The Hierarchy of Proinflammatory Cytokines in Ocular Inflammation.

PURPOSE: The concept of tissue-dependent cytokine hierarchy has been demonstrated in a number of diseases, but it has not been investigated in ophthalmic diseases. Here, we evaluated the functional hierarchy of interleukin-1ß (IL-1ß), IL-6, IL-17A, and tumor necrosis factor (TNF) in the induction of ocular inflammation. MATERIALS AND METHODS: We delivered adeno-associated virus (AAV) vectors expressing IL-1ß, IL-6, IL-17A, or TNF intravitreally in naïve C57/BL6 mice and compared and contrasted the inflammatory effects in the eye 5 weeks after AAV-mediated gene transfer. We also used an in vitro human system to test the effect of cytokines on barrier function. RESULTS: We found that IL-1ß had the highest ability to initiate ocular inflammation. The continuous overexpression of IL-1ß resulted in a significant upregulation of additional proinflammatory mediators in the eye. Using scanning laser ophthalmoscope and optical coherence tomography imaging techniques, we showed that a low dose of AAVIL-1ß was sufficient and was as pathogenic as a high dose of TNF in inducing vascular leakage, retinal degeneration, and cellular infiltration. Furthermore, only a marginal increase in IL-1ß was enough to cause cellular infiltration, thus confirming the highly pathogenic nature of IL-1ß in the eye. Contrary to our expectation, IL-6 or IL-17A had minimal or no effect in the eye. To examine the clinical relevance of our findings, we used an impedance assay to show that IL-1ß alone or TNF alone was able to cause primary human retinal endothelial cell barrier dysfunction in vitro. Again, IL-6 alone or IL-17A alone had no effect on barrier function; however, in the presence of IL-1ß or TNF, IL-17A but not IL-6 may provide additive proinflammatory effects. CONCLUSIONS: Our studies demonstrate the existence of a functional hierarchy of proinflammatory cytokines in the eye, and we show that IL-1ß is the most pathogenic when it is continuously expressed in the eye.

Evidence of protein-free homology recognition in magnetic bead force-extension experiments.

Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data.

Single molecule detection of direct, homologous, DNA/DNA pairing.

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the "default" option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

Demonstration that the shear force required to separate short double-stranded DNA does not increase significantly with sequence length for sequences longer than 25 base pairs.

We have measured the shear force for short double-stranded DNA sequences pulled by either the 3'3' or 5'5' ends and find that the shear force is independent of the pulling technique. For the 50% GC sequences examined, the force is a linear function of DNA length up to 20 base pairs (bp); however, we show that, as predicted by deGennes, the shear force approaches an asymptotic value in the limit where the number of base pairs approaches infinity, where the shear force for a 32 bp sequence is within 5% of the asymptotic value of 61.4 pN . Fits to deGennes' theory suggest that the shear force is distributed over fewer than 10 bp at the end of the sequence, with the rest of the sequence experiencing negligible shear force. The single base pair rupture force and the ratio of the backbone spring constant to the base pair spring constant determined from fits of the data to deGennes' theory are consistent with ab initio predictions.

Optical bistability at low light level due to collective atomic recoil.

We demonstrate optical nonlinearities due to the interaction of weak optical fields with the collective motion of a strongly dispersive ultracold gas. The combination of a recoil-induced resonance in the high gain regime and optical waveguiding within the dispersive medium enables us to achieve a collective atomic cooperativity of 275+/-50 even in the absence of a cavity. As a result, we observe optical bistability at input powers as low as 20 pW. The present scheme allows for dynamic optical control of the dispersive properties of the ultracold gas using very weak pulses of light. The experimental observations are in good agreement with a theoretical model.

Meiotic chromosomes move by linkage to dynamic actin cables with transduction of force through the nuclear envelope.

Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase telomere-led chromosome motion in budding yeast. Diverse findings reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed.

Measurement of the salt-dependent stabilization of partially open DNA by Escherichia coli SSB protein.

