Search

Nature Communications 11(1), 4511 (2020). doi:10.1038/s41467-020-18156-7, Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported., Published by Nature Publishing Group UK, [London]

In recent years, X-ray speckle-tracking techniques have emerged as viable tools for wavefront metrology and sample imaging applications. These methods are based on the measurement of near-field images. Thanks to their simple experimental setup, high angular sensitivity and compatibility with low-coherence sources, these methods have been actively developed for use with synchrotron and laboratory light sources. Not only do speckle-tracking techniques give the potential for high-resolution imaging, but they also provide rapid and robust characterization of aberrations of X-ray optical elements, focal spot profiles, and sample position and transmission properties. In order to realize these capabilities, software implementations are required that are equally rapid and robust. To address this need, a software suite has been developed for the ptychographic X-ray speckle-tracking technique, an X-ray speckle-based method suitable for highly divergent wavefields. The software suite is written in Python 3, with an OpenCL back end for GPU and multi-CPU core processing. It is accessible as a Python module, through the command line or through a graphical user interface, and is available as source code under Version 3 or later of the GNU General Public License.

Journal of applied crystallography 53(4), 927 - 936 (2020). doi:10.1107/S1600576720006925, The ever-increasing brightness of synchrotron radiation sources demands improved X-ray optics to utilize their capability for imaging and probing biological cells, nano-devices and functional matter on the nanometre scale with chemical sensitivity. Hard X-rays are ideal for high-resolution imaging and spectroscopic applications owing to their short wavelength, high penetrating power and chemical sensitivity. The penetrating power that makes X-rays useful for imaging also makes focusing them technologically challenging. Recent developments in layer deposition techniques have enabled the fabrication of a series of highly focusing X-ray lenses, known as wedged multi-layer Laue lenses. Improvements to the lens design and fabrication technique demand an accurate, robust, in situ and at-wavelength characterization method. To this end, a modified form of the speckle tracking wavefront metrology method has been developed. The ptychographic X-ray speckle tracking method is capable of operating with highly divergent wavefields. A useful by-product of this method is that it also provides high-resolution and aberration-free projection images of extended specimens. Three separate experiments using this method are reported, where the ray path angles have been resolved to within 4 nrad with an imaging resolution of 45 nm (full period). This method does not require a high degree of coherence, making it suitable for laboratory-based X-ray sources. Likewise, it is robust to errors in the registered sample positions, making it suitable for X-ray free-electron laser facilities, where beam-pointing fluctuations can be problematic for wavefront metrology., Published by Wiley-Blackwell, [S.l.]

A data-driven method for respiratory gating in PET has recently been commercially developed. We sought to compare the performance of the algorithm with an external, device-based system for oncologic 18F-FDG PET/CT imaging. Methods: In total, 144 whole-body 18F-FDG PET/CT examinations were acquired, with a respiratory gating waveform recorded by an external, device-based respiratory gating system. In each examination, 2 of the bed positions covering the liver and lung bases were acquired with a duration of 6 min. Quiescent-period gating retaining approximately 50% of coincidences was then able to produce images with an effective duration of 3 min for these 2 bed positions, matching the other bed positions. For each examination, 4 reconstructions were performed and compared: data-driven gating (DDG) (we use the term DDG-retro to distinguish that we did not use the real-time R-threshold-based application of DDG that is available within the manufacturer's product), external device-based gating (real-time position management (RPM)-gated), no gating but using only the first 3 min of data (ungated-matched), and no gating retaining all coincidences (ungated-full). Lesions in the images were quantified and image quality scored by a radiologist who was masked to the method of data processing. Results: Compared with the other reconstruction options, DDG-retro increased the SUVmax and decreased the threshold-defined lesion volume. Compared with RPM-gated, DDG-retro gave an average increase in SUVmax of 0.66 ± 0.1 g/mL (n = 87, P < 0.0005). Although the results from the masked image evaluation were most commonly equivalent, DDG-retro was preferred over RPM-gated in 13% of examinations, whereas the opposite occurred in just 2% of examinations. This was a significant preference for DDG-retro (P = 0.008, n = 121). Liver lesions were identified in 23 examinations. Considering this subset of data, DDG-retro was ranked superior to ungated-full in 6 of 23 (26%) cases. Gated reconstruction using the external device failed in 16% of examinations, whereas DDG-retro always provided a clinically acceptable image. Conclusion: In this clinical evaluation, DDG-retro provided performance superior to that of the external device-based system. For most examinations the performance was equivalent, but DDG-retro had superior performance in 13% of examinations, leading to a significant preference overall.

We report on the use of multilayer Laue lenses to focus the intense X-ray Free Electron Laser (XFEL) beam at the European XFEL to a spot size of a few tens of nanometers. We present the procedure to align and characterize these lenses and discuss challenges working with the pulse trains from this unique x-ray source.

