Multispectral fingerprinting for improved in vivo cell dynamics analysis.

BACKGROUND: Tracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC) as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks. RESULTS: We found that multicolor nuclear cell labeling and multispectral imaging led to improved resolution of in vivo NC cell identification by providing a unique spectral identity for each cell. NC cell spectral identity allowed for more accurate cell tracking and was consistent during short term time-lapse imaging sessions. Computer model simulations predicted significantly better object counting for increasing cell densities in 3-color compared to 1-color nuclear cell labeling. To better resolve cell contacts, we show that a combination of 2-color membrane and 1-color nuclear cell labeling dramatically improved the semi-automated analysis of NC cell interactions, yet preserved the ability to track cell movements. We also found channel versus lambda scanning of multicolor labeled embryos significantly reduced the time and effort of image acquisition and analysis of large 3D volume data sets. CONCLUSIONS: Our results reveal that multicolor cell labeling and multispectral imaging provide a cellular fingerprint that may uniquely determine a cell's position within the embryo. Together, these methods offer a spectral toolbox to resolve in vivo cell dynamics in unprecedented detail.

Impact of lincosamides antibiotics on the composition of the rat gut microbiota and the metabolite profile of plasma and feces.

The importance of the gut microorganisms and their wide range of interactions with the host are well-acknowledged. In this study, lincomycin and clindamycin were used to modulate microbial communities of Wistar rats to gain a comprehensive understanding of the implications of microbiome alterations. A metabolomics approach and taxonomic profiling were applied to characterize the effects of these antibiotics on the functionality of the microbiome and to identify microbiome-related metabolites. After treatment, the diversity of the microbial community was drastically reduced. Bacteroidetes and Verrucomicrobia were drastically reduced, Tenericutes and Deferribacteres completely disappeared, while abundance of Firmicutes and Proteobacteria were highly increased. Changes in plasma and feces metabolites were observed for metabolites belonging mainly to the class of complex lipids, fatty acids and related metabolites as well as amino acids and related compounds. Bile acid metabolism was markedly affected: taurocholic acid, glycochenodeoxycholic acid and cholic acid presented abrupt changes showing a specific metabolite pattern indicating disruption of the microbial community. In both plasma and feces taurocholic acid was highly upregulated upon treatment whereas glycochenodeoxycholic acid was downregulated. Cholic acid was upregulated in feces but downregulated in plasma. These results show that changes in the gut microbial community lead to alterations of the metabolic profile in blood and feces of the host and can be used to identify potentially microbiome-related metabolites. This implies that metabolomics could be a suitable tool to estimate the extent of changes induced in the intestinal microbiome with respect to consequences for the host.

Clinical trials in the elderly. Should we do more?

Much of the information on which treatment decisions are based in elderly patients is derived from studies involving younger adults. The benefit to risk ratio of any given intervention may be quite different in frail older patients with significant comorbidities, and the applicability of such study findings to routine geriatric medical practice is therefore limited. The recruitment of significant numbers of elderly patients into trials is necessary to enable clinicians to make informed and rational therapeutic decisions in this expanding population.

Cupric oxide needles for grazing cattle consuming low-copper, high-molybdenum forage and high-sulfate water.

Oxidized copper wire, commonly referred to as copper oxide needles (CuON), was evaluated using purebred Hereford cows and their calves. Thirty-seven cows were allocated to Cu treatments of 0, 25 or 50 g CuON (79.9% Cu in CuON) with 12, 12 and 13 cows per treatment, respectively; calves within cow treatments were allocated to treatment of 0 and 20 g CuON. Single oral doses of CuON were given at the start of a grazing trial that lasted 92 d. Cows and calves were weighed and blood samples were taken on d 0, 28, 63 and 92; liver biopsies were taken on d 0, 28 and 92 of the grazing trial. Cattle were consuming grass forage with mean concentrations on d 0, 28, 63 and 92 of the grazing trial ranging from 1.6 to 5.5 mg/kg DM for Cu, 2.5 to 5.5 mg/kg DM for Mo and 1.3 to 1.5 g/kg DM for total S. The water consumed by cattle contained 947 mg sulfate per liter (SE = 13.2, n = 4). Body weight of cows and calves was not influenced (P greater than .05) by CuON. Liver Cu was higher (P less than .01) in treated cows and calves but was not different (P greater than .05) between cows dosed with 25 or 50 g CuON. Treatment of cows and calves with CuON had no influence (P greater than .05) on the concentration of Fe or Mo in liver or plasma, the concentration of Cu and ceruloplasmin activity in plasma, or the concentration of Zn in liver. Plasma Zn did not differ (P greater than .05) in cows, but it was higher (P less than .05) in the calves suckling cows treated with CuON. It was concluded that dosing cows and calves with CuON resulted in a higher Cu content of liver but did not adversely influence the metabolism of Fe or Zn or modify the concentration of Mo in the plasma or liver of cows or calves.

Recovery of polysome function of T4-infected Escherichia coli after brief treatment with chloramphenicol and rifampin.

T4-infected Escherichia coli cells briefly exposed to rifampin, or to rifampin plus chloramphenicol, were capable of protein synthesis for some time after removal of the antibiotics, although ribonucleic acid synthesis was irreversibly inhibited. Partially completed peptides trapped on polysomes by high levels of chloramphenicol were eventually completed after removal of the drug, as demonstrated by subjecting labeled peptides from appropriate polysome regions to polyacrylamide disc gel electrophoresis. Thus, the effect of the drug appears to be reversible on the molecular as well as the cellular level.

Effect of ribosomal wash factors on inhibition by chloramphenicol.

In vitro protein-synthesizing systems from Escherichia coli can be categorized as either chloramphenicol-sensitive or chloramphenicol-insensitive. The chloramphenicol-sensitive systems used in this study required the presence of factors removed from ribosomes with 1.0 m NH(4)Cl when chromatographically purified ribosomes were used for amino acid incorporation. These ribosomal wash factors inhibited but did not eliminate amino acid incorporation in chloramphenicol-insensitive systems. For both systems, addition of increasing amounts of the ribosomal wash factors increased the sensitivity to chloramphenicol inhibition.