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Similarly to other species of ruminants, the river buffalo (2n=50) is characterized by the presence of all four casein fractions (??S1, ??, ??S2, and ??) encoded by the four linked autosomal genes (CSN1S1, CSN2, CSN1S2, and CSN3, respectively) mapped on chromosome 7. In particular, the CSN1S1 gene is characterized by an extremely split architecture with 19 exons, many of which of small size (24 bp). It encodes for a precursor of 214 amino acids with a signal peptide of 15 amino acid residues. In recent years, several polymorphisms at milk protein loci associated with traits of economic interest like milk coagulation properties or milk composition have been intensified. Despite that, so far, not any SNP within the milk protein loci was found to be associated with an important trait like milk yield. The aim of this study was to evaluate possible effects of the SNP c.628C>T, identified at position 89 of 17th exon of the CSN1S1 gene and responsible for the amino acid change p.Ser178Leu (A and B allele, respectively) (EMBL n° HE573919-20), on milk yield in Italian Mediterranean river buffaloes. A total of 7547 records for milk yield measured monthly on 1096 lactations of 552 buffaloes belonging to different farms located in Salerno and Caserta province (Southern Italy) were analyzed. Sampling and phenotypic collection data were carried out in collaboration with the Italian National Association of Buffalo Breeders (ANASB). The genotyping of examined animals was carried out by a method based on MboI-ACRS. The major allele (B) had a relative frequency of about 0.6 and ??2 values showed that there was no evidence of departure from the Hardy-Weinberg equilibrium (P???0.05). Association between CSN1S1 polymorphism and milk yield was investigated with a mixed linear model that included effects of parity, calving season and month of production. A significant association between the SNP c.628C>T and milk yield was found (P

This work arises as targets the genetic characterization at CSN1S1 and CSN3 loci, using techniques of molecular biology, of the agerolese cattle, an autochthonous genetic type reared in Campania, which is at risk of extinction.In particular, we proceeded to the identification of the CSN1S1 G and CSN3 B allele carriers, whose choice was dictated by the documented importance of their products (as1???CnG and k??? CnB).The molecular event that characterizes the CSN1S1 G allele is an insertion of a truncated retroposonlike element (LINE) of 371 bp realized between nucleotides 58 and 59 of 19th exon. Consequently, the 19th exon of bovine CSN1S1 G allele appears to be 756 bp long instead of 385 bp[1].The cause of a reduced efficiency of protein synthesis has been hypothesized to be an interaction between the LINE nucleotide sequence and the poly(A) sequence of the mature transcript, with consequent reduction of mRNA stability and its rapid degradation.The mutation responsible for the CSN3 B allele is a nucleotide substitution (GATAsp???GCTAla) at nucleotide 416 of 4th exon. This mutation is located relatively close to several glycosylation sites and probably affects the structure of the protein and glycosylation patterns[2]. It was reported that the CSN1S1 G allele is responsible for a considerable reduction of the content of as1 casein that involves a lower coagulation time and curd firmness of milk compared to that produced by cows bearers of the remaining alleles[3].Similarly, it was shown that the k???CnB variant influences the processes of cheesemaking, giving the milk a better attitude to cheese, with lower clotting times and firming, greater consistency of the curd and higher yield. From the milk obtained by the CSN3 BB cows it can get a higher yield in "Parmigiano Reggiano" equal to ~10%[4] The genomic DNA was extracted from 79 individual samples of bovine blood reared in different farms located in Campania. For the genotyping of CSN1S1 G allele carriers was applied a PCR protocol[1].The genotyping of CSN3 B allele was achieved by means of Hinf I PCR???RFLP[5] Indicating with N the other alleles at CSN1S1 and CSN3 loci, the genotype distribution of investigated population was 74N/N, 5N/G, 0G/G for CSN1S1 with a G allele frequency of 0.03 and 25N/N, 33N/B, 21B/B for CSN3 with a B allele frequency of 0.47 It can be assumed that the evidenced genetic variability at CSN3 locus for the agerolese cattle genetic type plays an important role in the determination of some fundamental parameters, both from the nutritional point of view that technological, for the production and quality of milk and of DOP products derived from it as the historians cheeses "Provolone del Monaco" and "Fior di latte???

