High-resolution three-dimensional structural microscopy by single-angle Bragg ptychography.

Coherent X-ray microscopy by phase retrieval of Bragg diffraction intensities enables lattice distortions within a crystal to be imaged at nanometre-scale spatial resolutions in three dimensions. While this capability can be used to resolve structure-property relationships at the nanoscale under working conditions, strict data measurement requirements can limit the application of current approaches. Here, we introduce an efficient method of imaging three-dimensional (3D) nanoscale lattice behaviour and strain fields in crystalline materials with a methodology that we call 3D Bragg projection ptychography (3DBPP). This method enables 3D image reconstruction of a crystal volume from a series of two-dimensional X-ray Bragg coherent intensity diffraction patterns measured at a single incident beam angle. Structural information about the sample is encoded along two reciprocal-space directions normal to the Bragg diffracted exit beam, and along the third dimension in real space by the scanning beam. We present our approach with an analytical derivation, a numerical demonstration, and an experimental reconstruction of lattice distortions in a component of a nanoelectronic prototype device.

Mechanism and dynamics of the reaction of XeF2 with fluorinated Si(100): possible role of gas phase dissociation of a surface reaction product in plasmaless etching.

Xenon difluoride is observed to react with Si-Si sigma-dimer and sigma-lattice bonds of Si(100)2 x 1 at 150 K by single and two atom abstraction at F coverages above 1 ML. As in the limit of zero F coverage, a measurable fraction of the scattered, gas phase product of single atom abstraction, XeF, is sufficiently internally excited to dissociate into F and Xe atoms before detection. Using the XeF internal energy and orientation distributions determined in the limit of zero coverage, the laws of conservation of momentum, energy, and mass are applied to the measured F velocity and angular distributions at higher coverage to simulate the Xe atom velocity and angular distributions and their intensities at higher coverage. The simulation predicts the observed Xe atom velocity and angular distributions at high coverage reasonably well, largely because the exothermicity channeled to XeF remains approximately constant as the coverage increases. This constancy is an opportune consequence of the trade-off between the attractiveness of the potential energy surface as the coverage is increased and the dynamics of the XeF product along the potential surface. The energy, momentum, and mass conservation analysis is also used to distinguish between Xe atoms that arise from XeF gas phase dissociation and Xe atoms that are produced by two atom abstraction. This distinction enables the calculation of percentages of the single and two atom abstraction pathways, as well as the percentages of the two pathways available to the Xe atom produced by two atom abstraction, inelastic scattering, and desorption. Finally, the simulation reveals that between 9% and 12% of F atoms produced by gas phase dissociation of XeF are scattered back toward the surface. These F atoms likely react readily with Si to form the higher fluorides that ultimately lead to etching. Gas phase dissociation of the scattered product of a surface reaction is a novel mechanism to explain the unique reactivity of XeF(2) to etch Si in the absence of a plasma.

Atom abstraction and gas phase dissociation in the interaction of XeF(2) with Si(100).

Xenon difluoride reacts with Si(100)2x1 by single atom abstraction whereby a dangling bond abstracts a F atom from XeF(2), scattering the complementary XeF product molecule into the gas phase, as observed in a molecular beam surface scattering experiment. Partitioning of the available reaction energy produces sufficient rovibrational excitation in XeF for dissociation of most of the XeF to occur. The resulting F and Xe atoms are shown to arise from the dissociation of gas phase XeF by demonstrating that the angle-resolved velocity distributions of F, Xe, and XeF conserve momentum, energy, and mass. Dissociation occurs within 2 A of the surface and within a vibrational period of the excited XeF molecule. Approximately an equal amount of the incident XeF(2) is observed to react by two atom abstraction, resulting in adsorption of a second F atom and scattering of a gas phase Xe atom. Two atom abstraction occurs for those XeF product molecules whose bond axes at the transition state are oriented within +/-60 degrees of the normal and with the F end pointed toward the surface.

An in vitro model system to study gene therapy in the human inner ear.

