The ITGAV rs3738919-C allele is associated with rheumatoid arthritis in the European Caucasian population: a family-based study

The integrin alpha(v)beta3, whose alpha(v) subunit is encoded by the ITGAV gene, plays a key role in angiogenesis. Hyperangiogenesis is involved in rheumatoid arthritis (RA) and the ITGAV gene is located in 2q31, one of the suggested RA susceptibility loci. Our aim was to test the ITGAV gene for association and linkage to RA in a family-based study from the European Caucasian population. Two single nucleotide polymorphisms were genotyped by PCR-restriction fragment length polymorphism in 100 French Caucasian RA trio families (one RA patient and both parents), 100 other French families and 265 European families available for replication. The genetic analyses for association and linkage were performed using the comparison of allelic frequencies (affected family-based controls), the transmission disequilibrium test, and the genotype relative risk.We observed a significant RA association for the C allele of rs3738919 in the first sample (affected family-based controls, RA index cases 66.5% versus controls 56.7%; P = 0.04). The second sample showed the same trend, and the third sample again showed a significant RA association. When all sets were combined, the association was confirmed (affected family-based controls, RA index cases 64.6% versus controls 58.1%; P = 0.005). The rs3738919-C allele was also linked to RA (transmission disequilibrium test, 56.5% versus 50% of transmission; P = 0.009) and the C-allele-containing genotype was more frequent in RA index cases than in controls (RA index cases 372 versus controls 339; P = 0.002, odds ratio = 1.94, 95% confidence interval = 1.3-2.9). The rs3738919-C allele of the ITGAV gene is associated with RA in the European Caucasian population, suggesting ITGAV as a new minor RA susceptibility gene.     

Improved BLAST searches using longer words for protein seeding

MOTIVATION: The blastp and tblastn modules of BLAST are widely used methods for searching protein queries against protein and nucleotide databases, respectively. One heuristic used in BLAST is to consider only database sequences that contain a high-scoring match of length at most 5 to the query. We implemented the capability to use words of length 6 or 7. We demonstrate an improved trade-off between running time and retrieval accuracy, controlled by the score threshold used for short word matches. For example, the running time can be reduced by 20-30% while achieving ROC (receiver operator characteristic) scores similar to those obtained with current default parameters. AVAILABILITY: The option to use long words is in the NCBI C and C++ toolkit code for BLAST, starting with version 2.2.16 of blastall. A Linux executable used to produce the results herein is available at: ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/protein_longwords     

Comparative genomic analysis of the odorant-binding protein family in 12 <Emphasis Type="Italic">Drosophila</Emphasis> genomes: purifying selection and birth-and-death evolution

Background  

Chemoreception is a widespread mechanism that is involved in critical biologic processes, including individual and social behavior. The insect peripheral olfactory system comprises three major multigene families: the olfactory receptor (Or), the gustatory receptor (Gr), and the odorant-binding protein (OBP) families. Members of the latter family establish the first contact with the odorants, and thus constitute the first step in the chemosensory transduction pathway.     

Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23–55 monosaccharides with 4–9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)_n, to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.Glycosylation is the most complex protein post-translational modification known today but unfortunately glycomics characterization is often kept apart from the proteomic characterization of proteins of biological tissues and samples. Such dual approaches may be complementary but also limiting because they will give neither the whole picture of all protein iso/glycoforms in a tissue nor the detailed structure of any of the single proteins included. Glycoproteomics approaches are now becoming available bridging these two fields by keeping the glycan and the peptide parts together in giving simultaneous and specific information on the glycan structures, their attachment sites and the identities of the core proteins (1). Here we report on new liquid chromatography-tandem MS (LC-MS/MS) protocols that can be used to decipher the structural heterogeneity of proteoglycans and even proteoglycan complexes, i.e. glycoproteins known to be inherently demanding to structurally characterize either alone or in mixtures. Because of the theoretically immense but biologically limited number of glycan structures appearing in nature, and given the limitations of MS analysis of glycopeptides (defining types, numbers and sequences of monosaccharides rather than monosaccharide identities, linkage positions and configurations), it is important to build on earlier experience from analyses of single isolated proteins and to use nomenclature, abbreviations and symbols that are well accepted and understood. Throughout this report we have, used the CFG nomenclature of glycan structures (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml) to increase the intelligibility of our findings (Fig. 1).Open in a separate windowFig. 1.Schematic depiction of the inter-α-trypsin inhibitor complex and its degradation by chondroitinase ABC. Both the linkage and cross-linkage regions are highlighted as well as the molecular details of the GalNAc to aspartic acid ester bond that cross-links the heavy chains to the CS chain of bikunin. In addition to CS-glycopeptides the endolytic activity of the chondroitinase ABC digestion is also expected to generate free disaccharides of the CS chain (faded region). The formation of unsaturated GlcA by chondrotinase ABC is shown below the complex and the monosaccharide symbols are explained in the upper left quadrant.Proteoglycans constitute a group of O-glycosylated proteins all carrying one or more complex glycan (glycosaminoglycan or GAG)¹ chains attached to Ser residues of the core proteins through a common linkage tetrasaccharide, called the GAG linkage region, which is composed of the innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) at the reducing end of the GAG chain (GlcA is glucuronic acid; Gal is galactose; and Xyl is xylose) (2).The structural difference between various subclasses of proteoglycans emanates from the repeated extension of the linkage tetrasaccharide by two monosaccharides; e.g. GlcAβ3GalNAcβ4 in chondroitin sulfate proteoglycans (CSPGs), GlcAβ4GlcNAcα4 in heparan sulfate proteoglycans (HSPGs) and from the extent and positions of O- and N-sulfations, GlcA epimerization and from the actual chain length of the glycosaminoglycan. The GAG chains may also be covalently attached, or cross-linked, to other proteins at the nonreducing end of the chain through a C-terminal aspartic acid ester linkage. In this report we concentrate on the structural characterization of the human inter-α-trypsin inhibitor CSPG complex.Chondroitin sulfate proteoglycans play a significant role in maintaining the structural integrity of most extracellular matrices. In addition to their structural role as matrix components, eukaryote CSPGs are known to be involved in more specialized functions such as signal transduction, morphogenesis and regulation of stem cell behavior and differentiation (3–9). In many cases, their biological activity is mediated by selective binding of protein ligands to distinct glycan structural variants (10, 11). Previous studies have made possible the analysis of the average fine structure of chondroitin sulfate (CS) chains facilitating the identification of discrete glycan domains likely involved in ligand interaction (12–16). The CSPG saccharide linkage region differs significantly from the structure of the rest of the glycan chain and its assembly has been shown to be essential for the regulation of the GAG biosynthesis (17–19).The glycomics approach for CSPGs structural analysis involves the release of the GAG chains from the core proteins, digestion of the released chain into disaccharides followed by their subsequent analysis by e.g. ion exchange chromatography, nuclear magnetic resonance spectroscopy or mass spectrometry. However, in this work-flow the identities of the core proteins as well as the attachment sites are lost hindering the assignment of specific structures to particular proteoglycan isoforms. In order to obtain integrated glycan-protein information, we recently developed a glycoproteomics approach allowing site-specific analysis of CSPG linkage region glycopeptides (20). Samples from human urine, plasma and cerebrospinal fluid were trypsinized and subjected to strong anion exchange (SAX) chromatography in order to enrich for anionic GAG-substituted glycopeptides. The fractions were then treated with chondroitinase ABC to depolymerize CS-chains into free disaccharides and residual hexasaccharide substituted peptides comprising the tetrasaccharide linkage region (21). The resulting CS-glycopeptides were finally characterized by reversed phase nLC-MS/MS run in positive mode using higher-energy collisional dissociation (HCD). In that study, 13 novel CSPGs were successfully identified and site-specific information regarding 13 already established CSPGs was obtained. However, the fine-structure analysis of the CS glycopeptides and their substitution patterns was hampered by the relatively weak intensities of sulfated and/or phosphorylated fragment ions obtained under the HCD conditions used. This problem became especially obvious for bikunin, the major component of the inter-α-trypsin inhibitor (IαI) complex and the most abundant and heterogeneous CSPG of those samples.Bikunin is a small acidic glycoprotein of about 40 kDa that is modified at Ser-10 by the attachment of one single CS chain. The linkage region of this GAG chain has been the target for several studies concluding a uniform galactose-4-sulfate tetrasaccharide structure (GlcAβ3Galβ3(4-O-SO₃)Galβ4Xylβ-O-) in both human plasma and urinary samples (22, 23). The IαI complex is a unique macromolecular arrangement of structurally related proteins, i.e. heavy chains, covalently linked to the CS chain of bikunin, also called the light chain of the IαI complex (Fig. 1) (24–27). In humans, one bikunin molecule is typically cross-linked to both heavy chain 1 (H1) and heavy chain 2 (H2) in the IαI-complex, but only to heavy chain 3 (H3) in the pre-α-trypsin inhibitor complex (28). Free bikunin is mainly found in urine and also known as the urinary trypsin inhibitor (UTI) (29). The plasma and urinary levels of bikunin are often raised during pathological processes such as chronic and acute inflammations, infections, cancer, renal diseases and diabetes (30–36). During inflammation, the CS chain may undergo changes in size and sulfation that correlate with the severity of the inflammatory response (37, 38).In the present work, we further developed our glycoproteomics approach to enable a detailed characterization and relative quantification of the IαI linkage and cross-linkage region glycopeptides derived from human plasma, urine and CSF samples. The use of alternating collisional energies proved to have a major impact on the fragmentation of CS glycopeptides and added valuable complementary structural information. The addition of sodium ions readily increased the stability of labile sulfate substituents upon HCD conditions allowing a more precise glycan characterization.The CS linkage region of bikunin was found to display a much larger heterogeneity than previously realized. The linkage region modifications were found to include sulfate, phosphate, fucose and sialic acid substitutions with notable differences across body fluids. We were also able to specifically identify glycopeptides derived from the cross-linking regions of H1 and H2. The heavy chains were often simultaneously attached to the same CS-chain, in close proximity to each other and sometimes even to adjacent GalNAc residues. Taken together, this study demonstrates that the linkage region of human CSPGs displays a vast greater structural complexity than previously perceived. Given that specific modifications of the linkage region may function as a molecular regulator of CS biosynthesis, these findings could also assist in further elucidating the mechanisms of CS chain elongation and substitution.     

Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

Tumor cellular senescence induced by genotoxic treatments has recently been found to be paradoxically linked to the induction of “stemness.” This observation is critical as it directly impinges upon the response of tumors to current chemo-radio-therapy treatment regimens. Previously, we showed that following etoposide (ETO) treatment embryonal carcinoma PA-1 cells undergo a p53-dependent upregulation of OCT4A and p21Cip1 (governing self-renewal and regulating cell cycle inhibition and senescence, respectively). Here we report further detail on the relationship between these and other critical cell-fate regulators. PA-1 cells treated with ETO display highly heterogeneous increases in OCT4A and p21Cip1 indicative of dis-adaptation catastrophe. Silencing OCT4A suppresses p21Cip1, changes cell cycle regulation and subsequently suppresses terminal senescence; p21Cip1-silencing did not affect OCT4A expression or cellular phenotype. SOX2 and NANOG expression did not change following ETO treatment suggesting a dissociation of OCT4A from its pluripotency function. Instead, ETO-induced OCT4A was concomitant with activation of AMPK, a key component of metabolic stress and autophagy regulation. p16ink4a, the inducer of terminal senescence, underwent autophagic sequestration in the cytoplasm of ETO-treated cells, allowing alternative cell fates. Accordingly, failure of autophagy was accompanied by an accumulation of p16ink4a, nuclear disintegration, and loss of cell recovery. Together, these findings imply that OCT4A induction following DNA damage in PA-1 cells, performs a cell stress, rather than self-renewal, function by moderating the expression of p21Cip1, which alongside AMPK helps to then regulate autophagy. Moreover, this data indicates that exhaustion of autophagy, through persistent DNA damage, is the cause of terminal cellular senescence.     

NDI‐Based Small Molecule as Promising Nonfullerene Acceptor for Solution‐Processed Organic Photovoltaics

A novel naphthalene diimide (NDI)‐based small molecule (BiNDI) is designed and synthesized by linking two NDI monomers via a vinyl donor moiety. The electronic structure of BiNDI is carefully investigated by ultraviolet photoelectron spectroscopy (UPS). Density functional theory (DFT) sheds further light on the molecular configuration and energy level distribution. Thin film transistors (TFT) based on BiNDI show a highest electron mobility of 0.365 cm² V^?1 s^?1 in ambient atmosphere. Organic photovoltaics (OPVs) by using BiNDI as the acceptor show a highest power conversion efficency (PCE) of 2.41%, which is the best result for NDI‐based small molecular acceptors. Transmission electron microscopy (TEM), atomic force microscopy (AFM), grazing incidence wide‐angle X‐ray diffraction (GIXD), and X‐ray photo­electron spectroscopy (XPS) characterization to understand the morphology and structure order of the bulk heterojunction film are performed. It is found that small amount of 1,8‐diiodooctane (DIO) (i.e., 0.5%) in the blended film facilitates the crystallization of BiNDI into fibrillar crystals, which is beneficial for the improvement of device performance.