Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group ,which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products

MWCNT/rGO/natural rubber latex dispersions for innovative, piezo‐resistive and cement‐based composite sensors

The present study is focused on the development and characterization of innovative cementitious-based composite sensors. In particular, multifunctional cement mortars with enhanced piezoresistive properties are realized by exploiting the concept of confinement of Multiwall Carbon Nanotubes (MWCNTs) and reduced Graphene Oxide (rGO) in a three-dimensional percolated network through the use of a natural-rubber latex aqueous dispersion. The manufactured cement-based composites were characterized by means of Inelastic Neutron Scattering to assess the hydration reactions and the interactions between natural rubber and the hydrated-cement phases and by Scanning Electron Microscopy and X-Ray diffraction to evaluate the morphological and mineralogical structure, respectively. Piezo-resistive properties to assess electro-mechanical behavior in strain condition are also measured. The results show that the presence of natural rubber latex allows to obtain a three-dimensional rGO/MWCNTs segregate structure which catalyzes the formation of hydrated phases of the cement and increases the piezo-resistive sensitivity of mortar composites, representing a reliable approach in developing innovative mortar-based piezoresistive strain sensors

Microenvironment inflammatory infiltrate drives growth speed and outcome of hepatocellular carcinoma: a prospective clinical study

In HCC, tumor microenvironment, heavily influenced by the underlying chronic liver disease, etiology and stage of the tissue damage, affects tumor progression and determines the high heterogeneity of the tumor. Aim of this study was to identify the circulating and tissue components of the microenvironment immune-mediated response affecting the aggressiveness and the ensuing clinical outcome. We analyzed the baseline paired HCC and the surrounding tissue biopsies from a prospective cohort of 132 patients at the first diagnosis of HCC for immunolocalization of PD-1/PD-L1, FoxP3, E-cadherin, CLEC2 and for a panel of 82 microRNA associated with regulation of angiogenesis, cell proliferation, cell signaling, immune control and autophagy. Original microarray data were also explored. Serum samples were analyzed for a panel of 19 cytokines. Data were associated with biochemical data, histopathology and survival. Patients with a more aggressive disease and shorter survival, who we named fast-growing accordingly to the tumor doubling time, at presentation had significantly higher AFP levels, TGF-β1 and Cyphra 21-1 levels. Transcriptomic analysis evidenced a significant downregulation of CLEC2 and upregulation of several metalloproteinases. A marked local upregulation of both PD-1 and PD-L1, a concomitant FoxP3-positive lymphocytic infiltrate, a loss of E-cadherin, gain of epithelial-mesenchymal transition (EMT) phenotype and extreme poor differentiation at histology were also present. Upregulated microRNA in fast-growing HCCs are associated with TGF-β signaling, angiogenesis and inflammation. Our data show that fast HCCs are characterized not only by redundant neo-angiogenesis but also by unique features of distinctively immunosuppressed microenvironment, prominent EMT, and clear-cut activation of TGFβ1 signaling in a general background of long-standing and permanent inflammatory state

HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer

The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient's immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment

Childhood-onset dystonia-causing KMT2B variants result in a distinctive genomic hypermethylation profile

Background: Dystonia is a clinically and genetically heterogeneous movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements and/or postures. Heterozygous variants in lysine methyltransferase 2B (KMT2B), encoding a histone H3 methyltransferase, have been associated with a childhood-onset, progressive and complex form of dystonia (dystonia 28, DYT28). Since 2016, more than one hundred rare KMT2B variants have been reported, including frameshift, nonsense, splice site, missense and other in-frame changes, many having an uncertain clinical impact. Results: We characterize the genome-wide peripheral blood DNA methylation profiles of a cohort of 18 patients with pathogenic and unclassified KMT2B variants. We resolve the “episignature” associated with KMT2B haploinsufficiency, proving that this approach is robust in diagnosing clinically unsolved cases, properly classifying them with respect to other partially overlapping dystonic phenotypes, other rare neurodevelopmental disorders and healthy controls. Notably, defective KMT2B function in DYT28 causes a non-random DNA hypermethylation across the genome, selectively involving promoters and other regulatory regions positively controlling gene expression. Conclusions: We demonstrate a distinctive DNA hypermethylation pattern associated with DYT28, provide an epigenetic signature for this disorder enabling accurate diagnosis and reclassification of ambiguous genetic findings and suggest potential therapeutic approaches

