Wi-Fi Live-Streaming Centrifuge Force Microscope for Benchtop Single-Molecule Experiments

The ability to apply controlled forces to individual molecules has been revolutionary in shaping our understanding of biophysics in areas as diverse as dynamic bond strength, biological motor operation, and DNA replication. However, the methodology to perform single-molecule experiments remains relatively inaccessible because of cost and complexity. In 2010, we introduced the centrifuge force microscope (CFM) as a platform for accessible and high-throughput single-molecule experimentation. The CFM consists of a rotating microscope with which prescribed centrifugal forces can be applied to microsphere-tethered biomolecules. In this work, we develop and demonstrate a next-generation Wi-Fi CFM that offers unprecedented ease of use and flexibility in design. The modular CFM unit fits within a standard benchtop centrifuge and connects by Wi-Fi to an external computer for live control and streaming at near gigabit speeds. The use of commercial wireless hardware allows for flexibility in programming and provides a streamlined upgrade path as Wi-Fi technology advances. To facilitate ease of use, detailed build and setup instructions, as well as LabVIEW-based control software and MATLAB-based analysis software, are provided. We demonstrate the instrument’s performance by analysis of force-dependent dissociation of short DNA duplexes of 7, 8, and 9 bp. We showcase the sensitivity of the approach by resolving distinct dissociation kinetic rates for a 7 bp duplex in which one G-C basepair is mutated to an A-T basepair.     

Persistence of vascular plants in a Norwegian boreal coniferous forest

Persistence, the tendency of a species to remain in its original position and not to colonize new sites, is studied for the most abundant forest vascular plants (25 species in spruce forest and 7 in pine forest) in Solhomfjell, Gjerstad, S Norway Data sets included presence/absence in 199 meso plots (1 m²) and 3184 subplots (1/16 m²), analyzed over a 5 yr interval, and a subset of 50 meso plots and 800 subplots, analyzed for six consecutive years Relationships between species variables (seedling frequency and mobility rate compiled from the literature, and cover and abundance means in the study area) are studied, and related to species optima along ecologically interpreted DCA ordination axes Vascular plant mobility may increase towards nutrient-poor sites Dominance in the boreal forest floor is mostly by clonal species Persistence was calculated for different temporal (1-5 yr) and spatial (1/16 and I m²) scales Persistence patterns in the spruce and pine forests were similar, but persistence decreased towards the xeric pine forests One main component of variation in persistence was demonstrated by PCA analyses the absolute level of persistence, which is related to seedling recruitment vs clonal growth, and within clonal species to ramet longevity, abundance, mobility, growth pattern and mode of surviving the infavourable season Minor components of variation in persistence were related to spatial scale and temporal scale Persistence characteristics were species-specific and varied little between years Numerous species characteristics were relevant to interpretation of variation in persistence, indicating a continuous, multidimensional variation in life history characteristics     

Dynamics of understory vegetation in an old-growth boreal coniferous forest, 1988–1993

Abstract. Understorey vegetation changes in a South Norwegian old-growth coniferous forest were studied between 1988 and 1993 in 200 1-m² vegetation plots. Our aims were to quantify the amount of between-year compositional change, and to elaborate the environmental basis for long-term vegetation change, including the previously identified gradient structure with a major gradient related to topography (and soil nutrient status and soil depth) and a minor gradient reflecting paludification and canopy coverage. Species richness (yearly mean and cumulative species number) and change in species richness differed between vascular plants and cryptogams, and between forest types. The number of vascular plant species decreased in pine forest in dry years; bryophyte species number increased in spruce forest. Statistically significant vegetation change, as tested by constrained ordination (CCA) with time as the constraining variable, is demonstrated for most one-year periods and for the five-year period in most forest types. Vegetation change along identified gradients, measured as plot displacement along DCA ordination axes, also occurred. The magnitude of year-to-year vegetation change was related neither to forest type nor to one-year period; different responses to climatic and environmental change were observed in each forest type. The largest average displacement observed, from medium-rich spruce forest towards poor spruce forest, was interpreted as a long-term trend. Humus-layer pH decreased by ca. 0.25 units from 1988 to 1993, most strongly in medium-rich spruce forest where exchangeable Ca decreased and Al and Mn increased strongly. Our study supports the hypothesis that vascular plants show a long-term and broad-scale response to soil acidification. Change in bryophyte composition is linked to some very long growing-seasons. Detailed analysis of short-term vegetation dynamics enhances the interpretation of long-term changes and stresses the complementarity of univariate and multivariate methods in the analysis of vegetation change.     

Consequences of abnormal CDK activity in S phase

Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1^? strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress.     

Ontogenesis of physiological responsiveness and guanine nucleotide sensitivity of cardiac muscarinic receptors during chick embryonic development

Atria isolated from 4-day chick embryos were much less responsive to the negative chronotropic effect of muscarinic agonists than were atria from 5- or 8-day embryos, even though the density of muscarinic acetylcholine receptors (mAChR) was similar at all these ages. The mAChR in hearts from 4-day embryos were also significantly less susceptible to regulation of receptor number by in vivo agonist treatment and required a 2-5-fold greater dose of the muscarinic agonist carbachol to achieve a decrease in receptor number equivalent to that observed in 5- or 8-day embryonic hearts. When 4-day atrial membranes were assayed in physiological buffers, agonist binding to the mAChR was not regulated by GTP unless a sulfhydryl reducing agent was present. Receptors from 5- and 8-day embryos did not require addition of a sulfhydryl reducing agent in order to see guanine nucleotide effects on agonist binding. Even in the presence of a sulfhydryl reducing agent, carbachol binding to the mAChR in 4-day membranes was much less sensitive to guanyl-5'-yl imidodiphosphate (GppNHp) than binding to mAChR in 5- or 8-day membranes. In addition, forskolin-activated adenylate cyclase activity was much less sensitive to inhibition by GppNHp in membranes from 4-day atria than from 5- and 8-day atria. The GTP-binding component (NI) which couples the mAChR to inhibition of adenylate cyclase activity was examined by covalent modification with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)