Detection and Quantification of Nocardia crassostreae, an Emerging Pathogen, in Mytilus galloprovincialis in the Mediterranean Sea Using Droplet Digital PCR.

Nocardia crassostreae is a novel pathogen responsible for infections in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis). N. crassostreae is also responsible for nocardiosis both in immunocompetent and immunocompromised patients. We investigated N. crassostreae DNA in mussels grown in marine sites of the Mediterranean Sea in the Campania Region. We examined 185 mussel pooled samples by droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR), each pool composed of 10 mussels and 149 individual mussels. ddPCR detected N. crassostreae DNA in 48 mussel pooled samples and in 23 individual mussel samples. qPCR detected N. crassostreae DNA in six pooled samples and six individual mussel samples. The two molecular assays for the detection of N. crassostreae DNA showed significant differences both in the pooled and in individual samples. Our study demonstrated that ddPCR outperformed real-time qPCR for N. crassostreae DNA detection, thus confirming that ddPCR technology can identify the pathogens in many infectious diseases with high sensitivity and specificity. Furthermore, in individual mussels showing histological lesions due to N. crassostreae, the lowest copy number/microliter detected by ddPCR of this pathogen was 0.3, which suggests that this dose could be enough to cause infections of N. crassostreae in mussels.

A Chemical Strategy for the Preparation of Multimodified Peptide Imaging Probes.

Multimodality probes appear of great interest for innovative imaging applications in disease diagnosis. Herein, we present a chemical strategy enabling site-specific double-modification and cyclization of a peptide probe exploiting native chemical ligation (NCL) and thiol-maleimide addition. The synthetic strategy is straightforward and of general applicability for the development of double-labeled peptide multimodality probes.

The Safety of Ethnic Foods in Campania Region: A Preliminary Study.

Ethnic food is produced by an ethnic group-using their familiarity with local ingredients of plants and/or animal origin-and it is consumed in a country other than the country of origin. In Italy, the ethnic food market has expanded over the last three decades. The aim of this study was to evaluate the correct labeling, the microbiological communities and the allergens present in 50 ethnic foods. The visual inspection of labels and microbiological and allergen analyses have been carried out for evaluating their food safety. The visual inspection of labels revealed the absence of labeling in Italian and/or a failure to specify the place of origin. Microbiological analyses showed the absence of pathogens (i.e., Salmonella spp., Listeria monocytogenes and E. coli 0157:H7) in all matrices, but the presence of process hygiene indicator bacteria (total bacterial count, Coagulase-positive Staphylococci, Bacillus cereus, coliforms, yeasts and molds) was found in 37 samples. With regard to allergens, 12 samples were non-compliant for the presence of at least one allergen, while only two products were of species different from those declared on the label. This research highlights the need to increase the control of ethnic foods and also to improve the labeling system by standardizing international regulations.

Chiral Fibers Formation Upon Assembly of Tetraphenylalanine Peptide Conjugated to a PNA Dimer.

Self-assembly of biomolecules such as peptides, nucleic acids or their analogues affords supramolecular objects, exhibiting structures and physical properties dependent on the amino-acid or nucleobase composition. Conjugation of the peptide diphenylalanine (FF) to peptide nucleic acids triggers formation of self-assembled structures, mainly stabilized by interactions between FF. In this work we report formation of homogeneous chiral fibers upon self-assembly of the hybrid composed of the tetraphenylalanine peptide (4F) conjugated to the PNA dimer adenine-thymine (at). In this case nucleobases seem to play a key role in determining the morphology and chirality of the fibers. When the PNA "at" is replaced by guanine-cytosine dimer "gc", disordered structures are observed. Spectroscopic characterization of the self-assembled hybrids, along with AFM and SEM studies is reported. Finally, a structural model consistent with the experimental evidence has also been obtained, showing how the building blocks of 4Fat arrange to give helical fibers.

Direct Synthesis of Fluorescent Oxazolo-phenoxazines by Copper-Catalyzed/Hypervalent Iodine(III)-Mediated Dimerization/Cyclization of 2-Benzylamino-phenols.

