LuxCDABE—Transformed Constitutively Bioluminescent Escherichia coli for Toxicity Screening: Comparison with Naturally Luminous Vibrio fischeri

We show that in vitro toxicity assay based on inhibition of the bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Photorhabdus luminescens is a versatile alternative to Vibrio fischeri Microtox™ test. Performance of two luxCDABE-transformed E. coli MC1061 constructs (pDNlux) and (pSLlux) otherwise identical, but having 100-fold different background luminescence was compared with the performance of V. fischeri. The microplate luminometer and a kinetic Flash-Assay test format was used that differently from Microtox test is also applicable for high throughput analysis. Toxic effects (30-s till 30-min EC50) of four heavy metals (Zn, Cd, Hg, Cu) and three organic chemicals (aniline, 3,5-dichloroaniline and 3,5-dichlorophenol) were studied. Both E. coli strains had comparable sensitivity and the respective 30-min EC50 values highly correlated (log-log R2 = 0.99; p < 0.01) showing that the sensitivity of the recombinant bacteria towards chemicals analyzed did not depend on the bioluminescence level of the recombinant cells. The most toxic chemical for all used bacterial strains (E. coli, V. fischeri) was mercury whereas the lowest EC50 values for Hg (0.04–0.05 mg/L) and highest EC50 values for aniline (1,300–1,700 mg/L) were observed for E. coli strains. Despite of that, toxicity results obtained with both E. coli strains (pSLlux and pDNlux) significantly correlated with V. fischeri results (log-log R2 = 0.70/0.75; p < 0.05/0.01). The use of amino acids (0.25%) and glucose (0.05%)-supplemented M9 medium instead of leucine-supplemented saline significantly (p < 0.05) reduced the apparent toxicity of heavy metals to both E. coli strains up to three orders of magnitude, but had little or no complexing effect on organic compounds. Thus, P. luminescens luxCDABE-transformed E. coli strains can be successfully used for the acute toxicity screening of various types of organic chemicals and heavy metals and can replace V. fischeri in certain cases where the thermostability of luciferase >30 °C is crucial. The kinetic Flash Assay test format of the bioluminescence inhibition assay facilitates high throughput analysis. The assay medium, especially in case of testing heavy metals should be a compromise: optimal for the viability/luminescence of the recombinant test strain and of minimum complexing potential

Regulation of interleukin-1Β–induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts

Objective To evaluate the efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin-1Β (IL-1Β)–induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP-1/CCL2), epithelial neutrophil–activating peptide 78 (ENA-78/CXCL5), growth-regulated oncogene Α (GROΑ/CXCL1), and matrix metalloproteinase 2 (MMP-2) activity in rheumatoid arthritis (RA) synovial fibroblasts. Methods Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP-1, ENA-78, and GROΑ produced in culture supernatants were measured by enzyme-linked immunosorbent assay. MMP-2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF-ΚB. Results EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 Μ M or 20 Μ M significantly inhibited IL-1Β–induced ENA-78, RANTES, and GROΑ, but not MCP-1 production in a concentration-dependent manner. EGCG at 50 Μ M caused a complete block of IL-1Β–induced production of RANTES, ENA-78, and GROΑ, and reduced production of MCP-1 by 48% ( P < 0.05). Zymography showed that EGCG blocked constitutive, IL-1Β–induced, and chemokine-mediated MMP-2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCΔ and inhibited the activation and nuclear translocation of NF-ΚB in IL-1Β–treated RA synovial fibroblasts. Conclusion These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55807/1/22023_ftp.pd

SHARK-NIR, the coronagraphic camera for LBT, moving toward construction

SHARK-NIR is one of the two coronagraphic instruments proposed for the Large Binocular Telescope. Together with SHARK-VIS (performing coronagraphic imaging in the visible domain), it will offer the possibility to do binocular observations combining direct imaging, coronagraphic imaging and coronagraphic low resolution spectroscopy in a wide wavelength domain, going from 0.5{\mu}m to 1.7{\mu}m. Additionally, the contemporary usage of LMIRCam, the coronagraphic LBTI NIR camera, working from K to L band, will extend even more the covered wavelength range. In January 2017 SHARK-NIR underwent a successful final design review, which endorsed the instrument for construction and future implementation at LBT. We report here the final design of the instrument, which foresees two intermediate pupil planes and three focal planes to accomodate a certain number of coronagraphic techniques, selected to maximize the instrument contrast at various distances from the star. Exo-Planets search and characterization has been the science case driving the instrument design, but the SOUL upgrade of the LBT AO will increase the instrument performance in the faint end regime, allowing to do galactic (jets and disks) and extra-galactic (AGN and QSO) science on a relatively wide sample of targets, normally not reachable in other similar facilities.Comment: 8 pages, 6 figures, AO4ELT5 conference proceeding

A Glycemia Risk Index (GRI) of Hypoglycemia and Hyperglycemia for Continuous Glucose Monitoring Validated by Clinician Ratings

