odds of recession in the latter months of 2020 or early 2021.
Bonds: Modest increase forecasted for longer-term yields. LPL Financial Research analysts' year-end 2020 forecast for the 10-year U.S. Treasury yield is a range of 2 to 2.25 percent. Continued Fed flexibility should provide enough support to the economy to foster a modest increase in longer-term yields.
Stocks: S&P 500 Index appears fairly valued around 3,250–3,300. Based on expectations for better earnings growth in 2020, along with anticipated continued economic growth, LPL Financial Research analysts expect support for stocks at current valuations. Their 2020 year-end S&P 500 fair value target is 3,250–3,300, which is calculated on a trailing price-to-earnings ratio (P/E) of roughly 18.75 multiplied by their 2020 S&P 500 earnings per share forecast of $175. LPL Financial Research analysts believe mild inflation and still-low interest rates support these valuations.
LPL Financial is a leader in the retail financial advice market and the nation's largest independent broker-dealer*. We serve independent financial advisors and financial institutions, providing them with the technology, research, clearing and compliance services, and practice management programs they need to create and grow thriving practices. LPL enables them to provide objective guidance to millions of American families seeking wealth management, retirement planning, financial planning and asset management solutions.
Securities offered through LPL Financial, member FINRA/SIPC.
*Based on total revenues, Financial Planning magazine June 1996-2019
Please see the LPL Financial Research Outlook 2020: "Bringing Markets Into Focus" for additional description and disclosure.
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There are total 10 letters in Lobulation, Starting with L and ending with N.
Lobulation is a scrabble word? Yes (12 Points) Lobulation has worth 12 Scrabble points.
In Lobulation L is 12th, O is 15th, B is 2nd, U is 21st, A is 1st, T is 20th, I is 9th, N is 14th letters in Alphabet Series.


Hola!! I am back after a vacation and still trying to shake off the lethargy that has engulfed my limbs. That's precisely why there have been no food posts on my blog for the longest time. I am just trying to scrape by with no fuss simple meals and doing something off beat had not even crossed my mind for the entire week. That is till last night.
The sudden craving for digging into some cake drove me crazy around midnight. But I had only sparse groceries on hand as I had finished off most of the stuff before going on vacation and had not yet replenished the pantry. And that's when I had the thought of trying something super healthy. Whole wheat cake with no butter and jaggery instead of sugar sounded tempting. Taking inspiration form the Odia Poda pitha, I added some ginger and cardamom to flavor it. Had initially planned on adding a bit of grated coconut but forgot it at the last minute as I was half asleep by the time I was done with it.
Just as I finished mixing everything and was about to pop it into the oven, tragedy struck. My trusted oven gave up and I ended up with a blown fuse and a husband who was threatening to go off any moment. Had to call the security guy and cajole him into fixing the fuse at the earliest. With the power restored, I had to focus on what to do with the cake mix I had on my hands as I could not risk switching on the oven. My first thought was to put it into the fridge and make some pancakes the next morning. But I wanted the cake real bad. Hence took a chance with baking it in my electric rice cooker. And thank God for it !!
Preparation - Take the whole wheat flour and baking powder in a bowl. Sift a few times using a sieve to remove lumps and ensure even mixing. This also improves the amount of air trapped into the flour which gives a spongier cake.
Mix the jaggery , water, ginger, coconut, cardamom, salt and vanilla essence in a bowl.
Sift in the flour into this liquid (use a sieve) and mix in using a heavy spoon. Mix in one direction only and avoid beating/whisking the mixture.
Finally add the oil and mix in as gently as possible.
Cooking - Preheat the oven at 180 degrees for 10 mins. Or if using electric rice cooker, pour 3 cups water into the pan and get it to a steaming stage.
Grease a tin with oil. Drizzle flour over it. Pour in the mixture.
Place the baking tin into the oven. Cook for 15 -20 mins till a toothpick comes out clean.
If rice cooker is used, it will switch off once the water is gone. The residual heat is enough to bake the cake thoroughly.
Wait till it cools down and then overturn the cake tin onto a plate.
The cake can be stored at room temperature for 3-4 days in a airtight container. If one needs to preserve it longer, it is best to refrigerate.
Note - The steamed version of this cake ( made in rice cooker ) is softer and spongier but does not have a proper crust.
I have used a lovely semi solid variety of jaggery that I got from Odisha. It has a wonderfully rich caramel flavour and gives a really intense color to the cake. While it is tough to get that variety in superstores, one can try using a mix of the light colored jaggery and some palm jaggery for a similar rich color.


Nearly 600 civilians fled the Islamic State group's last bastion in eastern Syria on Tuesday, as Kurdish-led forces and the US-backed SDF boxed holdout jihadists into an ever-shrinking pocket.
Turkey's military and its Syrian rebel allies have taken full control of northwest Syria's Afrin region, a Turkish army source said on Saturday.
Regime air strikes on Wednesday pushed the death toll from three days of bombing in Syria's rebel Eastern Ghouta to over 200 as Damascus pitched pro-government forces into the fray against Turkey in a Kurdish-held border region.
The US-led coalition fighting the Islamic State group said on Sunday it was working to create a 30,000-strong border security force in northern Syria.
After nearly a full year of predictions that the Islamic State was finished, it seems that the extremist group is indeed in full retreat.


going to see a man about a horse?
I guess I should mention that I have just started up a store on Society6 for t-shirts & mugs. I had hand silk screened t-shirts on the past, but as you can imagine this is a lot of additional work, limited to my skill, and 1 maybe 2 colours….so after some investigation and recommendations by fellow artist friends, I am giving Society6 a try.
I will continue to sell my books, original art & prints here on the site in my shiny new store as well.
I only have one design up so far, but will add more in the future. If there are any particular illustrations you would like to see there, just let me know!