A pipeline for targeted metagenomics of environmental bacteria.

BackgroundMetagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.MethodsCells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity.ResultsPure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade.ConclusionsWith the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract

Grasshopper Community Response to Climatic Change: Variation Along an Elevational Gradient

The impacts of climate change on phenological responses of species and communities are well-documented; however, many such studies are correlational and so less effective at assessing the causal links between changes in climate and changes in phenology. Using grasshopper communities found along an elevational gradient, we present an ideal system along the Front Range of Colorado USA that provides a mechanistic link between climate and phenology.This study utilizes past (1959-1960) and present (2006-2008) surveys of grasshopper communities and daily temperature records to quantify the relationship between amount and timing of warming across years and elevations, and grasshopper timing to adulthood. Grasshopper communities were surveyed at four sites, Chautauqua Mesa (1752 m), A1 (2195 m), B1 (2591 m), and C1 (3048 m), located in prairie, lower montane, upper montane, and subalpine life zones, respectively. Changes to earlier first appearance of adults depended on the degree to which a site warmed. The lowest site showed little warming and little phenological advancement. The next highest site (A1) warmed a small, but significant, amount and grasshopper species there showed inconsistent phenological advancements. The two highest sites warmed the most, and at these sites grasshoppers showed significant phenological advancements. At these sites, late-developing species showed the greatest advancements, a pattern that correlated with an increase in rate of late-season warming. The number of growing degree days (GDDs) associated with the time to adulthood for a species was unchanged across the past and present surveys, suggesting that phenological advancement depended on when a set number of GDDs is reached during a season.Our analyses provide clear evidence that variation in amount and timing of warming over the growing season explains the vast majority of phenological variation in this system. Our results move past simple correlation and provide a stronger process-oriented and predictive framework for understanding community level phenological responses to climate change

Signature of Electron Capture in Iron-Rich Ejecta of SN 2003du

Late-time near-infrared and optical spectra are presented for the normal-bright SN2003du. At about 300 days after the explosion, the emission profiles of well isolated [FeII] lines (in particular that of the strong 1.644mu feature) trace out the global kinematic distribution of radioactive material in the expanding. In SN2003du, the 1.644 mu [FeII] line shows a flat-topped, profile, indicative of a thick but hollow-centered expanding shell, rather than a strongly-peaked profile that would be expected from a ``center-filled'' distribution.Based on detailed models for exploding Chandrasekhar mass white dwarfs, we show that the feature is consistent with spherical explosion models.Our model predicts central region of non-radioactive electron-capture elements up to 2500--3000km/s as a consequence of burning under high density, and an extended region of 56Ni up to 9,000--10,000km/s. Furthermore our analysis indicates that the 1.644mu [FeII] profile is not consistent with strong mixing between the regions of electron- capture isotopes and the 56Ni layers as is predicted by detailed 3D models for nuclear deflagration fronts. We discuss the possibility that the flat-topped profile could be produced as a result of an infrared catastrophe and conclude that such an explanation is unlikely. We put our results in context to other SNeIa and briefly discuss the implications of our result for the use of SNe Ia as cosmological standard candles.Comment: 12 pages + 8 figures, ApJ (in press, Dec. 20, 2004) For high resolution figures send E-mail to [email protected]

Unilateral thoracoscopic surgical approach for diffuse emphysema

AbstractWe evaluated the use of a lateral thoracoscopic approach for lung reduction surgery in patients with diffuse emphysema. Sixty-seven patients with a mean age of 61.9 years underwent operation. Operative side was determined by preoperative imaging. The procedures were laser ablation in 10 patients and stapler resection in 57 patients. Ten patients, including six of the 10 patients in the laser-only group had poor outcome (death or hospitalization longer than 30 days), leading us to abandon the laser technique. Of the remaining 57 patients undergoing primary stapled resection, duration of chest tube placement averaged 13 days (range 3 to 53 days) with a mean hospital stay of 17 days (range 6 to 99 days). Seven patients required ventilation for longer than 72 hours, six patients underwent conversion of the procedure to open thoracotomy, four patients acquired arrhythmias, and three patients were treated for empyema. There was one early death (1.7%), from cardiopulmonary failure. Forty patients returned for 3-month evaluation. Significant ( p < 0.0001) improvements were seen in forced vital capacity (2.69 L after vs 2.26 L before) and forced expiration volume in 1 second (1.04 L after vs 0.82 L before), with 25 of 40 patients (63%) showing an improvement of more than 20%. Lung volume measures, in particular residual volume, fell significantly. Arterial blood gas analysis revealed that carbon dioxide tension fell significantly in patients with preoperative hypercapnia (carbon dioxide tension >45 mm Hg, p = 0.018). Six-minute walk test results improved (894 feet after vs 784 feet before, p = 0.002), and symptomatic benefit was confirmed by significant improvement in the dyspnea index. The combination of both hypercapnia and reduced single-breath diffusing capacity for carbon monoxide was significantly more frequent ( p = 0.0026) and was 86% specific (5 of 6 patients) in predicting serious postoperative risk. We conclude that the lateral thoracoscopic surgical approach to diffuse emphysema offers significant improvement in pulmonary mechanics and functional impairment. Patients with a combination of hypercapnia and reduced single-breath diffusing capacity for carbon monoxide should not be considered for this procedure because of significant perioperative risk. (J THORAC CARDIOVASC SURG 1996;111:308-16

