Orthogonal protection of peptides and peptoids for cyclization by the thiol-ene reaction and conjugation

Cyclic peptides and peptoids were prepared using the thiolene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization.

Polyphenol-rich foods in the Mediterranean diet are associated with better cognitive function in elderly subjects at high cardiovascular risk

Brain oxidative processes play a major role in age-related cognitive decline, thus consumption of antioxidant-rich foods might help preserve cognition. Our aim was to assess whether consumption of antioxidant-rich foods in the Mediterranean diet relates to cognitive function in the elderly. In asymptomatic subjects at high cardiovascular risk (n = 447; 52% women; age 55-80 y) enrolled in the PREDIMED study, a primary prevention dietary-intervention trial, we assessed food intake and cardiovascular risk profile, determined apolipoprotein E genotype, and used neuropsychological tests to evaluate cognitive function.We also measured urinary polyphenols as an objective biomarker of intake. Associations between energy-adjusted food consumption, urinary polyphenols, and cognitive scores were assessed by multiple linear regression models adjusted for potential confounders. Consumption of some foods was independently related to better cognitive function. The specific associations [regression coefficients (95% confidence intervals)] were: total olive oil with immediate verbal memory [0.755 (0.151-1.358)]; virgin olive oil and coffee with delayed verbal memory [0.163 (0.010-0.316) and 0.294 (0.055-0.534), respectively];walnuts with working memory [1.191 (0.061-2.322)]; and wine with Mini-Mental State Examination scores [0.252 (0.006-0.496)]. Urinary polyphenols were associated with better scores in immediate verbal memory [1.208 (0.236-2.180)]. Increased consumption of antioxidant-rich foods in general and of polyphenols in particular is associated with better cognitive performance in elderly subjects at high cardiovascular risk. The results reinforce the notion that Mediterranean diet components might counteract age-related cognitive decline.

On the existence and function of galanin receptor heteromers in the central nervous system

Galanin receptor (GalR) subtypes 1-3 linked to central galanin neurons may form heteromers with each other and other types of G protein-coupled receptors in the central nervous system (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR-5-HT1A heteromers likely exist with antagonistic GalR-5-HT1A receptor-receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1-5-HT1A heteromers in cellular models with trans-inhibition of the protomer signaling. A GalR1-GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1-GalR2-5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR-5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR-NPYY1 receptor interactions in putative GalR-NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1-GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1-GalR2-NPYY2 heterotrimer. Finally, putative GalR-α2-adrenoreceptor heteromers with antagonistic receptor-receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression

Coal deposition in carbonate rich shallow Lacustrine systems: The Calaf and Mequinenza sequences (Oligocene, eastern Ebro Basin, NE Spain)

Two main coal-bearing sequences developed during the Oligocene in the Tertiary Ebro Basin, the Calaf (early Oligocene) and Mequinenza (late Oligocene) coal basins. Coal deposition took place in shallow marsh-swamp-lake complexes which sometimes became closed and evolved under warm climatic conditions with fluctuating humidity. These shallow lacustrine systems are closely interrelated with the terminal parts of the distributive fluvial systems which spread from the tectonically active Ebro basin margins. Laterally extensive lignite-bearing sequences, including rather thin, lenticular autochthonous and/or hypautochthonous coal seams with high ash and sulphur contents, characterized coal deposition in the shallow lacustrine systems. Coal seam geometry, which makes them nearly subeconomic, resulted from the tectonic instability during basin margin evolution and the sometimes closed, arid conditions under which the lacustrine systems evolved. High ash and sulphur contents resulted from the inadequate isolation of peat forming environments from clastic influx and from the very low acidity and sometimes high sulphate contents of the lacustrine waters. Coal exploration in shallow lacustrine sequences similar to those described here must take into account that the spread of coal-forming environments and maxima of coal deposition are usually coincident with lake expansions and retraction or shifting of the terminal fluvial zones interrelated with the lacustrine areas.

