Differential Protein Expression in Berry Skin from Red Grapes with Varying Hybrid Character.

Protein expression from the berry skin of four red grape biotypes with varying hybrid character was compared at a proteome-wide level to identify the metabolic pathways underlying divergent patterns of secondary metabolites. A bottom-up shotgun proteomics approach with label-free quantification and MaxQuant-assisted computational analysis was applied. Red grapes were from (i) purebred Vitis vinifera (Aglianico cv.); (ii) V. vinifera (local Sciascinoso cv.) grafted onto an American rootstock; (iii) interspecific hybrid (V. vinifera × V. labrusca, Isabel), and (iv) uncharacterized grape genotype with hybrid lineage, producing relatively abundant anthocyanidin 3,5-O-diglucosides. Proteomics supported the differences between hybrids and purebred V. vinifera grapes, consistently with distinct phenotypic metabolite assets. Methanol O-anthraniloyltransferase, which catalyses the synthesis of methyl anthranilate, primarily responsible for the "foxy" odour, was exclusive of the Isabel hybrid grape. Most of the proteins with different expression profiles converged into coordinated biosynthetic networks of primary metabolism, while many possible enzymes of secondary metabolism pathways, including 5-glucosyltransferases expected for hybrid grapes, remained unassigned due to incomplete protein annotation for the Vitis genus. Minor differences of protein expression distinguished V. vinifera scion grafted onto American rootstocks from purebred V. vinifera skin grapes, supporting a slight influence of the rootstock on the grape metabolism.

Antibacterial potential of donkey's milk disclosed by untargeted proteomics.

Donkey's milk (DM) has been extensively investigated as a valuable substitute of breast milk, often suitable to manage cow's milk protein allergy in infants. DM exhibits potent inhibitory properties against numerous microbial species. Although oligosaccharides and lipids might contribute to the antimicrobial potential, the current inventory of proteins is not able to justify the low count of microorganisms generally observed in DM. The shotgun proteomic analysis of fractionated DM disclosed a set of 94 gene products, 41% of which have documented antimicrobial activity or are involved in transferring the passive immunity to the donkey offspring. The concerted action of lysozyme, lactoferrin, immunoglobulins provides the molecular basis for part of the DM antibacterial potential. The pH -4.6 insoluble fraction contained significant levels of L-amino acid oxidase, identified with 11 unique peptides matching the horse homologue gene product. This enzyme catalyses the oxidative deamination of amino acids into ketoacids, producing ammonia and H2O2. κ-casein, likely occurring as a fully O-glycosylated protein, may concur to inhibit the adhesion of pathogenic microorganisms, along with other glycoproteins. Proteomics supports the alimentary use of DM not only as a substitute of human milk in early infancy, but also for growing children, convalescent, elderly people and general population. SIGNIFICANCE: Donkey's milk (DM) is acquiring increasing popularity because it is a suitable substitute of the human milk, when breastfeeding is not possible and infants suffer from cow's milk allergy. DM is characterized by a much lower microbial load compared to ruminants' milk. This feature has been traditionally attributed to the high content of lysozyme. DM exhibits potent activity against a broad range of bacteria, viruses and fungi, suggesting that other protein components can be responsible of the antimicrobial potential. The gel-free proteomic analysis of pH 4.6-insoluble and soluble (whey) fractions demonstrated that DM contains a large number of gene products involved in antimicrobial mechanisms and in transferring passive immunity to the donkey offspring. DM contains relatively high levels of L-amino acid oxidase that catalyses the oxidative deamination of amino acid substrates into ketoacids, with production of ammonia and H2O2. In combination with lysozyme, lactoferrin and immunoglobulins, the presence of L-amino acid oxidase provides the molecular basis of the antibacterial potential observed for DM. Considered the low microbial load, DM can be sanitated at mild conditions, thereby preserving many of the native nutritional traits. Thus, DM can be considered a safe and nutritionally valid alimentary resource for growing children, convalescent, elderly people and general population. Data of this study represent the largest inventory of proteins identified in Equidae milk, so far.

Immunogenic Potential of Beer Types Brewed With Hordeum and Triticum spp. Malt Disclosed by Proteomics.

