Development of a Primary Human Cell Model for the Study of Human Cytomegalovirus Replication and Spread within Salivary Epithelium.

Various aspects of human cytomegalovirus (HCMV) pathogenesis, including its ability to replicate in specific cells and tissues and the mechanism(s) of horizontal transmission, are not well understood, predominantly because of the strict species specificity exhibited by HCMV. Murine CMV (MCMV), which contains numerous gene segments highly similar to those of HCMV, has been useful for modeling some aspects of CMV pathogenesis; however, it remains essential to build relevant human cell-based systems to investigate how the HCMV counterparts function. The salivary gland epithelium is a site of persistence for both human and murine cytomegaloviruses, and salivary secretions appear to play an important role in horizontal transmission. Therefore, it is important to understand how HCMV is replicating within the glandular epithelial cells so that it might be possible to therapeutically prevent transmission. In the present study, we describe the development of a salivary epithelial model derived from primary human "salispheres." Initial infection of these primary salivary cells with HCMV occurs in a manner similar to that reported for established epithelial lines, in that gH/gL/UL128/UL130/UL131A (pentamer)-positive strains can infect and replicate, while laboratory-adapted pentamer-null strains do not. However, while HCMV enters the lytic phase and produces virus in salivary epithelial cells, it fails to exhibit robust spread throughout the culture and persists in a low percentage of salivary cells. The present study demonstrates the utility of these primary tissue-derived cells for studying HCMV replication in salivary epithelial cells in vitroIMPORTANCE Human cytomegalovirus (HCMV) infects the majority of the world's population, and although it typically establishes a quiescent infection with little to no disease in most individuals, the virus is responsible for a variety of devastating sequelae in immunocompromised adults and in developing fetuses. Therefore, identifying the viral properties essential for replication, spread, and horizontal transmission is an important area of medical science. Our studies use novel human salivary gland-derived cellular models to investigate the molecular details by which HCMV replicates in salivary epithelial cells and provide insight into the mechanisms by which the virus persists in the salivary epithelium, where it gains access to fluids centrally important for horizontal transmission.

SAR11 lipid renovation in response to phosphate starvation.

Phytoplankton inhabiting oligotrophic ocean gyres actively reduce their phosphorus demand by replacing polar membrane phospholipids with those lacking phosphorus. Although the synthesis of nonphosphorus lipids is well documented in some heterotrophic bacterial lineages, phosphorus-free lipid synthesis in oligotrophic marine chemoheterotrophs has not been directly demonstrated, implying they are disadvantaged in phosphate-deplete ecosystems, relative to phytoplankton. Here, we show the SAR11 clade chemoheterotroph Pelagibacter sp. str. HTCC7211 renovates membrane lipids when phosphate starved by replacing a portion of its phospholipids with monoglucosyl- and glucuronosyl-diacylglycerols and by synthesizing new ornithine lipids. Lipid profiles of cells grown with excess phosphate consisted entirely of phospholipids. Conversely, up to 40% of the total lipids were converted to nonphosphorus lipids when cells were starved for phosphate, or when growing on methylphosphonate. Cells sequentially limited by phosphate and methylphosphonate transformed >75% of their lipids to phosphorus-free analogs. During phosphate starvation, a four-gene cluster was significantly up-regulated that likely encodes the enzymes responsible for lipid renovation. These genes were found in Pelagibacterales strains isolated from a phosphate-deficient ocean gyre, but not in other strains from coastal environments, suggesting alternate lipid synthesis is a specific adaptation to phosphate scarcity. Similar gene clusters are found in the genomes of other marine α-proteobacteria, implying lipid renovation is a common strategy used by heterotrophic cells to reduce their requirement for phosphorus in oligotrophic habitats.

Patient experiences with MedlinePlus.gov: a survey of internal medicine patients.

BACKGROUND: In 2004, the American College of Physicians joined with the National Library of Medicine in an effort to help patients find reliable health information free of commercial bias as the Web site MedlinePlus.gov. The aim of this study was to evaluate the usefulness of the MedlinePlus.gov site as perceived by patients who were referred to the site by their internist. MATERIALS AND METHODS: A 27-item questionnaire developed by the American College of Physicians Foundation was distributed between january and May 2005 to a convenience sample of 893 adult patient volunteers attending 34 internal medicine practices across the United States. RESULTS: The questionnaire revealed that although most patients (55%) routinely look up medical information, only 43% had used MedlinePlus.gov. Of those who had used the site, 95% were satisfied with the information they found there, and 94% said the information they found at MedlinePlus.gov would help them make better health decisions. DISCUSSION: Patients who used the MedlinePlus.gov site at the recommendation of their physician found it to use, informative, and felt it would help them make better health decisions. Directing patients to this high quality, noncommercial, educational resource online may be an important adjunct to patient education efforts by physicians.

Methane production by phosphate-starved SAR11 chemoheterotrophic marine bacteria.

The oxygenated surface waters of the world's oceans are supersaturated with methane relative to the atmosphere, a phenomenon termed the 'marine methane paradox'. The production of methylphosphonic acid (MPn) by marine archaea related to Nitrosopumilus maritimus and subsequent decomposition of MPn by phosphate-starved bacterioplankton may partially explain the excess methane in surface waters. Here we show that Pelagibacterales sp. strain HTCC7211, an isolate of the SAR11 clade of marine α-proteobacteria, produces methane from MPn, stoichiometric to phosphorus consumption, when starved for phosphate. Gene transcripts encoding phosphonate transport and hydrolysis proteins are upregulated under phosphate limitation, suggesting a genetic basis for the methanogenic phenotype. Strain HTCC7211 can also use 2-aminoethylphosphonate and assorted phosphate esters for phosphorus nutrition. Despite strain-specific differences in phosphorus utilization, these findings identify Pelagibacterales bacteria as a source of biogenic methane and further implicate phosphate starvation of chemoheterotrophic bacteria in the long-observed methane supersaturation in oxygenated waters.

Discovery of a SAR11 growth requirement for thiamin's pyrimidine precursor and its distribution in the Sargasso Sea.

Vitamin traffic, the production of organic growth factors by some microbial community members and their use by other taxa, is being scrutinized as a potential explanation for the variation and highly connected behavior observed in ocean plankton by community network analysis. Thiamin (vitamin B1), a cofactor in many essential biochemical reactions that modify carbon-carbon bonds of organic compounds, is distributed in complex patterns at subpicomolar concentrations in the marine surface layer (0-300 m). Sequenced genomes from organisms belonging to the abundant and ubiquitous SAR11 clade of marine chemoheterotrophic bacteria contain genes coding for a complete thiamin biosynthetic pathway, except for thiC, encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) synthase, which is required for de novo synthesis of thiamin's pyrimidine moiety. Here we demonstrate that the SAR11 isolate 'Candidatus Pelagibacter ubique', strain HTCC1062, is auxotrophic for the thiamin precursor HMP, and cannot use exogenous thiamin for growth. In culture, strain HTCC1062 required 0.7 zeptomoles per cell (ca. 400 HMP molecules per cell). Measurements of dissolved HMP in the Sargasso Sea surface layer showed that HMP ranged from undetectable (detection limit: 2.4 pM) to 35.7 pM, with maximum concentrations coincident with the deep chlorophyll maximum. In culture, some marine cyanobacteria, microalgae and bacteria exuded HMP, and in the Western Sargasso Sea, HMP profiles changed between the morning and evening, suggesting a dynamic biological flux from producers to consumers.