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Proteolysis degree, biological activities, and water-soluble peptide patterns were evaluated in 12 month-ripened Parmigiano Reggiano (PR) cheeses collected in different dairy farms and showing different salt and fat content. Samples classified in high-salt and high-fat group (HH) generally showed lower proteolysis degree than samples having low-salt and low-fat content (LL). This positive correlation between salt/fat reduction and proteolysis was also confirmed by the analysis of biological activities, as the LL group showed higher average values of angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. UHPLC/HR-MS allowed the identification of 805 unique peptides: LL and HH groups shared 59.3% of these peptides, while 20.9% and 19.9% were LL and HH specific, respectively. Frequency analysis of peptides identified a core of 183 peptides typical of 12-month ripened PR cheeses (corresponding to the 22.7% of total peptides), but no significant differences were detected in peptide patterns between LL and HH groups. Forty bioactive peptides, including 18 ACE-inhibitors and 12 anti-microbial peptides, were identified, of which 25 firstly found in PR cheese. Globally, this work contributed to unraveling the potentially healthy benefits of peptides fraction in PR cheese and provided prior evidence that PR with reduced fat/salt content showed the highest antihypertensive and antioxidant activities.

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The impact of salt and fat intake on human health drives the consumer's attention towards dairy food with reduced salt and fat contents. How changes in salt and fat content modulate dairy LAB population and the associated proteolytic activities have been poorly studied. Here, non-starter LAB populations from 12 Parmigiano Reggiano (PR) cheeses (12-month ripened), clustered in low salt and fat content (LL-PR) and high salt and fat content (HH-PR) groups, were investigated and identified at specie-level with molecular assays. Lactobacillus rhamnosus was dominant in HH-PR samples, whereas Lactobacillus paracasei in LL-PR samples. (GTG)5 rep-PCR analysis discriminated 11 and 12 biotypes for L. rhamnosus and L. paracasei isolates, respectively. Screening for proteolytic activity identified L. rhamnosus strains more proteolytic than L. paracasei, and, within L. rhamnosus species, HH-PR strains were generally more proteolytic than LL-PR strains. Two L. rhamnosus representatives, namely strain 0503 from LL-PR and strain 2006 from HH-PR, were functionally characterized in cow milk fermentation assay. HH-PR strain 2006 overcame LL-PR strain 0503 in acidification performance, leading to a fermented milk with higher angiotensin I-converting enzyme inhibitory and antioxidant activities. L. rhamnosus 2006 was more prone to release VPP, while L. rhamnosus 0503 released higher amount of IPP. This study provides evidences that salt/fat content affects NSLAB cultivable fraction and the associated proteolytic ability resulting in a complex occurrence of bioactive peptides featuring health-promoting properties.

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Non-starter lactic acid bacteria (NSLAB) inhabiting fermented food have been recently revised as source of probiotic strains. Here, we in vitro assessed the potential probiotic aptitude of a de-replicated set of NSLAB previously isolated from long ripened Parmigiano Reggiano cheeses (22 Lactobacillus rhamnosus, 18 Lactobacillus paracasei, 3 Lactobacillus casei, 2 Lactobacillus harbinensis, and 2 Lactobacillus fermentum). Most strains showed moderate to good resistance to biological barriers, including bile salts, lysozyme, and simulated gastric and pancreatic juices. Antimicrobial susceptibility tests against seven antimicrobials belonging to different categories showed that most strains are susceptible towards all the antibiotics, with the exception of vancomycin and streptomycin. The strains lost the streptomycin resistance when assayed on agar medium containing 0.5% bile salts, suggesting that detergent-like properties of cholic acids increase membrane permeability and mediate streptomycin susceptibility. No isolate showed bile salt hydrolase (BSH) activities, supporting that bile salts resistance and BSH activity are unpaired traits. Finally, NSLAB strains had moderate to high auto-aggregative and hydrophobic phenotypes, whereas two subsets of 22 and 8 strains co-aggregated with Escherichia coli and Salmonella enterica s. typhimurium, respectively. A multivariate analysis was effective to segregate one L. casei and two L. rhamnosus strains showing physiological characteristics compatible with probiotic properties.

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Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.

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The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)(5)-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.

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We report the synthesis of novel chelates of Gd and (68)Ga with DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin α(ν)ß(3) . Because α(ν)ß(3) is involved in neo-angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of (68)Ga complexation for the promising (68)Ga-PET technique; the AAZTA complex 15((68)Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.

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A set of phenyl-substituted Smac mimetics/IAP inhibitor analogues of lead compound 2a was synthesized, aiming to retain its strong cell-free potency while increasing its bioavailability. Seventeen compounds 2b-r were prepared and characterized in vitro, using cell-free and cellular assays. Among them, the p-CF(3) substituted analogue 2m showed the best permeability through cell membranes, and was selected for further in vitro and in vivo studies due to its strong, sub-micromolar cellular potency.

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In the last few years click chemistry reactions, and in particular copper-catalyzed cycloadditions, have been used intensively for the preparation of new bioconjugate molecules and materials applicable to biomedical and pharmaceutical areas. This review will be focused on conjugates of the tripeptide Arg-Gly-Asp formed by means of click chemistry reactions. This sequence is a well known binding motif for specific transmembrane proteins and is involved in cellular adhesion to the extracellular matrix, allowing the selective recognition of the biomolecule or polymer in which it is incorporated.

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Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.

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Alpha-glycosyl amides can be synthesized from the corresponding O-benzyl-alpha-glycosyl azides using a traceless Staudinger ligation with diphenylphosphanyl-phenyl esters 4. All the phosphines employed and their phenol precursors are stable to air at 4 degrees C for months. Fast intramolecular trapping of the reduction intermediates results in the direct formation of the amide link, which, in turn, prevents epimerisation and allows retention of configuration at the anomeric carbon. Yields and alpha-selectivity are high when the reaction is performed in polar aprotic solvents. Removal of the benzyl ether protecting groups is achieved by catalytic hydrogenation. Alpha-glycosyl amides represent a class of virtually unexplored nonhydrolyzable monosaccharide derivatives that may find a useful application as sugar mimics. Conformational studies by NMR spectroscopy confirm that deprotected alpha-glycosyl amides in the gluco, galacto, and fuco series retain the normal pyranose conformation of the monosaccharide. The reaction of phosphines 4 with tetra-O-acetyl-glycosyl azides is nonstereoconservative, and beta-glycosyl amides are obtained in good yields and with complete stereoselectivity starting from both alpha and beta azides.

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Alpha-glycosyl ureas can be synthesised directly from tetra-O-benzyl glycosyl azides and isocyanates, using a one-pot procedure that is simple and general in scope. The benzyl protecting groups are easily removed from the urea products by catalytic hydrogenation. The synthesised alpha-glycosyl ureas represent a new class of neo-glycoconjugates with the potential of being resistant towards carbohydrate processing enzymes.

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