From virtual screening hits targeting a cryptic pocket in BACE-1 to a nontoxic brain permeable multitarget anti-Alzheimer lead with disease-modifying and cognition-enhancing effects

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Ab42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognitionenhancing anti-AD lead

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Characteristics of Manchego cheese made from pasteurized ewes' milk inoculated with a bacteriocin-producing adjunct culture

The potential of using a bacteriocin-producing Lactococcus lactis strain to induce lysis of a Streptococcus thenmophilus culture, and to accelerate proteolysis and flavour formation in Manchego cheese, has been investigated. Two vats of cheese were manufactured from pasteurized ewes' milk in duplicate trials. Milk in the experimental vat was inoculated with L lactis subsp. lactis 415, a nisin Z and lacticin 481 producer. L lactis subsp. lactis INIA 415-2, a bacteriocinnonproducing mutant, was used as mesophilic starter in both vats. Cheeses were ripened at 12°C and 85% RH for 90 days. Microbiological, physicochemical, enzymatic, proteolytic, textural and sensory characteristics of cheeses were assessed S. thermophilus counts were lowered by the bacteriocin, aminopeptidase activity was slightly increased during early ripening, and proteolysis (estimated by the o-phthaldialdehyde method) was significantly higher throughout ripening. In spite of the enhanced proteolysis, textural and sensory characteristics of Manchego cheese were not significantly influenced by milk inoculation with the bacteriocin producer

Effect of milk inoculation with bacteriocin-producing lactic acid bacteria on a Lactobacillus helveticus adjunct cheese culture

The effect of eight strains of lactic acid bacteria (two strains of Enterococcus, one strain of Lactobacillus, and five strains of Lactococcus, which produce enterocin AS-48, enterocin 607, nisin A, nisin Z, plantaricin 684, lacticin 481, or nisin Z plus lacticin 481) on acid production and proteolytic activity of Lactobacillus helveticus LH 92 (a highly peptidolytic strain used as an adjunct in cheese making) was evaluated in mixed cultures in milk. Acid production by mixed cultures depended on the sensitivity of L. helveticus LH 92 to the different bacteriocins and on the acidification rates of bacteriocin-producing strains. Proteolysis values of mixed cultures were, in all cases, lower than those of L. helveticus LH 92 single culture (control). Cell-free aminopeptidase activity values after 9 h of incubation did not increase in the presence of enterocin producers or the nisin A producer, whereas in the presence of the nisin Z producer, cell-free aminopeptidase activity was, at most, 3.7-fold greater than the control value. In mixed cultures with the plantaricin producer, a progressive lysis of L. helveticus LH 92 took place, with cell-free aminopeptidase activity values after 9 h being, at most, 10.5-fold greater than the control value. The highest cell-free aminopeptidase activity values after 9 h were recorded in the presence of lacticin 481 producers, with the values being, at most, 25.1-fold greater than the control value. L. helveticus LH 92 was extremely sensitive to small variations in the concentration of the inoculum of the nisin Z plus lacticin 481 producer, with there being a narrow optimum for the release of intracellular aminopeptidases. Plantaricin and lacticin 481 producers seemed the most promising strains to be combined with L. helveticus LH 92 as lactic cultures for cheese manufacture, because of the accelerated release of intracellular aminopeptidases. Copyright ©, International Association for Food Protection

Fast induction of nisin resistance in Streptococcus thermophilus INIA 463 during growth in milk

Streptococcus thermophilus INIA 463 became nisin-resistant after exposure in skim milk to subminimal inhibitory concentrations of nisin (1-3 IU/ml) for less than 2 h. Addition of 20 IU/ml caused a 4 log unit decrease in S. thermophilus counts of a culture not exposed previously to nisin, whereas no decrease was observed in the culture exposed to nisin for 2 h. Transfer of immunity genes as responsible for nisin resistance was discarded. The presence of extracellular or intracellular specific nisin-degrading enzymes was not detected in the nisin-resistant variant of S. thermophilus INIA 463. Nisin resistance was caused by the induction of a resistance mechanism. Transmission electron microscopy (TEM) revealed that the nisin-resistant variant of S. thermophilus INIA 463 had a thickened cell wall compared to the wild strain. Resistance to nisin was lost after one transfer (4 h growth) in nisin-free skim milk. © 2004 Elsevier B.V. All rights reserved

Susceptibility of Clostridium perfringens to antimicrobials produced by lactic acid bacteria Reuterin and nisin

