Evaluation of the colorimetric tetrazolium assay for the cytotoxicity testing a commercial vanilla flavouring

The tetrazolium colorimetric assay (MTT-assay) has been used to evaluate the cytotoxicity of a vanilla flavouring found on the market. It was characterised by the use of 1,2-propylene glycol and glycerol as diluents and by the presence of vanillin, heliotropine and the corresponding 1,2-propylene and glycerol acetals. The method developed by Mosmann (J. Immunol. Methods 65, 55\u201363-1983) was used with some modifications. The same assay was applied to the diluent 1,2-propylene glycol in order to verify its possible influence on the toxic effect. This technique proved useful for preliminary screening useful to evidence doubts relating to the safety of flavouring preparations

GC/MS-MID Determination of Safrole in Soft Drinks

Safrole [1,3-benzodioxole, 5-(2-propenyl)-] is a limited substance according to the CE Directives N. 88/388 and 91/71 concerning \u201cflavourings for use in foodstuffs and to source materials for their production\u201d. At today the GC analytical methods proposed for the quantitative determination of safrole are critical as far as concerning reproducibility, recovery values and detection limits. This note describes an improved analytical method to quantify safrole in \u201csoft\u201d drinks. Average recoveries of safrole from samples spiked at levels from 30.0 to 80.0 microg/L ranged from 80 to 93% with good reproducibility (RSD=2.7 at 30.0 microg/Ll). Limits of detection (LOD) and quantification (LOQ) were 10.0 microg/L and 30.0 microg/L respectively. The proposed method is also suitable for routine analysis

Directed Evolution of Protein-Based Neurotransmitter Sensors for MRI

The production of contrast agents sensitive to neuronal signaling events is a rate-limiting step in the development of molecular-level functional magnetic resonance imaging (molecular fMRI) approaches for studying the brain. High-throughput generation and evaluation of potential probes are possible using techniques for macromolecular engineering of protein-based contrast agents. In an initial exploration of this strategy, we used the method of directed evolution to identify mutants of a bacterial heme protein that allowed detection of the neurotransmitter dopamine in vitro and in living animals. The directed evolution method involves successive cycles of mutagenesis and screening that could be generalized to produce contrast agents sensitive to a variety of molecular targets in the nervous system

Novel prefabricated panels for the repair of damaged interior RC beam-column joints

A novel prefabricated panel is introduced for the repair and strengthening of damaged interior RC beam-column joints. This prefabricated hybrid composite plate (HCP) is made of a Strain Hardening Cementitious Composite (SHCC) reinforced with CFRP materials. Besides the higher durability of this system when compared to EB-FRP, thanks to the high ductility of the SHCC, anchors can be used in combination with adhesive to attach the HCP to the concrete substrate. The first section of this paper reports the methodologies to apply HCP for the repair of two severely damaged interior RC beam-column systems. To evaluate the performance of this rehabilitation technique, the repaired specimens were subjected to the same loading pattern as it was imposed to their virgin states. Since both repaired specimens showed a higher lateral load and energy dissipation capacities than the corresponding values in their undamaged state, the effectiveness of the HCP for the repair of damaged interior beam-column joints is proved.Fundação para a Ciência e a Tecnologia (FCT

Is Corporate Social Responsibility an Agency Problem? Evidence from CEO Turnovers

We empirically examine two competing claims: first, if a firm’s Corporate Social Responsibility (CSR) activity is driven by its CEO’s private rent extraction (i.e. an agency problem), firms with higher CSR ratings are poorly governed and their managers are less likely to be dismissed for poor financial performance. In contrast, if CSR reflects owners’ preferences, CEOs of firms with higher CSR ratings are more likely to be removed in light of poor financial performance. We find that CEO turnover-financial performance sensitivity increases in firm CSR scores during the last years of both the outgoing CEO as well as his predecessor. Further, firm CSR ratings do not change following CEO turnover suggesting that CSR ratings are a firm characteristic. Our findings are consistent with the view that CSR is driven by shareholder preferences

Clonal interference of signaling mutations worsens prognosis in core-binding factor acute myeloid leukemia

Mutations in receptor tyrosine kinase/RAS signaling pathway genes are frequent in core-binding factor (CBF) acute myeloid leukemias (AMLs), but their prognostic relevance is debated. A subset of CBF AML patients harbors several signaling gene mutations. Genotyping of colonies and of relapse samples indicates that these arise in independent clones, thus defining a process of clonal interference (or parallel evolution). Clonal interference is pervasive in cancers, but the mechanisms underlying this process remain unclear, and its prognostic impact remains unknown. We analyzed a cohort of 445 adult and pediatric patients with CBF AML treated with intensive chemotherapy and with deep sequencing of 6 signaling genes (, , , , , ). A total of 152 (34%), 167 (38%), and 126 (28%) patients harbored no, a single, and multiple signaling clones (clonal interference), respectively. Clonal interference of signaling mutations was associated with older age ( = .004) and inv(16) subtype ( = .025) but not with white blood cell count or mutations in chromatin or cohesin genes. The median allele frequency of signaling mutations was 31% in patients with a single clone or clonal interference ( = .14). The repertoire of , , and / variants differed between groups. Clonal interference did not affect complete remission rate or minimal residual disease after 1-2 courses, but it did convey inferior event-free survival ( < 10), whereas the presence of a single signaling clone did not ( = .44). This inferior outcome was independent of clinical parameters and of the presence of specific signaling clones. Our results suggest that specific clonal architectures can herald distinct prognoses in AML

Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine

The development of molecular probes that allow in vivo imaging of neural signaling processes with high temporal and spatial resolution remains challenging. Here we applied directed evolution techniques to create magnetic resonance imaging (MRI) contrast agents sensitive to the neurotransmitter dopamine. The sensors were derived from the heme domain of the bacterial cytochrome P450-BM3 (BM3h). Ligand binding to a site near BM3h's paramagnetic heme iron led to a drop in MRI signal enhancement and a shift in optical absorbance. Using an absorbance-based screen, we evolved the specificity of BM3h away from its natural ligand and toward dopamine, producing sensors with dissociation constants for dopamine of 3.3–8.9 μM. These molecules were used to image depolarization-triggered neurotransmitter release from PC12 cells and in the brains of live animals. Our results demonstrate the feasibility of molecular-level functional MRI using neural activity–dependent sensors, and our protein engineering approach can be generalized to create probes for other targets.Charles A. Dana Foundation. Brain and Immuno-ImagingRaymond and Beverley Sackler FoundationNational Institutes of Health (U.S.) (grant R01-DA28299)National Institutes of Health (U.S.) (grant DP2-OD2441)National Institutes of Health (U.S.) (grant R01-GM068664)Jacobs Institute for Molecular Engineering for Medicine. Jacobs Institute for Molecular Engineering for MedicineNational Institutes of Health (U.S.) (grant R01-DE013023

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by 13C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively

Scoring System Prognostic of Outcome in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation for Myelodysplastic Syndrome

To develop a system prognostic of outcome in those undergoing allogeneic hematopoietic cell transplantation (allo HCT) for myelodysplastic syndrome (MDS)