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Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and 'bulging out' morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.

We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.

This paper focusses on the crosslink density and solvent-polymer interaction parameter of uncharged PMAA membranes. Crosslink densities and the solvent-polymer interaction parameter were obtained for a series of PMAA membranes which differed only in the ratio of crosslinker to monomer at the time of polymerization and crosslinking. Tensile force versus elongation, as well as polymer volume fraction in both the relaxed state (i.e. at the time of polymerization and crosslinking) and the swollen state (but not ionized - e.g. pH 3, 0.1M KCl) were measured. The crosslink density and interaction parameter for each of the membranes was calculated from the tensile force and polymer volume fraction data, using the method of Meissner and Janacek. The results were used to relate crosslink density to membrane permeability, which had previously been measured as a function of membrane stochiometry. For swollen state polymer volume fractions v s between 0.251 and 0.377 at 23.3°C, we found that the interaction parameter χ 1 could be approximated by a quadratic dependence on υ s . The χ 1 values obtained in this study are consistent with literature values for PMAA which had been polymerized and crosslinked in solutions; this χ 1 , can be used in the Flow swelling equation to calculate the crosslink density of PMAA gels whose equilibrium polymer volume fractions are known to be within the range spanned by the tensile measurements. However, the extreme sensitivity of the calculated crosslink density to small changes in χ 1 makes it difficult to use the swelling equation to calculate crosslink density without tensile measurements that cover the polymer volume fraction range of interest.

This paper focuses on the relationship between diffusive transport and membrane composition (crosslinker content and relaxed polymer volume fraction) in membranes whose permeability can be actively controlled by chemical or electrical stimuli. First, pH induced changes in permeability to uncharged fluorescent solutes were measured. Then, by correlating bath pH with membrane hydration, the transport properties of membranes of different crosslinker content and/or relaxed polymer volume fraction were compared at constant hydration. The membrane permeability was found to decrease as the amount of crosslinker added to the membrane formulation at the time of polymerization increased, while the permeability increased as the solvent content during polymerization increased. A free volume theory which was fit to the data shows good agreement, and predicts a monotonically decreasing porosity factor with increased crosslinker content.

The laser performance of rare earth doped crystals and glasses can be significantly improved by codoping with an additional ion which absorbs the pump light and efficiently transfers the energy to the lasing rare earth ion. It has been found recently that a fast energy transfer can occur at short times inconsistent with the standard Forster-Dexter model. Such anomalous behaviour may be due to additional mechanisms such as physical pairing of donors and acceptors resulting in additional short-range interactions which increase the transfer rate between donors and acceptors. The models developed are compared to the experimental results of energy transfer between Mn(II) and Tm(III) and Mn(II) and Nd(III) in fluoride glass.

Phase retrieval is a well-established method for the recovery of an object wave from its magnitude-only measurements, with applications in fields such as material science, biology, and astronomy. In order to guarantee a stable and global solution for phase retrieval, measurement diversity is frequently used. However, this process requires taking several measurements, which hinders some of the advantages of phase retrieval compared with interferometric techniques. Here we propose a novel diversity framework that we refer to as digital resampling diversity. The proposed resampling diversity along with sparsity-constrained object reconstruction enables improved object reconstruction from a single squared magnitude image, without using object support constraint. We demonstrate this framework with simulation and experimental results.

In this paper, we present a new approach providing super resolved images exceeding the geometrical limitation given by the detector pixel size of the imaging camera. The concept involves the projection of periodic patterns on top of the sample, which are then investigated under a microscope. Combining spatial scanning together with proper digital post-processing algorithm yields the improved geometrical resolution enhancement. This new method is especially interesting for microscopic imaging when the resolution of the detector is lower than the resolution due to diffraction.

In this manuscript we present a novel super resolving approach based upon projection of a random speckle pattern onto samples observed through a microscope. The projection of the speckle pattern is created by coherent illumination of the inspected pattern through a diffuser. Due to local interference of the coherent wave front with itself, a random speckle pattern is superimposed on the sample. This speckle pattern can be scanned over the object. A super-resolved image can be extracted from a temporal sequence of images by appropriate digital processing of the image stream. The resulting resolution is significantly higher than the diffraction limitation of the microscope objective. The new super-resolution method is demonstrated by application to fluorescence of biological samples.

