Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells

Regulation of plasma volume in male lowlanders during 4 days of exposure to hypobaric hypoxia equivalent to 3500 m altitude.

Acclimatization to hypoxia leads to a reduction in plasma volume (PV) that restores arterial O <sub>2</sub> content. Findings from studies investigating the mechanisms underlying this PV contraction have been controversial, possibly as experimental conditions were inadequately controlled. We examined the mechanisms underlying the PV contraction evoked by 4 days of exposure to hypobaric hypoxia (HH) in 11 healthy lowlanders, while strictly controlling water intake, diet, temperature and physical activity. Exposure to HH-induced an ∼10% PV contraction that was accompanied by a reduction in total circulating protein mass, whereas diuretic fluid loss and total body water remained unchanged. Our data support an oncotically driven fluid redistribution from the intra- to the extravascular space, rather than fluid loss, as the mechanism underlying HH-induced PV contraction. Extended hypoxic exposure reduces plasma volume (PV). The mechanisms underlying this effect are controversial, possibly as previous studies have been confounded by inconsistent experimental conditions. Here, we investigated the effect of hypobaric hypoxia (HH) on PV in a cross-over study that strictly controlled for diet, water intake, physical activity and temperature. Eleven males completed two 4-day sojourns in a hypobaric chamber, one in normoxia (NX) and one in HH equivalent to 3500 m altitude. PV, urine output, volume-regulating hormones and plasma protein concentration were determined daily. Total body water (TBW) was determined at the end of both sojourns by deuterium dilution. Although PV was 8.1 ± 5.8% lower in HH than in NX after 24 h and remained ∼10% lower thereafter (all P < 0.002), no differences were detected in TBW (P = 0.17) or in 24 h urine volumes (all P > 0.23). Plasma renin activity and circulating aldosterone were suppressed in HH during the first half of the sojourn (all P < 0.05) but thereafter similar to NX, whereas no differences were detected for copeptin between sojourns (all P > 0.05). Markers for atrial natriuretic peptide were higher in HH than NX after 30 min (P = 0.001) but lower during the last 2 days (P < 0.001). While plasma protein concentration was similar between sojourns, total circulating protein mass (TCP) was reduced in HH at the same time points as PV (all P < 0.03). Despite transient hormonal changes favouring increased diuresis, HH did not enhance urine output. Instead, the maintained TBW and reduced TCP support an oncotically driven fluid redistribution into the extravascular compartment as the mechanism underlying PV contraction

Comparative Genomic Analysis of the Class \u3ci\u3eEpsilonproteobacteria\u3c/i\u3e and Proposed Reclassification to Epsilonbacteraeota (phyl. nov.)

The Epsilonproteobacteria is the fifth validly described class of the phylum Proteobacteria, known primarily for clinical relevance and for chemolithotrophy in various terrestrial and marine environments, including deep-sea hydrothermal vents. As 16S rRNA gene repositories have expanded and protein marker analysis become more common, the phylogenetic placement of this class has become less certain. A number of recent analyses of the bacterial tree of life using both 16S rRNA and concatenated marker gene analyses have failed to recover the Epsilonproteobacteria as monophyletic with all other classes of Proteobacteria. In order to address this issue, we investigated the phylogenetic placement of this class in the bacterial domain using 16S and 23S rRNA genes, as well as 120 single-copy marker proteins. Single- and concatenated-marker trees were created using a data set of 4,170 bacterial representatives, including 98 Epsilonproteobacteria. Phylogenies were inferred under a variety of tree building methods, with sequential jackknifing of outgroup phyla to ensure robustness of phylogenetic affiliations under differing combinations of bacterial genomes. Based on the assessment of nearly 300 phylogenetic tree topologies, we conclude that the continued inclusion of Epsilonproteobacteria within the Proteobacteria is not warranted, and that this group should be reassigned to a novel phylum for which we propose the name Epsilonbacteraeota (phyl. nov.). We further recommend the reclassification of the order Desulfurellales (Deltaproteobacteria) to a novel class within this phylum and a number of subordinate changes to ensure consistency with the genome-based phylogeny. Phylogenomic analysis of 658 genomes belonging to the newly proposed Epsilonbacteraeota suggests that the ancestor of this phylum was an autotrophic, motile, thermophilic chemolithotroph that likely assimilated nitrogen from ammonium taken up from the environment or generated from environmental nitrate and nitrite by employing a variety of functional redox modules. The emergence of chemoorganoheterotrophic lifestyles in several Epsilonbacteraeota families is the result of multiple independent losses of various ancestral chemolithoautotrophic pathways. Our proposed reclassification of this group resolves an important anomaly in bacterial systematics and ensures that the taxonomy of Proteobacteria remains robust, specifically as genome-based taxonomies become more common