In mammals, the hormone prolactin (PRL) is best known for its role in the regulation of lactation (Lee et al. 2007). PRL affects multiple reproductive and metabolic functions through its receptors (PRLR), characterized by the ability to activate Janus kinase 2 and signal transducer and activator of transcription (Fleenor et al. 2006). PRL is an anterior pituitary peptide hormone involved in many endocrine activities essential for reproductive success (Vaclavicek et al. 2006). Several polymorphic sites have been detected within PRL and PRLR genes, and significant associations among their variants with milk production traits have been described in dairy cattle (Alfonso et al. 2012; Zhang et al. 2008). PRL (EMBL EF054878) and PRLR (EMBL GQ339914) genes consist of 5 and 9 exons, coding for a protein of 219 and 581 amino acids, respectively. A partial sequencing of PRL exon 5 and PRLR exon 9 performed on 10 Italian Mediterranean river buffaloes allowed us to identify 1 SNP for PRL (C108→T, numbering from the 1st nt of exon 5) and 7 SNPs for PRLR (G128→A, C374→T, G517→A, C520→A, C622→T, G626→A and T707→C, numbering from the 1st nt of exon 9). The PRL SNP is silent (Leu202) as well as 626 (Ala494) and 707 (Asp521) PRLR SNPs. The other 5 PRLR SNPs are responsible of the following amino acid changes: His328→Arg, Ala410→Val, Asp458→Asn, Gln459→Lys and Thr493→Met. 574 buffaloes of several farms in Salerno province (South Italy) were genotyped in out-sourcing (http://kbioscience.co.uk) for 108 PRL and 128 PRLR SNPs. The genotype distribution of investigated population was 10 C/C, 122 C/T and 442 T/T (frequency of T allele 0.87) for PRL and 9 G/G, 108 G/A and 457 A/A for PRLR (frequency of A allele 0.89). For both SNPs, the investigated population was in Hardy–Weinberg equilibrium. Such polymorphisms could represent useful genetic markers for association studies with quali-quantitative milk characteristics, but further studies are needed to evaluate their potential use.

This paper describes a method to significantly enhance single-molecule fluorescence detection in confocal microscopy, demonstrating that fractallike silver structures significantly improve dye stability and brightness. The experiments compared two immunoassay models based on the deposition of rabbit IgG on silver structures. The experiments were performed with the fluorophore-labeled protein at low picomolar concentrations. Well-separated bright spots were still easily distinguishable. Under standardized conditions we observed increased photostability and brightness enhancement for the dyes that were immobilized on the surface using a primary antibody. In contrast, when the unlabeled primary antibody was immobilized on the surface and the labeled secondary antibody was placed at a larger distance, we observed only a modest enhancement of fluorescence. Furthermore, based on backscattered reflected light images, it was proven that the observed fluorescence enhancements originate from the areas with deposited silver nanostructures. Fractal-like substrates are relatively easy to prepare. We believe that with their superior performance, they should find wide applications in single-molecule studies in which a longer observation time is required.

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti : sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM).