The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time.

Steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein that enhances steroid synthesis by facilitating the transfer of cholesterol to the inner membranes of mitochondria in hormonally regulated steroidogenic cells. It is currently assumed that StAR activity commences before or during StAR import into the mitochondrial matrix. The present study was designed to demonstrate that, once imported and becoming physiologically irrelevant, exhaustive accumulation of StAR must be limited by a rapid degradation of the protein to prevent potential damage to the organelles. The use of uncouplers and manipulation of the interior mitochondrial pH in hormone-induced ovarian granulosa cells and StAR-expressing COS cells suggests that StAR degradation is biphasic and involves two classes of proteases. During phase I, which normally lasts for the first approximately 2 h following import, StAR is rapidly degraded by a protease, or proteases, that can be arrested by a nonclassical action of proteasome inhibitors such as MG132. StAR molecules that evade phase I are subjected to a second class of protease(s), which is slower and MG132 resistant. A third proteolytic entity was revealed in studies with C-28 StAR, a loss-of-function mutant of StAR. Upon initiation of its import, C-28 StAR dissipates the inner membrane potential and causes swelling of the mitochondria. Degradation of C-28 StAR, probably by an intermembrane space protease, is extremely rapid and MG132 insensitive. Collectively, this study defines StAR as the first naturally occurring mitochondrial protein that can serve as a substrate to probe multiple proteolytic activities in mammalian mitochondria.

Tunable solid-state dye lasers can be prepared by incorporation of the dye during sol-gel formation. The current work presents measurements of dye photostability which were taken during laser operation over an extended time.. Fluorescence decay (photobleaching) rate was compared for the case of laser operation to the case where laser operation was suppressed, using identical excitation geometries. In addition, the confocal photobleaching experiments were repeated, using sol-gel glass, composite glass/polymer, and PMMA polymer host matrices.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

It has been proposed that the steroidogenic acute regulatory (StAR) protein controls hormone-stimulated steroid production by mediating cholesterol transfer to the mitochondrial inner membrane. This study was conducted to determine the effect of wild-type StAR and several modified forms of StAR on intramitochondrial cholesterol transfer. Forty-seven N-terminal or 28 C-terminal amino acids of the StAR protein were removed, and COS-1 cells were transfected with pCMV vector only, wild-type StAR, N-47, or the C-28 constructs. Lysates from the transfected COS-1 cells were then incubated with mitochondria from MA-10 mouse Leydig tumor cells that were preloaded with [3H]cholesterol. After incubation, mitochondria were collected and fractionated on sucrose gradients into outer membranes, inner membranes, and membrane contact sites, and [3H]cholesterol content was determined in each membrane fraction. Incubation of MA-10 mitochondria with wild-type StAR containing cell lysate resulted in a significant 34.9% increase in [3H]cholesterol content in contact sites and a significant 32.8% increase in inner mitochondrial membranes. Incubations with cell lysate containing N-47 StAR protein also resulted in a 16.4% increase in [3H]cholesterol in contact sites and a significant 26.1% increase in the inner membrane fraction. In contrast, incubation with the C-28 StAR protein had no effect on cholesterol transfer. The cholesterol-transferring activity of the N-47 truncation, in contrast to that of the C-28 mutant, was corroborated when COS-1 cells were cotransfected with F2 vector (containing cytochrome P450 side-chain cleavage enzyme, ferridoxin, and ferridoxin reductase) and either pCMV empty vector or the complementary DNAs of wild-type StAR, N-47 StAR, or C-28 StAR. Pregnenolone production was significantly increased in both wild-type and N-47-transfected cells, whereas that in C-28-transfected cells was similar to the control value. Finally, immunolocalization studies with confocal image and electron microscopy were performed to determine the cellular location of StAR and its truncated forms in transfected COS-1 cells. The results showed that wild-type and most of the C-28 StAR protein were imported into the mitochondria, whereas most of N-47 protein remained in the cytosol. These studies demonstrate a direct effect of StAR protein on cholesterol transfer to the inner mitochondrial membrane, that StAR need not enter the mitochondria to produce this transfer, and the importance of the C-terminus of StAR in this process.

