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Many autoimmune conditions are believed to result from chronic inflammation as a consequence of the interaction of genetic and environmental factors in susceptible individuals. One common feature in some autoimmune diseases is the decrease in terminal galactosylation of the constant region N-glycan of the total plasma immunoglobulin. To determine whether a similar pattern is characteristic for the autoimmune disorder myositis, we analyzed the antibody subclass specific glycosylation in patients with myositis, their asymptomatic siblings, and healthy unrelated age- and sex-matched controls. The antibody subclass specific glycosylation was determined from the LC-MS analyses of the IgG glycopeptides generated by trypsin digestion of the antibody heavy chain. The glycosylation profiles of the IgG subclasses were determined relative to the abundance of the monogalactosylated core-fucosylated glycoform, G1F. We found elevated amounts of glycoforms lacking terminal galactose in myositis patients. Pairwise statistical analyses reveals that galactosylation is statistically different between the myositis patients and control groups. Furthermore, the trend analysis for glycosylation indicates a pattern of decreasing galactosylation in the order: controls>siblings>myositis patients, suggesting the existence of a genetic, immune-related predisposition in the group of asymptomatic siblings that can be detected before the onset of clinical symptoms at the level of plasma proteins.
The idiopathic inflammatory myopathies (IIM), collectively referred to as the myositis syndromes, represent a rare group of systemic autoimmune disorders characterized by progressive muscle weakness and infiltration of multiple muscles and other tissues by immune cells. Within the group of IIM, with an overall prevalence of 0.001%, the best recognized disease subsets are dermatomyositis (DM), polymyositis (PM) and inclusion body myositis (IBM) 1, 2. While some myositis autoantigens, such as histidyl-tRNA synthetase (Jo-1) 3, 4, signal recognition particle 5 and the nuclear protein Mi-2, have been identified, the mechanisms leading to the breach in immune tolerance and disease pathogenesis in IIM remain largely unknown 3–8. The symptoms of myositis are non-specific, often resembling those of other diseases, and phenotypes may differ considerably among individuals. Although the diagnosis of myositis syndromes may be challenging, several helpful tests include muscle biopsy and measurements of autoantibody titers and muscle enzymes in serum7, 9–11. Growing evidence suggests that both genetic and environmental factors contribute to the development of myositis 12.
Despite the clinical heterogeneity among autoimmune diseases, substantial evidence indicates the existence of a common genetic component associated with alleles of the major histocompatibility complex region 13–15. Furthermore, gene expression analyses of family members discordant for autoimmune disorders revealed increased susceptibility of unaffected siblings and twins compared to the unrelated population. These studies found a distinct gene expression signature in the unaffected family members, which resembled, to a considerable extent, the pattern found in autoimmune patients, and to a lesser extent the pattern found in non-related controls 14, 16, 17.
At the protein level, the effector molecules of the immune system – the antibodies –undergo structural changes in autoimmune rheumatic disorders such as rheumatoid arthritis (RA) 18, 19, Sjøgrens's syndrome 20, psoriatic arthritis, and systemic lupus erythematosus (SLE) 21. These changes are mainly related to the glycoform distribution of the complex type N-linked glycan attached at Asn 297, the conserved glycosylation site on the antibody constant region. The shared feature among all these diseases is the elevation of the glycoforms lacking terminal galactose – collectively referred to as IgG Gal-0 – in patients compared to healthy controls.
The molecular mechanisms of the pro- and anti-inflammatory properties of serum IgG are largely modulated by its constant region glycosylation 22, 23. Until recently, it was believed that the Gal-0 portion of the glycoforms may trigger pro-inflammatory responses by activating the complement pathway through interaction of the unmasked GlcNAc residue with the mannan-binding lectin 24, 25. More recent studies, however, suggest that IgG-Gal 0 antibodies engage the classical Fcγ receptors pathways 26, and that the sialylated portion of the IgG is required and sufficient to suppress inflammation 27–29. In RA, the dynamic levels of IgG-Gal 0 correlate with disease progression, disease activity, and spontaneous remission of the disease in pregnancy and post-partum flares of disease 19, 30, 31. Although the pathogenic role of the IgG-Gal 0 glycoforms is not fully understood, the sugar signature of serum IgG was shown to be an useful indicator to differentiate between early RA and RA 32.
As part of a twin-sibling study aimed at identifying genetic and environmental risk factors of systemic rheumatic disorders 33, the subclass specific IgG glycosylation in fourteen patients with myositis, ten asymptomatic siblings of ten of the patients, and twelve healthy unrelated age-matched controls was characterized. Aims of the study were to determine: (i) whether a similar pattern of elevated IgG Gal-0, as seen in RA, is characteristic for myositis patients, and (ii) whether susceptibility can be defined at the protein level, from the phenotypes of IgG glycosylation in patients, siblings and controls. The methodology employed to address these questions combines Protein G affinity capture of plasma IgG, proteolytic degradation and high performance liquid chromatography – mass spectrometry (LC-MS) at the glycopeptide level 18, 34, 35. The main advantages of characterizing antibody glycopeptides by mass spectrometry are: (i) subclass specific changes in IgG glycosylation can be identified, and (ii) Fc and Fab glycans can be distinguished 34. The abundance values relative to the core fucosylated monogalactosyl glycoform in each subclass were determined for the eleven most abundant glycoforms observed by LC-MS. The results were statistically analyzed with regard to galactosylation, sialylation, bisecting GlcNAc, and lack of core fucosylation.
Dithiothreitol, ammonium bicarbonate, and 96% formic acid were purchased from Sigma-Aldrich (St. Louis, MO). Sequencing grade-modified porcine trypsin was obtained from Promega (Madison, WI). The Protein G-agarose kit was obtained from KPL (Washington DC). NuPage 4 – 12 % Bis-Tris pre-cast gels, sample loading and running buffers and Coomasie SimplyBlue were purchased from Invitrogen (Carlsbad, CA). Acetonitrile was purchased from Caledon Laboratories, Ltd. (Georgetown, Ontario). Purified water (17.8 MΩ) was obtained from an in-house Hydro Picopure 2 system. All chemicals were used without further purification unless otherwise specified.
The present study is part of the clinical study NCT00055055, aimed at identifying genetic and environmental risk factors in families with twins or siblings discordant for rheumatic disorders, including rheumatoid arthritis, systemic lupus erythematosus and myositis 33. The participants in this study were selected as follows: cases – adults or children with one of the above autoimmune conditions, who have a healthy twin or sibling of the same sex within 5 years of age; cases' unaffected twins or siblings, and unrelated controls – normal, age- and sex-matched volunteers. Blood samples were collected at a single time point. Out of these, plasma samples from myositis patients (M, n = 14), asymptomatic twins/siblings (S, n = 10) and unrelated age-matched controls (C, n = 12) were selected for the study of IgG glycosylation. All patients met the criteria for probable or definite PM/DM, as defined by Bohan and Peter 36 and modified by the International Myositis Assessment and Clinical Study Group (IMACS) 37. Physician global disease activity was assessed by a 100 mm visual analogue scale 38. The characteristics of the study population, including the disease activity assessed by the physician and medication at the time point of blood collection, are presented in Supplemental Table 1. The subjects in this study were followed with annual mailings of questionnaires asking about new diseases or medications for 3–4 years and none developed new autoimmune diseases. None of the subjects showed clinical or laboratory signs of other inflammatory diseases.
The isolation of plasma IgG was carried out in 0.5 mL compact reaction columns (CRCs), packed with agarose-bound Protein G, which binds all four human IgG subclasses. Washing/binding and elution buffers were provided in the Protein G-agarose kit and were used as suggested by the manufacturer. For each plasma sample, a 0.5 mL column was packed with ~ 200 μL drained agarose, as follows: 400 μL of slurry were mixed with 400 μL washing/binding buffer, transferred to the column, and allowed to flow by gravity. The packed affinity resin was equilibrated with 5 mL washing/binding buffer. Plasma samples (20 μL) were diluted to 100 μL with washing/binding buffer and applied on the resin. A volume of 200–300 μL of washing/binding buffer was subsequently added, in order to fill the remaining dead volume. A Nutator was used to mix the content of the column in a three dimensional, gentle rocking motion, for 45 minutes at room temperature. The non-bound protein fraction was removed by washing the resin with 5 mL washing/binding buffer, followed by 5 mL deionized water. Elution of the IgG was performed by adding 0.5 mL elution buffer and incubation for 15 minutes at room temperature. The eluted IgG was brought to physiologic pH by the addition of 150 μL of 5X washing/binding buffer.
Following elution and neutralization to pH 7.4, 30 μL of the solution containing the isolated IgG were mixed with 10 μL sample loading buffer containing 100 mM DTT. Reduction was carried out at 95°C for 10 minutes, followed by SDS-PAGE and automated in-gel digestion with trypsin of the heavy chain band, as described previously 35. To ensure complete trypsinization of the heavy chain, an additional digestion step was employed as follows: to each lyophilized sample, 25 μL ammonium bicarbonate (pH 7.4) containing 0.83 μg trypsin were added. The digestion was performed overnight at 37°C. The samples were stored at −80°C until further use.
The IgG glycopeptides were analyzed by LC/MS on a Waters Q-Tof Premier mass spectrometer equipped with a nanoAcquity UPLC system (Waters, Milford, MA). Analyses were performed on a 3 μm, 100 μm × 100 mm, Atlantis dC18 column (Waters, nanoAcquity), using a flow rate of 300 nL/min. A C18 trapping column (180 μm × 20 mm) with 5 μm particle size (Waters, nanoAcquity) was positioned in-line of the analytical column and upstream of a micro-tee union used both as a vent for trapping and as a liquid junction. Trapping was performed for 3 min at 5 μL/min flow rate, using the initial solvent composition. A 4 μL aliquot of the digest sample was injected onto the column. Peptides were eluted by using a linear gradient from 98% solvent A (0.1% formic acid in water (v/v)) and 2% solvent B (0.1% formic acid in acetonitrile (v/v)) to 40% solvent B over 90 minutes. Mass spectrometer settings for the MS analysis were: capillary voltage of 3.2 kV, cone voltage of 20 V, collision energy of 5.0 V and source temperature of 80°C. Mass spectra were acquired over the mass range 200 – 2000 Da. For subclass specific glycosylation analysis, triplicate analyses were acquired in the MS only mode. For calibration, an orthogonal reference spray (LockSpray) of a solution of Glu1-Fibrinopeptide B (500 fmol/μL) in water/acetonitrile 80:20 (v/v) and 0.1% formic acid, having a reference mass of 785.8496 (2+) was used.