The confined fluid-filled labyrinth of the human inner ear presents an opportunity for introduction of gene therapy reagents designed to treat hearing and balance dysfunction. Here we present a novel model system derived from the sensory epithelia of human vestibular organs and show that the tissue can survive up to 5 days in vitro. We generated organotypic cultures from 26 human sensory epithelia excised at the time of labyrinthectomy for intractable Meniere's disease or vestibular schwannoma. We applied multiply deleted adenoviral vectors at titers between 10(5) and 10(8) viral particles/ml directly to the cultures for 4-24 h and examined the tissue 12-96 h post-transfection. We noted robust expression of the exogenous transgene, green fluorescent protein (GFP), in hair cells and supporting cells suggesting both were targets of adenoviral transfection. We also transfected cultures with a vector that carried the genes for GFP and KCNQ4, a potassium channel subunit that causes dominant-progressive hearing loss when mutated. We noted a positive correlation between GFP fluorescence and KCNQ4 immunolocalization. We conclude that our in vitro model system presents a novel and effective experimental paradigm for evaluation of gene therapy reagents designed to restore cellular function in patients who suffer from inner ear disorders.

Dissociation of a product of a surface reaction in the gas phase: XeF2 reaction with Si.

Xenon difluoride interacts with Si(100)2 x 1 by atom abstraction, whereby a dangling bond abstracts a F atom from XeF2, scattering the complementary XeF. Partitioning of the reaction exothermicity produces sufficient XeF rovibrational excitation for dissociation to occur. The resulting F and Xe atoms are shown to arise from dissociation of XeF in the gas phase by demonstrating that the angle-resolved velocity distributions of F, Xe, and XeF conserve momentum, energy, and mass. This experiment documents the first observation of dissociation of a surface reaction product in the gas phase.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Two mechanisms for transducer adaptation in vertebrate hair cells.

Deflection of the hair bundle atop a sensory hair cell modulates the open probability of mechanosensitive ion channels. In response to sustained deflections, hair cells adapt. Two fundamentally distinct models have been proposed to explain transducer adaptation. Both models support the notion that channel open probability is modulated by calcium that enters via the transduction channels. Both also suggest that the primary effect of adaptation is to shift the deflection-response [I(X)] relationship in the direction of the applied stimulus, thus maintaining hair bundle sensitivity. The models differ in several respects. They operate on different time scales: the faster on the order of a few milliseconds or less and the slower on the order of 10 ms or more. The model proposed to explain fast adaptation suggests that calcium enters and binds at or near the transduction channels to stabilize a closed conformation. The model proposed to explain the slower adaptation suggests that adaptation is mediated by an active, force-generating process that regulates the effective stimulus applied to the transduction channels. Here we discuss the evidence in support of each model and consider the possibility that both may function to varying degrees in hair cells of different species and sensory organs.

Harvesting human hair cells.

The sensory hair cells of the inner ear are responsible for converting balance and hearing stimuli into electrical signals. Until recently, all previous studies of hair cell physiology had been performed on tissue obtained from non-mammals and rodents. In primates, hair cells are difficult to access, because they rest within the densest structure of the body, the otic capsule of the temporal bone. In this report, we describe a technique that we have used in physiological studies to harvest living human hair cells. We collected vestibular and cochlear tissue specimens from adult humans undergoing translabyrinthine and transotic surgical approaches for resection of lateral skull base tumors. Viable hair cells were identified and visualized with light microscopy. The ability to study normal hair cells from humans may further the study of normal and pathological human sensation, hair cell regeneration, and genetic causes of balance and hearing disorders.

Stimulus processing by type II hair cells in the mouse utricle.

