Plasmid copy number underlies adaptive mutability in bacteria.

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F'lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F'lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F'lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F'lac copies divide very little under selection but have enough energy to replicate their F'lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced

Acute dosing of latrepirdine (Dimebon), a possible Alzheimer therapeutic, elevates extracellular amyloid-beta levels in vitro and in vivo.

BACKGROUND: Recent reports suggest that latrepirdine (Dimebon, dimebolin), a retired Russian antihistamine, improves cognitive function in aged rodents and in patients with mild to moderate Alzheimer's disease (AD). However, the mechanism(s) underlying this benefit remain elusive. AD is characterized by extracellular accumulation of the amyloid-beta (Abeta) peptide in the brain, and Abeta-lowering drugs are currently among the most popular anti-amyloid agents under development for the treatment of AD. In the current study, we assessed the effect of acute dosing of latrepirdine on levels of extracellular Abeta using in vitro and in vivo experimental systems. RESULTS: We evaluated extracellular levels of Abeta in three experimental systems, under basal conditions and after treatment with latrepirdine. Mouse N2a neuroblastoma cells overexpressing Swedish APP were incubated for 6 hr in the presence of either vehicle or vehicle + latrepirdine (500pM-5 muM). Synaptoneurosomes were isolated from TgCRND8 mutant APP-overexpressing transgenic mice and incubated for 0 to 10 min in the absence or presence of latrepirdine (1 muM or 10 muM). Drug-naïve Tg2576 Swedish mutant APP overexpressing transgenic mice received a single intraperitoneal injection of either vehicle or vehicle + latrepirdine (3.5 mg/kg). Picomolar to nanomolar concentrations of acutely administered latrepirdine increased the extracellular concentration of Abeta in the conditioned media from Swedish mutant APP-overexpressing N2a cells by up to 64% (p = 0.01), while a clinically relevant acute dose of latrepirdine administered i.p. led to an increase in the interstitial fluid of freely moving APP transgenic mice by up to 40% (p = 0.01). Reconstitution of membrane protein trafficking and processing is frequently inefficient, and, consistent with this interpretation, latrepirdine treatment of isolated TgCRND8 synaptoneurosomes involved higher concentrations of drug (1-10 muM) and led to more modest increases in extracellular Abeta(x-42 )levels (+10%; p = 0.001); of note, however, was the observation that extracellular Abeta(x-40 )levels did not change. CONCLUSIONS: Here, we report the surprising association of acute latrepirdine dosing with elevated levels of extracellular Abeta as measured in three independent neuron-related or neuron-derived systems, including the hippocampus of freely moving Tg2576 mice. Given the reported association of chronic latrepirdine treatment with improvement in cognitive function, the effects of chronic latrepirdine treatment on extracellular Abeta levels must now be determined.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

A 96-well DNase I footprinting screen for drug–DNA interactions

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents

Double Spin Asymmetry of Electrons from Heavy Flavor Decays in p+p Collisions at sqrt(s)=200 GeV

We report on the first measurement of double-spin asymmetry, A_LL, of electrons from the decays of hadrons containing heavy flavor in longitudinally polarized p+p collisions at sqrt(s)=200 GeV for p_T= 0.5 to 3.0 GeV/c. The asymmetry was measured at mid-rapidity (|eta|<0.35) with the PHENIX detector at the Relativistic Heavy Ion Collider. The measured asymmetries are consistent with zero within the statistical errors. We obtained a constraint for the polarized gluon distribution in the proton of |Delta g/g(log{_10}x= -1.6^+0.5_-0.4, {mu}=m_T^c)|^2 < 0.033 (1 sigma), based on a leading-order perturbative-quantum-chromodynamics model, using the measured asymmetry.Comment: 385 authors, 17 pages, 15 figures, 5 tables. Submitted to Phys. Rev. D. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

Measurement of long-range angular correlation and quadrupole anisotropy of pions and (anti)protons in central dd++Au collisions at sNN\sqrt{s_{_{NN}}}=200 GeV

