Genomic Analysis Reveals Chromosomal Variation in Natural Populations of the Uncultured Psychrophilic Archaeon Cenarchaeum symbiosum

Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown. To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote Cenarchaeum symbiosum found living in association with a marine sponge, Axinella mexicana. Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from C. symbiosum. Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed. Homologous regions from the two variants were sequenced and compared in detail. The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region. Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.     

Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus

The influence of sporulation temperature (20, 30 and 37 °C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 °C were the most heat-resistant. However, spores sporulated at 20 °C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 °C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 °C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 °C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 °C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 °C, respectively.     

Determination of metformin in plasma by high-performance liquid chromatography after ultrafiltration

A rapid high-performance liquid chromatography (HPLC) method was developed for determination of metformin, an oral antidiabetic agent, in plasma. Sample preparation entailed a 30-min centrifugation of plasma through a micron filter with direct injection of the protein-free ultrafiltrate into an HPLC system consisting of a cation-exchange extraction column (7.5×4.6 mm), a column switching valve, and a cation-exchange analytical column (250×4.6 mm). The eluent was monitored at 232 nm. Metformin was well resolved at a retention time of about 5 min. There was less than 2% loss of metformin during ultrafiltration and good linearity was established from 0.10 to 40 mg/l of metformin hydrochloride. The lower limit of quantitation was about 0.05 mg/l, at which concentration the signal-to-noise was above 10. The intra- and inter-assay coefficients of variation at plasma concentrations of metformin hydrochloride between 0.25 and 25 mg/l were typically 0.8–1.4% and 3.5–6.4%, respectively. This method offers a rapid sample preparation time and achieves excellent sensitivity without resorting to extraction and evaporation techniques.     

Effect of oscillatory high hydrostatic pressure treatments on Byssochlamys nivea ascospores suspended in fruit juice concentrates

The effect of continuous (689 MPa with holding times of 5, 15 or 25 min) and oscillatory (one, three or five cycles at 689 MPa with holding times of 1 s) high hydrostatic pressure treatments on the viability of Byssochlamys nivea ascospores suspended in apple and cranberry juice concentrates adjusted by dilution to water activities (a_w) of 0·98 and 0·94 was evaluated at 21 and 60 °C. Inactivation of the initial spore inocula was achieved after three or five cycles of oscillatory pressurization at 60 °C when the a_w was 0·98 in both fruit juices. With a_w 0·94, the initial inocula were reduced by less than 1 log-cycle after five pressure cycles. Inactivation was not observed within 25 min with continuous pressurization at 60 °C. In treatments at 21 °C, no effect on spore viability was observed with continuous or oscillatory treatments.     

Spring migration phenology of birds in the Northern Prairie region is correlated with local climate change

ABSTRACT In apparent response to recent periods of global warming, some migratory birds now arrive earlier at stopover sites and breeding grounds. However, the effects of this warming on arrival times vary among locations and species. Migration timing is generally correlated with temperature, with earlier arrival during warm years than during cold years, so local variation in climate change might produce different effects on migration phenology in different geographic regions. We examined trends in first spring arrival dates (FADs) for 44 species of common migrant birds in South Dakota (1971–2006) and Minnesota (1964–2005) using observations compiled by South Dakota and Minnesota Ornithologists’ Unions. We found significant trends in FAD over time for 20 species (18 arriving earlier and two later) in South Dakota and 16 species (all earlier) in Minnesota. Of these species, 10 showed similar significant trends for both states. All 10 of these species exhibited significantly earlier arrival, and all were early spring migrants, with median FADs before 10 April in both states. Eighteen of the 44 species showed significant negative correlations of FADs with either winter (December–February) or spring (arrival month plus previous month) temperatures in one or both states. Interestingly, spring temperatures in both South Dakota and Minnesota did not warm significantly from 1971–2006, but winter temperatures in both states warmed significantly over the same time period. This suggests that the warmer winters disproportionately affected early spring migrants, especially those associated with aquatic habitats (seven of the 10 species showing significantly earlier spring arrival in both states). The stronger response to climate change by early spring migrants in our study is consistent with the results of several other studies, and suggests that migrants, especially early migrants, are capable of responding to local temperature conditions experienced on wintering grounds or along the migration route.     

Physical Activity Differentially Affects the Cecal Microbiota of Ovariectomized Female Rats Selectively Bred for High and Low Aerobic Capacity

The gut microbiota is considered a relevant factor in obesity and associated metabolic diseases, for which postmenopausal women are particularly at risk. Increasing physical activity has been recognized as an efficacious approach to prevent or treat obesity, yet the impact of physical activity on the microbiota remains under-investigated. We examined the impacts of voluntary exercise on host metabolism and gut microbiota in ovariectomized (OVX) high capacity (HCR) and low capacity running (LCR) rats. HCR and LCR rats (age = 27wk) were OVX and fed a high-fat diet (45% kcal fat) ad libitum and housed in cages equipped with (exercise, EX) or without (sedentary, SED) running wheels for 11wk (n = 7-8/group). We hypothesized that increased physical activity would hinder weight gain, increase metabolic health and shift the microbiota of LCR rats, resulting in populations more similar to that of HCR rats. Animals were compared for characteristic metabolic parameters including body composition, lipid profile and energy expenditure; whereas cecal digesta were collected for DNA extraction. 16S rRNA gene-based amplicon Illumina MiSeq sequencing was performed, followed by analysis using QIIME 1.8.0 to assess cecal microbiota. Voluntary exercise decreased body and fat mass, and normalized fasting NEFA concentrations of LCR rats, despite only running one-third the distance of HCR rats. Exercise, however, increased food intake, weight gain and fat mass of HCR rats. Exercise clustered the gut microbial community of LCR rats, which separated them from the other groups. Assessments of specific taxa revealed significant (p<0.05) line by exercise interactions including shifts in the abundances of Firmicutes, Proteobacteria, and Cyanobacteria. Relative abundance of Christensenellaceae family was higher (p = 0.026) in HCR than LCR rats, and positively correlated (p<0.05) with food intake, body weight and running distance. These findings demonstrate that exercise differentially impacts host metabolism and gut microbial communities of female HCR and LCR rats without ovarian function.     

Natural and artificial routes to pluripotency

Pluripotent cells of the blastocyst inner cell mass (ICM) and their in vitro derivatives, embryonic stem (ES) cells, contain genomes in an epigenetic state that are poised for subsequent differentiation. Their chromatin is hyperdynamic in nature and relatively uncondensed. In addition, a large number of genes are expressed at low levels in both ICM and ES cells. Also, the chromatin of naturally pluripotent cells contains specialized histone modification patterns such as bivalent domains, which mark genes destined for later developmentally-regulated expression states. Female pluripotent cells contain X chromosomes that have yet to undergo the process of X chromosome inactivation. Collectively, these features of very early embyronic chromatin are required for the successful specification and production of differentiated cell lineages. Artificial reprogramming methods such as somatic nuclear transfer (SCNT), ES cell fusion-mediated reprogramming (FMR), and induced pluripotency (iPS) yield pluripotent cells that recapitulate many features of naturally pluripotent cells, including many of their epigenetic features. However, the route to pluripotent epigenomic states in artificial pluripotent cells differs drastically from that of their natural counterparts. Here, we compare and contrast the differing routes to pluripotency under natural and artificial conditions. In addition, we discuss the intrinsically metastable nature of the pluripotent epigenome and consider epigenetic aspects of reprogramming that may lead to incomplete or inaccurate reprogrammed states. Artificial methods of reprogramming hold immense promise for the development of autologous cell graft sources and for the development of cell culture models for human genetic disorders. However, the utility of artificially reprogrammed cells is highly dependent on the fidelity of the reprogramming process and it is therefore critically important to assess the epigenetic similarities between embryonic and induced pluripotent stem cells.