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Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including αs1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro αs1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.

Pyroglutamic acid is present in high amounts (0.5g/100g) in many cheese varieties—and particularly in extensively ripened Italian cheeses such as Grana Padano and Parmigiano Reggiano.

In the present work, 67 strains of Lactobacillus helveticus isolated from whey starter cultures and cheeses were identified and grouped by genotypic and phenotypic methods. Strains were identified by sugar fermentation pattern, by cell-wall protein profile, and by probe hybridisation. Phenotypic diversity was evaluated by a chemometric model taking into account biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR, which provided strain-specific patterns and revealed the occurrence of different strains. The RAPD-PCR profiles were clustered according to their similarities; the groups obtained, together with the cell-wall protein profiling and the chemometric information, could be sometimes correlated with the type of cheese and/or dairy niches used as sources of strains. A computerised analysis of genotypic and phenotypic information could be successfully applied for rapid and reliable differentiation and characterisation of Lb. helveticus isolates occurring in different dairy products.

The ability of a biological control system to inhibit the outgrowth of Clostridium sporogenesspores during storage of mascarpone cheese under temperature-abuse conditions was investigated. Challenge studies were carried out on mascarpone cheese artificially contaminated with spores of C. sporogenes(10 cfu g−1), and with or without the coinoculum of a Streptococcus thermophilusstrain (105cfu g−1). During storage at 4, 12, and 25°C, the outgrowth of clostridia spores, the growth of S. thermophilus, and the pH changes were evaluated at 10, 20, 30, and 40 days. In mascarpone cheese stored at 4° and 12°C, S. thermophilusand C. sporogenesdid not show any growth. The initial pH (6·14) of the product also remained unchanged. During storage at 25°C S. thermophilusgrew up to about 107cfu g−1after 10 days, resulting in a pH decrease of mascarpone cheese to values close to 4·5. The cell number decreased progressively during storage reaching values near to 101cfu g−1after 40 days, whereas product acidity remained constant. C. sporogenes, when inoculated alone, also grew at 25°C. The cell number increased to levels of about 107cfu g−1after 20–40 days of storage according to the different mascarpone cheese lots used. No growth was found when C. sporogeneswas co-inoculated in mascarpone cheese with S. thermophilusand stored at 25°C. The study on the behaviour of C. sporogenes, known as a non-toxigenic variant of Clostridium botulinum, allowed us to obtain useful information for setting up an effective biological control system to inhibit growth of the toxigenic species as well. The use of an additional barrier, besides refrigerated storage, may help to maintain the safety of mascarpone cheese in the event it was exposed to elevated temperatures.

Aims:In the present study, a method based on SDS‐PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckiisubsp. bulgaricusand subsp. lactisdairy isolates.

Several industrial and artisan Italian cheeses were screened for the presence of vancomycin-resistant enterococci (VRE). One hundred and two strains, mostly belonging to the species Enterococcus faeciumafter species-specific PCR identification, were tested for antibiotic susceptibility by standard disc diffusion method and for resistance to vancomycin in liquid medium. About 25% of strains were resistant to both vancomycin and teicoplanin. PCR experiments showed that all resistant isolates harboured the vanAgene. Minimum inhibitory concentration (MIC) values of vancomycin for most of the vancomycin resistant E. faecium(VREF) ranged from 128 to 512 μg ml−1(high level resistance). PCR fingerprinting of VREF indicated that multiple isolation of the same strains did not occur. The importance of finding VRE in cheeses is discussed.

The influence of a traditional procedure of washing of smear Taleggio cheese on surface spreading of Listeria innocuawas studied. This practice is carried out during ripening to remove molds, to select the surface microflora, and to control the ripening process. One cheese, both of 2 (i) and 4 (ii) weeks of ripening, was surface-inoculated with approximately 3 log CFU of L. innocuaper entire cheese surface. The inoculated cheeses and others of the same age were weekly washed with brine solution. Listeriawas spread both on the surface of the inoculated cheese and on the other cheeses, and it was also found in the brines and on the wooden boxes where the cheeses were ripened. The time of ripening when contamination occurs influenced the behavior of Listeria. At the moment of contamination, the smear surface microflora of (i) cheese was ∼2 log CFU/g higher than of (ii) cheese. Listeriainoculated on 2-week-ripened cheese was able to colonize the entire surface of the cheese and to cross-contaminate the other cheeses. On the contrary, Listeriainoculated on a 4-week-ripened cheese was partially spread on the surface of the originally inoculated cheese, and the transfer of contamination by the washing procedure was restrained. Because a random distribution of Listeriaon cheese surface was observed, the importance of the mode of sampling was discussed. Because of the lack of critical control points during ripening of Taleggio cheese, the Listeriahazard needs to be controlled by taking appropriate control measures to break off the contamination cycle (cheese → brine → wooden boxes → cheese).

