Microbial Composition of Near-Boiling Silica-Depositing Thermal Springs throughout Yellowstone National Park

The extent of hyperthermophilic microbial diversity associated with siliceous sinter (geyserite) was characterized in seven near-boiling silica-depositing springs throughout Yellowstone National Park using environmental PCR amplification of small-subunit rRNA genes (SSU rDNA), large-subunit rDNA, and the internal transcribed spacer (ITS). We found that Thermocrinis ruber, a member of the order Aquificales, is ubiquitous, an indication that primary production in these springs is driven by hydrogen oxidation. Several other lineages with no known close relatives were identified that branch among the hyperthermophilic bacteria. Although they all branch deep in the bacterial tree, the precise phylogenetic placement of many of these lineages is unresolved at this time. While some springs contained a fair amount of phylogenetic diversity, others did not. Within the same spring, communities in the subaqueous environment were not appreciably different than those in the splash zone at the edge of the pool, although a greater number of phylotypes was found along the pool's edge. Also, microbial community composition appeared to have little correlation with the type of sinter morphology. The number of cell morphotypes identified by fluorescence in situ hybridization and scanning electron microscopy was greater than the number of phylotypes in SSU clone libraries. Despite little variation in Thermocrinis ruber SSU sequences, abundant variation was found in the hypervariable ITS region. The distribution of ITS sequence types appeared to be correlated with distinct morphotypes of Thermocrinis ruber in different pools. Therefore, species- or subspecies-level divergences are present but not detectable in highly conserved SSU sequences.     

Anti‐aging drugs reduce hypothalamic inflammation in a sex‐specific manner

Aging leads to hypothalamic inflammation, but does so more slowly in mice whose lifespan has been extended by mutations that affect GH/IGF‐1 signals. Early‐life exposure to GH by injection, or to nutrient restriction in the first 3 weeks of life, also modulate both lifespan and the pace of hypothalamic inflammation. Three drugs extend lifespan of UM‐HET3 mice in a sex‐specific way: acarbose (ACA), 17‐α‐estradiol (17αE2), and nordihydroguaiaretic acid (NDGA), with more dramatic longevity increases in males in each case. In this study, we examined the effect of these anti‐aging drugs on neuro‐inflammation in hypothalamus and hippocampus. We found that age‐associated hypothalamic inflammation is reduced in males but not in females at 12 months of age by ACA and 17αE2 and at 22 months of age in NDGA‐treated mice. The three drugs blocked indices of hypothalamic reactive gliosis associated with aging, such as Iba‐1‐positive microglia and GFAP‐positive astrocytes, as well as age‐associated overproduction of TNF‐α. This effect was not observed in drug‐treated female mice or in the hippocampus of the drug‐treated animals. On the other hand, caloric restriction (CR; an intervention that extends the lifespan in both sexes) significantly reduced hypothalamic microglia and TNF‐α in both sexes at 12 months of age. Together, these results suggest that the extent of drug‐induced changes in hypothalamic inflammatory processes is sexually dimorphic in a pattern that parallels the effects of these agents on mouse longevity and that mimics the changes seen, in both sexes, of long‐lived nutrient restricted or mutant mice.     

Signature Lipids and Stable Carbon Isotope Analyses of Octopus Spring Hyperthermophilic Communities Compared with Those of Aquificales Representatives

The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12, Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus, and Aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. The n-C_20:1 and cy-C₂₁ fatty acids dominated all of the Aquificales, while the alkyl glycerol ethers were mainly C_18:0. These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C₁₈ and C₂₀ alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C_20:1 and cy-C₂₁, plus a series of iso-branched fatty acids (i-C_15:0 to i-C_21:0), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in ¹³C relative to source water CO₂ by 10.9 and 17.2‰, respectively. The C_20–21 fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6‰, respectively. The biomass of T. ruber grown on CO₂ was depleted in ¹³C by only 3.3‰ relative to C source. In contrast, biomass was depleted by 19.7‰ when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3‰). The depletion in the C_20–21 fatty acids from the PSC indicates that Thermocrinis biomass must be similarly depleted and too light to be explained by growth on CO₂. Accordingly, Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.     

Advanced Granulation Technology (AGTTM). An alternate format for serum-free, chemically-defined and protein-free cell culture media

To overcome limitations of conventional milling technology, we investigated the application of fluid bed granulation for the production of dry-form nutrient media. Serum-free, protein-free and chemically-defined specialty media were produced in granulated format and compared with identical formulations manufactured by conventional methods. HPLC analysis of multiple lots of granulated materials demonstrated that biochemical constituents were precisely and homogeneously distributed throughout the granules and that nutrient levels were comparable to conventional formats. Comparison of medium performance in cell proliferation and biological production assays demonstrated equivalence with reference media. The fluid bed granulation process meets pharmaceutical quality requirements and may be applied to a broad range of nutrient formulations required for bioproduction applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of putative diuretic factors on in vivo urine production in the mosquito,Aedes aegypti

Changes in total urine production were measured in the mosquito, Aedes aegypti, following injection of 5-hydroxytryptamine (5-HT), Culex salinarius diuresin, culekinin depolarizing peptides (CDP-I, II and III) or the A. aegypti leucokinin peptides (ALP-I, II and III). All stimulated total urine production in a dose dependent manner. 5-HT was the least potent in urine production experiments with ED(50) values nearly 100-fold higher than other diuretic agonists. Doses greater than 2x10(-4) &mgr;moles inhibited urine production, suggesting either the occurrence of receptor down regulation, more than one type of 5-HT receptor, or increases in hindgut resorption of urine. The ALPs had relatively low ED(50) values compared to the CDPs suggesting that the endogenous peptides may have higher receptor binding affinities. Injection of mosquitoes with polyclonal antisera raised against either ALP-I or C. salinarius diuresin significantly reduced the response to injections of the respective peptides. The evidence presented above suggests that mosquito leucokinins and the C. salinarius diuresin function in the neuroendocrine regulation of urine production in the mosquito.     

Nucleic acid purification using microfabricated silicon structures

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.     

Enhanced-rate biodegradation of organophosphate neurotoxins by immobilized nongrowing bacteria

Pesticide wastes generated from livestock dipping operations containing the organophosphate (OP) insecticide coumaphos (CP) are well suited for disposal by biodegradation since they are highly concentrated (approximately 1 g/L), generally contained, and lack additional toxic components. In this study, a significantly enhanced efficiency of degrading CP in cattle dip waste (CDW) is reported using a dense, nongrowing cell population that functions without the addition of nutrients required for growing cell cultures. A recombinant strain of Escherichia coli containing the opd gene for organophosphate hydrolase (OPH), which is capable of active hydrolysis of OP neurotoxins including CP, was cultivated in a rich medium containing all essential nutrients. Cells were harvested and utilized in lab scale experiments in the form of either freely suspended cells or cells immobilized within a macroporous gel matrix, poly(vinyl alcohol) (PVA) cryogel. Significantly higher degradation rates were achieved with either suspended or immobilized OPH(+) cells compared to rates with the microbial consortium naturally present in CDW. Of the two nongrowing cell systems, the detoxification rate with immobilized cells was approximately twice that of freely suspended cells, and kinetic studies demonstrated that a higher maximum reaction rate was achieved with the immobilized cell system. A comparative study using both the CDW and pure CP substrates with free cells indicated that the CDW contained one or more factors that reduced the bioavailability of CP. The immobilized cells retained their activity over a 4-month period of use and storage, demonstrating both sustained catalytic activity and long-term mechanical stability.