First-principles calculation of DNA looping in tethered particle experiments

We calculate the probability of DNA loop formation mediated by regulatory proteins such as Lac repressor (LacI), using a mathematical model of DNA elasticity. Our model is adapted to calculating quantities directly observable in Tethered Particle Motion (TPM) experiments, and it accounts for all the entropic forces present in such experiments. Our model has no free parameters; it characterizes DNA elasticity using information obtained in other kinds of experiments. [...] We show how to compute both the "looping J factor" (or equivalently, the looping free energy) for various DNA construct geometries and LacI concentrations, as well as the detailed probability density function of bead excursions. We also show how to extract the same quantities from recent experimental data on tethered particle motion, and then compare to our model's predictions. [...] Our model successfully reproduces the detailed distributions of bead excursion, including their surprising three-peak structure, without any fit parameters and without invoking any alternative conformation of the LacI tetramer. Indeed, the model qualitatively reproduces the observed dependence of these distributions on tether length (e.g., phasing) and on LacI concentration (titration). However, for short DNA loops (around 95 basepairs) the experiments show more looping than is predicted by the harmonic-elasticity model, echoing other recent experimental results. Because the experiments we study are done in vitro, this anomalously high looping cannot be rationalized as resulting from the presence of DNA-bending proteins or other cellular machinery. We also show that it is unlikely to be the result of a hypothetical "open" conformation of the LacI tetramer.Comment: See the supplement at http://www.physics.upenn.edu/~pcn/Ms/TowlesEtalSuppl.pdf . This revised version accepted for publication at Physical Biolog

Concentration and Length Dependence of DNA Looping in Transcriptional Regulation

In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the J-factor for looping

Oxygen uptake and heart rate during simulated wildfire suppression tasks performed by Australian rural firefighters

Objective: Australian rural fire crews safeguard the nation against the annual devastation of wildfire. We have previously reported that experienced firefighters identified seven physically demanding tasks for Australian rural fire crews when suppressing wildfires. These firefighters rated the operational importance, typical duration, core fitness components, and likely frequency of the seven tasks. The intensity of these duties remains unknown. The aim of this study was to quantify the oxygen uptake (VO2), heart rate (HR) and movement speed responses during simulations of these physically demanding wildfire suppression tasks. Method: Twenty six rural firefighters (20 men, six women) performed up to seven tasks, during which time their HR and movement speed were recorded. The VO2 for each task was also calculated from the analysis of expired air collected in Douglas bags. Firefighters’ HR and movement speed were measured using HR monitors and portable global positioning system units, respectively. Results: The hose work tasks elicited a VO2 of 21-27 mL·kg-1·min-1 and peak HR of 77-87% age-predicted maximal HR (HRmax). Hand tool tasks were accompanied by VO2 of 28-34 mL·kg-1·min-1 and peak HR of 85-95%HRmax. Firefighters’ movement speed spanned 0.2 ± 0.1 to 1.8 ± 0.2 m·s-1 across the seven tasks. The cardiovascular responses in the hand tool tasks were, in most cases, higher (P < 0.05) than during those elicited by the hose work tasks. Conclusions: The cardiovascular responses elicited during simulations of physically demanding wildfire suppression approximated those reported for similar tasks in urban and forestry fire fighting jurisdictions. The findings may prompt Australian rural fire agencies to consider cardiovascular disease risk screenin

Measles virus causes immunogenic cell death in human melanoma

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host anti-tumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response

Post-synaptic action potentials are required for nitric oxide-dependent LTP in CA1 neurons of adult GluR1 knockout and wild-type mice

Neocortical long-term potentiation (LTP) consists of both presynaptic and postsynaptic components that rely on nitric oxide (NO) and the GluR1 subunit of the AMPA receptor, respectively. In this study, we found that hippocampal LTP, induced by theta-burst stimulation in mature (>8-week-old) GluR1 knock-out mice was almost entirely NO dependent and involved both the α splice variant of NO synthase-1 and the NO synthase-3 isoforms of NO synthase. Theta-burst induced LTP was also partly NO-dependent in wild-type mice and made up ∼50% of the potentiation 2 h after tetanus. Theta-burst stimulation reliably produced postsynaptic spikes, including a high probability of complex spikes. Inhibition of postsynaptic somatic spikes with intracellular QX314 or local TTX application prevented LTP in the GluR1 knock-out mice and also blocked the NO component of LTP in wild types. We conclude that theta-burst stimulation is particularly well suited to producing the postsynaptic somatic spikes required for NO-dependent LTP

Mesocorticolimbic monoamine correlates of methamphetamine sensitization and motivation.

Methamphetamine (MA) is a highly addictive psychomotor stimulant, with life-time prevalence rates of abuse ranging from 5-10% world-wide. Yet, a paucity of research exists regarding MA addiction vulnerability/resiliency and neurobiological mediators of the transition to addiction that might occur upon repeated low-dose MA exposure, more characteristic of early drug use. As stimulant-elicited neuroplasticity within dopamine neurons innervating the nucleus accumbens (NAC) and prefrontal cortex (PFC) is theorized as central for addiction-related behavioral anomalies, we used a multi-disciplinary research approach in mice to examine the interactions between sub-toxic MA dosing, motivation for MA and mesocorticolimbic monoamines. Biochemical studies of C57BL/6J (B6) mice revealed short- (1 day), as well as longer-term (21 days), changes in extracellular dopamine, DAT and/or D2 receptors during withdrawal from 10, once daily, 2 mg/kg MA injections. Follow-up biochemical studies conducted in mice selectively bred for high vs. low MA drinking (respectively, MAHDR vs. MALDR mice), provided novel support for anomalies in mesocorticolimbic dopamine as a correlate of genetic vulnerability to high MA intake. Finally, neuropharmacological targeting of NAC dopamine in MA-treated B6 mice demonstrated a bi-directional regulation of MA-induced place-conditioning. These results extend extant literature for MA neurotoxicity by demonstrating that even subchronic exposure to relatively low MA doses are sufficient to elicit relatively long-lasting changes in mesocorticolimbic dopamine and that drug-induced or idiopathic anomalies in mesocorticolimbic dopamine may underpin vulnerability/resiliency to MA addiction

Molecular techniques reveal cryptic life history and demographic processes of a critically endangered marine turtle

The concept of ‘effective population size’ (Ne), which quantifies how quickly a population will lose genetic variability, is one of the most important contributions of theoretical evolutionary biology to practical conservation management. Ne is often much lower than actual population size: how much so depends on key life history and demographic parameters, such as mating systems and population connectivity, that often remain unknown for species of conservation concern. Molecular techniques allow the indirect study of these parameters, as well as the estimation of current and historical Ne. Here, we use genotyping to assess the genetic health of an important population of the critically endangered hawksbill turtle (Eretmochelys imbricata), a slow-to-mature, difficult-to-observe species with a long history of severe overhunting. Our results were surprisingly positive: we found that the study population, located in the Republic of Seychelles, Indian Ocean, has a relatively large Ne, estimated to exceed 1000, and showed no evidence of a recent reduction in Ne (i.e. no genetic bottleneck). Furthermore, molecular inferences suggest the species' mating system is conducive to maintaining a large Ne, with a relatively large and widely distributed male population promoting considerable gene flow amongst nesting sites across the Seychelles area. This may also be reinforced by the movement of females between nesting sites. Our study underlines how molecular techniques can help to inform conservation biology. In this case our results suggest that this important hawksbill population is starting from a relatively strong position as it faces new challenges, such as global climate change

American political affiliation, 2003–43: a cohort component projection

The recent rise and stability in American party identification has focused interest on the long-term dynamics of party bases. Liberal commentators cite immigration and youth as forces which will produce a natural Democratic advantage in the future while conservative writers highlight the importance of high Republican fertility in securing Republican growth. These concerns foreground the neglect of demography within political science. This paper addresses this omission by conducting the first ever cohort component projection of American partisan populations to 2043 based on survey and census data. A number of scenarios are modeled, but, on current trends, we predict that American partisanship will shift much less than the nation’s ethnic composition because the parties’ age structures are similar. Still, our projections find that the Democrats gain two to three percentage points from the Republicans by 2043, mainly through immigration, though Republican fertility may redress the balance in the very long term