We present azimuthal angular correlations between charged hadrons and energy deposited in calorimeter towers in central $d$$+$Au and minimum bias $p$$+$$p$ collisions at $\sqrt{s_{_{NN}}}=200$ GeV. The charged hadron is measured at midrapidity $|\eta|<0.35$, and the energy is measured at large rapidity ($-3.7<\eta<-3.1$, Au-going direction). An enhanced near-side angular correlation across $|\Delta\eta| >$ 2.75 is observed in $d$$+$Au collisions. Using the event plane method applied to the Au-going energy distribution, we extract the anisotropy strength $v_2$ for inclusive charged hadrons at midrapidity up to $p_T=4.5$ GeV/$c$. We also present the measurement of $v_2$ for identified $\pi^{\pm}$ and (anti)protons in central $d$$+$Au collisions, and observe a mass-ordering pattern similar to that seen in heavy ion collisions. These results are compared with viscous hydrodynamic calculations and measurements from $p$$+$Pb at $\sqrt{s_{_{NN}}}=5.02$ TeV. The magnitude of the mass-ordering in $d$$+$Au is found to be smaller than that in $p$$+$Pb collisions, which may indicate smaller radial flow in lower energy $d$$+$Au collisions.Comment: 424 authors, 8 pages, and 4 figures. v2 is version accepted for publication in Phys. Rev. Lett. Published version will be at http://www.phenix.bnl.gov/phenix/WWW/info/pp1/161/ Plain text data tables will be at http://www.phenix.bnl.gov/papers.htm

Cold-nuclear-matter effects on heavy-quark production at forward and backward rapidity in d+Au collisions at sqrt(s_NN)=200 GeV

The PHENIX experiment has measured open heavy-flavor production via semileptonic decay muons over the transverse momentum range 1 < pT < 6 GeV/c at forward and backward rapidity (1.4 < |y| < 2.0) in d+Au and p+p collisions at ?sNN = 200 GeV. In central d+Au collisions an enhancement (suppression) of heavy-flavor muon production is observed at backward (forward) rapidity relative to the yield in p+p collisions scaled by the number of binary collisions. Modification of the gluon density distribution in the Au nucleus contributes in terms of anti-shadowing enhancement and shadowing suppression; however, the enhancement seen at backward rapidity exceeds expectations from this effect alone. These results, implying an important role for additional cold nuclear matter effects, serves as a key baseline for heavy-quark measurements in A+A collisions and in constraining the magnitude of charmonia breakup effects at the Relativistic Heavy Ion Collider and the Large Hadron Collider.Comment: 424 authors, 69 insitutions, 8 pages, 4 figures. Submitted to Physical Review Letters. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

Upsilon (1S+2S+3S) production in d+Au and p+p collisions at sqrt(s_NN)=200 GeV and cold-nuclear matter effects

The three Upsilon states, Upsilon(1S+2S+3S), are measured in d+Au and p+p collisions at sqrt(s_NN)=200 GeV and rapidities 1.2<|y|<2.2 by the PHENIX experiment at the Relativistic Heavy-Ion Collider. Cross sections for the inclusive Upsilon(1S+2S+3S) production are obtained. The inclusive yields per binary collision for d+Au collisions relative to those in p+p collisions (R_dAu) are found to be 0.62 +/- 0.26 (stat) +/- 0.13 (syst) in the gold-going direction and 0.91 +/- 0.33 (stat) +/- 0.16 (syst) in the deuteron-going direction. The measured results are compared to a nuclear-shadowing model, EPS09 [JHEP 04, 065 (2009)], combined with a final-state breakup cross section, sigma_br, and compared to lower energy p+A results. We also compare the results to the PHENIX J/psi results [Phys. Rev. Lett. 107, 142301 (2011)]. The rapidity dependence of the observed Upsilon suppression is consistent with lower energy p+A measurements.Comment: 495 authors, 11 pages, 9 figures, 5 tables. Submitted to Phys. Rev. C. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm