Pepscan (Netherlands)

[This corrects the article DOI: 10.3389/fmicb.2011.00021.].

A new type of hydrogel thin film that can be post-modified with biofunctional molecules is investigated for possible applications in evanescent wave optical affinity biosensors. This material is based on a polyisocyanide (PIC) copolymer that self-assembles into a fibrous polymer network. It is covalently tethered to a solid transducer surface, and its swelling gives rise to a hydrogel layer with a thickness up to several micrometers, as characterized by a combination of surface plasmon resonance and optical waveguide spectroscopy. Thin PIC-based hydrogel films with pendant functional biomolecules can be used in label-free affinity sensing strategies. In this work, we demonstrate the PIC-based affinity binding matrix through an avidin coupling of functional biomolecules and immunoassay-based detection, which can be readily expanded to other types of assays and target analytes. In addition, the stable PIC matrix proves to be resistant to fouling, even in the presence of complex biological fluids such as blood plasma or serum.

Mit flexiblen tetravalenten Gerüsten gelingt die Umwandlung linearer Peptide in tricyclische Peptide durch CLIPS/CuAAC-Ligation/Multicyclisierung. In der Zuschrift auf S. 510 zeigen J. H. van Maarseveen, P. Timmerman et al., dass es für die Art und Anzahl der Aminosäuren in den Schlaufen keinerlei Einschränkungen gibt. Die Methode ermöglicht die Hochdurchsatzsynthese tricyclischer Peptide gefolgt von Screenings zur Identifizierung wirksamer Peptid-Therapeutika.

Most serological assays detect antibody responses in biological samples through affinity of serum antibodies for antigens provided in the assay. Certain antigens, however, may be difficult to produce and/or may contain unwanted epitopes. In these cases, a practical alternative may be the use of peptides as representatives for specific epitopes. Peptides can be obtained after purification in large quantities for a modest price, but screening of a large set of peptides during development may be relatively expensive. To cut costs of screening peptides for a new serological assay, the concept was investigated of using cheap non-purified (crude) peptides instead of purified peptides.Peptides were selected that represent three well-described linear epitopes of viral proteins: VP2 of canine parvovirus (CPV), gp41 of human immunodeficiency virus (HIV) and E2 of classical swine fever virus (CSFV). Crude and purified biotinylated peptides with either a short or long spacer between the biotin and the epitope were used to test their capability to bind antibodies in a bead-based suspension array.The results show that, in a bead-based suspension array, crude peptides can function as antigen for specific monoclonal antibodies, and that the acquired signals are less than with purified peptides. CSFV-derived crude peptides were also able to detect specific antibodies in swine serum, indicating the applicability of crude peptides for pre-screening large numbers of different peptides during the development of serological peptide-based assays.

Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate. Here we investigate Trp-cage's relaxation dynamics in the amide I' spectral region (1530-1700 cm(-1)) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label ((13)C═(18)O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding-unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I' region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time- and frequency-resolved data. We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined α-helix precedes the rapid formation of the hydrophobic cage structure, implying a native-like folding intermediate, that mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone. We find that the main free-energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the α-helix, the 310-helix, and the Asp9-Arg16 salt bridge. Our results suggest that at low temperature (T ≪ Tm) a folding path via formation of α-helical contacts followed by hydrophobic clustering becomes more important.

This brief review focuses on the functional activities of plasma hemopexin recently recognized by several authors. In particular, the protease-like activity of hemopexin in vitro is linked with downregulation of the vascular angiotensin II receptor in vivo, leading to vascular expansion. Also a potential mechanism of inhibition of hemopexin activity by extracellular adenosine triphosphate is considered.

Although the cause of pre-eclampsia during pregnancy has not been elucidated yet, it is evident that placental and maternal endothelial dysfunction is involved. We previously demonstrated that in early onset pre-eclampsia placental calcyclin (S100A6) expression is significantly higher compared to controls ( De Groot , C. J. ; Clin. Proteomics 2007 , 1 , 325 ). In the current study, the results were confirmed and relatively quantified by using multiple reaction monitoring (MRM) on two peptide fragments of calcyclin. Cells were obtained from control (n = 5) and pre-eclamptic placental (n = 5) tissue collected by laser capture microdissection from formalin-fixed paraffin-embedded (FFPE) material treated with a solution to reverse formalin fixation. Two calcyclin peptides with an extra glycine inserted in the middle of the amino acid sequence were synthesized and used as an internal reference. Data presented show that MRM on laser microdissected material from FFPE tissue material is possible. The developed MRM assay to study quantitative levels of proteins in FFPE laser microdissected cells using nonisotopic-labeled chemical analogs of mass tagged internal references showed that in pre-eclamptic patients elevated levels of calcyclin is observed in placental trophoblast cells compared to normal trophoblast cells. By immunohistochemistry, we were able to confirm this observation in a qualitative manner.

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by beta-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.