A dimerization/cyclization reaction of 2-benzylamino-phenols for the direct synthesis of the oxazolo-phenoxazine skeleton is reported. The reaction occurs under copper catalysis in the presence of hypervalent iodine(III), giving selectively the 5H-oxazolo[4,5-b]phenoxazine compounds. The cascade process, which allows the conversion of the substrates into the tetracyclic products, involves three C-H functionalization steps. Initial oxidation of electron-rich arenes by the hypervalent iodine is essential for the dimerization of substrates, whereas the formation of the five-membered rings is promoted by the copper species. 1-Benzyl-2-phenyl-6-(aryl-benzyl)amino-benzimidazoles are regioselectively obtained using N,N'-dibenzyl-phenylenediamines as starting substrates. The fluorescence emission properties of these classes of products have been evaluated.

Impact of Freezing on the Microbiological Quality and Physical Characteristics of Buffalo Mozzarella Cheese.

Buffalo Mozzarella cheese from Campania is one of the most worldwide appreciated Italian dairy products. The increased demand for buffalo dairy products and the limited availability of the finest buffalo milk has prompted the diffusion of illicit practices, such as the use of milk, curd, or other products that are frozen or bought at low cost. The aim of this research was to provide preliminary results about the trend of the microbial communities of buffalo milk, curd and Buffalo Mozzarella cheese, during freezing storage of eleven months. At the same time, the alterations of physical properties and the presence of the molecular marker "γ4-casein", have been investigated. The results showed that freezing reduced the concentrations of the total bacterial count, Enterobacteriaceae, coliforms, Escherichia coli and yeasts in fresh milk and, the concentrations of the total bacterial count, coliforms, lactic acid bacteria and yeasts in mature curd. In the finished product, no notable decreases were observed, except for lactic acid bacteria. About the γ4-casein, no increase was observed in all matrices. These preliminary results allow us to conclude that the freezing process if properly carried out, does not compromise the microbiological quality and the physical properties of the Buffalo Mozzarella cheese.

Enantio- and Regioselective Palladium(II)-Catalyzed Dioxygenation of (Aza-)Alkenols.

An oxidative Pd-catalyzed intra-intermolecular dioxygenation of (aza-)alkenols has been reported, with total regioselectivity. To study the stereoselectivity, different chiral ligands as well as different hypervalent-iodine compounds have been compared. In particular, by using a C-6 modified pyridinyl-oxazoline (Pyox) ligand and hypervalent iodine bearing an aromatic ring, an excellent enantio- and diastereoselectivity has been achieved.

Synthesis of Cyclohepta[b]indoles by (4 + 3) Cycloaddition of 2-Vinylindoles or 4H-Furo[3,2-b]indoles with Oxyallyl Cations.

The synthesis of cyclohepta[b]indole derivatives through the dearomative (4 + 3) cycloaddition reaction of 2-vinylindoles or 4H-furo[3,2-b]indoles with in situ generated oxyallyl cations is reported. Oxyallyl cations are generated from α-bromoketones in the presence of a base and a perfluorinated solvent. Cyclohepta[b]indole scaffolds are obtained under mild reaction conditions, in the absence of expensive catalysts, starting from simple reagents, in good to excellent yields and with complete diasteroselectivity. Preliminary expansion of the scope to 3-vinylindoles and to aza-oxyallyl cations is reported.

Vancomycin-Iridium (III) Interaction: An Unexplored Route for Enantioselective Imine Reduction.

The chiral structure of antibiotic vancomycin (Van) was exploited as an innovative coordination sphere for the preparation of an IrCp* based hybrid catalysts. We found that Van is able to coordinate iridium (Ir(III)) and the complexation was demonstrated by several analytical techniques such as MALDI-TOF, UV, Circular dichroism (CD), Raman IR, and NMR. The hybrid system so obtained was employed in the Asymmetric Transfer Hydrogenation (ATH) of cyclic imines allowing to obtain a valuable 61% e.e. (R) in the asymmetric reduction of quinaldine 2. The catalytic system exhibited a saturation kinetics with a calculated efficiency of Kcat/KM = 0.688 h-1mM-1.

Evaluation of some microbiological and chemical parameters of Campania buffalo ricotta cheese.

Fresh ricotta (whey cheese), owing to its technological characteristics and intrinsic parameters (pH and aw), is an excellent substrate for the growth of spoilage and pathogenic microorganisms. On 19 July 2010, Campania buffalo ricotta cheese was assigned to the register of Protected Designations of Origin (PDO) by the European Commission, with EC Reg. 634/2010. It is mainly produced in the provinces of Caserta and Salerno around the rivers Garigliano and Volturno, the plain of the river Sele and the area of Cilento. This article reports the results of a 9-month monitoring (from May 2014 to January 2015) of ricotta samples aiming at evaluate the microbial status of some cheese factories, since ricotta is considered particularly suitable to judge the hygienic level of the dairy establishments. Four dairies were selected for sampling according to their different structural characteristics and the adequacy of both their premises and equipment.

