The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.     

The effect of lifestyle intervention in obesity on the soluble form of activated leukocyte cell adhesion molecule

Background

The aim of this study was to investigate the effect of a lifestyle intervention in obesity on the soluble form of the activated leukocyte cell adhesion molecule (sALCAM) and its association with metabolic parameters.

Methods

Twenty-nine obese subjects selected from the OPTIFAST®52 program. This program consisted into 2 crucial phases: an initial 12-week active weight reduction phase, followed by a 40-week weight maintenance phase. At baseline, after 12 weeks and at the end of the program, fasting glucose and insulin, total cholesterol, LDL-C, HDL-C, triglycerides, adiponectin, leptin, high sensitivity CRP, sALCAM, homeostasis model assessment-estimated insulin resistance (HOMA-IR) and leptin-to-adiponectin-ratio were determined. Oral glucose tolerance test (OGTT) was performed when indicated.

Results

At baseline, the serum concentration of sALCAM was increased and correlated positively with HOMA-IR and negatively with age. At the end of the program, sALCAM concentrations decreased significantly. Multivariate analysis showed that sALCAM significantly correlated with age, glucose concentration after 2 h OGTT and the HOMA-IR. A higher decrease of HOMA-IR during the study was observed in subjects with higher concentration of sALCAM at baseline.

Conclusions

sALCAM might be a novel biomarker in obesity that correlates and predicts insulin sensitivity improvement and that can be affected by lifestyle intervention.

     

Early‐breeding females experience greater telomere loss

Annual reproductive success is often highest in individuals that initiate breeding early, yet relatively few individuals start breeding during this apparently optimal time. This suggests that individuals, particularly females who ultimately dictate when offspring are born, incur costs by initiating reproduction early in the season. We hypothesized that increases in the ageing rate of somatic cells may be one such cost. Telomeres, the repetitive DNA sequences on the ends of chromosomes, may be good proxies of biological wear and tear as they shorten with age and in response to stress. Using historical data from a long‐term study population of dark‐eyed juncos (Junco hyemalis), we found that telomere loss between years was greater in earlier breeding females, regardless of chronological age. There was no relationship between telomere loss and the annual number of eggs laid or chicks that reached independence. However, telomere loss was greater when temperatures were cooler, and cooler temperatures generally occur early in the season. This suggests that environmental conditions could be the primary cause of accelerated telomere loss in early breeders.     

Integrating Batteries in the Future Swiss Electricity Supply System: A Consequential Environmental Assessment

Stationary batteries are projected to play a role in the electricity system of Switzerland after 2030. By enabling the integration of surplus production from intermittent renewables, energy storage units displace electricity production from different sources and potentially create environmental benefits. Nevertheless, batteries can also cause substantial environmental impacts during their manufacturing process and through the extraction of raw materials. A prospective consequential life cycle assessment (LCA) of lithium metal polymer and lithium‐ion stationary batteries is undertaken to quantify potential environmental benefits and drawbacks. Projections are integrated into the LCA model: Energy scenarios are used to obtain marginal electricity supply mixes, and projections about the battery performances and the recycling process are sourced from the literature. The roles of key parameters and methodological choices in the results are systematically investigated. The results demonstrate that the displacement of marginal electricity sources determines the environmental implications of using batteries. In the reference scenario representing current policy, the displaced electricity mix is dominated by natural gas combined cycle units. In this scenario, the use of batteries generates environmental benefits in 12 of the 16 impact categories assessed. Nevertheless, there is a significant reduction in achievable environmental benefits when batteries are integrated into the power supply system in a low‐carbon scenario because the marginal electricity production, displaced using batteries, already has a reduced environmental impact. The direct impacts of batteries mainly originate from upstream manufacturing processes, which consume electricity and mining activities related to the extraction of materials such as copper and bauxite.     

When and where plant‐soil feedback may promote plant coexistence: a meta‐analysis

Plant‐soil feedback (PSF) theory provides a powerful framework for understanding plant dynamics by integrating growth assays into predictions of whether soil communities stabilise plant–plant interactions. However, we lack a comprehensive view of the likelihood of feedback‐driven coexistence, partly because of a failure to analyse pairwise PSF, the metric directly linked to plant species coexistence. Here, we determine the relative importance of plant evolutionary history, traits, and environmental factors for coexistence through PSF using a meta‐analysis of 1038 pairwise PSF measures. Consistent with eco‐evolutionary predictions, feedback is more likely to mediate coexistence for pairs of plant species (1) associating with similar guilds of mycorrhizal fungi, (2) of increasing phylogenetic distance, and (3) interacting with native microbes. We also found evidence for a primary role of pathogens in feedback‐mediated coexistence. By combining results over several independent studies, our results confirm that PSF may play a key role in plant species coexistence, species invasion, and the phylogenetic diversification of plant communities.     

Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes

Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides.     

Patterns of sequence variability and divergence at the diminutive gene region of Drosophila melanogaster: complex patterns suggest an ancestral selective sweep

To identify putatively swept regions of the Drosophila melanogaster genome, we performed a microsatellite screen spanning a 260-kb region of the X chromosome in populations from Zimbabwe, Ecuador, the United States, and China. Among the regions identified by this screen as showing a complex pattern of reduced heterozygosity and a skewed frequency spectrum was the gene diminutive (dm). To investigate the microsatellite findings, nucleotide sequence polymorphism data were generated in populations from both China and Zimbabwe spanning a 25-kb region and encompassing dm. Analysis of the sequence data reveals strongly reduced nucleotide variation across the entire gene region in both the non-African and the African populations, an extended haplotype pattern, and structured linkage disequilibrium, as well as a rejection of neutrality in favor of selection using a composite likelihood-ratio test. Additionally, unusual patterns of synonymous site evolution were observed at the second exon of this locus. On the basis of simulation studies as well as recently proposed methods for distinguishing between selection and nonequilibrium demography, we find that this "footprint" is best explained by a selective sweep in the ancestral population, the signal of which has been somewhat blurred via founder effects in the non-African samples.     

Genetic evidence supporting caveolae microdomain regulation of calcium entry in endothelial cells

Various cellular signals initiate calcium entry into cells, and there is evidence that lipid rafts and caveolae may concentrate proteins that regulate transmembrane calcium fluxes. Here, using mice deficient in caveolin-1 (Cav-1) and Cav-1 knock-out reconstituted with endothelium-specific Cav-1, we show that Cav-1 is essential for calcium entry in endothelial cells and governs the localization and protein-protein interactions between transient receptor channels C4 and C1. Thus, Cav-1 is required for calcium entry in vascular endothelial cells and perhaps other specialized cell types containing caveolae.     

In vitro fermentation characteristics of selected glucose-based polymers by canine and human fecal bacteria

This research evaluated fermentation characteristics (short-chain fatty acid [SCFA] production, pH, and gas production) resulting from fermentation of glucose-based carbohydrates using canine (n = 3) and human (n = 3) fecal inoculum. Substrates included lyophilized canine ileal digesta containing maltodextrin, gamma-cyclodextrin, high molecular weight (MW) pullulan (MW 100000), or low MW pullulan (MW 6300) obtained from an in vivo experiment. Fermentation for 6 and 10 h with human fecal microflora resulted in higher gas and SCFA production than did canine fecal microflora. High MW pullulan fermentation resulted in the highest (p < 0.05) gas production and lowest (p < 0.05) pH for both dogs and humans. Total SCFA production was highest (p < 0.05) for low MW pullulan fermented by canine microflora, and for gamma-cyclodextrin, high MW pullulan, and low MW pullulan fermented by human microflora. Differences were noted in fermentation characteristics of substrates present in ileal digesta.