Growth and metabolite production of a grape sour rot yeast-bacterium consortium on different carbon sources.

The present study was designed to evaluate possible sugar-based trophic interactions between acetic acid bacteria (AAB) and non-Saccharomyces yeasts (NSY) involved in table grape sour rot, a disease in which berries spoilage is caused by the accumulation of several microbial metabolites. Acetobacter syzygii LMG 21419 (As) and Candida zemplinina CBS 9494 (Cz), a simplified AAB-NSY association responsible for table grape sour rot, grew differently in a minimal medium (YP) supplemented with glucose, ethanol, acetic and gluconic acid under monoculture conditions. In As -Cz co-culture media, after 24 h of incubation, As showed high relative abundance in YP-ethanol, whereas Cz was the dominant strain in YP-glucose medium. Co-culture in YP-glucose showed that glucose was converted into ethanol by Cz that, in turn, promoted the growth of As population. Gluconic acid was the main bacterial metabolite from glucose in monoculture, whereas acetic acid putatively derived from ethanol oxidation was found only in co-culture. However, gluconic acid showed inhibitory effect against As whereas acetic acid mainly inhibited Cz. Negative effects of both metabolites were mitigated in the glucose-supplemented medium. The results suggest a possible metabolic- based temporal succession between AAB and NSY during grape sour rot development. At the begin of sour rot, low glucose concentration promotes NSY producing ethanol, then, the AAB could take advantage from the oxidation of ethanol into acetic acid, becoming the dominant microbial sour rot population during the late stages of the process.

LKB1 loss is associated with glutathione deficiency under oxidative stress and sensitivity of cancer cells to cytotoxic drugs and γ-irradiation.

The liver kinase B1 (LKB1) gene is a tumor suppressor associated with the hereditary Peutz-Jeghers syndrome and frequently mutated in non-small cell lung cancer and in cervical cancer. Previous studies showed that the LKB1/AMPK axis is involved in regulation of cell death and survival under metabolic stress. By using isogenic pairs of cancer cell lines, we report here that the genetic loss of LKB1 was associated with increased intracellular levels of total choline containing metabolites and, under oxidative stress, it impaired maintenance of glutathione (GSH) levels. This resulted in markedly increased intracellular reactive oxygen species (ROS) levels and sensitivity to ROS-induced cell death. These effects were rescued by re-expression of LKB1 or pre-treatment with the anti-oxidant and GSH replenisher N-acetyl cysteine. This role of LKB1 in response to ROS-inducing agents was largely AMPK-dependent. Finally, we observed that LKB1 defective cells are highly sensitive to cisplatin and γ-irradiation in vitro, suggesting that LKB1 mutated tumors could be targeted by oxidative stress-inducing therapies.

Application of a recombinant laccase-chlorogenic acid system in protein crosslink and antioxidant properties of the curd.

Milk protein crosslink through the action of enzymes represents a feasible strategy to impart new functionalities to cheese. In this work we reported the effects of a laccase mediator system (LMS) on protein crosslink and antioxidant property of curd. The crosslinking activity of a purified recombinant laccase Ery4 and a commercial enzyme preparation (cLC), with three mediators was firstly evaluated in milk and then applied before curd manufacture. Only Ery4-LMS significantly increased curd weight compared to that of the control sample. SDS-PAGE revealed that similar high molecular weight bands produced by both LMSs in milk were also retained in curds. The antioxidant activity recorded in curds with Ery4-LMS was the highest among all samples both before and after gastro-pancreatic digestion. This is the first time that a CGA-based LMS is used in manufacture of curd with improved antioxidant properties. These results open new perspectives for dairy applications.

OVERVIEW OF NEUTRON MEASUREMENTS IN JET FUSION DEVICE.

The design and operation of ITER experimental fusion reactor requires the development of neutron measurement techniques and numerical tools to derive the fusion power and the radiation field in the device and in the surrounding areas. Nuclear analyses provide essential input to the conceptual design, optimisation, engineering and safety case in ITER and power plant studies. The required radiation transport calculations are extremely challenging because of the large physical extent of the reactor plant, the complexity of the geometry, and the combination of deep penetration and streaming paths. This article reports the experimental activities which are carried-out at JET to validate the neutronics measurements methods and numerical tools used in ITER and power plant design. A new deuterium-tritium campaign is proposed in 2019 at JET: the unique 14 MeV neutron yields produced will be exploited as much as possible to validate measurement techniques, codes, procedures and data currently used in ITER design thus reducing the related uncertainties and the associated risks in the machine operation.