Incompressibility of neutron-rich matter

The saturation properties of neutron-rich matter are investigated in a relativistic mean-field formalism using two accurately calibrated models: NL3 and FSUGold. The saturation properties density, binding energy per nucleon, and incompressibility coefficient are calculated as a function of the neutron-proton asymmetry α≡(N-Z)/A to all orders in α. Good agreement (at the 10% level or better) is found between these numerical calculations and analytic expansions that are given in terms of a handful of bulk parameters determined at saturation density. Using insights developed from the analytic approach and a general expression for the incompressibility coefficient of infinite neutron-rich matter, i.e., K0(α)=K0+Kτα2+ , we construct a hybrid model with values for K0 and Kτ as suggested by recent experimental findings. Whereas the hybrid model provides a better description of the measured distribution of isoscalar monopole strength in the Sn isotopes relative to both NL3 and FSUGold, it significantly underestimates the distribution of strength in 208Pb. Thus, we conclude that the incompressibility coefficient of neutron-rich matter remains an important open problem.

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins

Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

Correlation between charge transport and electroluminescence properties of Si-rich oxide/nitride/oxide-based light emitting capacitors.

The electrical and electroluminescence (EL) properties at room and high temperatures of oxide/ nitride/oxide (ONO)-based light emitting capacitors are studied. The ONO multidielectric layer is enriched with silicon by means of ion implantation. The exceeding silicon distribution follows a Gaussian profile with a maximum of 19%, centered close to the lower oxide/nitride interface. The electrical measurements performed at room and high temperatures allowed to unambiguously identify variable range hopping (VRH) as the dominant electrical conduction mechanism at low voltages, whereas at moderate and high voltages, a hybrid conduction formed by means of variable range hopping and space charge-limited current enhanced by Poole-Frenkel effect predominates. The EL spectra at different temperatures are also recorded, and the correlation between charge transport mechanisms and EL properties is discussed.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Cytosine-and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34 -bases long cytosine-rich sequence and its complementary guanin e-rich strand corresponding to the first intron of the n-mycgene were studied. Both sequences , not yet studied, contain a 12 - base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively ...

Diagnostic and prognostic value of antibodies against chimeric fibrin/filaggrin citrullinated synthetic peptides in rheumatoid arthritis

Introduction: Evidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target of anticitrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis (RA). We compared the diagnostic yield of three enzyme-linked immunosorbent assay (ELISA) tests by using chimeric fibrin/filaggrin citrullinated synthetic peptides (CFFCP1, CFFCP2, CFFCP3) with a commercial CCP2-based test in RA and analyzed their prognostic values in early RA. Methods: Samples from 307 blood donors and patients with RA (322), psoriatic arthritis (133), systemic lupus erythematosus (119), and hepatitis C infection (84) were assayed by using CFFCP- and CCP2-based tests. Autoantibodies also were analyzed at baseline and during a 2-year follow-up in 98 early RA patients to determine their prognostic value. Results: With cutoffs giving 98% specificity for RA versus blood donors, the sensitivity was 72.1% for CFFCP1, 78.0% for CFFCP2, 71.4% for CFFCP3, and 73.9% for CCP2, with positive predictive values greater than 97% in all cases. CFFCP sensitivity in RA increased to 80.4% without losing specificity when positivity was considered as any positive anti-CFFCP status. Specificity of the three CFFCP tests versus other rheumatic populations was high (> 90%) and similar to those for the CCP2. In early RA, CFFCP1 best identified patients with a poor radiographic outcome. Radiographic progression was faster in the small subgroup of CCP2-negative and CFFCP1-positive patients than in those negative for both autoantibodies. CFFCP antibodies decreased after 1 year, but without any correlation with changes in disease activity. Conclusions: CFFCP-based assays are highly sensitive and specific for RA. Early RA patients with anti-CFFCP1 antibodies, including CCP2-negative patients, show greater radiographic progression.

Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity.

