Electrogenerated indium tin oxide-coated glass surface with photosensitive interfaces: surface analysis

We present herein a photo-immobilization technique for the localized and specific conjugation of biochip platforms with different proteinaceous bioreceptors, such as antigen or antibodies. This methodology based on a photoactivable electrogenerated polymer film, pyrrole-benzophenone, allows the covalent immobilization of biomolecules through light mediation. The surface-conductive glass platform electropolymerized with poly(pyrrole-benzophenone) thin film may then be used to affinity-coat the chip with molecular recognition probes. This glass chip electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide (ITO). Thereafter, pyrrole-benzophenone monomers are electropolymerized onto the conductive metal oxide surface and then exposed to an antigen Staphylococcal Enterotoxin B (SEB)) solution and illuminated with UV light (wavelength approximately 345 nm) through a mask. As a result of the photochemical reaction, a pattern thin layer of the antigen was covalently bound to the benzophenone-modified surface. Then the sample to be analyzed, along with its specific target antibody (anti-SEB antibodies), is introduced onto the glass surface and left to react with the previously photo-immobilized antigen. When the immuno-reaction is completed, the specifically attached immunoglobulin analytes are detected by using secondary antibodies conjugated with Fluorescein isothiocyanate (FITC). The fluorescence signal emanating from the biochip surface is then quantified by two methods, using a filtered intensified charge-coupled device (CCD) camera and a grating spectrometer.     

Synthesis, antifungal and antimicrobial activity of alkylphospholipids

The antifungal, antibacterial and haemolytic activity of a series of alkylphosphocholines (e.g., miltefosine) and alkylglycerophosphocholines (e.g., edelfosine) has been investigated. These compound classes exhibit significant antifungal and moderate antibacterial activities. Several new alkylphosphocholine derivatives with amide or ester bonds in the alkyl chain have been synthesised. These compounds show much lower haemolytic activity than miltefosine. Alkylphosphocholines and alkylglycerophosphocholines show significant promise as novel orally available antifungal and antibacterial therapeutics.     

Altered specificity of a AAA+ protease

ClpXP, an ATP-dependent protease, degrades hundreds of different intracellular proteins. ClpX chooses substrates by binding peptide tags, typically displayed at the N or C terminus of the protein to be degraded. Here, we identify a ClpX mutant that displays a 300-fold change in substrate specificity, resulting in decreased degradation of ssrA-tagged substrates but improved degradation of proteins with other classes of degradation signals. The altered-specificity mutation occurs within "RKH" loops, which surround the entrance to the central pore of the ClpX hexamer and are highly conserved in the ClpX subfamily of AAA+ ATPases. These results support a major role for the RKH loops in substrate recognition and suggest that ClpX specificity represents an evolutionary compromise that has optimized degradation of multiple types of substrates rather than any single class.     

Tumor necrosis factor is critical to control tuberculosis infection

Tumor necrosis factor (TNF) is critical and non-redundant to control Mycobacterium tuberculosis infection and cannot be replaced by other proinflammatory cytokines. Overproduction of TNF may cause immunopathology, while TNF neutralization reactivates latent and chronic, controlled infection, which is relevant for the use of neutralizing TNF therapies in patients with rheumatoid arthritis.     

Individual LPS responsiveness depends on the variation of toll-like receptor (TLR) expression level

An individual's immune response is critical for host protection from many different pathogens, and the responsiveness can be assessed by the amount of cytokine production upon stimulating bacterial components such as lipopolysaccharide (LPS). The difference between individuals in their peripheral blood mononuclear cells (PBMC) responsiveness to LPS, a Gram-negative endotoxin, was investigated from 27 healthy individuals. We observed a large variation in IFNgamma production among different individuals. The PBMC of the consistently three highest and three lowest IFNgamma producers were investigated. Since previous studies described that a single point mutation in the coding region of TLR2 and TLR4 is linked to the individual responsiveness to pathogenic bacterial infections, we first examined the known point mutations in the coding region of TLR2Pro681His, TLR4Pro714His located in the cytoplasmic regions of the Toll-like domain as well as TLR4Asp299Gly located in the extracellular region. None of these mutations were associated with an individual's responsiveness to LPS, despite the presence of TLR4Asp299Gly mutation. Further investigation revealed that the variation of PBMC responsiveness to LPS among healthy individuals was due to constitutive expression levels of TLR4 and TLR2. This result is consistent with an aging-related low expression of Toll-like receptors in the mouse model of LPS responsiveness. The present study therefore suggests that the constitutive expression levels of TLR2 and TLR4 may contribute to the individual response to LPS.     

Divergent selection along climatic gradients in a rare central European endemic species,Saxifraga sponhemica

Background and Aims The effects of habitat fragmentation on quantitative genetic variation in plant populations are still poorly known. Saxifraga sponhemica is a rare endemic of Central Europe with a disjunct distribution, and a stable and specialized habitat of treeless screes and cliffs. This study therefore used S. sponhemica as a model species to compare quantitative and molecular variation in order to explore (1) the relative importance of drift and selection in shaping the distribution of quantitative genetic variation along climatic gradients; (2) the relationship between plant fitness, quantitative genetic variation, molecular genetic variation and population size; and (3) the relationship between the differentiation of a trait among populations and its evolvability.Methods Genetic variation within and among 22 populations from the whole distribution area of S. sponhemica was studied using RAPD (random amplified polymorphic DNA) markers, and climatic variables were obtained for each site. Seeds were collected from each population and germinated, and seedlings were transplanted into a common garden for determination of variation in plant traits.Key Results In contrast to previous results from rare plant species, strong evidence was found for divergent selection. Most population trait means of S. sponhemica were significantly related to climate gradients, indicating adaptation. Quantitative genetic differentiation increased with geographical distance, even when neutral molecular divergence was controlled for, and Q_ST exceeded F_ST for some traits. The evolvability of traits was negatively correlated with the degree of differentiation among populations (Q_ST), i.e. traits under strong selection showed little genetic variation within populations. The evolutionary potential of a population was not related to its size, the performance of the population or its neutral genetic diversity. However, performance in the common garden was lower for plants from populations with reduced molecular genetic variation, suggesting inbreeding depression due to genetic erosion.Conclusions The findings suggest that studies of molecular and quantitative genetic variation may provide complementary insights important for the conservation of rare species. The strong differentiation of quantitative traits among populations shows that selection can be an important force for structuring variation in evolutionarily important traits even for rare endemic species restricted to very specific habitats.     

Molecular dissection of the interactions of an antitumor interleukin‐2‐derived mutein on a phage display‐based platform

A mutein with stronger antitumor activity and lower toxicity than wild‐type human interleukin‐2 (IL‐2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL‐2 receptor alpha chain (thought to be critical for both IL‐2‐driven expansion of T regulatory cells and IL‐2‐mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL‐2‐derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage‐displayed IL‐2 variants to induce proliferation of the IL‐2‐dependent CTLL‐2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display‐based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL‐2 and related cytokines, accelerating the development of cytokine‐derived therapeutics. Copyright © 2015 John Wiley & Sons, Ltd.