BackgroundA composite metric for the quality of glycemia from continuous glucose monitor (CGM) tracings could be useful for assisting with basic clinical interpretation of CGM data.MethodsWe assembled a data set of 14-day CGM tracings from 225 insulin-treated adults with diabetes. Using a balanced incomplete block design, 330 clinicians who were highly experienced with CGM analysis and interpretation ranked the CGM tracings from best to worst quality of glycemia. We used principal component analysis and multiple regressions to develop a model to predict the clinician ranking based on seven standard metrics in an Ambulatory Glucose Profile: very low-glucose and low-glucose hypoglycemia; very high-glucose and high-glucose hyperglycemia; time in range; mean glucose; and coefficient of variation.ResultsThe analysis showed that clinician rankings depend on two components, one related to hypoglycemia that gives more weight to very low-glucose than to low-glucose and the other related to hyperglycemia that likewise gives greater weight to very high-glucose than to high-glucose. These two components should be calculated and displayed separately, but they can also be combined into a single Glycemia Risk Index (GRI) that corresponds closely to the clinician rankings of the overall quality of glycemia (r = 0.95). The GRI can be displayed graphically on a GRI Grid with the hypoglycemia component on the horizontal axis and the hyperglycemia component on the vertical axis. Diagonal lines divide the graph into five zones (quintiles) corresponding to the best (0th to 20th percentile) to worst (81st to 100th percentile) overall quality of glycemia. The GRI Grid enables users to track sequential changes within an individual over time and compare groups of individuals.ConclusionThe GRI is a single-number summary of the quality of glycemia. Its hypoglycemia and hyperglycemia components provide actionable scores and a graphical display (the GRI Grid) that can be used by clinicians and researchers to determine the glycemic effects of prescribed and investigational treatments

SHARK-NIR: from K-band to a key instrument, a status update

SHARK-NIR channel is one of the two coronagraphic instruments proposed for the Large Binocular Telescope, in the framework of the call for second generation instruments, issued in 2014. Together with the SHARK-VIS channel, it will offer a few observing modes (direct imaging, coronagraphic imaging and coronagraphic low resolution spectroscopy) covering a wide wavelength domain, going from 0.5μm to 1.7μm. Initially proposed as an instrument covering also the K-band, the current design foresees a camera working from Y to H bands, exploiting in this way the synergy with other LBT instruments such as LBTI, which is actually covering wavelengths greater than L' band, and it will be soon upgraded to work also in K band. SHARK-NIR has been undergoing the conceptual design review at the end of 2015 and it has been approved to proceed to the final design phase, receiving the green light for successive construction and installation at LBT. The current design is significantly more flexible than the previous one, having an additional intermediate pupil plane that will allow the usage of coronagraphic techniques very efficient in term of contrast and vicinity to the star, increasing the instrument coronagraphic performance. The latter is necessary to properly exploit the search of giant exo-planets, which is the main science case and the driver for the technical choices of SHARK-NIR. We also emphasize that the LBT AO SOUL upgrade will further improve the AO performance, making possible to extend the exo-planet search to target fainter than normally achieved by other 8-m class telescopes, and opening in this way to other very interesting scientific scenarios, such as the characterization of AGN and Quasars (normally too faint to be observed) and increasing considerably the sample of disks and jets to be studied. Finally, we emphasize that SHARK-NIR will offer XAO direct imaging capability on a FoV of about 15"x15", and a simple coronagraphic spectroscopic mode offering spectral resolution ranging from few hundreds to few thousands. This article presents the current instrument design, together with the milestones for its installation at LBT. <P /

Television, an Instrument for and a Mirror of Health and Health Services

This thematic issue of VIEW brings together articles that show how television has been an instrument for, as well as a mirror of, public service and specifically health services. Two approaches to this are featured and teased out. The first approach concerns health communication and campaigns, where information is diffused via television and strengthened or reinforced by visual and filmic means. The second concerns the structures that offer, manage and model norms of health and healthcare services. In introducing elements of the history of health, we hope to draw attention to the intersection of public health and television over the twentieth century, such that thinking about the relationship between them might change our understanding of both

Focus on the role of seed tannins and pectolytic enzymes in the color development of Pinot noir wine

Maceration techniques which promote the extraction of color pigments and tannin from grapes are often sought in Pinot noir winemaking to optimise color stability; alternatively, exogenous grape tannins may be included during fermentation. To examine the effect of seed-derived tannins and the use of pectolytic enzymes on color development in wines, conventional must preparations of Vitis vinifera L. cv Pinot noir grapes were compared with wines made using a supplementary addition of either a commercial seed tannin product or previously fermented seeds, while in a complementary experiment, seeds were sequentially removed during fermentation. After 6 months bottle aging, wines supplemented with either a commercial seed tannin solution (0.4 ​g/L), or fermented seeds (20% w/w seeds) had from 60% to 95% higher tannin concentration than the untreated wine, and up to 60% more monomeric anthocyanins. Conversely, when a third of the seeds were removed from the fermenting wine, the concentration of both tannin and non-bleachable pigments was 20–30% lower than in untreated wines and the wine hue had more red-purple tones. Exploration of the use of pectolytic enzymes in conjunction with seed removal was also found to have a significant impact on wine color parameters. Further insights on the timing of egress of tannin precursors from seeds was obtained from histochemical examination of the seeds that had been removed during alcoholic fermentation