Il paesaggio che cambia

Convegno: Paesaggio Rurale \u2013 Paesaggio Cultural

Electrochemical Immunosensor for Detection of IgY in Food and Food Supplements

Immunoglobulin Y is a water-soluble protein present in high concentration in hen serum and egg yolk. IgY has applications in many fields, e.g., from food stuff to the mass production of antibodies. In this work, we have implemented an electrochemical immunosensor for IgY based on templated nanoelectrodes ensembles. IgY is captured by the templating polycarbonate and reacted with anti-IgY labeled with horseradish peroxidase. In the presence of H2O2 and methylene blue as the redox mediator, an electrocatalytic current is generated which scales with IgY concentration in the sample. After optimizing the extracting procedure, the immunosensor was applied for analysis of fresh eggs and food integrators. The data obtained with the biosensor were validated by SDS-PAGE and Western blot measurements

Merging multi-OMICs with proteome integral solubility alteration unveils antibiotic mode of action

Antimicrobial resistance is responsible for an alarming number of deaths, estimated at 5 million per year. To combat priority pathogens, like Helicobacter pylori, the development of novel therapies is of utmost importance. Understanding the molecular alterations induced by medications is critical for the design of multi-targeting treatments capable of eradicating the infection and mitigating its pathogenicity. However, the application of bulk omics approaches for unraveling drug molecular mechanisms of action is limited by their inability to discriminate between target-specific modifications and off-target effects. This study introduces a multi-omics method to overcome the existing limitation. For the first time, the Proteome Integral Solubility Alteration (PISA) assay is utilized in bacteria in the PISA-Express format to link proteome solubility with different and potentially immediate responses to drug treatment, enabling us the resolution to understand target-specific modifications and off-target effects. This study introduces a comprehensive method for understanding drug mechanisms and optimizing the development of multi-targeting antimicrobial therapies

Nutrition and IBD: Malnutrition and/or Sarcopenia? A Practical Guide

Malnutrition is a major complication of inflammatory bowel disease (IBD). This mini review is focusing on main determinants of malnutrition in IBD, the most important components of malnutrition, including lean mass loss and sarcopenia, as an emerging problem. Each one of these components needs to be well considered in a correct nutritional evaluation of an IBD patient in order to build a correct multidisciplinary approach. The review is then focusing on possible instrumental and clinical armamentarium for the nutritional evaluation

The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus

Nanostructured Electrochemical immunosensors for the Identification of Egg Proteins: Immunoglobulin Y and Ovalbumin - Application to Cultural heritage and Food Analysis

Despite biosensors are widely used in many research fields and offer many advantages, as high selectivity and sensitivity, low costs and fast analytical procedures, they have been introduced in the cultural heritage field only recently. In this PhD project, two electrochemical immunosensors were developed, with Immunoglobulin Y (IgY) and Ovalbumin (OVA) as the target proteins. IgYin an antibody present in high concentration in egg yolk, while OVA is the main protein in egg white. To this aim, the development of sensors capable of identify these proteins provides complete information about samples from tempera paintings with egg as the organic binder. The electrochemical transducer used for these sensors is an ensemble of gold nanoelectrodes, prepared by electroless deposition of gold in track-etched nanoporous polycarbonate membranes. Thanks to the specific conformation of the NEEs, it is possible to exploit the superficial polycarbonate present in large amount to capture the target proteins and the chemical modification of the surface is not needed. In a first phase of this project, some parameters of the IgY-immunosensing procedure were optimised, to improve the applicability of the sensor in the cultural heritage field. Indeed, low amount of samples is a key role parameter in this research field. To this aim, the geometric area of the sensor was reduced to sub-millimetre dimensions, and it has been proved that this miniaturization does not affect the analytical performance of the sensors. Afterwards, the OVA immunosensor was developedex novo. The analytical procedure was optimised, focusing in particular on reducing aspecific interactions possibly cause by other proteins. The performances of the OVA-immunosensors were evaluated in terms on sensibility and selectivity. Both the IgY- and OVA-electrochemical immunosensors were applied in the identification of egg yolk and egg white in complex mock-up samples, preparedas cross-sections, that simulate multilayer painting. In particular the layers were prepared with whole egg or egg components separately, in a mixture with pigments and other organic binders, as linseed oil or animal glues. IgY-immunosensor was also applied in the study of food supplements that undergone an industrial process such as freeze-drying. Also in this case, the identification of the protein was successful. Measurements carried out with on extracts obtained by direct incubation of the cross-sections, successfully demonstrated the ability of the electrochemical immunosensors to detect the two target proteins (IgY and/or OVA) in most of the samples. With the aim of extending the applicability of the sensors to other research fields, finally, the IgY-immunosensors was exploited for the identification of the protein in food and food supplements based on egg yolk. The results obtained with the electrochemical immunosensor on these samples were compared with those obtained by electrophoretic and western blot analysis, indicating moreover a higher sensitivity of the sensor when samples with small amount of egg yolk were analysed

