Imaging of alkaline phosphatase activity in bone tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.     

A weak C' box renders U3 snoRNA levels dependent on hU3-55K binding

The rate of ribosome biogenesis, which is downregulated in terminally differentiated cells and upregulated in most cancers, regulates the growth rate and is linked to the cell's proliferative potential. The U3 box C/D small nucleolar RNP (snoRNP) is an integral component of the small subunit (SSU) processome and is essential for 18S rRNA processing. We show that U3 snoRNP assembly, and therefore U3 snoRNA accumulation, is regulated through the U3-specific protein hU3-55K. Furthermore, we report that the levels of several SSU processome components, including the U3 snoRNA but not other box C/D snoRNAs, are specifically downregulated during human lung (CaCo-2) and colon (CaLu-3) epithelial cell differentiation. c-Myc is reported to play an integral role in regulating ribosome production by controlling the expression of many ribosome biogenesis factors. Our data, however, indicate that this regulation is not dependent on c-Myc since the level of this protein does not change during epithelial cell differentiation. In addition, depletion of c-Myc had only a mild affect on the levels of SSU processome proteins. CaCo-2 cells are colon adenocarcinoma epithelial cells that are believed to revert to their precancerous state during differentiation. This suggests a significant increase in the levels of specific SSU processome components during tumorogenesis.     

Otopathogens Detected in Middle Ear Fluid Obtained during Tympanostomy Tube Insertion: Contrasting Purulent and Non-Purulent Effusions

Otitis media is a prominent disease among children. Previous literature indicates that otitis media is a polymicrobial disease, with Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis and Moraxella catarrhalis being the most commonly associated bacterial pathogens. Recent literature suggests that introduction of pneumococcal conjugate vaccines has had an effect on the etiology of otitis media. Using a multiplex PCR procedure, we sought to investigate the presence of the aforementioned bacterial pathogens in middle ear fluid collected from children undergoing routine tympanostomy tube placement at Wake Forest Baptist Medical Center during the period between January 2011 and March 2014. In purulent effusions, one or more bacterial organisms were detected in ~90% of samples. Most often the presence of H. influenzae alone was detected in purulent effusions (32%; 10 of 31). In non-purulent effusions, the most prevalent organism detected was A. otitidis (26%; 63 of 245). Half of the non-purulent effusions had none of these otopathogens detected. In purulent and non-purulent effusions, the overall presence of S. pneumoniae was lower (19%; 6 of 31, and 4%; 9 of 245, respectively) than that of the other pathogens being identified. The ratio of the percentage of each otopathogen identified in purulent vs. non-purulent effusions was >1 for the classic otopathogens but not for A. otitidis.     

Analysis of the distribution and evolution of the ATP‐dependent DNA ligases of bacteria delineates a distinct phylogenetic group ‘Lig E’

Prior to the discovery of a minimal ATP‐dependent DNA ligase in Haemophilus influenzae, bacteria were thought to only possess a NAD‐dependent ligase, which was involved in sealing of Okazaki fragments. We now know that a diverse range of bacterial species possess up to six of these accessory bacterial ATP‐dependent DNA ligases (b‐ADLs), which vary in size and enzymatic domain associations. Here we compare the domain structure of different types of b‐ADLs and investigate their distribution among the bacterial domain to describe possible evolutionary trajectories that gave rise to the sequence and structural diversity of these enzymes. Previous biochemical and genetic analyses have delineated three main classes of these enzymes: Lig B, Lig C and Lig D, which appear to have descended from a common ancestor within the bacterial domain. In the present study, we delineate a fourth group of b‐ADLs, Lig E, which possesses a number of unique features at the primary and tertiary structural levels. The biochemical characteristics, domain structure and inferred extracellular location sets this group apart from the other b‐ADLs. The results presented here indicate that the Lig E type ligases were horizontally transferred into bacteria in a separate event from other b‐ADLs possibly from a bacteriophage.     

Life tables for Down syndrome

Summary Life expectancy in Down syndrome was calculated to 68 years, using data for 1610 Down syndrome liveborn individuals among over 1.5 million consecutive British Columbia livebirths. Overall, although survival is significantly poorer than for the general population, over half of Down syndrome individuals can be expected to survive into their fifties, and 13.5% will still be alive at age 68. The data are presented as a life table, a practical format for the clinician and planner.     

Metal contamination in sediments and biota of the Bay of Quinte,Lake Ontario,Canada

The Bay of Quinte receives drainage from several large river systems, including the Moira River which carried sediment from mines into the Bay from the 1880s to the 1960s. We are investigating possible metal contamination of submerged weed beds and marsh biota which may contribute to the low diversity and biomass of macrophyte beds and Typha marshes in the Bay. In 1987, sediment, macrophytes and snails were sampled in wetlands close to the Moira River and at Hay Bay (part of the Bay of Quinte presumably unaffected by mine effluents) located 20 km from the Moira. Some element concentrations in sediment and biota were determined by neutron activation analysis (NAA) including Al, As, Br, Ca, Co, Cl, Cr, Cs, Fe, Hf, K, La, Na, Mg, Sb, Sc, Rb, Ta, Th, Ti, U, V and Zn. Other elements were analysed by acid dissolution and atomic absorption spectrophotometry (AAS) including Ag, As, Cu, Hg, Ni, Pb, and Zn. Levels of As in sediments and plants were higher close to the Moira River, whereas Cu and Ni showed the opposite pattern in sediments. The usefulness of species as bioassays differed: Stagnicola elodes Say accumulated significantly higher levels of Cu (35 vs 18 ppm) and V (l. l vs 0.5 ppm) than Planorbella trivolvis Say collected from the same sites. The macrophyte, Myriophyllum spicatum L. acted as an accumulator of Pb (up to 9.6 ppm), whereas Pb in Vallisneria americana Michx. at the same sites was undetectable.     

A Clinical Evaluation of Four Hypnotic Agents,Using a Latin-Square Design

A double-blind study with a Latin-square design was undertaken on 25 elderly patients, using a placebo and four hypnotic drugs: ethchlorvynol 500 mg., glutethimide 500 mg., chloral hydrate 500 mg., and secobarbital sodium 100 mg. The trial lasted for five weeks. The drugs were all effective compared with the placebo, differences in sleeping time being statistically significant. Differences between these four drugs were not statistically significant. Sleep was induced soonest by secobarbital and ethchlorvynol. Ethchlorvynol and glutethimide had a relatively somewhat longer period of activity than the others. Glutethimide produced most side effects, especially morning drowsiness. Ethchlorvynol and chloral hydrate produced relatively few cases of drowsiness.