Agricultural by-products with bioactive effects: A multivariate approach to evaluate microbial and physicochemical changes in a fresh pork sausage enriched with phenolic compounds from olive vegetation water

The use of phenolic compounds derived from agricultural by-products could be considered as an eco-friendly strategy for food preservation. In this study a purified phenol extract from olive vegetation water (PEOVW) was explored as a potential bioactive ingredient for meat products using Italian fresh sausage as food model. The research was developed in two steps: first, an in vitro delineation of the extract antimicrobial activities was performed, then, the PEOVW was tested in the food model to investigate the possible application in food manufacturing. The in vitro tests showed that PEOVW clearly inhibits the growth of food-borne pathogens such as Listeria monocytogenes and Staphylococcus aureus. The major part of Gram-positive strains was inhibited at the low concentrations (0.375–3 mg/mL). In the production of raw sausages, two concentrates of PEOVW (L1:0.075% and L2: 0.15%) were used taking into account both organoleptic traits and the bactericidal effects. A multivariate statistical approach allowed the definition of the microbial and physicochemical changes of sausages during the shelf life (14 days). In general, the inclusion of the L2 concentration reduced the growth of several microbial targets, especially Staphylococcus spp. and LABs (2 log10 CFU/g reduction),while the increasing the growth of yeasts was observed. The reduction of microbial growth could be involved in the reduced lipolysis of raw sausages supplemented with PEOVWas highlighted by the lower amount of diacylglycerols. Moisture and aw had a significant effect on the variability of microbiological features,while food matrix (the sausages' environment) can mask the effects of PEOVW on other targets (e.g. Pseudomonas). Moreover, the molecular identification of the main representative taxa collected during the experimentation allowed the evaluation of the effects of phenols on the selection of bacteria. Genetic data suggested a possible strain selection based on storage time and the addition of phenol compounds especially on LABs and Staphylococcus spp. The modulation effects on lipolysis and the reduction of several microbial targets in a naturally contaminated product indicates that PEOVW may be useful as an ingredient in fresh sausages for improving food safety and quality

Drilling constraints on lithospheric accretion and evolution at Atlantis Massif, Mid-Atlantic Ridge 30°N

Author Posting. © American Geophysical Union, 2011. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 116 (2011): B07103, doi:10.1029/2010JB007931.Expeditions 304 and 305 of the Integrated Ocean Drilling Program cored and logged a 1.4 km section of the domal core of Atlantis Massif. Postdrilling research results summarized here constrain the structure and lithology of the Central Dome of this oceanic core complex. The dominantly gabbroic sequence recovered contrasts with predrilling predictions; application of the ground truth in subsequent geophysical processing has produced self-consistent models for the Central Dome. The presence of many thin interfingered petrologic units indicates that the intrusions forming the domal core were emplaced over a minimum of 100–220 kyr, and not as a single magma pulse. Isotopic and mineralogical alteration is intense in the upper 100 m but decreases in intensity with depth. Below 800 m, alteration is restricted to narrow zones surrounding faults, veins, igneous contacts, and to an interval of locally intense serpentinization in olivine-rich troctolite. Hydration of the lithosphere occurred over the complete range of temperature conditions from granulite to zeolite facies, but was predominantly in the amphibolite and greenschist range. Deformation of the sequence was remarkably localized, despite paleomagnetic indications that the dome has undergone at least 45° rotation, presumably during unroofing via detachment faulting. Both the deformation pattern and the lithology contrast with what is known from seafloor studies on the adjacent Southern Ridge of the massif. There, the detachment capping the domal core deformed a 100 m thick zone and serpentinized peridotite comprises ∼70% of recovered samples. We develop a working model of the evolution of Atlantis Massif over the past 2 Myr, outlining several stages that could explain the observed similarities and differences between the Central Dome and the Southern Ridge