In type II and neonatal hair cells in the mouse utricle, the receptor potentials evoked by low-frequency sinusoidal deflections of the hair bundle are attenuated by adaptation of the mechanoelectrical transduction current and the voltage-dependent activation of a large potassium (K)-selective outwardly rectifying conductance, gDR. These processes may contribute to high-pass filtering of the responses of some utricular afferents to sinusoidal linear accelerations below 2 Hz. Depolarizing receptor potentials are more attenuated by gDR than are hyperpolarizing receptor potentials. It may therefore reduce nonlinear distortion introduced by mechanoelectrical transduction, which generates larger depolarizing currents than hyperpolarizing currents. The discharge properties of utricular afferents vary according to whether they innervate the striolar or extrastriolar zones of the sensory epithelium. Regional variation in hair-cell properties is likely to contribute. Preliminary results suggest that the outwardly rectifying K conductances of type II cells are slower and larger in the striola than in the extrastriola, consistent with regional variation in the relative numbers of delayed rectifier and A-current K channels.

Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.

To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function.

Ionic currents and electromotility in inner ear hair cells from humans.

The upright posture and rich vocalizations of primates place demands on their senses of balance and hearing that differ from those of other animals. There is a wealth of behavioral, psychophysical, and CNS measures characterizing these senses in primates, but no prior recordings from their inner ear sensory receptor cells. We harvested human hair cells from patients undergoing surgical removal of life-threatening brain stem tumors and measured their ionic currents and electromotile responses. The hair cells were either isolated or left in situ in their sensory epithelium and investigated using the tight-seal, whole cell technique. We recorded from both type I and type II vestibular hair cells under voltage clamp and found four voltage-dependent currents, each of which has been reported in hair cells of other animals. Cochlear outer hair cells demonstrated electromotility in response to voltage steps like that seen in rodent animal models. Our results reveal many qualitative similarities to hair cells obtained from other animals and justify continued investigations to explore quantitative differences that may be associated with normal or pathological human sensation.

Mechanoelectrical transduction and adaptation in hair cells of the mouse utricle, a low-frequency vestibular organ.

Hair cells of inner ear organs sensitive to frequencies above 10 Hz adapt to maintained hair bundle deflections at rates that reduce their responses to lower frequencies. Mammalian vestibular organs detect head movements at frequencies well below 10 Hz. We asked whether hair cells of the mouse utricle adapt, and if so, whether the adaptation was similar to that in higher frequency organs such as the frog saccule. Whole-cell transduction currents were recorded from hair cells in the epithelium of the mouse utricle. Hair bundles were deflected by a fluid jet or a stiff probe. The transduction currents evoked by step deflections adapted over 10-100 msec. The mean operating range was 1.5 micron (deflection of the tip of the bundle), approximately threefold larger than in frog saccule. Taller and more compact bundles of the mouse utricle account for this difference. As in frog saccular hair cells, adaptation shifted the current-deflection (I(X)) relation along the deflection axis. These adaptive shifts had time constants of 10-20 msec and reached 60-80% of stimulus amplitude. The adaptive shift and voltage-dependent bundle movement are consistent with the motor model of adaptation. When the fluid jet was used, adaptation also broadened the I(X) relation and reduced the maximum current. Adaptation attenuated the transduction currents evoked by sinusoidal bundle deflections below 5 Hz, within the frequency range of the utricle, but because it was incomplete, substantial responses remained. Moreover, the adaptive shift mechanism preserves sensitivity even in the presence of large stimuli that would otherwise saturate transduction.

Lack of correlation between phenotype and genotype for the polymorphically expressed dihydropyrimidine dehydrogenase in a family of Pakistani origin.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in pyrimidine catabolism. DPD deficiency is associated with an increased risk of toxicity in cancer patients receiving 5-fluorouracil (5-PU) treatment. DPD deficiency causes an inborn error of metabolism called thymine-uraciluria that is in some instances associated with convulsive disorders and developmental delay in children. We have studied the molecular mechanism accounting for DPD deficiency in a Pakistani pedigree having 2-year-old child with thymine-uraciluria and exhibiting some degree of motor impairment and developmental delay. A common splice mutation was found in the patient's dihydropyrimidine dehydrogenase (DPYD) gene that produces a mutant mRNA resulting in the complete lack of DPD protein and activity in lymphocytes and primary fibroblast. This trait segregated in the family following a typical Mendelian distribution. Surprisingly, the patient's brother also had thymine-uraciluria and was homozygous for the splicing mutation but was clinically asymptomatic. Sequence tagged sites (STS) linkage analyses within 5 megabases of telomeric and centromeric DNA surrounding the DPYD gene revealed no allelic polymorphism between the two brothers. These results suggest that DPD deficiency might not be the only cause of the more severe clinical phenotypes observed in certain thymine-uraciluria patients and that an incomplete correlation between phenotype and genotype is present in the population.

Calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links.

Mechanically gated "transduction" channels in inner ear hair cells are thought to be connected to tip links stretched between adjacent stereocilia. To locate active channels, calcium-green fluorescence in single stereocilia was measured with two-photon laser scanning microscopy. Bundle deflection increased fluorescence in many but not all stereocilia; the increase was blocked by depolarization. The number of stereocilia responding was proportional to the transduction current, consistent with Ca2+ influx through transduction channels. Fluorescence rose first in the tips of stereocilia and then in the bases, in agreement with channel localization at the tips. Some of the shortest stereocilia in a bundle showed a fluorescence increase, as did some of the tallest, indicating that transduction channels can be at either or both ends of tip links.

Inwardly rectifying currents of saccular hair cells from the leopard frog.

1. Inwardly rectifying currents were characterized in sensory hair cells isolated from the saccules of leopard frogs, using the whole cell configuration of the patch-clamp technique in voltage-clamp mode. 2. Two types of inwardly rectifying currents were distinguishable based on their ionic selectivity, activation and deactivation kinetics, voltage dependence, dependence on external K+ and sensitivity to divalent cations. 3. One inwardly rectifying current displayed K+ selectivity, rapid monoexponential activation (tau approximately 1 ms at -120 mV), steep voltage dependence, dependence of the activation voltage range on external K+ and block by external Ba2+. We refer to this current as IK1, consistent with the terminology used for a similar current in cardiac cells. In 5 mM external K+, IK1 activated negative to -60 mV, was half-activated at -86 mV and fully activated by -110 mV. 4. The other inwardly rectifying current was a mixed K+/Na+ current with slow sigmoidal activation (slow tau approximately 100 ms at -120 mV) and deactivation, shallow voltage dependence and no dependence of the activation curve on external K+ and which was blocked by external Cd2+. This current was called Ih because of its similarities to Ih of photoreceptors. Ih activated negative to -50 mV, was half-activated at -90 mV and was fully activated at -130 mV. 5. A correlation between cell shape and the type of inwardly rectifying current was noted; the more spherical cells had Ih alone and the more cylindrically shaped cells had Ih and IK1. 6. The mean resting potential of 115 cells with IK1 and Ih was -68 +/- 0.5 mV (mean +/- SE) and that of 53 cells with Ih alone was -50 +/- 0.5 mV. This suggests that IK1 contributes to the more negative resting potential of the cylindrical cells. 7. In current-clamp mode, the voltage responses to current steps of the two cell populations differed. Small negative current steps evoked faster, smaller responses in cells with IK1 and Ih than in cells with Ih alone. In cells with Ih alone, long (> 100 ms) negative current steps evoked a hyperpolarization that partly repolarized as Ih activated. Cells with Ih alone showed electrical resonance at rest whereas cells with IK1 resonated only in response to positive current steps. 8. A model developed to explain electrical resonance in bullfrog saccular hair cells was adapted to include Ih or IK1 and Ih.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterologous regulation of inositol lipid hydrolysis in human breast cancer cells by oestradiol 17 beta, bombesin and fluoroaluminate.