Transcript levels of ten caste-related genes in adult diploid males of Melipona quadrifasciata (Hymenoptera, Apidae) - A comparison with haploid males, queens and workers

In Hymenoptera, homozygosity at the sex locus results in the production of diploid males. In social species, these pose a double burden by having low fitness and drawing resources normally spent for increasing the work force of a colony. Yet, diploid males are of academic interest as they can elucidate effects of ploidy (normal males are haploid, whereas the female castes, the queens and workers, are diploid) on morphology and life history. Herein we investigated expression levels of ten caste-related genes in the stingless bee Melipona quadrifasciata, comparing newly emerged and 5-day-old diploid males with haploid males, queens and workers. In diploid males, transcript levels for dunce and paramyosin were increased during the first five days of adult life, while those for diacylglycerol kinase and the transcriptional co-repressor groucho diminished. Two general trends were apparent, (i) gene expression patterns in diploid males were overall more similar to haploid ones and workers than to queens, and (ii) in queens and workers, more genes were up-regulated after emergence until day five, whereas in diploid and especially so in haploid males more genes were down-regulated. This difference between the sexes may be related to longevity, which is much longer in females than in males

Intercomparison of stratospheric chemistry models under polar vortex conditions

Several stratospheric chemistry modules from box, 2-D or 3-D models, have been intercompared. The intercomparison was focused on the ozone loss and associated reactive species under the conditions found in the cold, wintertime Arctic and Antarctic vortices. Comparisons of both gas phase and heterogeneous chemistry modules show excellent agreement between the models under constrained conditions for photolysis and the microphysics of polar stratospheric clouds. While the mean integral ozone loss ranges from 4-80% for different 30-50 days long air parcel trajectories, the mean scatter of model results around these values is only about +/-1.5%. In a case study, where the models employed their standard photolysis and microphysical schemes, the variation around the mean percentage ozone loss increases to about +/-7%. This increased scatter of model results is mainly due to the different treatment of the PSC microphysics and heterogeneous chemistry in the models, whereby the most unrealistic assumptions about PSC processes consequently lead to the least representative ozone chemistry. Furthermore, for this case study the model results for the ozone mixing ratios at different altitudes were compared with a measured ozone profile to investigate the extent to which models reproduce the stratospheric ozone losses. It was found that mainly in the height range of strong ozone depletion all models underestimate the ozone loss by about a factor of two. This finding corroborates earlier studies and implies a general deficiency in our understanding of the stratospheric ozone loss chemistry rather than a specific problem related to a particular model simulation

Simultaneous genome sequencing of symbionts and their hosts

Second-generation sequencing has made possible the sequencing of genomes of interest for even small research groups. However, obtaining separate clean cultures and clonal or inbred samples of metazoan hosts and their bacterial symbionts is often difficult. We present a computational pipeline for separating metazoan and bacterial DNA in silico rather than at the bench. The method relies on the generation of deep coverage of all the genomes in a mixed sample using Illumina short-read sequencing technology, and using aggregate properties of the different genomes to identify read sets belonging to each. This inexpensive and rapid approach has been used to sequence several nematode genomes and their bacterial endosymbionts in the last year in our laboratory and can also be used to visualize and identify unexpected contaminants (or possible symbionts) in genomic DNA samples. We hope that this method will enable researchers studying symbiotic systems to move from gene-centric to genome-centric approaches

Permanent draft genome sequences of the symbiotic nitrogen fixing Ensifer meliloti strains BO21CC and AK58

Ensifer (syn. Sinorhizobium) meliloti is an important symbiotic bacterial species that fixes nitrogen. Strains BO21CC and AK58 were previously investigated for their substrate utilization and their plant-growth promoting abilities showing interesting features. Here, we describe the complete genome sequence and annotation of these strains. BO21CC and AK58 genomes are 6,985,065 and 6,974,333 bp long with 6,746 and 6,992 genes predicted, respectively. © retained by original authors