The protein/peptide composition of five beer kinds, including two experimental beer-like products brewed with einkorn (Triticum monococcum), a beer labeled as "gluten-free," a traditional all-barley malt and a wheat (T. aestivum) containing beer, was characterized with HPLC-ESI MS/MS-based proteomics. To enlarge the characterization of the components, the polypeptides were fractionated according to their molecular size (cut-off 6 kDa). All the beer types contained a variety of polypeptides arising from all the gliadin subfamilies (α-/ß-, γ-, and ω-gliadins) able to induce an immune response in celiac disease (CD) patients in addition to a panel of IgE-reactive food allergens. Wheat storage proteins were heavily hydrolyzed in the beer samples brewed with einkorn. The presence of gluten-like fragments, also including the 25-mer and 33-mer-like of α-gliadin, was confirmed in beer brewed with barley and wheat malt as well as in the gluten-free beer. Both CD-toxic and allergenic peptides of all beer samples were drastically degraded when subjected to a simulated gastroduodenal (GD) digestion. After in vitro digestion, the level of gluten-like peptides assayed with the G12 competitive ELISA, was below the threshold (20 ppm) for a food to be considered as "gluten-free." A few gliadin-derived epitopes occurred in the digests of beers crafted with wheat or Norberto-ID331 line of einkorn. In contrast, digests of all barley malt and gluten-free beers did not contain detectable gluten-like epitopes, but only minor fragments of hordeins and IgE-reactive food allergens. All beer samples evoked a weak immune response on gliadin-reactive celiac T cells isolated from intestinal biopsies of celiac patients. Compared to undigested polypeptides, the response was markedly reduced by GD digestion. Although the consumption of a moderate amount of beer brewed with barley or einkorn could deliver a relatively low amount of CD-toxic epitopes, the findings of this study emphasize the urgent need of a reliable and accurate quantification of gluten epitopes in all types of beer, also including the gluten-free one, to compute realistically the contribution of beer to the overall gluten intake, which can be responsible of intestinal tissue damages in celiacs.

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

Proteomic analysis of eucalyptus leaves unveils putative mechanisms involved in the plant response to a real condition of soil contamination by multiple heavy metals in the presence or absence of mycorrhizal/rhizobacterial additives.

Here we report on the growth, accumulation performances of, and leaf proteomic changes in Eucalyptus camaldulensis plants harvested for different periods of time in an industrial, heavy metals (HMs)-contaminated site in the presence or absence of soil microorganism (AMs/PGPRs) additives. Data were compared to those of control counterparts grown in a neighboring nonpolluted district. Plants harvested in the contaminated areas grew well and accumulated HMs in their leaves. The addition of AMs/PGPRs to the polluted soil determined plant growth and metal accumulation performances that surpassed those observed in the control. Comparative proteomics suggested molecular mechanisms underlying plant adaptation to the HMs challenge. Similarly to what was observed in laboratory-scale investigations on other metal hyperaccumulators but not on HMs-sensitive plants, eucalyptus grown in the contaminated areas showed an over-representation of enzymes involved in photosynthesis and the Calvin cycle. AMs/PGPRs addition to the soil increased the activation of these energetic pathways, suggesting the existence of signaling mechanisms that address the energy/reductive power requirement associated with augmented growth performances. HMs-exposed plants presented an over-representation of antioxidant enzymes, chaperones, and proteins involved in glutathione metabolism. While some antioxidant enzymes/chaperones returned to almost normal expression values in the presence of AMs/PGPRs or in plants exposed to HMs for prolonged periods, proteins guaranteeing elevated glutathione levels were constantly over-represented. These data suggest that glutathione (and related phytochelatins) could act as key molecules for ensuring the effective formation of HMs-chelating complexes that are possibly responsible for the observed plant tolerance to metal stresses. Overall, these results suggest potential genetic traits for further selection of phytoremediating plants based on dedicated cloning or breeding programs.

Rapid peptidomic profiling of peritoneal fluid by MALDI-TOF mass spectrometry for the identification of biomarkers of endometriosis.