The effectiveness as antimicrobials of lactic acid bacteria produced compounds reuterin and nisin was assessed against vegetative cells and spores of Clostridium perfringens isolates (from ovine milk obtained in farms with diarrheic lambs) and C.perfringens CECT 486 (type A toxin producer). We also tested the inhibitory effect of lysozyme and sodium nitrite on Clostridium. Minimal inhibitory concentrations (MIC) of antimicrobials were determined in modified RCM (mRCM) and milk by using a broth microdilution method, after 7d at 37°C under anaerobic conditions. The sensitivity of C.perfringens to the tested antimicrobials was strain and culture medium-dependent. In general, vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 2.03-16.25mM) inhibited the growth of vegetative cells and the outgrowth of spores of all tested C.perfringens, both in mRCM and milk, with higher resistance in milk. Nisin (MIC values 0.78-12.5μg/ml) was also effective against vegetative cells and spores of tested C.perfringens in both culture media. However, lysozyme (up to 400μg/ml) did not control the growth of any of the tested Clostridium. Sodium nitrite only inhibited the outgrowth of spores of two C.perfringens isolates at the maximum concentration assayed (300μg/ml) exclusively in mRCM medium. These results suggest that reuterin and nisin have the potential to control the growth of C.perfringens, and might help to ensure safety at different stages of the food chain. Future studies in food/feed products would be necessary to further corroborate this hypothesis. © 2014 Elsevier Ltd

Influence of a bacteriocin-producing lactic culture on the volatile compounds, odour and aroma of Hispánico cheese

The effect of milk inoculation with a bacteriocin-producing lactic culture on Hispánico cheese volatile compounds, odour and aroma was investigated in duplicate experiments, each consisting of two 50-L vats. Milk for experimental cheese was inoculated with 0.5% Lactococcus lactis subsp. lactis INIA 415, a bacteriocin-producing (Bac+) strain harbouring the structural genes of nisin Z and lacticin 481, 0.5% L. lactis subsp. lactis INIA 415-2, a Bac- mutant, and 2% TA052, a commercial Streptococcus thermophilus culture. Lactic cultures for control cheese were 1% L. lactis subsp. lactis INIA 415-2 and 2% TA052. Milk inoculation with bacteriocin-producing culture enhanced the formation of hexanal, 2-methyl-1-propanol, 3-methyl-1-butanol, acetone, 2-pentanone, 2-hexanone and 2-heptanone, but decreased the formation of acetaldehyde, ethanol, 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, ethyl acetate, ethyl butanoate, ethyl hexanoate, 2-butanone, 2,3-butanedione, 2,3-pentanedione and 3-hydroxy-2-butanone. Experimental cheese received higher scores than control cheese for the odour descriptor "clean cheese" and for the aroma descriptor "sheepy", but quality and intensity of odour and aroma were not significantly influenced by milk inoculation with the bacteriocin-producing culture. © 2004 Elsevier Ltd. All rights reserved

Characteristics of Manchego cheese manufactured from raw ewes' milk inoculated with a bacteriocin-producing adjunct culture

The effect of Lactococcus lactis subsp. lactis INIA 415, a nisin Z and lacticin 481 producer, on the characteristics of raw milk Manchego cheese has been studied. Experimental cheese was made from milk inoculated with the bacteriocin producer, and control cheese from milk without it. Bacteriocin-nonproducing mutant L. lactis subsp. lactis INIA 415-2 and a bacteriocin-sensitive Streptococcus thermophilus culture were added to both vats. Cheeses were ripened at 12°C and 85% RH for 90 days. Lysis of S. thermophilus was not enhanced by the bacteriocin, and aminopeptidase activity was lower in experimental cheese than in control cheese. Proteolysis values (estimated by the o-phthaldialdehyde method) of experimental and control cheeses did not differ, and textural characteristics (fracturability, elasticity and hardness) were not influenced either by the bacteriocin producer. Odour, aroma and taste of experimental cheese received higher quality and intensity scores than those of control cheese on day 30, but differences gradually disappeared afterwards

Inhibitory activity of reuterin, nisin, lysozyme and nitrite against vegetative cells and spores of dairy-related Clostridium species

The butyric acid fermentation, responsible for late blowing of cheese, is caused by the outgrowth in cheese of some species of Clostridium, resulting in texture and flavor defects and economical losses. The aim of this study was to evaluate the effectiveness of different antimicrobial compounds against vegetative cells and spores of C. tyrobutyricum, C. butyricum, C. beijerinckii and C. sporogenes strains isolated from cheeses with late blowing defect. Minimal inhibitory concentration (MIC) for reuterin, nisin, lysozyme and sodium nitrite were determined against Clostridium strains in milk and modified RCM (mRCM) after 7. d exposure. Although the sensitivity of Clostridium to the tested antimicrobials was strain-dependent, C. sporogenes and C. beijerinckii generally had higher MIC values than the rest of Clostridium species. The majority of Clostridium strains were more resistant to antimicrobials in milk than in mRCM, and vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 0.51-32.5. mM) and nisin (MIC values 0.05-12.5. μg/ml) were able to inhibit the growth of vegetative cells and spores of all assayed Clostridium strains in milk and mRCM. Strains of C. tyrobutyricum exhibited the highest sensitivity to lysozyme (MIC values. <. 0.20-400. μg/ml) and sodium nitrite (MIC values 18.75-150. μg/ml). These results suggest that reuterin and nisin, with a broad inhibitory activity spectrum against Clostridium spp. spores and vegetative cells, may be the best options to control Clostridium growth in dairy products and to prevent associated spoilage, such as late blowing defect of cheese. However, further studies in cheese would be necessary to validate this hypothesis. © 2013 Elsevier B.V