The LC-MS data were analyzed using MassLynx 4.1 software (Waters, Milford, MA). The monosaccharide and glycoform annotations used in the following sections are as follows: G – galactose, F – fucose, B – bisecting N-acetyl glucosamine, SA – sialic acid. For example, the annotation G1FSA refers to the core fucosylated, complex biantennary glycan, bearing one galactose and one terminal sialic acid residue. Because no species containing two sialic acid or two fucose moieties were observed, the presence/absence of SA or F in the annotation will indicate the presence of one/zero residues of that kind. The obtained LC-MS/MS data do not provide unambiguous linkage information to differentiate between isobaric structures, such as triantennary glycans and those containing bisecting GlcNAc. Because of the considerable number of previous studies of human IgG glycosylation18,19,34, we assigned those structures containing an extra GlcNAc moiety in addition to the two antennas, as glycans with bisecting GlcNAc. The IgG subclass specific glycosylation profiles were determined by averaging the MS scans over the chromatographic retention time in which glycopeptides from a specific subclass eluted. The IgG tryptic glycopeptides ions were observed as the +2 and +3 charge states. For each of the eleven most abundant glycoforms (shown in Figure 1B) the abundances of each species were summed and normalized to the abundance of the fucosyl monogalactosyl glycoform (G1F) within each IgG subclass. From these analyses, it was not possible to differentiate between the IgG2 and IgG3 subclasses, because the glycopeptides have identical peptide backbones.
Statistical analyses were performed using the statistical software package SAS (version 9.1). All variables analyzed are defined as ratios of their total ion abundance to the sum of the abundances of the eleven most abundant glycoforms observed in the LC-MS analyses. The data were normalized to the sum of all glycoforms so that statistical analyses of each individual variable can be performed. Pairwise comparisons among patients (M), asymptomatic siblings (S) and unrelated controls (C) in the IgG1 and IgG2–3 subclasses were performed for each of the glycoforms as well as the following summed variables: Gal-0 (agalactosylation) defined as the sum (G0F + G0FB + G0); bisecting GlcNAc (B) defined as the sum (G0FB + G1FB + G2FB); sialic acid (SA) defined as the sum (G1FSA + G2FSA); and Fuc-0 (afucosylation) defined as (G0 + G1 + G2). All pairwise comparisons were performed by treating matched groups and subjects nested within groups as random effects to account for any correlations among the data within subject and within matched groups. We performed standard mixed effects analysis of variance in SAS 9.1 using the SAS procedure called PROC MIXED. A comparison of variables was determined to be statistically significant at a P<0.05. Since the patients, their asymptomatic siblings, and the unrelated controls may be viewed as ordered groups, we performed a trend analysis, using ORIOGEN methodology39,40, to investigate the changes in variable over these ordered categories. The expectation is that there is a continuum from patients to asymptomatic siblings to unrelated controls with the asymptomatic siblings serving as the middle group. Because the IgG4 glycopeptides were observed with low relative abundance, and in some samples they were not detected above the noise level, no statistical analyses of the IgG4 glycosylation were performed.
Human IgG contains four distinct IgG subclasses – IgG1, IgG2, IgG3 and IgG4 – sharing more than 95% sequence homology. All four subclasses can be isolated from serum using Protein G affinity capture 18. Their concentration in human plasma may vary considerably among individuals, with IgG1 (10–12 mg/mL) and IgG2 (2–6 mg/mL) having the highest concentrations, followed by IgG3 (0.5–1 mg/mL) and IgG4 (0.2–1 mg/mL) 41. Structural differences are found in the hinge region, disulfide bridges, and in the CH2 domain of the heavy chain, around the consensus site for N-glycosylation, Asn 297. The minimal constant region tryptic peptide containing the N-linked site has the sequence EEQX297NSTXR, where X represents characteristic residues for each IgG subclass, as follows: X = both Y for IgG1, both F for IgG2–3, and simultaneously F296/Y300 for IgG4. As a result of these amino acid variations, glycopeptides from each subclass exhibit differences in their hydrophobic character, a feature which was used in the present study to selectively determine the glycoform distribution within each subclass. Glycopeptides derived from IgG2 and IgG3 are identical and could not be characterized separately. However, because the concentration of IgG2 is typically higher than that of IgG3, it is reasonable to assume that the major glycoform contribution for the EEQF297NSTFR glycopeptides is derived from IgG2.
Because the complex type glycan moieties attached to peptides are potentially labile under ESI conditions, the extracted ion current (EIC) for the protonated N-acetyl glucosamine (GlcNAc+, m/z 204.1) 35, 42–46 facilitated identification of glycopeptides in LC-MS without enrichment of the sugar containing species (as shown in Figure 1A for one patient sample). In our experience, glycopeptides always show some level of in-source decomposition, which can be roughly estimated from the abundance of sugar oxonium ions, such as m/z 204.1, 366.1, relative to that of non-glycopeptide ions. For each IgG subclass, glycopeptide ions containing partial glycan structures, arising from in source decomposition, can be detected in the high m/z region (see the discussion of Figure 1B). Their abundance relative to that of non-glycopeptide ions is also indicative of the extent of in-source decay. For the semi-quantitative determination of the IgGs, it is desirable to minimize the extent of in-source decay, and this can be achieved by careful adjustment of the mass spectrometer settings. The cone voltage, defined in the Waters QT of Premier configuration as the potential offset between capillary voltage and the counter electrode, was found to have a dramatic effect on both in-source decomposition and sensitivity. Therefore, its value was adjusted such that good sensitivity for the detection of all glycoforms, and low in-source decomposition were achieved. Although the low abundance of oxonium ions resulted in higher noise in the EIC of m/z 204.1, this did not impact negatively the detection of glycopeptides.
Glycopeptides derived from IgG1 were found to elute first, followed by those derived from IgG4 and lastly IgG2–3 (Figure 1A). Within each subclass, neutral glycopeptides elute earlier than those containing terminal sialic acid. In order to obtain the picture of glycoform distribution for each subclass, the MS scans were averaged over the retention time window in which the glycopeptide ions characteristic of each specific subclass were detected. As an example, Figure 1B shows the averaged MS data obtained for the IgG1 glycopeptides with the sequence EEQY297NSTYR. From these data, the most abundant glycoforms observed are G0–2F, followed by those containing bisecting GlcNAc and terminal sialic acid. The glycans lacking the core fucose were detected with lowest abundance. A species arising from in-source decomposition of IgG1 glycopeptides, assigned to the partial glycan GlcNAc3Man3Fuc, was detected as the ion m/z 1216.063. Ions corresponding to this glycan structure were detected for all IgG subclasses. As described above for the ion of m/z 204.1, the abundance of this ion can be minimized through adjustment of source parameters. For the quantitative analyses, the abundances of the observed charge states for each glycoform (+2 and +3 charge state) were summed to determine the total abundance of each glycoform. The relative abundances of each glycoform was then determined relative to the total abundance of all the glycoforms. In several instances the IgG4 glycopeptides were of low abundance and poor S/N ratio, therefore, their relative glycoform distributions are not presented and further statistical analyses were not performed.
To more easily visualize these data, a bar graph representing the relative abundance of each glycoform of the IgG1 subclass observed in one plasma sample set is shown in Figure 1C. The bars represent the mean of triplicate LC-MS analyses and the error bars are the result of three technical replicates of each individual sample. Of the most abundant glycoforms observed (G0F, G1F, and G2F), a considerable increase in the relative abundance of the G0F glycoform in the IgG1 subclass was observed for the myositis patient compared to either the asymptomatic sibling or the unrelated control. Interestingly, the observed abundance for the G0F glycoform for the sibling was intermediate between the myositis patient and the unrelated control. A similar trend was observed for the G1F glycoform, however, the differences in abundance of this glycoform where less among the three samples. For the G2F glycoform, the asymptomatic sibling had the highest level compared to the patient or control.
To evaluate the entire data set, the relative abundances for each glycoform from the IgG1 and IgG2–3 subclasses were averaged across each sample group (patient, sibling, control). The resulting average for each glycoform in the IgG1 subclass and IgG2–3 subclass is shown in Figure 2 and Supplemental Table 2. In Figure 2, the bar graphs correspond to the average glycoform relative abundance and the error bars represent the standard deviation across all samples within that specific group (myositis, sibling, or control). The triplicate LC-MS analyses of the relative abundances showed very good reproducibility with an average standard deviation of 0.004 for all the variables analyzed (Supplemental Table 2). From these graphs, one can determine that the glycan profile in patients suffering from myositis tend to lack terminal galactose residues. The relative abundance of the G0F glycoform in both the IgG1 and IgG2–3 subclasses is elevated relative to the control. This observation correlates with findings reported for the glycan profile of rheumatoid arthritis patients21. Furthermore, the asymptomatic siblings exhibit G0F glycan levels intermediate between the myositis patients and the controls in both subclasses. To ascertain whether the measured differences in the relative abundances of the glycoforms are significant, a statistical evaluation was applied to all variables from the LC/MS analyses.