99mTc by 99Mo produced at the ENEA-FNG facility of 14MeV neutrons.

A severe supply crisis of 99Mo, precursor of 99mTc a diagnostic radionuclide largely used in Nuclear Medicine, occurred in 2008-2009 due to repeated shut-down of the two main (aged) fission reactors. An alternative route for producing 99Mo by 100Mo(n,2n)99Mo reaction was investigated at ENEA. The experiment, designed according to Monte Carlo simulations performed with the Fluka code, produced 99Mo by irradiating a natural Molybdenum powdered target with 14MeV neutrons produced at the Frascati Neutron Generator. The 99Mo specific activity was measured at metrological level by γ-ray spectrometry.

Efficacy of gaseous ozone to counteract postharvest table grape sour rot.

This work aims at studying the efficacy of low doses of gaseous ozone in postharvest control of the table grape sour rot, a disease generally attributed to a consortium of non-Saccharomyces yeasts (NSY) and acetic acid bacteria (AAB). Sour rot incidence of wounded berries, inoculated with 8 NSYstrains, or 7 AAB, or 56 yeast-bacterium associations, was monitored at 25 °C up to six days. Sour rot incidence in wounded berries inoculated with yeast-bacterium associations resulted higher than in berries inoculated with one single NSY or AAB strain. Among all NSY-AAB associations, the yeast-bacterium association composed of Candida zemplinina CBS 9494 (Cz) and Acetobacter syzygii LMG 21419 (As) showed the highest prevalence of sour rot; thus, after preliminary in vitro assays, this simplified As-Cz microbial consortium was inoculated in wounded berries that were stored at 4 °C for ten days under ozone (2.14 mg m-3) or in air. At the end of cold storage, no berries showed sour-rot symptoms although ozonation mainly affected As viable cell count. After additional 12 days at 25 °C, the sour rot index of inoculated As-Cz berries previously cold-stored under ozone or in air accounted for 22.6 ± 3.7% and 66.7 ± 4.5%, respectively. Molecular analyses of dominant AAB and NSY populations of both sound and rotten berries during post-refrigeration period revealed the appearance of new strains mainly belonging to Gluconobacter albidus and Hanseniaspora uvarum species, respectively. Cold ozonation resulted an effective approach to extend the shelf-life of table grapes also after cold storage.

Test of "crab-waist" collisions at the DAPhiNE Phi factory.

The electron-positron collider DAPhiNE, the Italian Phi factory, has been recently upgraded in order to implement an innovative collision scheme based on large crossing angle, small beam sizes at the crossing point, and compensation of beam-beam interaction by means of sextupole pairs creating a "crab-waist" configuration in the interaction region. Experimental tests of the novel scheme exhibited an increase by a factor of 3 in the peak luminosity of the collider with respect to the performances reached before the upgrade. In this Letter we present the new collision scheme, discuss its advantages, describe the hardware modifications realized for the upgrade, and report the results of the experimental tests carried out during commissioning of the machine in the new configuration and standard operation for the users.

Irreversible CYP3A inhibition accompanied by plasma protein-binding displacement: a comparative analysis in subjects with normal and impaired liver function.

In this study, quinine was used as a probe substrate and erythromycin as a prototypical irreversible inhibitor of CYP3A to ascertain whether, like reversible CYP inhibition, the magnitude of irreversible inhibition is also strictly dependent on the status of liver function. The effect of erythromycin on oral quinine disposition was studied in 10 healthy subjects and in 20 patients with cirrhosis of the liver who had varying degrees of liver dysfunction. This effect was shown to be the result of two types of interaction: (i) irreversible inhibition of CYP3A-mediated quinine metabolism, the extent of which proved to be independent of liver function, and (ii) displacement of quinine from plasma protein-binding sites, the magnitude of the displacement increasing dramatically as liver function worsened. Such an interaction causes limited increases in the total concentration of the displaced drug but disproportionate increases in its free concentration; the latter increases are magnified by liver dysfunction, thereby requiring that the monitoring of free drug concentrations be made mandatory.

Hyperthermic isolated perfusion with low-dose TNFalpha and doxorubicin in patients with locally advanced soft tissue limb sarcomas.

We report here the results of 27 patients who underwent hyperthermic isolated limb perfusion with low-dose TNFalpha (1 mg) and doxorubicin (8.5 mg/l of limb volume) for locally advanced soft tissue sarcomas. A tumor response was observed in 85% of cases. After a median follow-up of 30 months, limb salvage and local disease control were achieved in 82 and 85% of patients, respectively. Locoregional toxicity was low or mild in 14 patients, while 2 patients had severe limb toxicity. Systemic side effects were negligible. The perfusate/plasma area under the curve (AUC) ratio for TNFalpha was 56. HILP with low-dose TNFalpha and DXR proved to be an active neoadjuvant drug regimen against limb-threatening STS.