Common fragile sites are chromosomal loci prone to breakage and rearrangement, hypothesized to provide targets for foreign DNA integration. We cloned a simian virus 40 integration site and showed by fluorescent in situ hybridization analysis that the integration event had occurred within a common aphidicolin-induced fragile site on human chromosome 7, FRA7H. A region of 161 kb spanning FRA7H was defined and sequenced. Several regions with a potential unusual DNA structure, including high-flexibility, low- stability, and non-B-DNA-forming sequences were identified in this region. We performed a similar analysis on the published FRA3B sequence and the putative partial FRA7G, which also revealed an impressive cluster of regions with high flexibility and low stability. Thus, these unusual DNA characteristics are possibly intrinsic properties of common fragile sites that may affect their replication and condensation as well as organization, and may lead to fragility., link_to_OA_fulltext

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

A photoacoustic spectrometer is described which has been constructed to measure the spectra of photovoltaic materials in the ultra violet, visible and near infra red regions. The range of this photoacoustic spectrometer is from 0.3 to 1.8 micrometers. This technique has the advantage of being non- intrusive and non-destructive. Measurements have been made of a variety of photovoltaic materials, including a CdS/CuIn 1- x Ga x Se 2 heterojunction and an InP/InGaAsP quantum well layer system. Bands arising from intrinsic impurity subgap levels were observed beyond the band edges.

A novel two dimensional optical position sensor based on confinement of luminescence within a flat panel is described. The basis of the sensor is a flat luminescent panel, such as those used in luminescent solar energy concentrators. In these concentrators, a glass or plastic plate is either coated or impregnated with a fluorescent material. The incident radiation produces luminescent emission, much of which is confined within the plate through total internal reflection, and is thus guided to solar cells which line the thin edges of the plate. In order to use such plates as a position sensor, four photodiodes are placed along two orthogonal axes at the edges of the plate, and the plate is illuminated with a spot of light. The spot of light induces luminescence, which is detected by the four photodiodes. The photodiodes' output is processed to provide position information. Large area position sensors based on the above idea were constructed, using fluorescent plexiglass which was obtained from commonly available fluorescent clipboards. The photodiode output was combined using a self-normalizing function to eliminate sensitivity to non-uniformity in the optical characteristics of the fluorescent plates. This paper presents the results obtained to date, together with suggestions for further improvement in the sensor based on these results.

Otefin is a 45-kDa nuclear envelope protein with no apparent homology to other known proteins. It includes a large hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of 17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the nuclear envelope. Chemical extraction of nuclei from Drosophila embryos revealed that otefin is a peripheral protein whose association with the nuclear envelope is stronger than that of lamin. Deletion mutants of otefin were expressed in order to identify regions that direct otefin to the nuclear envelope. These experiments revealed that the hydrophobic sequence at the carboxyl terminus is essential for correct targeting to the nuclear envelope, whereas additional regions in the hydrophilic domain of otefin are required for its efficient targeting and stabilization in the nuclear envelope.

Quantitative digital microscopy and analysis of fluorescence gels are examples of applications where images are acquired, usually with a video camera and frame grabber, for further processing and analysis. The use of a standard video camera and frame grabber creates problems in the subsequent data analysis, due to the nonlinear relation between the camera output and the optical input, and due to pixel jitter. In addition, the data are degraded by conversion to a standard video format followed by sampling at a rate determined by the frame grabber's image memory. In this work, an image processing workstation is described which uses a low cost ($850) 8-bit digital CCD camera in place of the video camera/frame grabber combination. The system has excellent linearity and high dynamic range. Proper setup and calibration of this system is essential, and procedures which were developed for this purpose are described. The results which were obtained are compared to those of a standard video camera. The tradeoffs involved in the use of this low cost system are discussed, and examples of data obtained with the system are presented. Although this system cannot replace a 12-bit or 16-bit digital camera in all applications, it can do so in many applications, at a price that makes widespread use of image quantitation systems practical.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A novel two dimensional optical position sensor based on confinement of luminescence within a flat panel is described. The basis of the sensor is a flat luminescent panel, such as those used in luminescent solar energy concentrators. In these concentrators, a glass or plastic plate is either coated or impregnated with a fluorescent material. The incident radiation produces luminescent emission, much of which is confined within the plate through total internal reflection, and is thus guided to solar cells which line the thin edges of the plate. In order to use such a plate as a position sensor, four detectors are placed along two orthogonal axes at the edges of the plate, and the plate is illuminated with a spot of light. The spot of light induces luminescence, which is detected by the four detectors. The detectors' output is processed to provide position information. Position sensors based on the above idea were constructed, using both microscope slides coated with a fluorescent dye, and poly(methyl methacrylate) plates which were cast in the presence of a fluorescent dye so that they exhibited fluorescence throughout their volume. The results showed that the detector output varied monotonically with position. This paper presents the results obtained to date, together with a theory which relates the detector output to position. Suggestions for the improvement of the sensor, particularly in the areas of materials processing and sensor geometry, are provided.

