A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments

correction on the stability of manganese tris β diketonate complexes as redox mediators in dsscs

Correction for 'On the stability of manganese tris(β-diketonate) complexes as redox mediators in DSSCs' by Stefano Carli et al., Phys. Chem. Chem. Phys., 2016, 18, 5949–5956

Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa

Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS) platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent- labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of ANANAS in inflamed tissues supports the potential of this platform as a targeted carrier for bioactive moieties in the treatment of inflammatory bowel disease

Studio dei meccanismi molecolari coinvolti nell'attività antiproliferativa dell'olio essenziale di Pistacia lentiscus

The phytocomplex from Pistacia lentiscus, a shrub of the Anacardiaceae family, is an essential oil obtained by hydrodistillation of leaves, fruits or from a trunk exudate (mastic gum). The mastic gum has been known to be effective in several gastric diseases, against Helicobacter Pylori and for its antibacterial and antifungine activities. Furthermore, Pistacia oil's major chemical constituents are monoterpenes with chemiopreventive and chemiotherapic properties. We investigated the antiproliferative properties of the volatile oil from Pistacia lentiscus twigs and leaves using human cell lines from ovarian (2008 and cis-platinum resistant, C13*) and colon (LoVo) adenocarcinoma, and human stable fibroblast line (HFFF2) as in vitro models. The MTT test showed that, after 3-hour treatment, phytocomplex (about 150 µg/ml) was able to inhibit the growth of all adenocarcinoma cell lines. After 24 hour treatment the IC50 on 2008 and LoVo cells resulted 3 times lower. On fibroblast line the phytocomplex was active only after 72 hour treatment. Western blotting analysis confirmed the oil capability to reduce carcinoma cell growth by decreasing the expression of p-ERK, MAPKs induced by mitogenic stimuli. In treated cells: vacuolisation, decreasing cellular size and brightness, directly proportional to reduction of cell viability, were observed by optical microscope. Using annexin V with propidium iodide we observed that oil was able to stimulate apoptosis in a dose-dependent manner. ROS has been recognized as an important mediator of the stress response; in particular, by regulating the loss of mitochondrial membrane potential. Furthermore upstream of ROS generation there is a respiratory chain block that leads even an increase of mitochondrial membrane potential. Analysis of mitochondrial membrane potential, with Rhodamine123, and ROS generation, with H2DCF-DA, showed the oil capability to activate mitochondrial apoptotic pathway. Oil-treatment also induced alteration on H+ gradient and interruption of electron flow between respiratory chain complexes III and IV, thereby causing loss of ATP. In the initiation of apoptosis caspases play critical roles. They can be grouped into "apoptotic initiators", for example caspase 8 and 9, and "apoptotic effectors", such as caspase 3, according to their substrate specificities and target proteins. Our data indicated that Pistacia lentiscus oil caused programmed cell death via a caspase-dependent pathway. In fact, after 3 hour treatment and 21 hour incubation, caspase 3 activity level, resulted higher than in the control, especially for the highest dose used. We also performed a flow cytometry-based cell cycle analysis, observing that the phytocomplex induced dose-dependent arrest in G2/M phase by decreasing cyclin B1 levels on all adenocarcinoma lines, especially on ovarian cells, and acting on acetylated tubulin and microtubules' polymerization/depolymerization.Il fitocomplesso ricavato da Pistacia lentiscus, una pianta arbustiva della famiglia delle Anacardiaceae, è un olio essenziale ottenuto per idrodistillazione da foglie, frutti o dall'essudato del tronco (mastice). Il mastice (il più noto dei fitocomplessi) è risultato essere efficace verso vari disturbi a livello gastrico e contro l'Helicobacter Pylori ed inoltre di avere attività  antibatterica ed antifungina. I principali costituenti degli oli ottenuti dal genere Pistacia sono i monoterpeni che presentano proprietà  preventive e chemioterapiche. Oggetto di studio di questi tre anni di dottorato è un olio essenziale di Pistacia lentiscus, ottenuto per idrodistillazione da foglie e ramoscelli, testato su una linea cellulare di adenocarcinoma del colon (cellule LoVo), una di adenocarcinoma dell'ovaio (cellule 2008) e la loro variante cis-platino resistente (cellule C13*) ed una linea di fibroblasti umani (cellule HFFF2). Il test dell'MTT dimostra che dopo 3 ore di trattamento il fitocomplesso, alla dose di 150 µg/ml circa, è in grado di inibire la proliferazione di tutte le linee di adenocarcinoma. Dopo 24 ore di trattamento, invece, l'IC50 risulta 3 volte inferiore per le linee LoVo e 2008. Per contro, sulla linea non tumorale di fibroblasti umani il fitocomplesso si dimostra attivo solo dopo 72 ore di trattamento. L'analisi mediante Western blotting conferma la capacità  dell'olio di ridurre la crescita cellulare delle cellule di adenocarcinoma diminuendo in maniera dose-dipendente l'espressione delle p-ERK, MAP chinasi indotte da stimoli mitogeni. Mediante microscopia ottica, inoltre, si può apprezzare come le cellule trattate risultino più piccole, rotondeggianti e meno "luminose", ad indicare una loro minore vitalità . Approfondendo i meccanismi d'azione dell'olio (mediante l'utilizzo di annexina V e ioduro di propidio) si osserva che il trattamento è in grado di attivare nelle cellule tumorali meccanismi di morte cellulare programmata. E' noto che i ROS sono degli importanti mediatori della risposta agli stress cellulari, in particolare attraverso la mediazione della perdita del potenziale della membrana mitocondriale, e che, a monte della produzione di ROS, c'è un blocco della catena respiratoria che porta ad un concomitante aumento del potenziale della membrana mitocondriale. Le analisi di questo, mediante Rodamina 123, e della generazione di ROS, utilizzando H2DCF-DA, dimostrano la capacità  dell'olio di attivare la via apoptotica mitocondriale. Il trattamento è quindi anche in grado di indurre un'alterazione nel gradiente protonico ed un'interruzione del passaggio di elettroni attraverso la catena respiratoria causando così anche la diminuzione della produzione di ATP. Nell'attivazione del processo apoptotico le caspasi giocano un ruolo cruciale. Questi enzimi, a seconda della specificità  di substrato e del target, possono essere raggruppati in "niziatori" o "effettori" di apoptosi, tra questi ultimi la caspasi 3 risulta essere sicuramente la più importante. I nostri risultati indicano che l'olio di Pistacia lentiscus è in grado di attivare la morte cellulare programmata attraverso la via caspasi-dipendente. Infatti, dopo 3 ore di trattamento e 21 di incubazione, i livelli della caspasi 3 si dimostrano più alti rispetto a quelli del controllo, specialmente per la dose maggiore adottata. E' stata effettuata anche un'analisi citofluorimetrica del ciclo cellulare che ha permesso di osservare come il fitocomplesso induca un arresto del ciclo in fase G2/M in maniera dose-dipendente. Questo arresto avviene attraverso una diminuzione dei livelli citoplasmatici di ciclina B1, specialmente nelle linee di adenocarcinoma ovarico, e agendo sui livelli di tubulina acetilata e sui meccanismi di polimerizzazione/depolimerizzazione dei microtubuli

