Development of a biosensor for copper detection in aqueous solutions using an Anemonia sulcata recombinant GFP.

Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu(2+), Cd(2+), Pb(2+) and Ni(2+) concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1-1 µM). Moreover, starting from these data, using the combination of molecular biology techniques and optical setup, we developed a device for the detection of Cu(2+) in water solutions. The quenching effect detected with the device showed that the relative attenuation of the signal (0.46 ± 0.02 AU) was slightly larger than the data measured by fluorescence spectra (0.65 ± 0.03 AU). The good sensitivity in the span of two orders of the magnitude of Cu(2+) concentration, the fact that the instrument is made up of low-cost and sturdy parts and the selective quenching of rAsGFP to copper ions make this setup suited as a low cost, on-the-field, copper ion-specific biosensor.

A rapid and sensitive method to detect specific human lymphocyte antigen (HLA) class II alleles associated with celiac disease.

BACKGROUND: Celiac disease (CD) is a complex disorder triggered by gluten affecting genetically predisposed individuals. More than 90% of patients carry human lymphocyte antigen (HLA)-DQ2 (DQA1*05, DQB1*02) and/or HLA-DQ8 (DQA1*03, DQB1*0302). We propose the use of the DQ-CD Typing kit that allows identification of the HLA class II alleles (DQA1*0201,*03,*05, DQB1*02,*0302, DRB1*03,*04,*07) selected to be informative in the CD risk evaluation and of a second kit, namely DQ-CD Zygosis, for DQB1*02 homozygosity determination. METHODS: The study was performed on a cohort of 100 individuals previously HLA typed with commercial kits. Fresh blood or previously extracted DNA was amplified in a unique PCR program using allele-specific primers and visualized on agarose gel. RESULTS: DNA amplification yielded strong and clear products without non specific signals or ghost bands. All the samples showed the expected alleles in accordance with the previous HLA typing. CONCLUSIONS: The DQ-CD Typing and Zygosis kits are fast, simple, economical and accurate tools that can be used to determinate the HLA-DQ2/DQ8 status in laboratory practice addressed for the diagnosis of CD. Molecular HLA testing is considered a valid support in the confirmation/exclusion of CD, especially in high-risk groups, such as CD relatives, or when serological and histological data are ambiguous.

Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines.

OBJECTIVE: Little is known about the interaction of gliadin with intestinal epithelial cells and the mechanism(s) through which gliadin crosses the intestinal epithelial barrier. We investigated whether gliadin has any immediate effect on zonulin release and signaling. MATERIAL AND METHODS: Both ex vivo human small intestines and intestinal cell monolayers were exposed to gliadin, and zonulin release and changes in paracellular permeability were monitored in the presence and absence of zonulin antagonism. Zonulin binding, cytoskeletal rearrangement, and zonula occludens-1 (ZO-1) redistribution were evaluated by immunofluorescence microscopy. Tight junction occludin and ZO-1 gene expression was evaluated by real-time polymerase chain reaction (PCR). RESULTS: When exposed to gliadin, zonulin receptor-positive IEC6 and Caco2 cells released zonulin in the cell medium with subsequent zonulin binding to the cell surface, rearrangement of the cell cytoskeleton, loss of occludin-ZO1 protein-protein interaction, and increased monolayer permeability. Pretreatment with the zonulin antagonist FZI/0 blocked these changes without affecting zonulin release. When exposed to luminal gliadin, intestinal biopsies from celiac patients in remission expressed a sustained luminal zonulin release and increase in intestinal permeability that was blocked by FZI/0 pretreatment. Conversely, biopsies from non-celiac patients demonstrated a limited, transient zonulin release which was paralleled by an increase in intestinal permeability that never reached the level of permeability seen in celiac disease (CD) tissues. Chronic gliadin exposure caused down-regulation of both ZO-1 and occludin gene expression. CONCLUSIONS: Based on our results, we concluded that gliadin activates zonulin signaling irrespective of the genetic expression of autoimmunity, leading to increased intestinal permeability to macromolecules.