The rezipping force of two complementary DNA strands under tension has been measured in the presence of Escherichia coli single-stranded-binding proteins under salt conditions ranging from 10- to 400 mM NaCl. The effectiveness of the binding protein in preventing rezipping is strongly dependent on salt concentration and compared with the salt dependence in the absence of the protein. At concentrations less than 50 mM NaCl, the protein prevents complete rezipping of lambda-phage on the 2-s timescale of the experiment, when the ssDNA is under tensions as low as 3.5 +/- 1 pN. For salt concentrations greater than 200 mM NaCl, the protein inhibits rezipping but cannot block rezipping when the tension is reduced below 6 +/- 1.8 pN. This change in effectiveness as a function of salt concentration may correspond to salt-dependent changes in binding modes that were previously observed in bulk assays.

Direct measurements of the stabilization of single-stranded DNA under tension by single-stranded binding proteins.

The unzipping and rezipping of a double-stranded DNA molecule is carried out in the presence of two single-stranded binding proteins T4 gp32 and E. Coli SSB protein to determine the effect of the proteins on the stability of single- and double-stranded DNA. The proteins do not have a significant effect on unzipping, indicating that the two proteins do not destabilize the double-stranded DNA; however, both proteins inhibit rezipping. At protein concentrations where the rezipping force response is saturated, E. Coli SSB protein reduces the rezipping force to 5.5+/-1.5 pN , while T4 gp32 completely blocks rezipping on the time scale of the experiment.

Measurements of the hysteresis in unzipping and rezipping double-stranded DNA.

Complete unzipping and rezipping of lambda -phage double-stranded DNA is achieved by applying a constant force. A strong hysteresis is observed at all tested time scales and temperatures. Hysteresis also occurs for partial unzipping, indicating stability for the partially open state over a force range of 2- 5pN . Results are compared to nearest-neighbor model simulations, and reasonable agreement is found.

Effects of temperature on the mechanical properties of single stranded DNA.

We present the first measurements of the temperature dependent extension of single stranded DNA. At forces between 2 and 10 pN the extension increases with temperature. This increase in extension is consistent with the disruption of hairpins, and a simple theory that includes hairpin formation shows good agreement with the data at these low forces. In contrast, at forces above 10 pN and temperatures higher than 40 degrees C, the extension decreases rapidly with temperature in a manner not consistent with predictions.

Long phase coherence time and number squeezing of two Bose-Einstein condensates on an atom chip.

We measure the relative phase of two Bose-Einstein condensates confined in a radio frequency induced double-well potential on an atom chip. We observe phase coherence between the separated condensates for times up to approximately 200 ms after splitting, a factor of 10 longer than the phase diffusion time expected for a coherent state for our experimental conditions. The enhanced coherence time is attributed to number squeezing of the initial state by a factor of 10. In addition, we demonstrate a rotationally sensitive (Sagnac) geometry for a guided atom interferometer by propagating the split condensates.

Comparison of the measured phase diagrams in the force-temperature plane for the unzipping of two different natural DNA sequences.

In this work, we consider the critical force required to unzip two different naturally occurring sequences of double-stranded DNA (dsDNA) at temperatures ranging from 20 degrees C to 50 degrees C, where one of the sequences has a 53% average guanine-cytosine (GC) content and the other has a 40% GC content. We demonstrate that the force required to separate the dsDNA of the 53% GC sequence into single-stranded DNA (ssDNA) is approximately 0.5 pN, or approximately 5% greater than the critical force required to unzip the 40% GC sequence at the same temperature. In the temperature range between 20 and 40 degrees C the measured critical forces correspond reasonably well to predictions based on a simple theoretical homopolymeric model, but at temperatures above 40 degrees C the measured critical forces are much smaller than the predicted forces. The correspondence between theory and experiment is not improved by using Monte Carlo simulations that consider the heteropolymeric nature of the sequences.

Pause point spectra in DNA constant-force unzipping.

Under constant applied force, the separation of double-stranded DNA into two single strands is known to proceed through a series of pauses and jumps. Given experimental traces of constant-force unzipping, we present a method whereby the locations of pause points can be extracted in the form of a pause point spectrum. A simple theoretical model of DNA constant-force unzipping is presented, which generates theoretical pause point spectra through Monte Carlo simulation of the unzipping process. The locations of peaks in the experimental and theoretical pause point spectra are found to be nearly coincident below 6000 basepairs for unzipping the bacteriophage lambda-genome. The model only requires the sequence, temperature, and a set of empirical basepair binding and stacking energy parameters, and the good agreement with experiment suggests that pause point locations are primarily determined by the DNA sequence. The model is also used to predict pause point spectra for the bacteriophage phi X174 genome. The algorithm for extracting the pause point spectrum might also be useful for studying related systems which exhibit pausing behavior such as molecular motors.