La celiachia è una particolare intolleranza alimentare che comporta un’enteropatia autoimmune cronica a danno dell’intestino tenue. È una malattia particolarmente diffusa nel mondo occidentale e sebbene ci sia una chiara predisposizione familiare, l’ereditarietà, non segue i classici pattern mendeliani, suggerendo un’eziopatogenesi multigenica e/o multifattoriale fondamentalmente legata a due fattori: uno intrinseco al paziente, rappresentato dalla predisposizione genetica, che è per lo più legata al gene HLA DQ2/8; uno esogeno o, meglio, ambientale, legato all’assunzione, attraverso la dieta, di glutine, componente proteica contenuta in diversi cereali. La frazione tossica di tale proteina è rappresentata dalla componente alcool solubile: la gliadina, caratterizzata dalla frequente ricorrenza del tetrapeptide -gln-gln-gln-pro-, ora considerato l’agente lesivo della malattia. Infatti, le gravi lesioni alla mucosa dell’intestino tenue causate dall’intolleranza al glutine, regrediscono solo dopo l’eliminazione di tale nutriente dalla dieta. Oggi, la diagnosi definitiva di celiachia può essere ottenuta solo attraverso l’esame istopatologico della mucosa digiunale volto a rilevare l’atrofia dei villi intestinali. Tuttavia, la possibilità di avere metodi più pratici e meno invasivi rispetto a quelli istopatologici, può rappresentare una prospettiva importante al fine di eseguire “screening” di massa e per il “follow-up” della malattia celiaca. Da questo punto di vista, diversi sistemi ELISA (Enzyme Linked Immunosorbent Assay) sono stati messi a punto per il dosaggio di anticorpi anti-transglutaminasi tissutale (Ab anti-tTG), rappresentanti un marker sierologico per la celiachia (Dieterich W. et al., 1997; 2001). Perciò, lo sviluppo di un sistema di analisi quantitativo più semplice e rapido, quale un biosensore proteico, che sfrutti i principi della fluorescenza, per la determinazione degli Ab anti-tTG, è da considerarsi molto utile e innovativo tanto da rappresentare l’obiettivo prioritario del seguente lavoro di ricerca. L’aspetto chiave di tale tecnologia è racchiuso nella capacità di produrre un segnale luminoso, quando sono rilevati tali anticorpi nel siero o nella saliva dei pazienti affetti, tanto da metterli in condizione di seguire il “follow-up” della malattia in modo semplice e immediato. In particolare, tale lavoro ha previsto la messa a punto di un saggio fluorescente competitivo basato sulla simultanea applicazione di due metodologie: la “FRET” (Fluorescence Resonance Energy Transfer) e la tecnica “molecular beacons”. Queste metodiche utilizzano la presenza di opportune sonde fluorescenti coniugate a due differenti molecole biologiche, nel caso specifico la transglutaminasi da fegato di Guinea pig (gptTG) e gli anticorpi anti-tTG da coniglio e tengono conto della “distanza di Förster” in cui avviene trasferimento di energia tra i fluorofori utilizzati. In un primo momento i saggi di FRET, volti a studiare le interazioni Ag-Ab in soluzione, sono stati eseguiti mediante l’acquisizione di misure di fluorescenza statica (steady-state) e risolta nel tempo (lifetime). Dopo di che, al fine di simulare i regolari saggi immuno-enzimatici eseguiti su piastre multi-pozzetto, tali misure sono state effettuate sui campioni marcati, immobilizzati in maniera non covalente su vetrino, in modo tale da sviluppare un saggio fluorescente di tipo “front-face”. Tale configurazione con l’ausilio della Microscopia Confocale in Fluorescenza ha permesso di studiare le dinamiche interazionali di singole molecole di Ag legate agli Ab anti-tTG. Inoltre, al fine di ottimizzare la tecnica di fluorescenza, in modo da ottenere un basso “background”, un’alta sensibilità e specificità, i campioni sono stati immobilizzati su vetrini prima ricoperti con particelle metalliche d’argento, formanti strutture ramificate visibili a occhio nudo (fractal-like structures). Questo perché è stato osservato, che le caratteristiche spettrali dei fluorofori, possono cambiare in maniera rilevante quando si trovano in stretta vicinanza di una superficie metallica, tanto da produrre un aumento dell’emissione del segnale fluorescente che può essere utilizzato per migliorare la determinazione quantitativa di bioanaliti. Parallelamente, al fine di abbattere gli alti costi delle preparazioni di gptTG commercialmente disponibili, l’attività sperimentale ha previsto la realizzazione di un’opportuna strategia di clonaggio del gene della tTG da Guinea pig e di un efficiente sistema di espressione della proteina ricombinante in un ceppo batterico di Escherichia coli. I risultati ottenuti hanno dimostrano che la scelta dei fluorofori utilizzati per marcare l’Ag commerciale e gli Ab-anti tTG da coniglio è stata corretta. Infatti, si è verificato il trasferimento di energia di risonanza tra gli immunoreagenti selezionati, fenomeno su cui si basa il saggio a fluorescenza proposto. Inoltre, il principio della fluorescenza aumentata dai metalli è stato utile a migliorare la rilevazione del bioanalita d’interesse, registrando un aumento considerevole dell’emissione di fluorescenza di singole molecole di Ag marcato, immobilizzato su vetrino ricoperto da nanostrutture d’argento ramificate, migliorando la sensibilità del saggio fluorescente progettato. Per contro, sono necessari altri studi per ottimizzare le condizioni di espressione della gptTG ricombinante in E. coli, poiché la quantità di enzima solubile prodotta, in presenza di chaperoni chimici, non è sufficiente per passare alla procedura di purificazione. Perciò, l’uso di chaperoni molecolari esogeni, come supporto per indurre un corretto ripiegamento della struttura proteica, potrebbe essere una buona strategia per ottenere elevate quantità di proteina solubile ricombinante in E. coli.

In this work the thermophilic trehalose/maltose-binding protein from Thermococcus litoralis is presented as a probe for the design of a high stable fluorescence biosensor for glucose. In particular, we show the possibility of modulating the protein specificity by changing temperature. In addition to glucose sensing, we also report on the possibility of utilizing odorant-binding proteins as a probe for the development of optical sensors for analytes of environmental interests.

Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Forster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor–acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.

Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2–3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from localized areas of silver nanostructures.