Because decreased levels of galactosylation were first noted in autoimmune patients suffering from rheumatoid arthritis19, the galactosylation levels (G0F) in myositis patients was of particular interest. In addition to changes in the individual glycoforms in the IgG1 and IgG2–3 subclasses, changes in specific glycan features were also evaluated. It is known, for example, that fucose and sialic acid are sugars involved in various pathological processes47,48,49. To determine whether changes in a specific glycan feature could be observed in myositis patients, the individual glycoforms representing a specific glycan feature were added together. These features were evaluated to account for the total agalactosylation (Gal-0), total bisecting N-acetylglucosamine (GlcNAc), total sialic acid (SA), and total afucosylation (Fuc-0). These summed variables are defined as Gal-0=(G0F + G0FB + G0), bisecting GlcNAc=(G0FB + G1FB + G2FB), SA=(G1FSA + G2FSA), and Fuc-0=(G0 + G1 + G2). The relative abundances for each glycoform in the IgG1 and IgG2–3 subclasses as well as the summed glycoforms are shown in Supplemental Table 2.
To determine the statistical significance of the glycosylation changes, pairwise comparisons among the three sample groups (patients, siblings, and controls) were performed using standard mixed effects analysis of variance in SAS 9.1 using the SAS procedure called PROC MIXED. Of the different individual and summed glycoforms analyzed, the variables representing agalactosylation showed significant differences (P < 0.05) among the sample groups in the pairwise comparisons (Table 1). In general, higher levels of G0F were found in myositis patients compared to either siblings or controls (Figure 3). For the G0F values, highly statistically significant differences (P < 0.0001) were observed between the groups of myositis patients and controls for both IgG1 and IgG2–3. The groups of myositis patients and their asymptomatic siblings were also found statistically different with regard to G0F in the IgG1 subclass (P = 0.0098), whereas the statistical significance for G0F between these groups in the IgG2–3 subclass was borderline significant (P = 0.0504). No significant difference (P > 0.05) was observed for the IgG1 G0F variable between the groups of siblings and unrelated controls, however, the IgG2–3 G0F variable was observed as statistically significant between these two groups (P = 0.0175).
To account for the presence of all galactose deficient sugars, the variable Gal-0 = (G0F + G0FB + G0) among the three groups was evaluated. Similarly, the two extreme groups of myositis patients (M) and controls (C) were highly statistically different (P < 0.0001) with regard to Gal-0 in both the IgG1 and the IgG2–3 subclasses. Additionally, Gal-0 was found statistically different between the groups of myositis patients (M) and their asymptomatic siblings (S) in both the IgG1 subclass (P = 0.0105) and the IgG2–3 subclass (P = 0.0221). Similar to what was observed for G0F between the siblings (S) and control (C) groups, no significant difference (P > 0.05) was observed for Gal-0 in the IgG1 subclass, but this variable was significantly different in the IgG2–3 subclass (P = 0.0071). In addition to G0F, the G0FB and G0 variables may contribute to the statistical significance of Gal-0. Of these two variables in either IgG subclass which showed pairwise statistical relevance was the G0FB variable in IgG1 subclass (Table 1). These results indicate that the major component of the Gal-0 sum is most likely represented by the G0F variable.
As an extension of the statistical analyses, because the siblings in this clinical study were asymptomatic, we grouped the siblings and the controls into one "healthy" group and performed a comparison of this group with the patient group. From this analysis, the agalactosylation variables that were observed as significant between either the patients vs siblings or the patients vs controls were found to be highly statistically different between the patient group vs the combined healthy group for both the IgG1 and IgG2–3 subclasses (Table 1).
A previous study demonstrated that IgG Gal-0 can be an useful prognostic indicator to differentiate between early RA and RA 32. Furthermore, genetic data suggested that unaffected siblings of autoimmune individuals have gene expression signatures that share more similarities with the affected family members than with unrelated controls 16. As noted earlier in the Experimental Section, the study groups were ordered with the asymptomatic siblings serving as the intermediate group. Given this order, it is reasonable to expect the responses to exhibit a trend over the groups (increasing or decreasing). Hence, we employed statistical trend analysis to determine whether there was a pattern of decreasing IgG Gal-0 among the patient, siblings, and control groups. Trend tests have been employed over the past several decades in areas ranging from toxicology to clinical trials50–55. In situations where the explanatory variable is ordinal, as is the present situation with the three groups, trend tests might provide useful interpretation of the data. Often there is a tendency for the mean response to increase (or decrease) gradually over ordered categories and classical pairwise tests cannot always detect such gradual systematic changes. The variables that were determined to be statistically significant among the three sets (Table 1) were interrogated for the existence of a trend using the ORIOGEN methodology described previously39,40. The G2F variable (galactose-containing glycan) was also included in the trend analyses as this variable would be complementary to the agalactosylation variables. The results of the trend analysis are shown in Table 2 and suggest a significant decreasing pattern (P < 0.001) of the mean G0F and Gal-0 responses in the order myositis>sibling>control in both IgG1 and IgG2–3, which remained significant even after adjusting for multiple testing using the Bonferroni correction (P < 0.002). A bar graph representation of the trend analysis results of the G0F variable for IgG1 and IgG2–3 are shown in Figure 3. Although it appears that Gal-0 is largely dominated by G0F, the second component of Gal-0, G0FB, also displayed a significant decreasing trend (P < 0.001) in the same direction in the IgG1 data (Table 2). This suggests that, despite the fact that IgG1 Gal-0 is dominated by G0F, the term G0FB contributes significantly to the sum as well. Similar to the pairwise comparisons, the trend analysis of the G0FB variable was not statistically significant in the IgG2–3 subclass data set (P > 0.05 without adjustment for multiple testing). The decreasing trends determined for G0F and Gal-0 in the order myositis>sibling>control are validated by the increasing trend of G2F in the opposite direction in both IgG1 and IgG2–3 data. The existence of the trend towards increasing agalactosylation of serum IgG in the order control<sibling<myositis is further evidence that IgG Gal-0 may be a useful predictor to assess the susceptibility of an individual to develop an autoimmune condition before the onset of clinical symptoms. Although the healthy siblings have not been monitored to the point when they showed disease symptoms, this finding is consistent with previous studies of SLE and diabetes in patients and their asymptomatic siblings, showing that the development of autoantibodies in the last group preceded clinical manifestations of the disease.
As described for agalactosylation, similar statistical pairwise and trend analyses were performed for the glycoforms containing terminal sialic acid (SA), bisecting GlcNAc, and for those lacking the core fucose (Fuc-0). In each case, all glycoforms containing the distinctive monosaccharide were analyzed together, regardless of the number of galactose residues. In the statistical analyses, no significant changes were found for any of the above variables in either IgG1 or IgG2–3 subclass.
It is known that physiological variations, such as age and gender, can influence plasma IgG glycosylation56,57,58. Additionally, a correlation between the use of prednisone and the levels of IgG2 galactosylation was found recently for RA patients19. Therefore, we performed multiple regression analysis to evaluate the effect of various factors, namely age, diagnosis (DM/PM), medication (Yes/No), and disease activity, on each variable in the IgG1 and IgG2–3 subclasses and found none of the factors to be significant at P < 0.05 for any of the variables in the two data sets. Because the statistical power of these analyses is limited by the number of cases in the study, however, these analyses could be more meaningful for a larger number of patients.
The unique design of this study also provides an opportunity to investigate the potential role that genes and the environment have on the glycosylation profile of IgGs. Because one sample set was obtained from monozygotic twins discordant for myositis, the IgG glycosylation pattern of genetically identical individuals could be interrogated. One can thus assume that the discordance for myositis is due to an environmental influence. Although only one sample set was available from monozygotic twins, the results are interesting and, thus, will be mentioned briefly. From the relative abundance of the IgG1 glycoforms from this sample set, it was determined that the IgG1 G0F abundance was higher in the patient vs their asymptomatic sibling (Supplemental Figure 1). These data reflect the same trend observed for the averages of all patients and all siblings (Figure 2A). Although only one sample set from monozygotic twins was available, these IgG glycosylation data may indicate that different serologic phenotypes may be associated with discordance of monozygotic twins. A larger cohort of monozygotic twins, however, would be necessary. This hypothesis is also supported by previous studies of monozygotic twins which revealed a link between the phenotypic differences observed in identical twins discordant for SLE, RA, and DM, and environmental epigenetic events at the level of DNA methylation affecting expression of genes associated with the immune function 14, 59, 60.
Since the initial observation that galactosylation of serum IgG antibodies is decreased in RA patients 21, there has been a great amount of effort devoted to improve the understanding of the implications of the conserved immunoglobulin glycosylation in physiologic and pathologic states. Although the altered IgG glycosylation was initially regarded as a feature related only to RA, it became clear that the decrease in terminal galactosylation is associated not only with RA, but it may represent a shared feature among several autoimmune and non-autoimmune diseases32.