Baseline Chromatin Modification Levels May Predict Interindividual Variability in Ozone-Induced Gene Expression

Traditional toxicological paradigms have relied on factors such as age, genotype, and disease status to explain variability in responsiveness to toxicant exposure; however, these are neither sufficient to faithfully identify differentially responsive individuals nor are they modifiable factors that can be leveraged to mitigate the exposure effects. Unlike these factors, the epigenome is dynamic and shaped by an individual’s environment. We sought to determine whether baseline levels of specific chromatin modifications correlated with the interindividual variability in their ozone (O3)-mediated induction in an air–liquid interface model using primary human bronchial epithelial cells from a panel of 11 donors. We characterized the relationship between the baseline abundance of 6 epigenetic markers with established roles as key regulators of gene expression—histone H3 lysine 4 trimethylation (H3K4me3), H3K27 acetylation (H3K27ac), pan-acetyl H4 (H4ac), histone H3K27 di/trimethylation (H3K27me2/3), unmodified H3, and 5-hydroxymethylcytosine (5-hmC)—and the variability in the O3-induced expression of IL-8, IL-6, COX2, and HMOX1. Baseline levels of H3K4me3, H3K27me2/3, and 5-hmC, but not H3K27ac, H4ac, and total H3, correlated with the interindividual variability in O3-mediated induction of HMOX1 and COX2. In contrast, none of the chromatin modifications that we examined correlated with the induction of IL-8 and IL-6. From these findings, we propose an “epigenetic seed and soil” model in which chromatin modification states between individuals differ in the relative abundance of specific modifications (the “soil”) that govern how receptive the gene is to toxicant-mediated cellular signals (the “seed”) and thus regulate the magnitude of exposure-related gene induction

Phage Orf family recombinases:conservation of activities and involvement of the central channel in DNA binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination

Occupational Exposure to Pfiesteria Species in Estuarine Waters Is Not a Risk Factor for Illness

BACKGROUND: Exposure to the dinoflagellate Pfiesteria has, under certain circumstances, been associated with deficits in human learning and memory. However, uncertainties remain about the health risk of chronic, low-level exposures (as seen among occupationally exposed commercial fishermen), particularly in light of studies suggesting that Pfiesteria strains are widespread in the estuarine environment in the U.S. mid-Atlantic region. METHODS: We selected an initial cohort of 152 persons, including 123 persons with regular, occupational exposure to the Chesapeake Bay; 107 of the cohort members were followed for the full four summer “seasons” of the study. Cohort members were questioned biweekly about symptoms, and data were collected about the areas of the bay in which they worked. These latter data were matched with data on the presence or absence of Pfiesteria in each area, based on polymerase chain reaction analysis of > 3,500 water samples. Cohort members underwent neuropsychological testing at the beginning and end of each summer season. RESULTS: No correlation was found between work in an area where Pfiesteria was identified and specific symptomatology or changes on neuropsychological tests. CONCLUSIONS: Although high-level or outbreak-associated exposure to Pfiesteria species (or specific strains within a species) may have an effect on health, routine occupational exposure to estuarine environments in which these organisms are present does not appear to pose a significant health risk

Asymmetric Explosion of Type Ia Supernovae as Seen from Near Infrared Observations

We present near-infrared spectra of late phase (>200 d) Type Ia supernovae (SNe Ia) taken at the Subaru telescope. The [Fe II] line of SN 2003hv shows a clear flat-topped feature, while that of SN 2005W show less prominent flatness. In addition, a large shift in their line center, varying from -3000 to 1000 (km/s) with respect to the host galaxies, is seen. Such a shift suggests the occurrence of an off-center, non-spherical explosion in the central region, and provides important, new constraints on the explosion models of SNe Ia.Comment: 8 pages, 10 figures. Accepted for publication in the Astrophysical Journal (Letters); some corrections don