Cyclic pyrrole-imidazole polyamides targeted to the androgen response element

Hairpin pyrrole−imidazole (Py-Im) polyamides are a class of cell-permeable DNA-binding small molecules that can disrupt transcription factor−DNA binding and regulate endogenous gene expression. The covalent linkage of antiparallel Py-Im ring pairs with an γ-amino acid turn unit affords the classical hairpin Py-Im polyamide structure. Closing the hairpin with a second turn unit yields a cyclic polyamide, a lesser-studied architecture mainly attributable to synthetic inaccessibility. We have applied our methodology for solution-phase polyamide synthesis to cyclic polyamides with an improved high-yield cyclization step. Cyclic 8-ring Py-Im polyamides 1−3 target the DNA sequence 5′-WGWWCW-3′, which corresponds to the androgen response element (ARE) bound by the androgen receptor transcription factor to modulate gene expression. We find that cyclic Py-Im polyamides 1−3 bind DNA with exceptionally high affinities and regulate the expression of AR target genes in cell culture studies, from which we infer that the cycle is cell permeable

‘Fixed-axis’ magnetic orientation by an amphibian: non-shoreward-directed compass orientation, misdirected homing or positioning a magnetite-based map detector in a consistent alignment relative to the magnetic field?

Experiments were carried out to investigate the earlier prediction that prolonged exposure to long-wavelength (>500 nm) light would eliminate homing orientation by male Eastern red-spotted newts Notophthalmus viridescens. As in previous experiments, controls held in outdoor tanks under natural lighting conditions and tested in a visually uniform indoor arena under full-spectrum light were homeward oriented. As predicted, however, newts held under long-wavelength light and tested under either full-spectrum or long-wavelength light (>500 nm) failed to show consistent homeward orientation. The newts also did not orient with respect to the shore directions in the outdoor tanks in which they were held prior to testing. Unexpectedly, however, the newts exhibited bimodal orientation along a more-or-less `fixed' north-northeast—south-southwest magnetic axis. The orientation exhibited by newts tested under full-spectrum light was indistinguishable from that of newts tested under long-wavelength light, although these two wavelength conditions have previously been shown to differentially affect both shoreward compass orientation and homing orientation. To investigate the possibility that the `fixed-axis' response of the newts was mediated by a magnetoreception mechanism involving single-domain particles of magnetite, natural remanent magnetism (NRM) was measured from a subset of the newts. The distribution of NRM alignments with respect to the head—body axis of the newts was indistinguishable from random. Furthermore, there was no consistent relationship between the NRM of individual newts and their directional response in the overall sample. However, under full-spectrum, but not long-wavelength, light, the alignment of the NRM when the newts reached the 20 cm radius criterion circle in the indoor testing arena (estimated by adding the NRM alignment measured from each newt to its magnetic bearing) was non-randomly distributed. These findings are consistent with the earlier suggestion that homing newts use the light-dependent magnetic compass to align a magnetite-based `map detector' when obtaining the precise measurements necessary to derive map information from the magnetic field. However, aligning the putative map detector does not explain the fixed-axis response of newts tested under long-wavelength light. Preliminary evidence suggests that, in the absence of reliable directional information from the magnetic compass (caused by the 90° rotation of the response of the magnetic compass under long-wavelength light), newts may resort to a systematic sampling strategy to identify alignment(s) of the map detector that yields reliable magnetic field measurements

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

A behavioral perspective on the biophysics of the light-dependent magnetic compass: a link between directional and spatial perception?

In terrestrial organisms, sensitivity to the Earth's magnetic field is mediated by at least two different magnetoreception mechanisms, one involving biogenic ferromagnetic crystals (magnetite/maghemite) and the second involving a photo-induced biochemical reaction that forms long-lasting, spin-coordinated, radical pair intermediates. In some vertebrate groups (amphibians and birds), both mechanisms are present; a light-dependent mechanism provides a directional sense or 'compass', and a non-light-dependent mechanism underlies a geographical-position sense or 'map'. Evidence that both magnetite- and radical pair-based mechanisms are present in the same organisms raises a number of interesting questions. Why has natural selection produced magnetic sensors utilizing two distinct biophysical mechanisms? And, in particular, why has natural selection produced a compass mechanism based on a light-dependent radical pair mechanism (RPM) when a magnetite-based receptor is well suited to perform this function? Answers to these questions depend, to a large degree, on how the properties of the RPM, viewed from a neuroethological rather than a biophysical perspective, differ from those of a magnetite-based magnetic compass. The RPM is expected to produce a light-dependent, 3-D pattern of response that is axially symmetrical and, in some groups of animals, may be perceived as a pattern of light intensity and/or color superimposed on the visual surroundings. We suggest that the light-dependent magnetic compass may serve not only as a source of directional information but also provide a spherical coordinate system that helps to interface metrics of distance, direction and spatial position.National Science Foundation, USA IOB 06-47188 ; IOB 07-48175info:eu-repo/semantics/publishedVersio

Plans for a 10-m Submillimeter-wave Telescope at the South Pole

A 10 meter diameter submillimeter-wave telescope has been proposed for the NSF Amundsen-Scott South Pole Station. Current evidence indicates that the South Pole is the best submillimeter-wave telescope site among all existing or proposed ground-based observatories. Proposed scientific programs place stringent requirements on the optical quality of the telescope design. In particular, reduction of the thermal background and offsets requires an off-axis, unblocked aperture, and the large field of view needed for survey observations requires shaped optics. This mix of design elements is well-suited for large scale (square degree) mapping of line and continuum radiation from submillimeter-wave sources at moderate spatial resolutions (4 to 60 arcsecond beam size) and high sensitivity (milliJansky flux density levels). the telescope will make arcminute angular scale, high frequency Cosmic Microwave Background measurements from the best possible ground-based site, using an aperture which is larger than is currently possible on orbital or airborne platforms. Effective use of this telescope will require development of large (1000 element) arrays of submillimeter detectors which are background-limited when illuminated by antenna temperatures near 50 K.Comment: 12 pages, 3 figure

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs

Detection of Lymph Node Metastases in Penile Cancer

Penile cancer (PC) is a relatively rare malignancy in the United States (US) but a greater concern in developing nations. Lymph node imaging remains critical to the staging and treatment of this disease as metastases develop in a predictable, anatomic fashion. Early surgical intervention remains a mainstay in treatment and imaging often aids in decision making. This review highlights the indications for imaging in both low-stage and advanced disease. Furthermore, we discuss the benefits and limitations of currently available imaging for staging of inguinal and pelvic lymph nodes in PC and novel modalities in development