Jerusalem College of TechnologyJerusalem, IsraelABSTRACTAn azo dye, methyl red (MR), was introduced into glass films and bulks and into polymer films. The glasses wereeither pure silicate or composed of a silicate network with the addition of long chain organic molecules. Someglasses were composites of silicate and methyl methacrylate (MMA) or poly(vinyl acetate) (PVAc). The polymerfilms, which consisted of PVAc or poly(vinyl alcohol) (PVA), were deposited on glass plates.The induced transparency of the samples was measured by a pump and probe method, using the 488 nm line of anargon-ion laser for both the pump and probe beams. The nonlinear effect was observed with the neutral and basicforms of MR (orange and yellow respectively). The magnitude of the effect decreased with increasing density of thehost as follows: polymer matrices, composite glass, sol-gel glass. These results, as well as measurements of theexcited state spectra, are consistent with the hypothesis that the observed nonlinearities are the result of a trans-cisisomerization of MR. for optical excitation in the milliwatt range.1. INTRODUCTIONThird order nonlinear optical processes can be used to produce effects such as optical phase conjugation, volumeholograms and optical switching."2 Organic dyes have been shown to exhibit large third order nonlinear opticaleffects)'2 Since organic dyes are easily introduced into polymers and glasses, high quality solid samples or thinfilms containing organic dyes are easily fabricated. In this paper, the nonlinear absorption of an azo dye, methyl red(MR), is presented. MR is a color indicator and its spectral properties have been extensively investigated.3 It existsin two isomeric forms, cis and trans, which are shown in Figure 1. The trans isomer is the more stable of the two.

A necessary part of digital image processing is segmentation of the images into a set of objects which exist on some background. We are interested in a class of objects whose distinguishing characteristic is their color. Such objects occur in many applications, such as microscopy, printing, production line monitoring, etc. In this work, a general method of rapid color-based segmentation is presented. The only hardware requirement is that look up tables (LUT) be available. Most image processing hardware contains either LUTs or processors capable of rapidly performing table lookup. The method presented allows simultaneous application of constraints on both hue and saturation. In addition, it allows for use of different color transformations. As such, it constitutes a general approach to analysis of images consisting of three spectral components. Because of the speed of LUT operations, this approach is suitable for many applications which are time sensitive. The major drawback of using LUTs is that the gray level resolution per color is limited by the size of the LUT. This method was implemented on a Matrox MVP-AT image processor, which is capable of processing 12 bit images (4 bits/color). In many cases, this resolution is adequate, as can be seen from examples which are presented.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Measurement of the time-response of the excited-state concentration of manganese has confirmed our previous hypothesis of fast energy transfer between manganese and neodymium in fluoride glasses.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The laser performance of rare earth doped crystals and glasses can be significantly improved by codoping with an addiontal ion which absorbs the pump light and efficiently transfers the energy to the lasing rare earth ion. Such methods have been used in chromium-neodymium doped gadolinium scandium gallium garnets, glasses and glass ceramics, and manganese-thulium doped lead zinc gallium fluoride glasses. Energy transfer between donors and acceptors in such materials has been traditionally modeled by the Foerster-Dexter equation. However, it has been found recently that an anomalous energy transfer can occur at very short times inconsistent with the above model. We propose that such transfer can occur due to two additional causes: 1 . the physical pairing of donors and acceptors in the materials and 2. additional short-range interactions which noticeably increase the transfer rate between donors and acceptors. Models for both these cases are presented and compared with experimental results.