Pyroglutamic acid is present in many cheese varieties and particularly in high amounts (0.5 g/100g of cheese) in extensively ripened Italian cheeses (Grana Padano and Parmigiano Reggiano) that are produced with thermophilic lactic acid bacteria as starters. The mechanism of pyroglutamic acid formation in cheese seems to be mostly enzymatic, as demonstrated by the presence of only l-pyroglutamic acid enantiomer. Thermophilic lactobacilli are involved in pyroglutamic acid production, as suggested by the low pyroglutamic acid content found in Bagos, a ripened Italian mountain cheese produced without addition of starter. Because milk pasteurization did not influence the pyroglutamic acid content in the ripened Grana Padano cheese, the formation of pyroglutamic acid mainly depends on the whey starter microflora rather than that of raw milk. Pyroglutamic acid concentration is linearly correlated (R2= 0.94) with the age of Grana Padano cheese.

Sixty-two strains ofLactobacillus helveticusisolated from whey starter cultures for Grana Padano cheese and from the cheese curd after moulding were grouped by PCR fingerprinting and SDS-;PAGE fingerprinting of cell wall proteins. Strain clustering allowed discrimination of five groups ofL. helveticusstrains, distributed differently between the whey starter and the cheese. In particular, two clearly distinguished biotypes respectively dominated theL. helveticuspopulations of the whey starter and the cheese curd after moulding. These two groups of strains also showed a peculiar cell wall protein profile, thus strengthening differences between biotypes alternatively dominating in whey starter and cheese curd. This study showed that the community ofL. helveticusdominating in Grana cheese is composed of different biotypes. Cheese technology parameters may play a major role in selecting dominant strains.

Lactic acid bacteria (LAB) counts, PepX activity towards H‐Phe‐Pro‐βNA, and aminopeptidase activity towards H‐Arg‐βNA.HCl, H‐Lys‐βNA, H‐Leu‐βNA, H‐Pro‐βNA, H‐Glu‐βNA derivatives have been evaluated in 32 commercial samples of cheese, one processed cheese, and one yoghurt. The presence of intracellular exo‐peptidase activities in cheese extracts free from bacterial cells was detected, even after 1 year of ripening. An inverse ratio between the presence of viable lactic microflora and peptidase activity in the cheese extracts was observed. The importance of LAB starter exo‐peptidases in the degradation of casein oligopeptides, and the key role of autolysis in the release of peptidases in the cheese, are discussed.

Surface-smear micro-organisms isolated from Taleggio cheese were screened for their ability to inhibitListeria monocytogenes. Most of the isolates showing antilisterial activity (19% of the total) consisted of coryneform bacteria, mainly belonging to theMicrobacterium lacticumspecies. The inhibitory activity was observed also after growth at 5°C and pH 6·0. After cross-inhibition tests among inhibitory strains and between these and other pigmented, non-inhibitory strains, two bacterial mixtures were assayed as surface-smear starter of Taleggio cheese. At different ripening stages (1, 20 and 40 days), the cheese surface was contaminated with 2·5×102cfu cm−2L. monocytogenes(Ohio strain) and the growth evaluated over 15 days of incubation at 5°C. Contrary to the laboratory experiments,Listeriacould not be completely inhibited on the cheese surface. With 5·5–6·0 pH range of the cheese rind, (lower than usual values), the growth of surface-smear bacteria was delayed, or even stopped. Nevertheless, a listeriostatic effect was achieved on the surface of cheese samples contaminated at the end of ripening. This seemed to confirm the essential role played by surface-smear bacteria, within the rind microflora, in controlling the development of contaminant micro-organisms, and by competitive bacteria in reducing the overall risks associated with soft cheese. The mechanisms involved in the selection and the growth of competing bacteria on the cheese surface are also discussed.

SDS–PAGE fingerprinting of cell‐wall proteins extracted from 119 strains belonging to different species of lactic acid bacteria have been compared. The method of extraction and electrophoretic separation utilized in this work was found to be a reliable and rapid way for characterizing thermophilic lactobacilli species and strains. A protein of approximately 50 kDa was found to be characteristic for all theLact. helveticusstrains, and two cell‐wall proteins of about 20 and 30 kDa were typical of the species Lact. delbrueckii, but the discrimination between the subspecies lactisand bulgaricuswas not possible by the electrophoretic technique used. The other thermophilic species studied in this work presented cell‐wall protein patterns that permitted their differentiation from both Lact. helveticusand Lact. delbrueckii.

This study investigated the evolution of the Lactobacillusspecies and the microbial composition of the starter during Grana cheese making. The early stages of cheese making were studied, and both the composition of the natural whey starter and its modification in response to curd cooking were considered. The growth and distribution of the thermophilic lactobacilli in the cheese at 48h after molding was affected by the temperature gradient between the external and internal cheese zones. Growth was maximum between 0 and 6h in the cheese exterior and between 6 and 24h in the core. This variation occurred because the cheese interior was around 52°C 6h after molding, which is far from the optimum for the thermophilic lactobacilli growth. Dot-blot hybridization experiments allowed the identification of up to 280 isolates. Lactobacillus helveticuspredominated in the natural whey starter and in the external cheese zones. Distribution of Lactobacillus delbrueckiiand the heterofermentative lactobacilli, which slowly increased from the molding until 48h, was more variable in the internal regions than in the external regions of the cheese. This study demonstrates that the thermophilic lactobacilli behave differently during the technological process than during experiments using laboratory models.

We have studied the growth and acidification activities of strains of the most important species present in natural whey starters for Grana cheese under a temperature gradient that was similar to that used in the first 24h of cheese making. Results showed that strains can be classified according to their ability to adapt to cheese-making conditions. Some strains that had good acidification activity at optimal temperature were not able to decrease pH under technological conditions of cheese manufacture.

We studied the incidence of inducible Lactobacillus helveticusstrains among 74 isolates from natural whey starter cultures that are used for the production of Italian Grana and Provolone cheeses. Mitomycin C was used for induction of cultures. Of the 77 strains tested, 35 strains (47%) released supernatant particles in the culture that were able to inhibit up to a maximum of 47 (61%) sensitive indicator strains. The presence of bacteriophages in culture supernatants of L. helveticusstrains LH38 and LH60 was verified by electron microscopy. None of the positive culture supernatants formed plaques, and inhibition zones were observed only on agar plates that had been inoculated with the indicator strains. Fourteen of the 17 positive culture supernatants were able to propagate on some indicator strains, indicating the presence of completely infective virions in liquid medium. The particles were insensitive to proteolytic enzymes, were unable to pass through a microfiltration membrane, were sensitive to heat, and were sedimented by ultracentrifugation. Some of these particles showed killer activity against nonproliferating cells of indicator strains. The lysogenic state of the L. helveticusstrains was related to a stronger resistance of these strains to the products of the induction; higher prevalence of indicator strains were found among the noninducible strains.

The potential performances and limitations of an all-analogue implementation of a posteriori-probability (APP) decoders in a standard complementary-metal-oxide semiconductor (CMOS) technology are investigated. In particular, the accuracy and speed trade-off related to the usage of MOS transistors in the weak inversion (w.i.) region is analysed in depth. Transistor level simulations of a (18, 9, 5) tail-biting decoder are reported and contrasted with the results of the software implementation of the same decoding algorithm.

LB100 does not sensitize CML stem cells to tyrosine kinase inhibitor–induced apoptosis.

This paper investigates the electroluminescence characteristics of InGaN-based LEDs at extremely low current levels (down to 500 pA), i.e. in and below the region where recombination dynamics are governed by Shockley-Read-Hall recombination. Two different regimes are identified in the current-voltage characteristics, a first one below 100 nA associated to emission at wavelength below midgap, and a second one below 100 ?A with a dominant emission at the quantum well wavelength. The experimental findings are interpreted by considering that, at extremely low current, carriers can tunnel towards states deeper than midgap, thus undergoing radiative recombination through defects.

Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance, disease evolution and innate anti-cancer immunity. In CML, persistence of ABL tyrosine kinase inhibitor (TKI)-resistant quiescent LSCs, disease development and inhibition of natural killer (NK) cell growth and anti-cancer activity depend on cell-autonomous and bone marrow (BM)-generated signals.

Deininger: Novartis: Other: Consulting or Advisory Role, Research Funding; BMS: Other: Consulting & Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role. Milojkovic:BMS: Honoraria; ARIAD Pharmaceuticals Inc.: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

No relevant conflicts of interest to declare.

The problems that arise when trying to port pacemaker analogue blocks to submicrometre technology are discussed. A circuit topology is proposed for a programmable voltage multiplier that can handle the relatively high voltages required to stimulate the heart, while still being able to operate from an almost exhausted pacemaker battery.

No relevant conflicts of interest to declare.

Abstract 35

Abstract 773

No relevant conflicts of interest to declare.

Shacham: Karyopharm: Equity Ownership. Kauffman:Karyopharm: Equity Ownership.

No relevant conflicts of interest to declare.

Abstract 2494

Abstract 2738

Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Abstract 2901

Verstovsek: Incyte Corporation: Research Funding.

Cortes: Novartis: Research Funding.

Cortes: Novartis: Research Funding. Cancelas:CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

No relevant conflicts of interest to declare.

Abstract 855