The present study represents the first investigation of the myositis syndromes with regard to glycosylation of serum IgG, from the perspective of a twin/sibling clinical study. Characterization of the IgG glycopeptides by LC-MS showed that myositis patients have statistically significant elevated amounts of glycoforms lacking galactose compared to their healthy siblings and unrelated controls, which is consistent with the previous findings involving RA. Statistical significant differences in the glycosylation between patients with dermatomyositis and those with polymyositis were not observed, nor could we find any correlation with disease activity. A potential limitation of this study is the small number of patients analyzed and their heterogeneity within the group, derived from the differential diagnosis and the use of medication. The novelty, however, comes from the group of asymptomatic siblings for which a galactosylation phenotype intermediate between the patients and controls was found. The overall incidence of myositis (0.001% incidence of all syndromes) and the qualification criteria for candidates of this clinical study (i.e. patients must have a healthy twin or sibling of the same sex within 5 years of age) render selection of possible subjects difficult. Despite this fact, statistically significant differences were observed between patients and controls as well as the patients and their asymptomatic siblings for the abundances of the G0F and Gal-0 glycoforms. In addition, trend analysis indicates an increasing pattern in IgG Gal-0 in the order controls<siblings<patients in both IgG1 and IgG2–3. This result is validated by the significantly decreasing trend of the digalactosylated glycoform G2F in the reverse order. This observation is consistent with results from previous genetic studies which revealed similarities in the gene expression profiles between autoimmune patients and first degree relatives 16. Hence, in addition to genome, susceptibility can be defined at the proteome level as well. Because IgG Gal-0 was found elevated in several autoimmune disorders, we believe it represents a more general indicator for the presence of an inflammatory condition rather than a disease-specific marker. The usefulness of IgG Gal-0 as a potential prediction tool of an autoimmune condition needs to be further explored in order to define intervals characteristic for disease, susceptibility and normal levels. This would imply the analysis of a larger cohort of autoimmune patients and their siblings in different age brackets. In the emerging era of individualized medicine, such a test measuring the IgG galactosylation could be routinely employed not only as a complimentary diagnostic tool, but also in preventive care. With the knowledge of a certain serum IgG glycosylation pattern together with the individual's family history, prophylactic strategies can be adopted to prevent onset of clinical symptoms in susceptible individuals. While autoantibodies are usually heterogeneous and characteristic only for a limited number of individuals, determining serum IgG galactosylation may compliment the diagnosis of autoimmune diseases. In conclusion, this study indicates that myositis patients have increased levels of core-fucosylated agalactosyl glycans and that susceptibility in asymptomatic siblings can be identified at the level of serum proteins in the pattern of IgG glycosylation. We believe that the present study of myositis, although performed on a small cohort, sets an important foundation for future studies of this and other autoimmune diseases.
This research was supported by the Intramural Research Program of the NIH, National Institutes of Environmental Health Sciences, projects ES050171, ES101074-07 DIR, ES045005-14. The authors would like to acknowledge the technical assistance of Dr. Terrance O'Hanlon (NIEHS/NIH) in the preparation of the patient samples, Dr. Jason Williams (NIEHS/NIH) and Dr. Alina Zamfir (University of Aurel Vlaicu Arad) for critical review of the manuscript, and the NIEHS Protein Microcharacterization Core Facility for assistance with in-gel digestions.
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Beachhead - What Is the Value of Software?
As someone who has spent a fair amount of time on or around the sea, I always enjoy looking at boats and ships—and comparing them with canned corn.
Canned corn is a commodity. Although we may appreciate what companies go through to select the corn, clean it, can it and move it to store shelves, for the most part, one can of corn is exactly like another can of corn. No consumers spend too much time standing in front of the canned corn shelves at their store, wondering whether a particular brand would be better for their family than another one. They select a can and move on. As I said, canned corn is a commodity.
This is not the case with boats. Although most people would expect any given boat to float, different designs of boats meet different needs. There are sailboats, power boats, tugboats, and different models of each of these types. Each model of boat has a slightly different design based on the needs of the captain, crew and business.
Just as most people would not spend hours in front of a shelf deciding on which can of corn to buy, most people would not run down to the yacht broker, throw a suitcase of money on the dock and say that they would take any boat the broker had in stock.
And, although the total cost of ownership of the boat must be taken into consideration, the real determining factor in buying a boat is whether it meets your needs, the pay-back period on the purchase, and whether you could modify the boat to meet future desires.
In other words, the boat is not a commodity.
These days, you hear a lot about the operating system being a "commodity", or that "no one cares about the operating system". I believe that this broad generalization, like a lot of broad generalizations, fails under many circumstances. Let's see where this particular generalization runs aground.
A bank in Brazil had automatic teller machines (ATMs). The operating system that it used in these ATMs was OS/2. The company that made OS/2 was "retiring" OS/2, and the bank would no longer be able to get the support it needed for new motherboards, device drivers and so forth. The ATMs used Intel 386, 486 and low-end Pentiums as CPUs, with varying amounts of main memory, disk sizes and other differences, reflecting a long time line of development and deployment.
The bank decided to use Linux as its new operating system, because it supported all the different CPUs, and the bank would be able to maintain it or expand on it into the far future. Here, the value of the operating system was in its potential longevity.
A lottery system, also in Brazil, decided to use Linux and free and open-source software inside the lottery terminals. It was replacing software from a proprietary vendor who had taken months to deploy requested changes to the lottery system. With a local development team, these deployment schedules dropped from months to weeks. This generated millions of dollars of additional revenue, as the new features could be delivered faster. The value here was the speed in making changes.
A public transportation authority was spending millions of dollars a year on proprietary software that was used only to write letters, do presentations and run simple spreadsheets. By moving to OpenOffice.org, the authority was able to save those millions of dollars and spend that money on hiring new cleaners for the transportation system. The manager of the transportation authority said that nobody cared which software was used to type the letters, but that everyone mentioned how the transportation vehicles were being kept very clean. The value was in re-deploying the money that would have been spent on royalties to services people desired.
A company in Rio de Janeiro needed a system that would work in Portuguese, but the software it could buy was only available in English. A free software developer duplicated the functionality of the English-only software using MySQL, Python, FreeGIS software, Gnuplot and other free and open-source software. The combined software was designed to prompt and report in Portuguese, and it cost less to develop than the packaged system. The value was having software in the company's own language and creating a local programming job.
So, you see that the value of the software, as with a boat, cannot be measured by the cost of building it, but by how well it meets the needs of the user. And, although I have seen many, many families that can eat the same can of corn, I have never, even one time, seen exactly the same business problem.
Jon "maddog" Hall is the Executive Director of Linux International (www.li.org), a nonprofit association of end users who wish to support and promote the Linux operating system. During his career in commercial computing, which started in 1969, Mr Hall has been a programmer, systems designer, systems administrator, product manager, technical marketing manager and educator. He has worked for such companies as Western Electric Corporation, Aetna Life and Casualty, Bell Laboratories, Digital Equipment Corporation, VA Linux Systems and SGI. He is now an independent consultant in Free and Open Source Software (FOSS) Business and Technical issues.
Trending Topics
Practical Task Scheduling Deployment
July 20, 2016 12:00 pm CDT
One of the best things about the UNIX environment (aside from being stable and efficient) is the vast array of software tools available to help you do your job. Traditionally, a UNIX tool does only one thing, but does that one thing very well. For example, grep is very easy to use and can search vast amounts of data quickly. The find tool can find a particular file or files based on all kinds of criteria. It's pretty easy to string these tools together to build even more powerful tools, such as a tool that finds all of the .log files in the /home directory and searches each one for a particular entry. This erector-set mentality allows UNIX system administrators to seem to always have the right tool for the job.
Cron traditionally has been considered another such a tool for job scheduling, but is it enough? This webinar considers that very question. The first part builds on a previous Geek Guide, Beyond Cron, and briefly describes how to know when it might be time to consider upgrading your job scheduling infrastructure. The second part presents an actual planning and implementation framework.
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|Jul 28, 2016
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- Stunnel Security for Oracle
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- Managing Linux Using Puppet
- My +1 Sword of Productivity
- SUSE LLC's SUSE Manager
- Parsing an RSS News Feed with a Bash Script
- Doing for User Space What We Did for Kernel Space
- SuperTuxKart 0.9.2 Released
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Geek Guides
With all the industry talk about the benefits of Linux on Power and all the performance advantages offered by its open architecture, you may be considering a move in that direction. If you are thinking about analytics, big data and cloud computing, you would be right to evaluate Power. The idea of using commodity x86 hardware and replacing it every three years is an outdated cost model. It doesn't consider the total cost of ownership, and it doesn't consider the advantage of real processing power, high-availability and multithreading like a demon.
This ebook takes a look at some of the practical applications of the Linux on Power platform and ways you might bring all the performance power of this open architecture to bear for your organization. There are no smoke and mirrors here—just hard, cold, empirical evidence provided by independent sources. I also consider some innovative ways Linux on Power will be used in the future.Get the Guide