There are many indications that interaction of serum proteins with intravenously injected cationic lipoplexes disturbs lipofection in vitro and in vivo. However, transfection with certain lipid compositions such as N-[1- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol appears to be more resistant to serum and more efficacious. We investigated the mechanism of interaction between fluorescently labeled lipoplexes of the above composition and fluorescently labeled serum proteins. Fluorescence resonance energy transfer measurements in vitro indicate that serum proteins interact instantly and closely with the DOTAP/cholesterol lipoplexes. In accord with this, preinjection of fluorescently labeled serum into mice before injection of lipoplexes showed an immediate association of proteins with lipoplexes. Serum proteins colocalized with the lipoplexes in the lung vasculature; however, they dissociated from the cationic lipid as soon as 1 hr postinjection, probably because of displacement of serum proteins from lipoplexes by extracellular proteoglycans. Indeed, this displacement was imitated by heparin, a typical glycosaminoglycan, and could be explained by the inability of weakly acidic serum proteins to neutralize the DOTAP/cholesterol electrical surface potential psi0. The stability of the cationic lipid psi0 in serum could be a key reason for the high lung association and transfection efficiency with this formulation.

A microwave-assisted reaction between Mo(CO)6 and Se has yielded MoSe2 nanoparticles. Nanorods of MoSe2 of lengths ranging from 45 to 55 nanometers are identified in the product mixture. The prepared MoSe2 has been characterized by X-ray powder diffraction measurements, FTIR spectroscopy, photoacoustic spectroscopy (PAS), transmission electron microscopy (TEM) and high resolution TEM (HRTEM).

Growth arrest-specific 2-like 3 (Gas2l3) is a newly discovered cell cycle protein and a cytoskeleton orchestrator that binds both actin filament and microtubule networks. Studies of cultured mammalian cells established Gas2l3 as a regulator of the cell division process, in particular cytokinesis and cell abscission. Thus far, the role of Gas2l3 in vivo remains entirely unknown. In order to investigate Gas2l3 in developing vertebrates, we cloned the zebrafish gene. Spatiotemporal analysis of gas2l3 expression revealed a ubiquitous maternal transcript as well as a zygotic transcript primarily restricted to brain tissues. We next conducted a series of loss-of-function experiments, and searched for developmental anomalies at the end of the segmentation period. Our analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos. This signature phenotype could be rescued by elevated levels of gas2l3 RNA. At the tissue level, gas2l3 downregulation interferes with cell proliferation, suggesting that the cell cycle activities of Gas2l3 are essential for brain tissue homeostasis. Altogether, this study provides the first insight into the function of gas2l3 in vivo, demonstrating its essential role in brain development.

Acylated pardaxin is translocated through the cytoplasmic membrane and is accumulated in the nucleoli of NG108-15 and chromaffin cells. The uptake is time- and dose-dependent and temperature-sensitive. However, the binding of acylated 125I-pardaxin cannot be reduced by competition with pardaxin acylated with Rudinger's reagent. In this respect, acylated pardaxin resembles the Tat protein 37–71 fragment. Metabolic inhibitors do not significantly reduce the uptake of acylated 125I-pardaxin. Acylated pardaxin might be useful as a vector to translocate other molecules.

Conjugates between anti-tetanus F(ab′)2 fragments and the (37–72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108-15 neurohybridoma cells and Rev-2-T-6 lymphoma cells. The uptake could not be inhibited by competition with (37–72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.

Characterization and control of membrane-based separation or drug delivery systems require the development of noninvasive methods for measurement of membrane hydration during the process of interest. In this paper, real time fluorescence methods which were developed in order to study such membrane systems are described, and data which demonstrate their sensitivity and speed are presented. Sensitivity and speed were sufficient that transient phenomena such as diffusion lag time and Donnan distribution effects caused by changes in fixed charge density were well resolved.