Synthesis and Biological Evaluation of New Natural Phenolic (2E,4E,6E)-Octa-2,4,6-trienoic Esters.

In the present study the esterification of the OH groups of resveratrol, caffeic acid, ferulic acid, and ß-sitosterol with an antioxidant polyconjugated fatty acid, (2E,4E,6E)-octa-2,4,6-trienoic acid, was achieved. As the selective esterification of OH groups of natural compounds can affect their biological activity, a selective esterification of resveratrol and caffeic acid was performed by an enzymatic approach. The new resulting compounds were characterized spectroscopically (FT-IR, NMR mono, and bidimensional techniques); when necessary the experimental data were integrated by quantum chemical calculations. The antioxidant, anti-inflammatory and proliferative activity was evaluated. The good results encourage the use of these molecules as antioxidant and/or anti-inflammatory agents in dermocosmetic application.

Tandem Tetrahydroisoquinoline-4-carboxylic Acid/ß-Alanine as a New Construct Able To Induce a Flexible Turn.

Tetrahydroisoquinoline-4-carboxylic acid, a constrained ß2 -amino acid named ß-TIC, was synthesised for the first time in enantiopure form. The biocatalytic route applied herein represents one of the few successful examples of enzymatic resolution of ß2 -amino acids. Model tetrapeptides, namely, Fmoc-l-Ala-ß-TIC-ß-Ala-l-Val-OBn (Fmoc=fluorenylmethyloxycarbonyl, Bn=benzyl), containing both isomers of ß-TIC, were prepared. Both computational and NMR spectroscopy studies were performed. A reverse-turn conformation was observed in the case of (R)-ß-TIC enantiomer that was obtained in 99 % enantiomeric excess by enzymatic resolution. The ß-TIC/ß-Ala construct represents the first example of a flexible turn mimetic containing a cyclic and an acyclic ß-amino acid. Furthermore, the presence of an aromatic ring of ß-TIC could facilitate non-covalent interactions to increase the potential of this scaffold for the preparation of protein-protein interaction modulators.

Study of the Population Dynamics of Listeria Monocytogenes and Pseudomonas Fluorescens in Buffalo Mozzarella by Means of Challenge Testing.

Campania's buffalo mozzarella is a greatly appreciated cheese in Italy and worldwide. From a microbiological standpoint, it is a highly perishable food and potentially at risk of contamination by pathogens such as Listeria monocytogenes (L. monocytogenes). The present paper reports the results of a challenge test carried out to assess the population dynamics of L. monocytogenes, alone and in presence of Pseudomonas fluorescens (P. fluorescens), in buffalo mozzarella. For this purpose buffalo mozzarella samples were contaminated with L. monocytogenes alone or combined with P. fluorescens. In samples where L. monocytogenes was inoculated alone, the bacterial load remained unchanged. On the contrary, in samples contaminated with L. monocytogenes and P. fluorescens, the growth of L. monocytogenes increased.

1H-Azepine-4-amino-4-carboxylic acid: a new α,α-disubstituted ornithine analogue capable of inducing helix conformations in short Ala-Aib pentapeptides.

A very efficient synthesis of orthogonally protected 1H-azepine-4-amino-4-carboxylic acid, abbreviated as Azn, a conformationally restricted analogue of ornithine, was realized. It was obtained on a gram scale in good overall yield in five steps, three of which did not require isolation of the intermediates, starting from the readily available 1-amino-4-oxo-cyclohexane-4-carboxylic acid. Both enantiomers were used for the preparation of pentapeptide models containing Ala, Aib, and Azn. Conformational studies using both spectroscopic techniques (NMR, CD) and molecular dynamics on model 5-mer peptides showed that the (R)-Azn isomer possesses a marked helicogenic effect.

Characterisation of bubaline coronavirus strains associated with gastroenteritis in water buffalo (Bubalus bubalis) calves.