subset: commoncrawl


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Jan 23
Sep 6
Raleigh Times Bar hopscotch day party
Feb 21
More info soon
Aug 17
The Bleeding Hearts CD Release show for their new album "Divorcing New York"!
Dec 28
Sounds Like: Cheap Trick, The Ramones, AC/DC, KISS
Label: None
Bio: Reformed with original members; Joe Yerry, James"Jimbo" Britt, Sam Madison and the addition of drummer extraordinaire and teen sensation, Bill Eagen! We will still be playing a few choice songs from the first two albums but have a "shit ton" of new material we will be showcasing in the next few m...See Full Bio


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Upton In Second Slot As Tigers Begin Exhibition Schedule"Upton tends to get on base more. You want as many guys on base in front of Miggy, Victor and J.D. (Martinez)."
Tigers Should Pay JD, But How Much? And Who Else? [BLOG]Less than eleven months ago, I became the first person in Metro Detroit to jump fully aboard the J.D. Martinez bandwagon
Austin Jackson Shows Off New Suit In Hysterical Fashion [VIDEO]Is this the best lining of a suit you have seen?
Tigers Appear To Have Great Team Chemistry At Team Barbecue [PHOTOS]One observation I've made about the Detroit Tigers these past few years is that this team is truly like one big family.


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New Congress: Fewer moderates make deals harder
By ALAN FRAM
Associated Press
WASHINGTON — When the next Congress cranks up in January, there will be more women, many new faces and 11 fewer tea party-backed House Republicans from the class of 2010 who sought a second term.
Overriding those changes, though, is a thinning of pragmatic, centrist veterans in both parties. Among those leaving are some of the Senate's most pragmatic lawmakers, nearly half the House's centrist Blue Dog Democrats and several moderate House Republicans.
That could leave the parties more polarized even as President Barack Obama and congressional leaders talk up the cooperation needed to tackle complex, vexing problems such as curbing deficits, revamping tax laws and culling savings from Medicare and other costly, popular programs.
"This movement away from the center, at a time when issues have to be resolved from the middle, makes it much more difficult to find solutions to major problems," said William Hoagland, senior vice president of the Bipartisan Policy Center, a private group advocating compromise.
In the Senate, moderate Scott Brown, R-Mass., lost to Democrat Elizabeth Warren, who will be one of the most liberal members. Another GOP moderate, Richard Lugar of Indiana, fell in the primary election. Two others, Kay Bailey Hutchison of Texas and Olympia Snowe of Maine, are retiring.
Moderate Democratic senators such as Kent Conrad of North Dakota, Herb Kohl of Wisconsin, Ben Nelson of Nebraska, and Jim Webb of Virginia are leaving, as is Democratic-leaning independent Joe Lieberman.
While about half the incoming 12 Senate freshmen of both parties are moderates, new arrivals include tea party Republican Ted Cruz of Texas, conservative Deb Fischer of Nebraska, and liberals such as Tammy Baldwin of Wisconsin and Hawaii's Mazie Hirono.
There's a similar pattern in the House, where 10 of the 24 Democratic Blue Dogs lost, are retiring or, in the case of Rep. Joe Donnelly, D-Ind., are moving to the Senate. That will further slash a centrist group that just a few years ago had more than 50 members, though some new freshmen might join.
Among Republicans, moderates like Reps. Judy Biggert of Illinois and New Hampshire's Charles Bass were defeated while others such as Reps. Jerry Lewis of California and Steven LaTourette of Ohio decided to retire.
"Congress seems to be going in the opposite direction of the country, just as the country is screaming for solutions to gridlock," said Democratic strategist Phil Singer.
Whether the changes are good is often in the eye of the beholder.
Seventy-one of the 83 House GOP freshmen of 2010 were re-elected Nov. 6, but 11 lost, including one of the group's highest profile members, conservative Rep. Allen West, R-Fla. Another faces a runoff in December.
"Some of the people who are the anti-government ideologues, some of them are gone," said House Minority Leader Nancy Pelosi, D-Calif. "And that message has been rejected by the American people."
Sal Russo, strategist for the Tea Party Express, said such departures would be balanced by newly elected conservatives, including the Senate's Cruz and GOP Reps.-elect Ted Yoho of Florida and Mark Meadows of North Carolina.
"Pretty much everybody that ran in 2012 was talking about the economic woes we face, stopping excessive spending, controlling unsustainable debt," he said.
Overall, the new House is on track for a 234-201 Republican majority, a narrowing of their 242-193 advantage today, which includes five vacancies. Democrats will control the Senate 55-45, up from 53-47.
A dozen of the 100 senators and at least 81 of the 435 House members, almost one-fifth, will be in their first term, slightly above historic averages. The Associated Press hasn't declared winners in two House races.
Many newcomers, in Washington for orientation sessions after their election, described a need to compromise. Some also made it clear there will be plenty of fuel for partisan clashes.
"I'm going in open-minded," said conservative Rep.-elect Roger Williams, R-Texas. "But I have certain core values like we all do and I'm not going to waver on that."
All together, there will be 73 women in the House and 20 in the Senate. Both are records.
For the first time, more than half of House Democrats — 105, in this case — will not be white males.
One white male will be Rep.-elect Joseph Kennedy III, a Massachusetts Democrat whose father was Rep. Joe Kennedy, D-Mass., and grandfather was New York Sen. and Democratic presidential candidate Robert Kennedy. When the newest Kennedy takes office, it will end the only two years since 1947 without a member of his family in Congress.
Those leaving include several who have been in the middle of recent years' policy battles.
Among them are Sen. Jon Kyl, R-Ariz., the GOP's No. 2 Senate leader; Indiana's Lugar, a longtime GOP power on foreign policy; North Dakota's Conrad, the Senate Budget Committee chairman and one of his party's chief deficit foes; and Jeff Bingaman, D-N.M., who heads the Senate Energy and Natural Resources Committee.
Gone from the House will be California Democratic Reps. Pete Stark, a major force on health issues, and Howard Berman, long influential in foreign affairs, plus liberal Massachusetts stalwart Barney Frank, whose name is on the new law overhauling the government's regulation of banks and other financial institutions.
Also leaving: House Rules Committee Chairman David Dreier, R-Calif., and Ron Paul, 77, who charted his own libertarian course in Congress and long-shot campaigns for the GOP presidential nomination in 2008 and 2012.
"The status quo will continue," Paul, who sees little difference between the two political parties, said of the new Congress. As for his own departure, he said, "Nobody will notice."
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Merillat PartsIf you don't see the part you need listed, please contact us. We will be adding items as needed.
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Sync Cable
Crutchfield Customer Review (What's this?)
This is a decent sync cable for your iPhone or iPod if you want to connect it to the head unit in your vehicle. The coiled cable stays up out of the way when not in use. I have it in a 1999 Tacoma and use the vacant opening where a factory CD unit would have gone to hold the iPhone while driving. Works great without having a longer cable getting in the way when I'm shifting gears.
Pros: Coiled feature is nice. cable appears to be robust.
Cons: None that I can see, other than a bit pricey but what isn't these days?