IgY - electrochemical immunosensor based on nanoelectrodes ensembles

3Abstract It has been recently demonstrated that chicken egg-yolk immunoglobulin Y (IgY) has many immunological properties not only on avian, but also on mammalians, such as human [1]. Since it is easy to insulate (directly from egg yolk – where it is present at a concentration of 10 mg/mL), recently scientists proposed new immunotherapies based on the addiction of IgY to medicine or food both for animals and humans [1]. In addition, eggs are very common ingredients in all diets, so it is important to understand if it is possible to obtain such immunological properties from every day foods, or if IgY degrades during cooking procedures and temperature changes. For this reason, we developed an electrochemical immunosensors based on Au-nanoelectrodes ensemble (NEE) as transducer, able to identify IgY in different matrices. NEEs are prepared via template electroless deposition of gold in a polycarbonate (PC) membrane. Polycarbonate is affine to proteins and antibodies and, for these reason NEEs are very suitable to be used both as electrodes and as protein-receiver material [2]. IgY, the biological molecular recognition element, is immobilized directly on the PC surface of the electrode. It is recognized by the secondary antibody anti-IgY labelled with HRP, an electroactive enzyme that reacts with his substrate (H2O2) and a redox mediator (methylene blue), originating the electrocatalytic cycle. Only the reduction of the oxidated form of methylene blue is an electrochemical reaction, so it is possible to observe an electrocatalytic peak. The proposed immunosensor was tested on different samples, starting from standard IgY, then on analytes collected from yolk or egg-white, and following from more complex food samples containing egg yolk or full cooked eggs. In this communication, the obtained results are presented and discussed.reservedmixedGaetani, Chiara; Ugo, Paolo; Moretto, Ligia MariaGaetani, Chiara; Ugo, Paolo; Moretto, LIGIA MARI

Differences in the structure of drinking, cart expression and dopamine turnover between polydipsic and non polydipsic rats in the quinpirole model of psychotic polydipsia

Rationale Dopaminergic D2/D3 agonist quinpirole (QNP) elicits nonregulatory drinking in rats, a model of psychotic polydipsia. Why only a fraction of QNP-treated rats responds to the treatment becoming polydipsic is still unclear. Objectives To unveil possible factors contributing to such variability, we analyzed drinking microstructure in saline and QNP-treated rats, the hypothalamic expression of the cocaine and amphetamine regulated transcript (CART), and the monoaminergic turnover in selected brain areas. Methods Rats were daily treated with saline or QNP 0.5 mg/kg, and their 5-h water intake was measured for five consecutive days. The number of bouts and episodes of licking, and their duration, were also measured. Brain CART expression was measured by in situ hybridization and monoamines turnover by HPLC analysis of tissue extracts. Based on the amount of water ingested during the 5-h session, QNP-treated rats were post hoc grouped in polydipsic (PD) and in nonpolydipsic (NPD) rats, and the results compared accordingly. Results The number of drinking bouts and episodes increased in PD rats, while NPD rats behaved as the controls. CART expression decreased in the arcuate nucleus of the hypothalamus of the PD rats. In contrast, both PD and NPD rats showed a reduction of DA turnover in both ventral tegmental area (VTA) and nucleus accumbens (NAcc). No difference was detected in the turnover of 5HT and NA. Conclusions Microstructure analysis confirms that QNP acts on the appetitive component of drinking behavior, making it compulsive. CART expression reduction in response to dopaminergic hyperstimulation might sustain excessive drinking in PD rats. © 2014 Springer-Verlag Berlin Heidelberg