Stable enzyme biosensors based on chemically synthesized Au-polypyrrole nanocomposites

This work describes development and optimization of a generic method for the immobilization of enzymes in chemically synthesized gold polypyrrole (Au-PPy) nanocomposite and their application in amperometric biosensors. Three enzyme systems have been used as model examples: cytochrome c, glucose oxidase and polyphenol oxidase. The synthesis and deposition of the nanocomposite was first optimized onto a glassy carbon electrode (GCE) and then, the optimum procedure was used for enzyme immobilization and subsequent fabrication of glucose and phenol biosensors. The resulting nanostructured polymer strongly adheres to the surface of the GCE electrode, has uniform distribution and is very stable. The method has proved to be an effective way for stable enzyme attachment while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of pyrrole and enzyme and the Au-PPy matrix avoids enzyme leaking. The preparation conditions, Michaelis-Menten kinetics and analytical performance characteristics of the two biosensors are discussed. Optimization of the experimental parameters was performed with regard to pyrrole concentration, enzyme amount, pH and operating potential. These biosensors resulted in rapid, simple, and accurate measurement of glucose and phenol with high sensitivities (1.089 mA/M glucose and 497.1 mA/M phenol), low detection limits (2 x 10(-6)M glucose and 3 x 10(-8)M phenol) and fast response times (less than 10s). The biosensors showed an excellent operational stability (at least 100 assays) and reproducibility (R.S.D. of 1.36%).     

Lipid transfer proteins in coffee: isolation of Coffea orthologs,Coffea arabica homeologs,expression during coffee fruit development and promoter analysis in transgenic tobacco plants

The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5′ deletions, were fused to the reporter gene β-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.     

In vitro resistance mechanisms of Neisseria meningitidis against neutrophil extracellular traps

Neisseria meningitidis (Nm) is a leading cause of septicemia in childhood. Nm septicemia is unique with respect to very quick disease progression, high in vivo bacterial replication rate and its considerable mortality. Nm circumvents major mechanisms of innate immunity such as complement system and phagocytosis. Neutrophil extracellular traps (NETs) are formed from neutrophils during systemic infection and are suggested to contain invading microorganisms. Here, we investigated the interaction of Nm with NETs. Both, meningococci and spontaneously released outer membrane vesicles (SOMVs) were potent NET inducers. NETs were unable to kill NET bound meningococci, but slowed down their proliferation rate. Using Nm as model organism we identified three novel mechanisms how bacteria can evade NET‐mediated killing: (i) modification of lipid A of meningococcal LPS with phosphoethanolamine protected Nm from NET‐bound cathepsin G; (ii) expression of the high‐affinity zinc uptake receptor ZnuD allowed Nm to escape NET‐mediated nutritional immunity; (iii) binding of SOMVs to NETs saved Nm from NET binding and the consequent bacteriostatic effect. Escape from NETs may contribute to the most rapid progression of meningococcal disease. The induction of NET formation by Nm in vivo might aggravate thrombosis in vessels ultimately directing to disseminated intravascular coagulation (DIC).     

Recognition of Neisseria meningitidis by the Long Pentraxin PTX3 and Its Role as an Endogenous Adjuvant

Long pentraxin 3 (PTX3) is a non-redundant component of the humoral arm of innate immunity. The present study was designed to investigate the interaction of PTX3 with Neisseria meningitidis. PTX3 bound acapsular meningococcus, Neisseria-derived outer membrane vesicles (OMV) and 3 selected meningococcal antigens (GNA0667, GNA1030 and GNA2091). PTX3-recognized microbial moieties are conserved structures which fulfil essential microbial functions. Ptx3-deficient mice had a lower antibody response in vaccination protocols with OMV and co-administration of PTX3 increased the antibody response, particularly in Ptx3-deficient mice. Administration of PTX3 reduced the bacterial load in infant rats challenged with Neisseria meningitidis. These results suggest that PTX3 recognizes a set of conserved structures from Neisseria meningitidis and acts as an amplifier/endogenous adjuvant of responses to this bacterium.     

Amyloidogenic Propensity of a Natural Variant of Human Apolipoprotein A-I: Stability and Interaction with Ligands

A number of naturally occurring mutations of human apolipoprotein A-I (apoA-I) have been associated with hereditary amyloidoses. The molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here we examined the effects of the Arg173Pro point mutation in apoA-I on the structure, stability, and aggregation propensity, as well as on the ability to bind to putative ligands. Our results indicate that the mutation induces a drastic loss of stability, and a lower efficiency to bind to phospholipid vesicles at physiological pH, which could determine the observed higher tendency to aggregate as pro-amyloidogenic complexes. Incubation under acidic conditions does not seem to induce significant desestabilization or aggregation tendency, neither does it contribute to the binding of the mutant to sodium dodecyl sulfate. While the binding to this detergent is higher for the mutant as compared to wt apoA-I, the interaction of the Arg173Pro variant with heparin depends on pH, being lower at pH 5.0 and higher than wt under physiological pH conditions. We suggest that binding to ligands as heparin or other glycosaminoglycans could be key events tuning the fine details of the interaction of apoA-I variants with the micro-environment, and probably eliciting the toxicity of these variants in hereditary amyloidoses.     

Electroencephalographic Patterns in Chronic Pain: A Systematic Review of the Literature

The main objective of this study is to review and summarize recent findings on electroencephalographic patterns in individuals with chronic pain. We also discuss recent advances in the use of quantitative Electroencephalography (qEEG) for the assessment of pathophysiology and biopsychosocial factors involved in its maintenance over time. Data collection took place from February 2014 to July 2015 in PubMed, SciELO and PEDro databases. Data from cross-sectional studies and longitudinal studies, as well as clinical trials involving chronic pain participants were incorporated into the final analysis. Our primary findings related to chronic pain were an increase of theta and alpha EEG power at rest, and a decrease in the amplitude of evoked potentials after sensory stimulation and cognitive tasks. This review suggests that qEEG could be considered as a simple and objective tool for the study of brain mechanisms involved in chronic pain, as well as for identifying the specific characteristics of chronic pain condition. In addition, results show that qEEG probably is a relevant outcome measure for assessing changes in therapeutic studies.