Dynamical instability of a doubly quantized vortex in a Bose-Einstein condensate.

Doubly quantized vortices were topologically imprinted in /F=1> 23Na condensates, and their time evolution was observed using a tomographic imaging technique. The decay into two singly quantized vortices was characterized and attributed to dynamical instability. The time scale of the splitting process was found to be longer at higher atom density.

Measurement of the phase diagram of DNA unzipping in the temperature-force plane.

We separate double stranded lambda phage DNA by applying a fixed force at a constant temperature ranging from 15 to 50 degrees C, and measure the minimum force required to separate the two strands. The measurements also offer information on the free energy of double stranded DNA (dsDNA) at temperatures where dsDNA does not thermally denature in the absence of force. While parts of the phase diagram can be explained using existing models and free energy parameters, others deviate significantly. Possible reasons for the deviations between theory and experiment are considered.

Coherent matter-wave manipulation in the diabatic limit.

Guided systems for coherent matter waves are expected to offer substantial improvements over unguided systems, but adiabatic coupler proposals have proven difficult to realize. We outline instead considerations for a coherence-preserving diabatic approach enabling filters, couplers, and interferometers that can accept multimode guide inputs of up to magneto-optical-trap temperatures.

Enhancement of phase space density by increasing trap anisotropy in a magneto-optical trap with a large number of atoms.

The phase space density of dense, cylindrical clouds of atoms in a 2D magneto-optic trap is investigated. For a large number of trapped atoms (>10(8)), the density of a spherical cloud is limited by photon reabsorption. However, as the atom cloud is deformed to reduce the radial optical density, the temperature of the atoms decreases due to the suppression of multiple scattering leading to an increase in the phase space density. A density of 2 x 10(-4) has been achieved in a magneto-optic trap containing 2 x 10(8) atoms.

Measuring the forces involved in polyvalent adhesion of uropathogenic Escherichia coli to mannose-presenting surfaces.

Mechanisms of bacterial pathogenesis have become an increasingly important subject as pathogens have become increasingly resistant to current antibiotics. The adhesion of microorganisms to the surface of host tissue is often a first step in pathogenesis and is a plausible target for new antiinfective agents. Examination of bacterial adhesion has been difficult both because it is polyvalent and because bacterial adhesins often recognize more than one type of cell-surface molecule. This paper describes an experimental procedure that measures the forces of adhesion resulting from the interaction of uropathogenic Escherichia coli to molecularly well defined models of cellular surfaces. This procedure uses self-assembled monolayers (SAMs) to model the surface of epithelial cells and optical tweezers to manipulate the bacteria. Optical tweezers orient the bacteria relative to the surface and, thus, limit the number of points of attachment (that is, the valency of attachment). Using this combination, it was possible to quantify the force required to break a single interaction between pilus and mannose groups linked to the SAM. These results demonstrate the deconvolution and characterization of complicated events in microbial adhesion in terms of specific molecular interactions. They also suggest that the combination of optical tweezers and appropriately functionalized SAMs is a uniquely synergistic system with which to study polyvalent adhesion of bacteria to biologically relevant surfaces and with which to screen for inhibitors of this adhesion.

Guiding neutral atoms on a chip

We demonstrate the guiding of neutral atoms by the magnetic fields due to microfabricated current-carrying wires on a chip. Atoms are guided along a magnetic field minimum parallel to and above the current-carrying wires. Two guide configurations are demonstrated: one using two wires with an external magnetic field, and a second using four wires without an external field. These guide geometries can be extended to integrated atom optics circuits, including beam splitters.

Localization of metastable atom beams with optical standing waves: nanolithography at the heisenberg limit

The spatially dependent de-excitation of a beam of metastable argon atoms, traveling through an optical standing wave, produced a periodic array of localized metastable atoms with position and momentum spreads approaching the limit stated by the Heisenberg uncertainty principle. Silicon and silicon dioxide substrates placed in the path of the atom beam were patterned by the metastable atoms. The de-excitation of metastable atoms upon collision with the surface promoted the deposition of a carbonaceous film from a vapor-phase hydrocarbon precursor. The resulting patterns were imaged both directly and after chemical etching. Thus, quantum-mechanical steady-state atom distributions can be used for sub-0.1-micrometer lithography.