COMPLESSI NANOASSEMBLATI DI ACIDI NUCLEICI, AVIDINA E COMPOSTI BIOTINILATI PER USO COME VETTORI PER IL TRASPORTO INTRACELLULARE

L???invenzione \ue8 relativa all???uso come vettori per il trasporto intracellulare di molecole, uguali o diverse tra di loro, scelte tra molecole per uso diagnostico e molecole bioattive per uso terapeutico, quali ad esempio cromofori o fluorofori, radiotraccianti farmaci, anticorpi, peptidi, proteine, enzimi, oligonucleotidi a singola o doppia catena e loro analoghi (PNA, LNA)

The \u201cANANAS\u201d project

The ANANAS Technology Platform The Avidin Nucleic Acid Nano Assemblies (ANANAS) are soft biocompatible and biodegradabole poly-avidin nanoparticles (NP) obtained through a proprietary Nature-guided process of assembly, which guarantees stoichiometric composition and performance reproducibility. In practical terms, the ANANAS are biocompatible polymers of avidin with superior performance, applicable in both drug delivery and immunodiagnostics. The NPs are multifunctionalizable in one pot reactions, they have colloidal stability in physiological buffer which permits several applications in nanomedicine. Intellectual Property: the ANANAS technology is patented worldwide

Characterization of multi-functional nanosystems based on the avidin-nucleic acid interaction as signal enhancers in immuno-detection

The Avidin-Nucleic-Acids-Nano-Assembly (ANANAS) is a kind of soft poly-avidin nanoparticle originating from the high affinity interaction between avidin and the nucleic acids. In this work we investigated the possibility of transforming ANANAS cores into stoichiometrically controlled multi-functional nanoparticles through a \u201cone-pot\u201d procedure, and we measured in a quantitative way their ability to work as reagents for enhanced immunodiagnostic detection. Initially, we measured the ANANAS loading capability for biotinylated proteins of different nature. About 200 molecules of biotin-horseradish-peroxidase (40KDa b-HRP) and 60 molecules of biotin-immunoglobulin-G (150KDa b-IgG) could be accommodated onto each nanoparticle, showing that steric limitations dictate the number of loadable entities. Stoichiometrically controlled functional assemblies were generated by mixing core particles with sub-saturating amounts of b-HRP and b-IgG. When applied as detection reagents in an Enzyme-Linked-ImmunoSorbed-Assay (ELISA), these assemblies were up to two-orders of magnitude more sensitive than commercial HRP-based reagents. Assemblies of different composition displayed different efficacy, indicating that the system functionality can be fine-tuned. Within-assay variability (CV%), measured to assess if the assembly procedure is reproducible, was within 10%. Stability experiments demonstrated that the functionalyzed assemblies are stable in solution for more than one week. In principle, any biotinylated function can be loaded onto the core particle, whose high loading capacity and tunability may open the way towards further application in biomedicine