Protected maleimide building blocks for the decoration of peptides, peptoids and peptide nucleic acids

Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides into polyamides such as peptides, peptide nucleic acids, and peptoids were prepared, as well as the corresponding oligomers. Suitable maleimide deprotection conditions were established in each case. The stability of the adducts generated by Michael-type maleimide-thiol reaction and Diels-Alder cycloaddition to maleimide deprotection conditions was exploited to prepare a variety of conjugates from peptide and PNA scaffolds incorporating one free and one protected maleimide. The target molecules were synthesized by using two subsequent maleimide-involving click reactions separated by a maleimide deprotection step. Carrying out maleimide deprotection and conjugation simultaneously gave better results than performing the two reactions subsequently.

Straightforward synthesis of cyclic and bicyclic peptides

Cyclic peptide architectures can be easily synthesized from cysteine-containing peptides with appending maleimides, free or protected, through an intramolecular Michael-type reaction. After peptide assembly, the peptide can cyclize either during the trifluoroacetic acid treatment, if the maleimide is not protected, or upon deprotection of the maleimide. The combination of free and protected maleimide moieties and two orthogonally protected cysteines gives access to structurally different bicyclic peptides with isolated or fused cycles.

Prediction of Antibacterial Activity from Physicochemical Properties of Antimicrobial Peptides

Consensus is gathering that antimicrobial peptides that exert their antibacterial action at the membrane level must reach a local concentration threshold to become active. Studies of peptide interaction with model membranes do identify such disruptive thresholds but demonstrations of the possible correlation of these with the in vivo onset of activity have only recently been proposed. In addition, such thresholds observed in model membranes occur at local peptide concentrations close to full membrane coverage. In this work we fully develop an interaction model of antimicrobial peptides with biological membranes; by exploring the consequences of the underlying partition formalism we arrive at a relationship that provides antibacterial activity prediction from two biophysical parameters: the affinity of the peptide to the membrane and the critical bound peptide to lipid ratio. A straightforward and robust method to implement this relationship, with potential application to high-throughput screening approaches, is presented and tested. In addition, disruptive thresholds in model membranes and the onset of antibacterial peptide activity are shown to occur over the same range of locally bound peptide concentrations (10 to 100 mM), which conciliates the two types of observations

Membrane interaction of a new synthetic antimicrobial lipopetide sp-85 with broad spectrum activity

Antimicrobial peptides offer a new class of therapeutic agents to which bacteria may not be able todevelop genetic resistance, since their main activity is in the lipid component of the bacterial cell mem-brane. We have developed a series of synthetic cationic cyclic lipopeptides based on natural polymyxin,and in this work we explore the interaction of sp-85, an analog that contains a C12 fatty acid at theN-terminus and two residues of arginine. This analog has been selected from its broad spectrum antibac-terial activity in the micromolar range, and it has a disruptive action on the cytoplasmic membrane ofbacteria, as demonstrated by TEM. In order to obtain information on the interaction of this analog withmembrane lipids, we have obtained thermodynamic parameters from mixed monolayers prepared withPOPG and POPE/POPG (molar ratio 6:4), as models of Gram positive and Gram negative bacteria, respec-tively. LangmuirBlodgett films have been extracted on glass plates and observed by confocal microscopy,and images are consistent with a strong destabilizing effect on the membrane organization induced bysp-85. The effect of sp-85 on the membrane is confirmed with unilamelar lipid vesicles of the same com-position, where biophysical experiments based on fluorescence are indicative of membrane fusion andpermeabilization starting at very low concentrations of peptide and only if anionic lipids are present.Overall, results described here provide strong evidence that the mode of action of sp-85 is the alterationof the bacterial membrane permeability barrier.

Altered nitrogen balance and decreased urea excretion in male rats fed cafeteria diet are related to arginine availability

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids.We analyzed whether reduced urea excretion was a consequence of higherNO ; (nitrite,nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion.There were no differences in plasma nitrate or nitrite. NO and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithinewhen comparedwith controls,whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.