Relevance of visco-plastic theory in a multi-directional inhomogeneous granular flow

We confront a recent visco-plastic description of dense granular flows [P. Jop et al, Nature, {\bf 441} (2006) 727] with multi-directional inhomogeneous steady flows observed in non-smooth contact dynamics simulations of 2D half-filled rotating drums. Special attention is paid to check separately the two underlying fundamental statements into which the considered theory can be recast, namely (i) a single relation between the invariants of stress and strain rate tensors and (ii) the alignment between these tensors. Interestingly, the first prediction is fairly well verified over more than four decades of small strain rate, from the surface rapid flow to the quasi-static creep phase, where it is usually believed to fail because of jamming. On the other hand, the alignment between stress and strain rate tensors is shown to fail over the whole flow, what yields an apparent violation of the visco-plastic rheology when applied without care. In the quasi-static phase, the particularly large misalignment is conjectured to be related to transient dilatancy effects

Numerical simulation of 2D steady granular flows in rotating drum: On surface flows rheology

13 pages, 14 figures, 61 references, submitted to Phys. FluidsThe rheology of 2D steady surface flow of cohesionless cylinders in a rotating drum is investigated through {\em Non Smooth Contact Dynamics} simulations. Profile of volume fraction, translational and angular velocity, rms velocity, strain rate and stress tensor were measured at the midpoint along the length of the surface flowing layer where the flow is generally considered as steady and homogeneous. Analysis of these data and their inter-relations suggest the local inertial number - defined as the ratio between local inertial forces and local confinement forces - to be the relevant dimensionless parameter to describe the transition from the quasi-static part of the packing to the flowing part at the surface of the heap. Variations of the components of the stress tensor as well as the ones of rms velocity as a function of the inertial number are analysed within both the quasi-static and the flowing phases. Their implications are discussed

Yeasts and wine off-flavours: a technological perspective

Review article. Part of the special issue "Wine microbiology and safety: from the vineyard to the bottle (Microsafety Wine)", 19-20 Nov. 2009, ItalyIn wine production, yeasts have both beneficial and detrimental activities. Saccharomyces cerevisiae is the yeast mainly responsible for turning grape juice into wine but this species and several others may also show undesirable effects in wines. Among such effects, technologists are particularly concerned with the production of offflavours that may occur during all stages of winemaking. Typical spoiling activities include the production of ethyl acetate by apiculate yeasts before fermentation, hydrogen sulphide by S. cerevisiae during fermentation phases, acetaldehyde by film-forming yeasts during bulk storage, and volatile phenols by Dekkera bruxellensis during storage or after bottling. The occurrence of these hazards depends on the technological operations designed to obtain a given type of wine and most can be avoided by current preventive or curative measures. On the contrary, good manufacturing practices must be strengthened to deal with the problem of volatile phenol production in red wines. Appropriate monitoring of D. bruxellensis populations and quantification of 4-ethylphenol is advised during storage, particularly when oak barrels are used, and absence of viable cells must be guaranteed in bottled wines. This work, which is based on our experience at winery level, aims to provide information on appropriate technological strategies to deal with the problem of off-flavours produced by yeasts

Oenological characterisation of indigenous strains of S. cerevisiae isolated in a biodynamic winery in the Cortona DOC area

Genotypic and technological characterisation of the S. cerevisiae population isolated in a biodynamic winery in the Cortona DOC area was performed to gain better knowledge of the variables that influence winemaking. The oenological performance of 11 S. cerevisiae strains was evaluated with physiological tests; strain typing was performed through analysis of interdelta sequences and 26S rDNA sequencing. The analysis revealed a remarkable variability in terms of S. cerevisiae strains, despite the homogeneity of wine features, underlining the high levels of biodiversity characterising biodynamic agriculture. Some strains were found in wines of different vintages, suggesting the presence of an established microbiota in the winery. Oenological tests demonstrated that while some yeasts provided reliable oenological performance, other strains were not able to accomplish prompt and effective alcoholic fermentation, or were characterised by spoilage characteristics, such as excessive production of volatile phenols or acetic acid. Indigenous strains of S. cerevisiae could be a useful instrument for reliable winemaking without altering the native microbiota of each oenological environment. However, characterisation of their oenological suitability, and the application of practices able to drive the evolution of microbiota, must be employed to reduce the risk of wine spoilage

High performance liquid chromatography tandem mass spectrometry dual extraction method for identification of green tea catechin metabolites excreted in human urine