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Full Metal Alchemist
Full Metal Alchemist
Nov 05, 2004 7:19PM PST
Full Metal ALchemist, One of my favorite animes, is going to be on adult swim on Saturday at Midnight est time. Its a great series and it will be interesting to see how well the english vocal cast is. Hopefully they'll do as well as the Japanese cast did. I suggest anyone who likes anime to make sure to watch it.
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(Save the dates: RealShare Apartments East comes to the Hyatt Regency in Miami, FL, on February 26, 2013, and RealShare Los Angeles comes to the Hyatt Regency Century Plaza in Los Angeles, CA, on March 27, 2013.)
LOS ANGELES-Dallas-based Axiometric's jobs/multifamily-permitting report showed that Los Angeles ranked fifth among the top 10 MSAs for multifamily property permitting for trailing 12 months ending November, 2012. According to the report, permits for 8,582 units were issued in the L.A. market.
The top 10 MSAs listed in the report were:
- Houston (11,192 units)
- New York (13,664 units)
- Dallas (12,574 units)
- Austin (10,242 units)
- Los Angeles (8,582 units)
- Washington, DC (8,415 units)
- Seattle (8,309 units)
- Denver (6,799 units)
- Raleigh (5,919 units)
Nationally, November saw an increase in jobs and a decrease in the unemployment rate, while on the multifamily property-permitting side, the number stood at 261,232, the 13th consecutive month during which that figure has topped 200,000.
On the jobs front, however, while the US Bureau of Labor Statistics saw monthly nonfarm payrolls increasing by 146,000 jobs in November, September and October, job numbers were revised downward—from 148,000 to 132,000 and from 171,000 to 138,000, respectively. These revisions took away 49,000 jobs that were originally reported. Furthermore, private payrolls rose by 147,000 jobs, slightly lower than the average of 155,000 jobs seen from January through October 2012. For more information on the report, click here.


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Hotels in Havana
- Approximate price: Cheapest | Luxury
- Stars: 1 star | 5 stars
- First name: A-z | Z-a
- Central: More central | Less central
- Services: More services
- User evaluation: Best rated
- BestSelling: Most popular
Quinta Avenida Habana
5ta A Avenida e/ 76 y 80, Miramar, Playa, Havana, Cuba
Quinta Avenida Habana
Havana, 8 km from the city centre. 5ta A Avenida e/ 76 y 80, Miramar, Playa see on map
The Hotel is located in the residential area. Its location also has the benefit of providing access to an area where shops and diplomatic...
Nacional de Cuba
Calle 0 esq. 21, Vedado, 10400 Havana, Cuba
Nacional de Cuba
Havana, 2.5 km from the city centre. Calle 0 esq. 21, Vedado see on map
Means pride for people from Havana and admiration from foregners. This landmark hotel stores the still warm memories of the...
from ¥9,975
Approximate price
NH Capri La Habana
Calle 21 Esquina N Y O, Havana, Cuba
NH Capri La Habana
Havana, 1.9 km from the city centre. Calle 21 Esquina N Y O see on map
NH Capri has two bars and three restaurants serving international and Italian dishes. There is complimentary wireless internet, a...
Ignacio Agramonte No. 267, Habana Vieja, Havana, Cuba
Havana, 1.1 km from the city centre. Ignacio Agramonte No. 267, Habana Vieja see on map
Originally the mansion of the Marquis of Pinar del Río that was turned into a hotel in 1909, guests are offered a welcoming...
Calle 0, Esquina 25 - Vedado, Havana, Cuba
Havana, 1.7 km from the city centre. Calle 0, Esquina 25 - Vedado see on map
The hotel, with it's two towers, was built in a sober, elegant English style, during the popular building...
Ave. 3ra y calle 84, Playa, Havana, Cuba
Havana, 8.6 km from the city centre. Ave. 3ra y calle 84, Playa see on map
This historic hotel was fully renovated in the year 2006 and features a garden as well as a terrace. It comprises 2 buildings with rooms...
Mercure Sevilla Havane
Trocadero 55 e Prado y Zulueta, 260 Havana, Cuba
Mercure Sevilla Havane
Havana, 1.2 km from the city centre. Trocadero 55 e Prado y Zulueta see on map
The facades of the city hotel shine in the evening sun, displaying shades of soft colours, sensual and improved by age. It comprises a...
Roc Presidente
Calzada 110 - Esquina Avenida de los Presidentes, 10400 Havana, Cuba
Roc Presidente
Havana, 3 km from the city centre. Calzada 110 - Esquina Avenida de los Presidentes see on map
The city hotel soars up into the sky with 10 historic floors and affords distinctive colours. The hotel occupies an impressive building,...
from ¥5,197
Approximate price
Club Acuario
Calle 248 y 5ta Avenida Marina Hemingway, Santa Fé, Playa, 10400 Havana, Cuba
Club Acuario
Havana, 13.9 km from the city centre. Calle 248 y 5ta Avenida Marina Hemingway, Santa Fé, Playa see on map
Located at the exclusive residential neighbourhood of Marina Hemingway, near Habana´s International Conference Centre, to the West of...
Habana Riviera
Paseo Malecón, Vedado, 10400 Havana, Cuba
Habana Riviera
Havana, 3.9 km from the city centre. Paseo Malecón, Vedado see on map
The hotel has seen more than 40 years of history and was built and decorated in the 50?s. Many celebrities in the spheres of politics and...
from ¥4,014
Approximate price
About HavanaVilla de San Cristobel de Havana, better known as Havana, is the capital of the Republic of Cuba and is considered a World Heritage Site by UNESCO. Founded in 1519, it is formed by 15 municipalities and the city main history and legends are accumulated in Havanas old historical center, one of the main points of interest for tourists to the city. The citys enchantment can be seen in its streets in the multitude of architectonic styles that literally emanate history, and in the vibrant life that is seen in each doorway in every corner of the city of Havana.
Havana has a bay and strolling along it is to experience one of the most pleasant and entertaining walkways in the world. The port of Havana has long equipped the city with an important naval infrastructure that often resulted in parallel development throughout the city, binding it to the culture of the sea and the waves.
The main places of interest are Old Havana, the most important colonial district of all Central America. Declared a World Heritage Site 1982, a stroll through Old Havana under the street lights reveals historical museums, churches, parks and buildings, all bearing witness to the citys splendor. Central Havana is the most populated municipality; for that reason it is characterized by noise and a spirit of plurality. The most remarkable sites are the old Presidential Palace, Paseo del Prado, the palace of Fine Arts and the National Capitol. The levee is an area for wandering and reflection where one can relax and enjoy the tranquility of the sea lapping along the entire coastline of Cubas capital. In this area, the colonial era is not as visible as in Old Havana, paving the way for new structures from the 20th and 21st centuries; the city as a whole has opened up to the new sculptural, architectonic and artistic currents of the present era.
If you dont have a lot of time to enjoy several days walking through the citys music and flavor, we recommend losing yourself in the environs of Havana by visiting the botanical garden, the zoological garden, the Church of San Lazaro or Lenin Park. Because there are so many things to see in this city, it is advisable to stay in its streets and among its people for as long as possible. And for those who wish to take full advantage of their stay and mix the typical Caribbean beach, sun and relaxation, a visit to Havana and Varadero will delight all of those interested in Cuban culture.
Points of Interest in Havana
In Havana we will have scattered clouds, a rise in temperature is expected; dress in light attire.
- Thursday
- Friday
Flights to HavanaAre you reserving a hotel room in Havana and have no way to get there? Book your flight now with Destinia!
Top Hotel Deals
Central America - Caribbean
Ciudad de la Habana
Beach Hotels
- Salou
- Benidorm
- Gandia
- Torremolinos
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- Penyiscola
- Zahara de los Atunes
- Cadiz
- Valencia
- Mojacar
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- Conil de la Frontera
- Alicante
- Roses
- Cullera
- Chiclana de la Frontera
- San Sebastian
- Fuengirola
- Isla Cristina
- El Puerto de Santa Maria
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Bollywood actor Kunal Khemu was injured when an angry mob pelted stones at the actor and the crew who were shooting for a romantic single in Lucknow. The incident took place in the wee hours of Saturday, June 11, when the unit was shooting at the historical 'Bada Imambara' in old Lucknow.
Dhoondte Reh Jaaoge - Title Song
Watch the Title song of Dhoondte Reh Jaaoge, an upcoming movie under the UTV Motion Pictures banner starring Kunal Khemu, Sonu Sood and Soha Ali Khan.
kunal khemu
In arranged marriages, the family looks for the bride to be younger than the groom, but this is no longer the trend or norm. There are men who fall in love and marry older women and vice-versa. Bollywood actors Bipasha Basu and Karan Singh Grover fell in love on the sets of their film 'Alone'. Bi
Recently, it was reported that Bollywood couple Kunal Khemu and Soha Ali Khan are headed for a divorce. But Kunal has strongly reacted to the reports and dismissed them as rumours. The 'Bhaag Johnny' actor took to Twitter and rubbished the reports of his trouble in his marriage with Soha.


