Characterization of novel nonacid glycosphingolipids as biomarkers of human gastric adenocarcinoma

Changes in glycosphingolipid structures have been shown to occur during the development of several types of human cancers, generating cancer-specific carbohydrate structures that could be used as biomarkers for diagnosis and therapeutic targeting. In this study, we characterized nonacid glycosphingolipids isolated from a human gastric adenocarcinoma by mass spectrometry, enzymatic hydrolysis, and by binding with a battery of carbohydrate-recognizing ligands. We show that the majority of the complex nonacid glycosphingolipids had type 2 (Galβ4GlcNAc) core chains (neolactotetraosylceramide, the Le^x, H type 2, x₂, and the P1 pentaosylceramides, and the Le^y, A type 2, and neolacto hexaosylceramides). We also found glycosphingolipids with type 1 (Galβ3GlcNAc) core (lactotetraosylceramide and the H type 1 pentaosylceramide) and globo (GalαGal) core chains (globotriaosylceramide and globotetraosylceramide). Interestingly, we characterized two complex glycosphingolipids as a P1 heptaosylceramide (Galα4Galβ4GlcNAcβ3Galβ4GlcNAcβ3Gal β4Glcβ1Cer) and a branched P1 decaosylceramide (Galα4Gal β4GlcNAcβ3(Galα4Galβ4GlcNAcβ6)Galβ4GlcNAcβ3Galβ4Glc β1Cer). These are novel glycosphingolipid structures and the first reported cases of complex glycosphingolipids larger than pentaosylceramide carrying the P1 trisaccharide. We propose that these P1 glycosphingolipids may represent potential biomarkers for the early diagnosis of gastric cancer.     

Cerebrospinal fluid concentrations of peptides derived from chromogranin B and secretogranin II are decreased in multiple sclerosis

Novel biomarkers for multiple sclerosis (MS) could improve diagnosis and provide clues to pathogenesis. In this study surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze protein expression in CSF from 46 MS patients, 46 healthy siblings to the patients, and 50 unrelated healthy controls. Twenty-four proteins in the mass range 2–10 kDa were expressed at significantly different levels ( p  < 0.01) in a robust manner when comparing the three groups. Identities of three proteins were determined using biochemical purification followed by tandem mass spectrometric analysis. Immunoprecipitation experiments confirmed the identities for two peptides derived from chromogranin B ( m / z 6252) and from secretogranin II ( m / z 3679). These peptides were all decreased in MS when compared with siblings or controls. Radioimmunoassays specific for each peptide confirmed these differences. The lowered concentrations did not correlate to the axonal damage marker neurofilament light protein and may thus reflect functional changes rather than neurodegeneration. Further studies will investigate the involvement of these peptides in MS pathogenesis.     

Long-term response of <Emphasis Type="Italic">Dreissena polymorpha</Emphasis> larvae to physical and biological forcing in a shallow lake

Müggelsee, a shallow eutrophic lake (Berlin, Germany), has been subject to global warming and concurrent reductions of anthropogenic nutrient loading during the past decades. Here, we focus on the recent increase in abundance of Dreissena polymorpha larvae. We aimed at ascertaining whether the change in abundance of larvae was driven by changes in climate, especially by climate warming, and/or by the concurrent changes in trophic state of the lake. Both the numbers of small, newly developed larvae and their lengths have increased in recent years, suggesting that conditions for overall reproductive success have improved. The timing of the increase in abundance of larvae was matched by changes in nutrient loading and phytoplankton biomass, induced by a reduced inflow of nutrients into the lake. Besides a correlation between the first appearance of larvae each year and the timing of the requisite temperature for first spawning (12°C), no relationship between changes in water temperature and abundance, length and survival rates of larvae was found. However, a sudden drop in abundance of larvae in 2003 may be primarily attributed to low dissolved oxygen conditions during an unusually long period of stratification, induced by anomalous meteorological conditions. The increase in length and survival rates of larvae was most likely due to changes in food composition, which followed the decrease in nutrient availability, and to changes in the occurrence of planktivorous fish. The results suggest that the first appearance of larvae per year and the decline in abundance of larvae in 2003 were driven by climatic influences, while the overall increase in abundance and length of D. polymorpha larvae in Müggelsee was more likely caused by changes in the trophic state of the lake rather than by climate warming. Priority programme of the German Research Foundation—contribution 8.     

Sensitive protein detection via triple-binder proximity ligation assays

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.     

Haptoglobin-α1, -α2, vitamin D-binding protein and apolipoprotein C-III as predictors of etanercept drug response in rheumatoid arthritis