Recently, a coronavirus strain (179/07-11) was isolated from water buffalo (Bubalus bubalis) and the virus which displayed a strict genetic and biological relatedness with bovine coronavirus (BCoV) was referred to as bubaline coronavirus (BuCoV). Here, we report the characterisation of four BuCoVs strains identified in the faeces or intestinal contents of water buffalo calves with acute gastroenteritis. Single BuCoV infections were detected in all but one cases from which two clostridia species were also isolated. Sequence and phylogenetic analyses of the 5' end of the spike-protein gene showed that three BuCoVs were closely related to the prototype strain 179/07-11, whereas the fourth isolate (339/08-C) displayed a higher genetic identity to recent BCoV reference strains. Three strains adapted to the in vitro grow on human rectal tumour cells were also evaluated for their ability to replicate in a bovine cell line (Madin Darby bovine kidney) and to cause haemagglutination of chicken erythrocytes and all displayed biological properties similar to those already described for the prototype BuCoV. The present report shows that albeit genetically heterogeneous, the different BuCoV strains possess a common biological pattern which is different from most BCoV and BCoV-like isolates.

Prevalence of antibodies to selected viral and bacterial pathogens in wild boar (Sus scrofa) in Campania Region, Italy.

Serum samples were collected from wild boars (Sus scrofa) harvested during the 2005-2006 hunting season in Campania, southern Italy. Samples were tested for antibodies to Leptospira interrogan, Brucella spp., Salmonella spp., Aujeszky disease virus (ADV), porcine reproductive and respiratory stress syndrome virus (PRRSV), porcine parvovirus (PPV), classical swine fever virus (CSFV), and swine vesicular disease virus (SVDV). Of the 342 serum samples tested, 15 (4.4%) were seropositive to Brucella spp., nine (2.6%) were seropositive to L. interrogans, 66 (19.3%) were seropositive for Salmonella spp., 105 (30.7%) were seropositive for ADV, 27 (7.9%) were seropositive for PPV, and 129 (37.7%) were seropositive for PRRSV. All sera tested seronegative for SVDV and CSFV antibodies. These results, recorded for the first time in Campania, support the hypothesis that wild boar are reservoirs of certain infectious agents, but some infections in wild boars originate from their domestic counterparts.

Structural elucidation of the Rifaximin Ph. Eur. Impurity H.

Rifaximin, a semisynthetic, rifamycin-based non-systemic antibiotic is used in the treatment of acute and chronic gastrointestinal disorders. The aim of this study was the elucidation of the molecular structure of the 802 Dalton impurity which was found in Rifaximin industrial batches and reported with an erroneous structure in European Pharmacopoeia 6.5 (2009) [7] monograph as Rifaximin Impurity H. This impurity was isolated from Rifaximin by preparative HPLC and purified by column chromatography. The molecular structure was evidenced by means of (1)H and (13)C NMR spectroscopy, mass spectrometry and FT-IR.

Malignant catarrhal fever in a captive American bison (Bison bison) in Italy.

Malignant catarrhal fever (MCF) is a fatal, systemic disease of cattle and other domestic and wild ruminants that, in Europe, is caused by Ovine herpesvirus 2 (OvHV-2). American bison (Bison bison) are highly susceptible to the disease. An adult American bison, housed in a zoo in southern Italy in close cohabitation with a group of domestic sheep (Ovis aries aries) displayed clinical signs that resembled the acute form of MCF. By real-time polymerase chain reaction, OvHV-2 DNA was detected intravitam in blood, in nasal and ocular swabs, and postmortem in tissue samples of the bison. By indirect fluorescent antibody test, high MCF antibody titers were found in the bison serum. Ovine herpesvirus 2 DNA and antibodies were also found in blood samples from the domestic sheep, thus suggesting a potential role of these animals as a source of the infection. To the authors' knowledge, this is the first MCF case in captive ruminants in Italy and the second confirmed case in captive bison of European zoos.

Semisynthesis of new D-seco-C-nor-taxane derivatives containing a polyfunctionalized furanosyl or cyclopentenyl or cyclopentyl C-ring.

The synthesis of new D-seco-C-nor-taxane derivatives in which the D-ring has been deleted and the C-ring has been transformed into a new pentatomic ring, i.e., the polyfunctionalized tetrahydrofuranosyl and cyclopentenyl or cyclopentyl ring, was performed starting from baccatin III derivatives. The synthetic strategy adopted took advantage of the oxetane ring opening and disconnection of the C4-C5 bond, followed by an intramolecular condensation. The formation of furanosyl or cyclopentyl rings is strictly dependent on the presence of unprotected or protected oxygen at C-7 in the starting material. The reactions proceeded with good diastereoselectivity with control of the stereochemistry of one or two stereocenters.