Peptidomic profiling of peritoneal fluid by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) may represent a promising, suitable, rapid method for early diagnosis and staging of endometriosis. In a case-control study, peritoneal fluid was collected from 23 patients affected by endometriosis (eight minimal/mild endometriosis and 15 moderate/severe endometriosis) and six "endometriosis free" women undergoing laparoscopy. MALDI-TOF mass spectra of the peptide fraction extracted from peritoneal fluid samples lead to identify biomarkers potentially suitable for discriminating between peritoneal fluid samples from women affected by minimal/mild endometriosis and those from women affected by moderate/severe endometriosis. Peptidomic analysis of peritoneal fluid samples may define putative peptide biomarkers suitable for staging endometriosis and improve our understanding of the pathogenesis of endometriosis.

Chest tuberculosis with mediastinal asymptomatic lymphadenitis without lung involvement in an immunocompetent patient.

Tuberculosis remains the major cause of morbidity and mortality by a single infectious agent, particularly in developing countries. In recent years, we have witnessed the emergence of uncommon radiographic patterns of chest tuberculosis. Lymphadenitis is the most common extrapulmonary tuberculosis (TB) manifestation which, in developed countries, occurs more frequently in childhood, but also among adult immigrants from endemic countries and in HIV-infected people. Isolated and asymptomatic mediastinal lymphadenitis is uncommon in immunocompetent adults. We report a case of a young adult man from Senegal affected by sovraclavear and mediastinal TB lymphadenitis, which contains some uncommon elements: no compromised immunity, especially no HIV-infection, no lung lesions, no symptoms of infection or of mediastinum involvement, and rapid response to therapy in terms of mass size reduction. Examination of extra-thoracic lymph nodes and the patient's characteristics guided our diagnostic process to suspect TB. Surgical biopsy and subsequent histopathological and microbiological examinations of lymph material, first by Lowestein-Jensen and BACTEC cultures that remain the gold standard of diagnosis, confirmed the diagnosis. Chest X-ray was inconclusive; however, CT played an important role in the diagnostic course and in the management of the patient, particularly in determining disease activity, offering mediastinum and parenchymal details, as well as in identifying typical features of tuberculous lymph nodes and also of active/non active disease. Six months of antimycobacterial regimen is the recommended treatment in TB lymphadenitis of HIV-negative adults.

Proteomics for the elucidation of cold adaptation mechanisms in Listeria monocytogenes.

Listeria monocytogenes, one of the major food-related pathogens, is the aetiological agent of listeriosis, a potentially life-threatening illness. It is able to survive in hostile environments and stress conditions such as those encountered in food-processing technologies (high salt concentration, wide range of pH and temperature, low water availability) and it also thrives at temperatures ranging from -0.4 to 45 °C. In this study, expression proteomics was applied to gain insight into key cellular events that allow L. monocytogenes to survive and multiply even at refrigeration temperatures. Interestingly, we observed that the adaptation processes mainly affect biochemical pathways related to protein synthesis and folding, nutrient uptake and oxidative stress. Furthermore, proteins implicated in metabolic pathways for energy production, such as glycolysis and Pta-AckA pathway, were present to a higher level in the cells grown at 4 °C. This suggests that, on the whole, cells exhibit an enhanced demand for energy to sustain cold growth. Proteomics may represent a key tool in deciphering specific mechanisms underlying cold adaptation response and, more widely, cell machinery.

Simultaneous detection of N-acetyl-L-cysteine and physiological low molecular mass thiols in plasma by capillary electrophoresis.

N-acetyl-L-cysteine (NAC) is a therapeutic drug widely used as mucolytic agent in the treatment of respiratory diseases. Recently it has been proposed that NAC administration may modify the plasma levels of low molecular weight thiols (LMW) like cysteine, homocysteine and glutathione, though it has been still debated if their plasma concentration increases or decreases during the therapy. Therefore research calls for methods able to analyze simultaneously NAC and the other plasma LMW thiols in order to evaluate if NAC is able to modify plasma thiols concentration and in particular to reduce homocysteine levels in hyperhomocysteinemia. In this paper we present a new capillary electrophoresis method that allows a baseline separation of plasma NAC from the physiological thiols. The proposed method has been utilized to measure the drug and the physiological LMW thiols in NAC administered chronic obstructive broncho-pneumopathy (COPB) disease patients.