Increasing the bandwidth of resonant gravitational antennas: the case of explorer.

Resonant gravitational wave detectors with an observation bandwidth of tens of hertz are a reality: the antenna Explorer, operated at CERN by the ROG Collaboration, has been upgraded with a new readout. In this new configuration, it exhibits an unprecedented useful bandwidth: in over 55 Hz about its center operating frequency of 919 Hz the spectral sensitivity is better than 10(-20) Hz(-1/2). We describe the detector and its sensitivity and discuss the foreseeable upgrades to even larger bandwidths.

In vivo antitumor activity and host toxicity of methoxymorpholinyl doxorubicin: role of cytochrome P450 3A.

Methoxymorpholinyl doxorubicin (MMDX; PNU 152243) is a promising doxorubicin derivative currently undergoing clinical evaluation. Previous in vitro studies suggested that the compound undergoes hepatic biotransformation by cytochrome P450 (CYP) 3A into a more cytotoxic metabolite(s). The present study examined the role of CYP3A-mediated metabolism in the in vivo antitumor activity and host toxicity of MMDX in the mouse model and investigated the potential for increasing the therapeutic effectiveness of the drug by inducing its hepatic CYP-catalyzed activation. We found that MMDX cytotoxicity for cultured M5076 tumor cells was potentiated 22-fold by preincubating the drug with NADPH-supplemented liver microsomes from untreated C57BL/6 female mice. A greater (50-fold) potentiation of MMDX cytotoxicity was observed after its preincubation with liver microsomes isolated from animals pretreated with the prototypical CYP3A inducer pregnenolone-16alpha-carbonitrile. In contrast, in vivo administration of the selective CYP3A inhibitor troleandomycin (TAO) reduced both potentiation of MMDX cytotoxicity and the rate of CYP3A-catalyzed N-demethylation of erythromycin by isolated liver microsomes (55.5 and 49% reduction, respectively). In vivo antitumor activity experiments revealed that TAO completely suppressed the ability of 90 microg/kg MMDX i.v., a dose close to the LD10, to delay growth of s.c. M5076 tumors in C57BL/6 mice and to prolong survival of DBA/2 mice with disseminated L1210 leukemia. Moreover, TAO administration markedly inhibited the therapeutic efficacy of 90 microg/kg MMDX i.v. in mice bearing experimental M5076 liver metastases; a complete loss of MMDX activity was observed in liver metastases-bearing animals receiving 40 microg/kg MMDX i.v. plus TAO. However, pregnenolone-16alpha-carbonitrile pretreatment failed to enhance MMDX activity in mice bearing either s.c. M5076 tumors or experimental M5076 liver metastases. Additional experiments carried out in healthy C57BL/6 mice showed that TAO markedly inhibited MMDX-induced myelosuppression and protected the animals against lethal doses of MMDX. Taken together, these findings demonstrate that an active metabolite(s) of MMDX synthesized via CYP3A contributes significantly to its in vivo antitumor activity and host toxicity.

Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases.

The possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 microg kg(-1) MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 microg kg(-1) free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection.

Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity.

In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposi's sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis.

CD45 regulates apoptosis induced by extracellular adenosine triphosphate and cytotoxic T lymphocytes.

Several lines of evidence implicate protein tyrosine phosphatases (PTP) in the regulation of apoptotic cell death. We have evaluated the role of CD45, the major PTP of hematopoietic cells, in apoptosis induced by extracellular ATP (ATPe) and cytotoxic T lymphocytes (CTL). We observed that two CD45- clones obtained by mutagenesis of the Fas- cell line L1210, exhibit a higher susceptibility to apoptosis induced by ATPe, which was also evident in Ca(2+)-free conditions, when compared to the parental cell line or CD45+ variants. The CD45- cells were also more susceptible to death mediated by an alloreactive CTL clone. When the cytotoxic assay was performed in the presence of EGTA, a Ca2+ chelator, which prevents cytotoxic granule exocytosis and perforin polymerization on target cell membranes, only the CD45- target cells were killed by the CTL clone. These results suggest that a cytotoxic pathway other than the secretory or Fas-dependent pathways was responsible for the enhanced susceptibility of CD45- cells to death, and therefore provide further evidence for the role of ATPe as a possible mediator of Ca(2+)-independent target cell destruction by CTL.