Inositol lipid turnover has been implicated in the action of oestradiol 17 beta and bombesin-related peptides on the human breast cancer cell line MCF-7. In the present study, in addition to measuring inositol lipid turnover as indicated by inositol monophosphate (IP) accumulation, we have also monitored the effect of oestradiol on the incorporation of both 3H-inositol and 14C-glycerol into MCF-7 cell phospholipids. Pre-treatment of MCF-7 cells with oestradiol (10 nM) for 48 hr stimulated a 4.3-fold increase in IP production. This was similarly accompanied by a 3.4-fold increase in the incorporation of 3H-inositol into total phosphoinositides and a 40% increase in cell growth. The oestrogen antagonist LYI 17018 completely attenuated these effects. Oestradiol also stimulated 14C-glycerol incorporation into phosphatidyl inositol, -choline and -ethanolamine by 97%, 82% and 99%, respectively. IP production in response to bombesin was potentiated by oestradiol in a dose-dependent fashion. Fluoroaluminate (AlF4-) stimulated a dose-dependent increase in IP production and oestradiol pre-treatment increased the sensitivity of this IP response to AlF4-. Medroxyprogesterone acetate inhibited bombesin-stimulated IP production but had no effect on the response to AlF4-. Our data suggest that the oestrogenic action on basal IP production in MCF-7 cells may reflect an effect on inositol lipid synthesis rather than turnover. However, the potentiation by oestradiol of both bombesin- and AlF4(-)-stimulated inositol lipid hydrolysis suggests the operation of a post-receptor regulatory mechanism(s) which is independent of the inositol lipid pool size.

Interaction of paracrine factors during labour: interleukin-1 beta causes amplification of decidua cell prostaglandin F2 alpha production in response to bradykinin and epidermal growth factor.

Inflammatory mediators have been implicated in the stimulation of decidual prostaglandin (PG) production during infection-driven preterm labour. The aim of the present study is to investigate a potential interaction between interleukin-1 beta (IL-1) and bradykinin (BK) in the regulation of decidual PGF2 alpha production and PG precursor release. Pretreatment of primary decidua cell cultures with IL-1 significantly enhanced PGF2 alpha production in response to BK. This effect was accompanied by an increase in unesterified arachidonic acid and an enhanced turnover of arachidonoyl phosphatidate. Bradykinin also stimulated arachidonic acid release in decidual fibroblasts, an effect which was potentiated in the presence of epidermal growth factor (EGF), but which was not accompanied by an increase in PGF2 alpha production. Decidual fibroblasts pretreated with IL-1 manifest a 10-fold increase in PGF2 alpha production in response to BK and EGF. These observations provide the first description of synergism between cytokine, kinin and growth factor in the uterine decidua which may partly mediate the in vivo increase in prostaglandin production associated with decidual activation and infection-driven labour.

Human decidua is a target tissue for bradykinin and kallikrein: phosphoinositide hydrolysis accompanies arachidonic acid release in uterine decidua cells in vitro.

The endocrine and intracellular mechanisms regulating prostaglandin precursor release in the uterine decidua during labor are unknown. This in vitro study investigates a potential role for a kallikrein-kinin system in the activation of phospholipid hydrolysis and arachidonic acid release in human decidua cells. Primary cultures of human decidua cells were prelabeled with [3H]inositol or [14C]arachidonic acid to monitor phosphoinositide hydrolysis and prostaglandin precursor release, respectively. Bradykinin (100 nmol/L) stimulated a rapid release of arachidonic acid (within 2 min) associated with an increase in inositol trisphosphate which was detectable after 20 s. Protein kinase C activation by phorbol ester enhanced arachidonic acid release in response to both bradykinin and the Ca++ ionophore A23187 but inhibited bradykinin-stimulated phosphoinositide hydrolysis. Epidermal growth factor also enhanced arachidonate release in response to both bradykinin and A23187. Kallikrein stimulated both phosphoinositide hydrolysis and arachidonic acid release in decidua cells. Kallikrein action was inhibited by the kallikrein protease inhibitor aprotinin and D-Arg[Hyp3Thi5,8,D-Phe7] bradykinin, a B2 receptor antagonist. Bradykinin also stimulated prostaglandin F2 alpha production in both primary decidua cell cultures and fibroblasts in the presence of interleukin-1 beta. These findings are consistent with a mediatory role for bradykinin in the action of kallikrein on decidua cells and suggest that inositol phospholipid hydrolysis is instrumental for arachidonic acid release in response to bradykinin in these cells. This study supports a novel role for a kallikrein-kinin system in the human uterine decidua.