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Most Recent Articles In Memo Pad
Latest Memo Pad Articles
- Sonia Rykiel Taps Glen Luchford for Fall Campaign
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LIGHTS, CAMERA, ACTION: Harmony Korine has directed the spot for the Dior Addict Eau de Toilette fragrance campaign, starring Sasha Luss in an fantastic castle setting.
The advertisement comes in a 20-second version destined for television and a 90-second online iteration.
Ryan McGinley was signed on to lens the print advertising, which will appear in single and double pages. The scent — due out starting in Italy on May 13 — includes notes of Sicilian mandarin, jasmine sambac, Tunisian neroli and vanilla, plus essence of sandalwood.


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Research Article
The mentalizing network orchestrates the impact of parochial altruism on social norm enforcement
Version of Record online: 13 MAY 2011
DOI: 10.1002/hbm.21298
Copyright © 2011 Wiley-Liss, Inc.
Human Brain Mapping
Volume 33, Issue 6, pages 1452–1469, June 2012
Additional Information
How to Cite
Baumgartner, T., Götte, L., Gügler, R. and Fehr, E. (2012), The mentalizing network orchestrates the impact of parochial altruism on social norm enforcement. Hum. Brain Mapp., 33: 1452–1469. doi: 10.1002/hbm.21298
Publication History
- Issue online: 10 MAY 2012
- Version of Record online: 13 MAY 2011
- Manuscript Accepted: 10 FEB 2011
- Manuscript Revised: 24 JAN 2011
- Manuscript Received: 31 AUG 2010
Funded by
- Swiss National Science Foundation
- parochial altruism;
- ingroup favoritism;
- outgroup hostility;
- third-party punishment;
- punishment network;
- justification;
- mentalizing network;
- fMRI;
- neural circuitry;
- social neuroscience;
- neuroeconomics
Parochial altruism—a preference for altruistic behavior towards ingroup members and mistrust or hostility towards outgroup members—is a pervasive feature in human society and strongly shapes the enforcement of social norms. Since the uniqueness of human society critically depends on the enforcement of norms, the understanding of the neural circuitry of the impact of parochial altruism on social norm enforcement is key, but unexplored. To fill this gap, we measured brain activity with functional magnetic resonance imaging (fMRI) while subjects had the opportunity to punish ingroup members and outgroup members for violating social norms. Findings revealed that subjects' strong punishment of defecting outgroup members is associated with increased activity in a functionally connected network involved in sanction-related decisions (right orbitofrontal gyrus, right lateral prefrontal cortex, right dorsal caudatus). Moreover, the stronger the connectivity in this network, the more outgroup members are punished. In contrast, the much weaker punishment of ingroup members who committed the very same norm violation is associated with increased activity and connectivity in the mentalizing-network (dorsomedial prefrontal cortex, bilateral temporo-parietal junction), as if subjects tried to understand or justify ingroup members' behavior. Finally, connectivity analyses between the two networks suggest that the mentalizing-network modulates punishment by affecting the activity in the right orbitofrontal gyrus and right lateral prefrontal cortex, notably in the same areas showing enhanced activity and connectivity whenever third-parties strongly punished defecting outgroup members. Hum Brain Mapp, 2011. © 2011 Wiley-Liss, Inc.


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Ultra-wideband (UWB) Analog Front-End (AFE) /Analog- to-Digital Converter (ADC)(9703)
Award Information
Small Business Information
1069 Elkton Drive, Colorado Springs, CO, 80907
Principal Investigator:
Director of Engineering
(719) 590-1112
Director of Engineering
(719) 590-1112
Business Contact:
Thomas Linnenbrink
President and Technical Director
(719) 590-1112
President and Technical Director
(719) 590-1112
Research Institution:
AbstractQ-DOT proposes an innovative analog front-end (AFE) and analog-to-digital converter (ADC) for ultra-wideband (UWB) systems. Modern communications and radar signals combine complex modulation with wide bandwidth to reduce power and lower the probability of intercept. Current digital receivers do not offer both high dynamic range and wide bandwidth needed to detect and characterize these signals. The proposed AFE includes an LNA and novel track-and-hold to facilitate direct conversion to over 10 GHz. Two ADCs are proposed: a novel parallel UWB ADC, and a Nyquist ADC with unique circuits to provide high-resolution UWB performance. Each offers > 10 times the bandwidth of present devices with comparable resolution. Rather than channelizing using filters and mixers, the parallel ADC uses frequency domain encoding to spread the UWB input among baseband ADCs. Digital processing reconstructs the entire input bandwidth with high dynamic range. The Nyquist ADC uses a novel front end to reduce complexity and power while providing UWB performance. The proposed AFE/ADC is modular, expandable, and adaptable. After conceptual design, prototype high resolution UWB AFE/ADC chips can be realized during Phase II. The UWB AFE/ADC will help realize small, low-power, high-performance digital receivers for future surveillance and instrumentation systems.
* information listed above is at the time of submission.
An Official Website of the United States Government


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During spring semester several Palm Beach Atlantic University faculty, staff and students worked as the QEP Research Team to review data and hear presentations about possible areas of focus for PBA's second Quality Enhancement Plan (QEP). This five-year project to improve student learning is
As a freshman at Palm Beach Atlantic University four years ago, Korey Bricker faced a tough decision. Should she, a talented graduate of her high school's International Baccalaureate program, enroll in PBA's Frederick M. Supper Honors Program? Or should she devote herself entirely to nursing,
Five Palm Beach Atlantic University students received scholarships from the Executive Women of the Palm Beaches. The students recently were recognized at the 33rd annual Women in Leadership Luncheon. Lauren Ayres of Palm Beach Shores received the Executive Women Scholarship. She is a third-year
While acknowledging that there is much "disunity" in the United States today, celebrated neurosurgeon and former presidential candidate Dr. Ben Carson sounded a hopeful tone as he delivered the commencement address to more than 500 Palm Beach Atlantic University graduates and hundreds of guests at
At 9 years old, Carly Chapman's life went from sports and dance classes to hospitals and chemotherapy. But even as the fourth-grader battled osteosarcoma, an aggressive form of bone cancer, she found herself surrounded by a skilled, compassionate medical team at Riley Hospital for Children in
Surviving cancer was Mary Reynolds' first battle. Next came the extreme physical exhaustion and other symptoms that her doctors diagnosed as Cancer-Related Fatigue. For eight weeks this spring, Reynolds found the strength to come to Palm Beach Atlantic University's Greene Complex for Sports and
Palm Beach Atlantic University junior Emily Hochheiser found a profitable way to combine her dreams of starting a business with her love of sun and surf. She wrote a plan for a company called Mahalo, a mobile rental and retail business for various beach-related products, from chairs and
When 20 Palm Beach Atlantic students stepped into an ultra-secure Federal Bureau of Investigation (FBI) building to meet with recruiters and tour the facility, they had to capture the experience with only their memories – no cell phones allowed. Through Career Development's Career Treks
Bank of America has awarded a 15,000 corporate community grant to support Palm Beach Atlantic University's Peer-to-Peer Mentor program during the 2016-17 academic year. "We are honored to continue our partnership with Palm Beach Atlantic University and the Peer-to-Peer Mentor program. Through
The Office of Assessment, Accreditation and Research at Palm Beach Atlantic University has announced the recipients of the University's 2016-2017 Quality Initiative Faculty and Student Research Grants. The grant cycle runs from July 1, 2016, to June 30, 2017. Dr. Stephanie Bennett, professor of


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Unger Sanitary / Scrubbing Brushes with Squeegees
Fast and Easy Cleaning with a Sanitary Brush
A sanitary brush will make the cleaning process in your kitchen, dining room, restroom, or hallway more thorough and efficient. These items enable users to scrub, clean, and dry various floor types with the same tool. Plus, each scrubbing brush with a squeegee comes with colored dot stickers that will show employees which room the tool can be used in, which will help prevent cross-contamination.Also make sure to check out other great items from Unger.


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15-16 Honors Reception Recap
Big Weekend for Track & Field and XC Clubs at NIRCA Nationals!!!
NATIONAL RUNNERS-UP! Men's Volleyball Club Serves Up Historic Finish
Hiring for 2016-2017 Club Sports Supervisor is Open
Club Sports Home Events: March 18-20 Weekend
Congrats THON 2016 Club Sports Dancers!!!
FALL 2015 CHAMPIONSHIP RECAP: 1 National Title, 3 Runner-Ups and More!!!
Athletic Training Services for Club Sports Athletes
Upcoming First Aid/CPR/AED Classes
Club Sports Weight Room Hours
Over 75 Club Sport Orgs to choose from!
Check on the 2016-2017 Activation Checklist to see what your club needs to become active for the Fall 2016 & Spring 2017 semester.
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About Registering for the HSCG Conference
The HSCG 2016 Annual Conference is open to members and non-members of the Handcrafted Soap & Cosmetic Guild. Members, however, are entitled to a discount on the Conference Registration fee (see below for pricing details).
What Comes With Your Registration?
Every registration includes:
- Access to all speaker topics and sessions.
- Access to all special events and networking parties.
- All conference meals and breaks.
- Detailed preparation information (online)
- Conference Registration pack.
- All HSCG gifts and goodies
- Industry goodie bag
- All sponsored gifts and goodies
- Access to the Exhibitor room
- Entry to the Awards Dinner on May 21, 2016
- Entry into at least one raffle
- Lots of networking time with other soap and cosmetic makers and industry experts
- Tons of fun and information!
Registration DOES NOT include:
- Guestrooms at the Saddlebrook Resort. (However, attendees ARE entitled to a discounted room rate.)
- Ground transportation to and from the hotel.
- Any meals outside of those outlined in the conference schedule.
Registration Fees and Deadlines
|Rate Class
In order to make it easier for you to pay for your conference registration, the HSCG is be able to offer PayPal Credit which gives you up to 6 months to pay without interest (subject to credit approval).
If you want to bring a guest with you who won't be attending the conference but wants to join you for meals, you may purchase a guest meal ticket. The cost is $319.00 and covers all meals, the Awards Dinner and any special parties. Purchase Guest Meal Ticket.
An HSCG member may bring up to 4 associates (family members, business partners, staff, etc.) to the conference under his/her member number and take advantage of the member rate for the associates.
Cancellation Policy
For fully paid registration fees, guest tickets or exhibitor tables canceled on or before November 30, 2015 the HSCG will issue a refund for 50% the conference registration fee paid. No refunds will be given for cancellations received after November 30, 2015. Cancellations must be made in writing either via email to or by regular mail to HSCG, 178 Elm St, Saratoga Springs, NY 12866.
This cancellation policy will remain the same regardless of whether or not the conference has open spots, sells out or has a waiting list. If a Waiting List is created due to a sold out conference, the HSCG shall follow the Wait Lists Policy of June 30, 2009 (as revised October 7, 2013).Sign Up Now!


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Crunchy Chicken
Total Time
Prep 10 mins
Cook 20 mins
This is my way of cooking: easy-peasy, quick and good tasting. This recipe was sent to me by the Campbells website. I hope you like it.
Ingredients Nutrition
- 2 tablespoons grated parmesan cheese
- 1 cup herb seasoned stuffing mix, crushed into crumbs
- 4 boneless chicken breast halves
- 1 egg, beaten
- 2 tablespoons margarine or 2 tablespoons butter, melted
- Mix stuffing and cheese on plate.
- Dip chicken into beaten egg.
- Then coat with stuffing/cheese mixture.
- Place chicken on baking sheet.
- Drizzle with margarine.
- BAKE at 400°F.
- for 20 min.
- or until chicken is done.
- The original recipe calls for serving this with canned/jarred chicken gravy-- just a suggestion!
Most Helpful
I took Merlot's suggestion and made nuggets out of these and they are wonderful,Crispy little appy's that we dipped in sweet and sour sauce,and enjoyed EVERY BITE!!!! Thanks,Darlene
The stuffing and cheese makes an excellent tasting breading for the chicken!!! It seals in the flavor of the chicken which turns out very tender and moist. The next time I make this, I will try it with chicken nuggests and use as an appetizer. This is a winner, Bekah.


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Frostenden Holiday Cottages
Please find below a selection of self catering holiday cottages to rent in or near Frostenden.
Our call centre is currently closed.
We open today at 9am. You can also send us an e-mail or request a callback.Request a callback
Please find below a selection of self catering holiday cottages to rent in or near Frostenden.
Your search returned 19 cottages.
A fantastic base for exploring North Suffolk and South Norfolk. Ideal for friends and families. Dog-friendly. WiFi. A short drive from Southwold, the broads and wonderful coastline within 25 minutes' walk. (Ref. 933427)
7 nights from £263Book Now
Excellent family holiday cottage in quiet village of Pakefield. Enclosed garden with games room. Beach 2 mins walk. Lowestoft 2 miles. (Ref. 25132)
7 nights from £309Book Now
Lovely mid-terrace cottage in a quiet location, in the village of Pakefield. En-suite bedroom. Enclosed courtyard. Short walk to a dog-friendly beach. Superb promenade walks. Lowestoft 1 mile. (Ref. 923949)
7 nights from £279Book Now
Spacious semi-detached cottage in Lowestoft. Character holiday home with variety of bedrooms, spacious living areas, enclosed garden. Short walk to town amenities. (Ref. 925624)
7 nights from £363Book Now
A lovely mid-terrace cottage close to the centre of Lowestoft, close to the beach, pier and shops. WiFi. Enclosed garden. Perfect base from which to explore both Norfolk and Suffolk. Norwich 26 miles. (Ref. 925708)
7 nights from £314Book Now
Fabulous, stone-built, Grade II listed cottage on the former Thorington Hall Estate in High Street near Thorington. En-suite facilities. Woodburning stove. Feature beams. WiFi. Thorington 2.8 miles. (Ref. 913819)
7 nights from £272Book Now
This character, end-terrace cottage is ideal for exploring the Suffolk Heritage Coast, between Aldeburgh and Southwold. Woodburner. Enclosed lawned garden with furniture. Minsmere nature reserve nearby. Southwold 9 miles. Aldeburgh 11 miles. (Ref. 29701)
7 nights from £293Book Now
A lovely semi-detached property located in the peaceful hamlet of St Michael South Elmham. Historic, beamed cottage. Woodburning stove. Enclosed garden. Bungay 4 miles. (Ref. 22136)
7 nights from £360Book Now
Fabulous period cottage. Charming and characterful interior with heavy beams, inglenook fireplace, warming woodburning stove. Spacious and comfortable accommodation. Surrounded by beautiful rural countryside. Bungay 4 miles. (Ref. 24120)
7 nights from £573Book Now


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The templates documentation NEEDS to be updated!
Just a few of the tags not working with (!)default template as is:
Haven't even tried the rest because I gave up on template and came here for (non-existing) help.
Either hardcode the tags and classes in the template file, or make them work with at least the default template. In either case UPDATE THE TEMPLATE DOCUMENTATION
I had such high hopes for this plugin.
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Qualifying for two seats on the Pendergrass City Council runs from 8:30 a.m. Monday to 4:30 p.m. Wednesday at Pendergrass City Hall, 75 Glenn Gee Road. Seats held by John Pethel and Judy Stowe are up for election Nov. 2.
Qualifying will take place in City Clerk Renee Martinez's office. The fee is $35, and candidates must have lived in the city for at least a year and be qualified to vote in Pendergrass municipal elections.
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Freddie Mercury Official Birthday Party Update: The Refreshments
NEW Queen + Adam Lambert Tickets Released!
New seats made available for ALL THREE Queen + Adam Lambert shows at Hammersmith next month! There are number of reserved tickets that have now been released for sale - snap them up quick!
Queen + Adam Lambert play Hammersmith Apollo on July 11th, 12th and 14th.
For tickets go to www.hmvapollo.com / www.ticketmaster.co.uk or call HMV Tickets 08432 221 0100 / Ticketmaster 08448 444 748.
More News
Freddie Mercury Official Birthday Party Update: Peter Phoebe Freestone To Compere The Event Once More