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

Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic-aromatic interactions

Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional π-systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic-aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic-aromatic interactions at this interface suggested that substitution of residue TyrB26 by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [TrpB26]insulin analog (determined as a T3Rf3 zinc hexamer at a resolution of 2.25 Å) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the in vitro lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the TrpB26-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly

NEXUS/Physics: An interdisciplinary repurposing of physics for biologists

In response to increasing calls for the reform of the undergraduate science curriculum for life science majors and pre-medical students (Bio2010, Scientific Foundations for Future Physicians, Vision & Change), an interdisciplinary team has created NEXUS/Physics: a repurposing of an introductory physics curriculum for the life sciences. The curriculum interacts strongly and supportively with introductory biology and chemistry courses taken by life sciences students, with the goal of helping students build general, multi-discipline scientific competencies. In order to do this, our two-semester NEXUS/Physics course sequence is positioned as a second year course so students will have had some exposure to basic concepts in biology and chemistry. NEXUS/Physics stresses interdisciplinary examples and the content differs markedly from traditional introductory physics to facilitate this. It extends the discussion of energy to include interatomic potentials and chemical reactions, the discussion of thermodynamics to include enthalpy and Gibbs free energy, and includes a serious discussion of random vs. coherent motion including diffusion. The development of instructional materials is coordinated with careful education research. Both the new content and the results of the research are described in a series of papers for which this paper serves as an overview and context.Comment: 12 page

A double-sided silicon micro-strip super-module for the ATLAS inner detector upgrade in the high-luminosity LHC

The ATLAS experiment is a general purpose detector aiming to fully exploit the discovery potential of the Large Hadron Collider (LHC) at CERN. It is foreseen that after several years of successful data-taking, the LHC physics programme will be extended in the so-called High-Luminosity LHC, where the instantaneous luminosity will be increased up to 5 × 1034 cm−2 s−1. For ATLAS, an upgrade scenario will imply the complete replacement of its internal tracker, as the existing detector will not provide the required performance due to the cumulated radiation damage and the increase in the detector occupancy. The current baseline layout for the new ATLAS tracker is an all-silicon-based detector, with pixel sensors in the inner layers and silicon micro-strip detectors at intermediate and outer radii. The super-module is an integration concept proposed for the strip region of the future ATLAS tracker, where double-sided stereo silicon micro-strip modules are assembled into a low-mass local support structure. An electrical super-module prototype for eight double-sided strip modules has been constructed. The aim is to exercise the multi-module readout chain and to investigate the noise performance of such a system. In this paper, the main components of the current super-module prototype are described and its electrical performance is presented in detail

Measurement of the rare decay pi0 -> e+ e-

The branching ratio of the rare decay pi0 -> e+ e- has been measured precisely, using the complete data set from the KTeV E799-II experiment at Fermilab. We observe 794 candidate pi0 -> e+ e- events using K0_L -> 3pi0 as a source of tagged pi0's. The expected background is 52.7 +- 11.2 events, predominantly from high e+ e- mass pi0 -> e+ e- gamma decays. We have measured B[(pi0 -> e+ e-), (m_e+e-/m_pi0)^2 > 0.95] = 6.44 +- 0.25(stat) +- 0.22(syst) x10^(-8), which is above the unitary bound from pi0 -> gamma gamma and within the range of theoretical expectations from the standard model.Comment: 6 pages, 4 figure

A double-sided, shield-less stave prototype for the ATLAS upgrade strip tracker for the high luminosity LHC

A detailed description of the integration structures for the barrel region of the silicon strips tracker of the ATLAS Phase-II upgrade for the upgrade of the Large Hadron Collider, the so-called High Luminosity LHC (HL-LHC), is presented. This paper focuses on one of the latest demonstrator prototypes recently assembled, with numerous unique features. It consists of a shortened, shield-less, and double sided stave, with two candidate power distributions implemented. Thermal and electrical performances of the prototype are presented, as well as a description of the assembly procedures and tools

Measurement of Direct Emission in the KL->pi+pi-gamma Decay Mode

In this paper the KTeV collaboration reports the analysis of 112.1*10^3 candidate KL->pi+pi-gamma decays including a background of 671+/-41 events with the objective of determining the photon production mechanisms intrinsic to the decay process. These decays have been analyzed to extract the relative contributions of the CP violating bremsstrahlung process and the CP conserving M1 and CP violating E1 direct photon emission processes. The M1 direct photon emission amplitude and its associated vector form factor parameterized as |g_M1|(1+ (a_1/a_2)/(M(rho)^2-M(K)^2+2M(K)*E(gamma)) have been measured to be |g_M1|=1.198 +/- 0.035(stat) +/- 0.086(syst) and a_1/a_2 = -0.738 +/- 0.007(stat) +/- 0.018 (syst) GeV^2/c^2 respectively. An upper limit for the CP violating E1 direct emission amplitude |g_E1| < 0.21 (90% CL) has been found. The overall ratio of direct photon emission (DE) to total photon emission including the bremsstrahlung process (IB) has been determined to be DE/(DE +IB) = 0.689 +/- 0.021 for E(gamma) > 20 MeV.Comment: REVTeX 3.1, 4 pages, 4 figures submitted to Physical Review Letter

Cost-per-diagnosis as a metric for monitoring cost effectiveness of HIV testing programmes in low income settings in southern Africa : health economic and modelling analysis

Introduction: As prevalence of undiagnosed HIV declines, it is unclear whether testing programmes will be cost effective. To guide their HIV testing programmes,countries require appropriatemetrics that can be measured. The cost-per-diagnosisis potentially a useful metric. Methods:We simulated a series of setting-scenarios for adult HIV epidemics and ART programmes typical of settings in southern Africa using an individual-based model and projected forward from 2018 under two policies: (i) a minimum package of “core” testing (i.e. testing in pregnant women, for diagnosis of symptoms, in sex workers, and in men coming forward for circumcision) is conducted, and (ii) “core” testing as above plus “additional-testing”, for which we specify different rates of testing and various degrees to which those with HIV are more likely to test than thosewithout HIV. We also considered a plausible range of unit test costs. The aim was to assess the relationship between cost-per-diagnosisand the incremental cost-effectiveness ratio(ICER) of the additional-testingpolicy. Discount rate 3%; costs in 2018 $US. Results:There was a strong graded relationship between the cost-per-diagnosisand the ICER. Overall, the ICERwas below$500 per-DALY-averted (the cost effectiveness threshold used in primary analysis) so long as thecost-per-diagnosiswas below $315. This thresholdcost-per-diagnosiswas similar according to epidemic and programmatic features including the prevalence of undiagnosed HIV, the HIV incidence and a measure of HIV programme quality (the proportion of HIV diagnosed people having a viral load <1000 copies/mL). However, restrictingto women, additional-testingdid not appear cost-effective even at acost-per-diagnosisof below$50, while restrictingto men additional-testingwas cost effective up to a cost-per-diagnosisof $585. Thethreshold cost for testing in men fell to$256 when the cost effectiveness threshold was $300instead of$500, and to $81 when considering a discount rate of 10% perannum.Conclusions:For testing programmesin low income settings in southern African there is an extremely strong relationship between the cost-per-diagnosisand the cost per DALY averted, indicating that the cost-per-diagnosiscan be used to monitor the cost effectiveness of testing programmes