IntroductionThe introduction of tumor necrosis factor-alpha (TNF-α) antagonists has substantially improved patient’s clinical outcome in rheumatoid arthritis (RA). However, nearly 20% to 40% of RA patients do not respond to anti-TNF-α treatment strategies. To identify valid predictors of TNF-α antagonist response in RA, serum proteome profiles from responders (R) and non-responders (NR) to etanercept, a soluble recombinant TNF-α receptor/IgG Fc fusion protein receptor, were compared in a prospective cohort study.MethodsIn this clinical study 50 RA patients with inadequate response to conventional DMARDs were included and treated with etanercept. The primary efficacy endpoint was response according to the European League against Rheumatism (EULAR) improvement criteria. Serum samples collected prior to initiation and after six months of etanercept therapy were cleared of the most abundant major proteins by immunoaffinity chromatography. After separation by two-dimensional differential gel electrophoresis (2D-DIGE) and identification by mass spectrometry (MS) data were validated by Western blot analysis.ResultsAfter six months of etanercept treatment 62% (n = 31) of RA patients achieved response. Haptoglobin-α1 (Hp-α1) and -α2 (Hp-α2) and vitamin D-binding protein (VDBP) were found to be significantly upregulated in responder sera (P ≤0.02) at study entry. In contrast, apolipoprotein C-III (ApoC-III) showed significantly higher levels in non-responders (P = 0.0162). At study end ApoA-II, Hp-α1, Hp-α2 and VDBP were identified to be expressed at significantly higher levels (P <0.05) in responder sera.ConclusionsBy application of clinical proteomics in immunodepleted sera we could identify and validate for the first time Hp-α1, -α2, VDBP and ApoC-III as potential biomarkers for prediction of etanercept drug response in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0553-1) contains supplementary material, which is available to authorized users.     

Dietary Fish Oil Inhibits Pro-Inflammatory and ER Stress Signalling Pathways in the Liver of Sows during Lactation

Lactating sows have been shown to develop typical signs of an inflammatory condition in the liver during the transition from pregnancy to lactation. Hepatic inflammation is considered critical due to the induction of an acute phase response and the activation of stress signaling pathways like the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), both of which impair animal´s health and performance. Whether ER stress-induced UPR is also activated in the liver of lactating sows and whether dietary fish oil as a source of anti-inflammatory effects n-3 PUFA is able to attenuate hepatic inflammation and ER stress-induced UPR in the liver of sows is currently unknown. Based on this, two experiments with lactating sows were performed. The first experiment revealed that ER stress-induced UPR occurs also in the liver of sows during lactation. This was evident from the up-regulation of a set of genes regulated by the UPR and numerically increased phosphorylation of the ER stress-transducer PERK and PERK-mediated phosphorylation of eIF2α and IκB. The second experiment showed that fish oil inhibits ER stress-induced UPR in the liver of lactating sows. This was demonstrated by decreased mRNA levels of a number of UPR-regulated genes and reduced phosphorylation of PERK and PERK-mediated phosphorylation of eIF2α and IκB in the liver of the fish oil group. The mRNA levels of various nuclear factor-κB-regulated genes encoding inflammatory mediators and acute phase proteins in the liver of lactating sows were also reduced in the fish oil group. In line with this, the plasma levels of acute phase proteins were reduced in the fish oil group, although differences to the control group were not significant. In conclusion, ER stress-induced UPR is present in the liver of lactating sows and fish oil is able to inhibit inflammatory signaling pathways and ER stress-induced UPR in the liver.     

Identification of the Molecular and Genetic Basis of PX2, a Glycosphingolipid Blood Group Antigen Lacking on Globoside-deficient Erythrocytes

The x₂ glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P^k-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-β-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcβ3Gal, as x₂. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P₁^k phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x₂ and sialylated forms of x₂, whereas x₂ is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x₂. Knockdown experiments with siRNA against B3GALNT1 diminished x₂ levels. We conclude that x₂ fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x₂ joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P₁^k or P₂^k erythrocyte units are preferentially selected for transfusion to P^k patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels.     

Giant clams in shallow reefs: UV-resistance mechanisms of Tridacninae in the Red Sea

The photosymbiosis of tropical giant clams (subfamily Tridacninae) with unicellular algae (Symbiodiniaceae) restricts their distribution to the sunlit, shallow waters of the euphotic zone where organisms are additionally exposed to potentially damaging levels of solar UV radiation. Metabolic and physiological responses of Red Sea Tridacna maxima clams, including net calcification and primary production, as well as valvometry (i.e., shell gaping behavior) were assessed when exposed to simulated high radiation levels received at 3 and 5 m underwater. The two levels of radiation included exposure treatments to photosynthetically active radiation (PAR; 400–700 nm) alone and to both, PAR and ultraviolet-B radiation (UV-B; 280–315 nm). The valvometry data obtained using flexible magnetic sensors indicated that specimens under PAR + UV-B exposure significantly reduced the proportion of their exposed mantle area, a potential photo-protective mechanism which, however, reduces the overall amount of PAR received by the algal symbionts. Consequently, specimens under PAR + UV-B displayed a slight, although non-significant, reduction in primary production rates but no signs of additional oxidative stress, changes in symbiont densities, chlorophyll content, or levels of mycosporine-like amino acids. Net calcification rates of T. maxima were not affected by exposure to UV-B; however, calcification was positively correlated with incident PAR levels. UV-B exposure changes the valvometry, reducing the exposed mantle area which consequently diminishes the available PAR for the photosymbionts. Still, T. maxima maintains high rates of primary production and net calcification, even under high levels of UV-B. This provides experimental support for a recently described, effective UV-defensive mechanism in Tridacninae, in which the photonic cooperation of the associated algal symbionts and giant clam iridocytes is assumed to establish optimal conditions for the photosynthetic performance of the clams’ symbionts.