The simultaneous analysis of free-form and conjugated flavonoids in the same sample is difficult but necessary to properly estimate their bioavailability. A method was developed to optimise the extraction of both free and conjugated forms of catechins and metabolites in a biological sample following the consumption of green tea. A double-blind randomised controlled trial was performed in which 26 volunteers consumed daily green tea and vitamin C supplements and 24 consumed a placebo for 3 months. Urine was collected for 24h at 4 separate time points (pre- and post-consumption) to confirm compliance to the supplementation and to distinguish between placebo and supplementation consumption. The urine was assessed for both free and conjugated metabolites of green tea using LC-MS2 analysis, after a combination extraction method, which involved an ethyl acetate extraction followed by an acetonitrile protein precipitation. The combination method resulted in a good recovery of EC-O-sulphate (91±7%), EGC-O-glucuronide (94±6%), EC (95±6%), EGC (111±5%) and ethyl gallate (74±3%). A potential total of 55 catechin metabolites were investigated, and of these, 26 conjugated (with methyl, glucuronide or sulphate groups) and 3 free-form (unconjugated) compounds were identified in urine following green tea consumption. The majority of EC and EGC conjugates significantly increased post-consumption of green tea in comparison to baseline (pre-supplementation) samples. The conjugated metabolites associated with the highest peak areas were O-methyl-EC-O-sulphate and the valerolactones M6/M6'-O-sulphate. In line with previous studies, EC and EGC were only identified as conjugated derivatives, and EGCG and ECG were not found as mono-conjugated or free-forms. In summary, the method reported here provides a good recovery of catechin compounds and is appropriate for use in the assessment of flavonoid bioavailability, particularly for biological tissues that may contain endogenous deconjugating enzymes

Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot

Aims: To identify ascomycetous yeasts recovered from sound and damaged grapes by the presence of honeydew or sour rot. Methods and Results: In sound grapes, the mean yeast counts ranged from 3.20 ± 1.04 log CFU g-1 to 5.87 ± 0.64 log CFU g-1. In honeydew grapes, the mean counts ranged from 3.88 ± 0.80 log CFU g-1 to 6.64 ± 0.77 log CFU g-1. In sour rot grapes counts varied between 6.34 ± 1.03 and 7.68 ± 0.38 log CFU g-1. Hanseniaspora uvarum was the most frequent species from sound samples. In both types of damage, the most frequent species were Candida vanderwaltii, H. uvarum and Zygoascus hellenicus. The latter species was recovered in high frequency because of the utilization of the selective medium DBDM (Dekkera ⁄ Brettanomyces differential medium). The scarce isolation frequency of the wine spoilage species Zygosaccharomyces bailii (in sour rotten grapes) and Zygosaccharomyces bisporus (in honeydew affected grapes) could only be demonstrated by the use of the selective medium ZDM (Zygosaccharomyces differential medium). Conclusions: The isolation of several species only from damaged grapes indicates that damage constituted the main factor determining yeast diversity. The utilization of selective media is required for eliciting the recovery of potentially wine spoilage species. Significance and Impact of the Study: The impact of damaged grapes in the yeast ecology of grapes has been underestimate

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer

Imatinib in combination with phosphoinositol kinase inhibitor buparlisib in patients with gastrointestinal stromal tumour who failed prior therapy with imatinib and sunitinib: a Phase 1b, multicentre study

Background The majority of patients with advanced gastrointestinal stromal tumours (GISTs) develop resistance to imatinib and sunitinib, the standard of care for these patients. This study evaluated the combination of buparlisib, an oral phosphoinositide 3-kinase (PI3K) inhibitor, with imatinib in patients with advanced GIST, who have failed prior therapy with imatinib and sunitinib. Methods This Phase 1b, multicentre, open-label study aimed to determine the maximum tolerated dose (MTD) and/or a recommended Phase 2 dose of buparlisib in combination with 400 mg of imatinib through a dose-escalation part and a dose-expansion part, and also evaluated the clinical profile of the combination. Results Sixty patients were enrolled, including 25 in the dose-escalation part and 35 in the dose-expansion part. In the combination, MTD of buparlisib was established as 80 mg. No partial or complete responses were observed. The estimated median progression-free survival was 3.5 months in the expansion phase. Overall, 98.3% of patients had treatment-related adverse events (AEs), including 45% with grade 3 or 4 AEs. Conclusions Buparlisib in combination with imatinib provided no additional benefit compared with currently available therapies. Due to the lack of objective responses, further development of this combination was not pursued for third-line/fourth-line advanced/metastatic GIST.Experimentele farmacotherapi

Next-Generation Sequencing Reveals Significant Bacterial Diversity of Botrytized Wine

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies