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A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.

Variant histones that differ in amino acid sequence from S-phase histones are widespread in eukaryotes, yet the structural changes they cause to nucleosomes and how those changes affect relevant cellular processes have not been determined. H2A.F/Z is a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av was widely distributed in the genome and not limited to sites of active transcription. H2Av was present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody precipitated both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. The distribution of H2Av was not uniform. The complex banding pattern of H2Av in polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density of H2Av measured by immunoprecipitation varied between different sequences. Of the sequences assayed, H2Av was least abundant on 1.688 satellite sequences and most abundant on the hsp70 genes. Finally, transcription caused, to an equivalent extent, both H2Av and H2A to be less tightly associated with DNA.The basic unit of chromatin in eukaryotes is the nucleosome. A nucleosome consists of 146 base pairs of DNA wrapped around an octamer of histone proteins H2A, H2B, H3, and H4 (1). Although chromatin is a highly reiterative structure, cells create heterogeneity in the structure of nucleosomes to facilitate and regulate DNA-mediated processes such as transcription. Heterogeneity is created, in part, by posttranslational modifications of histone proteins, including acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation (2-4). Acetylation status of the amino termini of histones H3 and H4, in particular, plays a important role in transcriptional regulation (2, 5).Heterogeneity in nucleosome structure also results from incorporation of variant histone proteins into the nucleosome. In contrast to the canonical histones, which are multicopy genes expressed during S-phase of the cell cycle, variant histones are encoded by single copy genes that differ in amino acid sequence from S-phase histones and whose expression is not limited to S-phase (6, 7). Variant histones allow specialization of nucleosome structure for specific purposes. Sperm-specific variant histones, for example, facilitate the dramatic compaction of DNA that occurs during spermatogenesis (4,8). A specific variant of histone H3 is incorporated specifically at centromeres creating a specialized chromatin structure required for proper function of the kinetochore (9 -14), and macro-H2A, a histone H2A variant, localizes preferentially to the inactive X chromosome in mammals and may alter chromatin in a way that helps suppress transcription (15). These and other variants have been identified for histones H2A,...

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Recent EU directives and regulations for quality control and authentication of food products have prompted the development of new methods for large-scale tests to ensure the protection of consumers. In view of this, an innovative method based on MALDI-TOF mass spectrometry has been developed and successfully applied to fish authentication. Highly specific mass spectrometric profiles from 25 different fish species were obtained. Signals generated from proteins with molecular weights of about 11 kDa have been selected as specific biomarkers for unambiguous discrimination. This method is also suitable for verifying commercial product authenticity and to rapidly discriminate species subjected to fraudulent substitutions, such as those belonging to Gadidae and Pleuronectiformes. For example, biomarkers for fillets of sole (m/z 11975.21), European plaice (m/z 11351.73, 11763.63) and Greenland halibut (m/z 11432.38) were defined. Structural characterization by mass spectrometry of several proteins generating biomarker signals allowed us to identify them as parvalbumins, known to be among the major fish allergens.

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Probiotics are live microorganisms that confer health benefits on the host. However, in recent years, several concerns on their use have been raised. In particular, industrial processing and storage of probiotic products are still technological challenges as these could severely impair cell viability. On the other hand, safety of live microorganisms should be taken into account, especially when administered to vulnerable people, such as the elderly and immunodeficient individuals. These drawbacks have enhanced the interest toward new products based on non-viable probiotics such as paraprobiotics and postbiotics. In particular, paraprobiotics, defined as “inactivated microbial cells (non-viable) that confer a health benefit to the consumer,” hold the ability to regulate the adaptive and innate immune systems, exhibit anti-inflammatory, antiproliferative and antioxidant properties and exert antagonistic effect against pathogens. Moreover, paraprobiotics can exhibit enhanced safety, assure technological and practical benefits and can also be used in products suitable for people with weak immunity and the elderly. These features offer an important opportunity to prompt the market with novel functional foods or nutraceuticals that are safer and more stable. This review provides an overview of central issues on paraprobiotics and highlights the urgent need for further studies aimed at assessing safety and efficacy of these products and their mechanisms of action in order to support decisions of regulatory authorities. Finally, a definition is proposed that unambiguously distinguishes paraprobiotics from postbiotics.

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant α-gliadin (r-α-gliadin) showing the most conserved sequence among the fraction of α-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-α-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-α-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120–139) and p23 (aa 220–239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-α-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-γ secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.