Optica 8(1), 15 - 23 (2021). doi:10.1364/OPTICA.410851, Single particle imaging at x-ray free electron lasers (XFELs) has the potential to determine the structure and dynamics of single biomolecules at room temperature. Two major hurdles have prevented this potential from being reached, namely, the collection of sufficient high-quality diffraction patterns and robust computational purification to overcome structural heterogeneity. We report the breaking of both of these barriers using gold nanoparticle test samples, recording around 10 million diffraction patterns at the European XFEL and structurally and orientationally sorting the patterns to obtain better than 3-nm-resolution 3D reconstructions for each of four samples. With these new developments, integrating advancements in x-ray sources, fast-framing detectors, efficient sample delivery, and data analysis algorithms, we illuminate the path towards sub-nanometer biomolecular imaging. The methods developed here can also be extended to characterize ensembles that are inherently diverse to obtain their full structural landscape., Published by OSA, Washington, DC

The deterioration of both the signal-to-noise ratio and the spatial resolution in the electron-density distribution reconstructed from diffraction intensities collected at different orientations of a sample is analysed theoretically with respect to the radiation damage to the sample and the variations in the X-ray intensities illuminating different copies of the sample. The simple analytical expressions and numerical estimates obtained for models of radiation damage and incident X-ray pulses may be helpful in planning X-ray free-electron laser (XFEL) imaging experiments and in analysis of experimental data. This approach to the analysis of partially coherent X-ray imaging configurations can potentially be used for analysis of other forms of imaging where the temporal behaviour of the sample and the incident intensity during exposure may affect the inverse problem of sample reconstruction.

Methods are described for processing XFEL data from individual crystalline fibrils. The methods are applied to data collected at the Linac Coherent Light Source from an amyloid-forming oligopeptide from the adenovirus shaft., Serial diffraction data collected at the Linac Coherent Light Source from crystalline amyloid fibrils delivered in a liquid jet show that the fibrils are well oriented in the jet. At low fibril concentrations, diffraction patterns are recorded from single fibrils; these patterns are weak and contain only a few reflections. Methods are developed for determining the orientation of patterns in reciprocal space and merging them in three dimensions. This allows the individual structure amplitudes to be calculated, thus overcoming the limitations of orientation and cylindrical averaging in conventional fibre diffraction analysis. The advantages of this technique should allow structural studies of fibrous systems in biology that are inaccessible using existing techniques.

Journal of applied crystallography 53(3), 760 - 780 (2020). doi:10.1107/S1600576720005567, A method is presented for the measurement of the phase gradient of a wavefront by tracking the relative motion of speckles in projection holograms as a sample is scanned across the wavefront. By removing the need to obtain an undistorted reference image of the sample, this method is suitable for the metrology of highly divergent wavefields. Such wavefields allow for large magnification factors that, according to current imaging capabilities, will allow for nanoradian angular sensitivity and nanoscale sample projection imaging. Both the reconstruction algorithm and the imaging geometry are nearly identical to that of ptychography, except that the sample is placed downstream of the beam focus and that no coherent propagation is explicitly accounted for. Like other X-ray speckle tracking methods, it is robust to low-coherence X-ray sources, making it suitable for laboratory-based X-ray sources. Likewise, it is robust to errors in the registered sample positions, making it suitable for X-ray free-electron laser facilities, where beam-pointing fluctuations can be problematic for wavefront metrology. A modified form of the speckle tracking approximation is also presented, based on a second-order local expansion of the Fresnel integral. This result extends the validity of the speckle tracking approximation and may be useful for similar approaches in the field., Published by Wiley-Blackwell, [S.l.]

We report experimental results on the diffractive imaging of three-dimensionally aligned 2,5-diiodothiophene molecules. The molecules were aligned by chirped near-infrared laser pulses, and their structure was probed at a photon energy of 9.5 keV (λ ≈ 130 pm) provided by the Linac Coherent Light Source. Diffracted photons were recorded on the Cornell-SLAC pixel array detector, and a two-dimensional diffraction pattern of the equilibrium structure of 2,5-diiodothiophene was recorded. The retrieved distance between the two iodine atoms agrees with the quantum-chemically calculated molecular structure to be within 5%. The experimental approach allows for the imaging of intrinsic molecular dynamics in the molecular frame, albeit this requires more experimental data, which should be readily available at upcoming high-repetition-rate facilities.

Optics express 27(26), 37816 - (2019). doi:10.1364/OE.27.037816, An outstanding question in X-ray single particle imaging experiments has been thefeasibility of imaging sub 10-nm-sized biomolecules under realistic experimental conditionswhere very few photons are expected to be measured in a single snapshot and instrumentbackground may be significant relative to particle scattering. While analyses of simulated datahave shown that the determination of an average image should be feasible using Bayesian methodssuch as the EMC algorithm, this has yet to be demonstrated using experimental data containingrealistic non-isotropic instrument background, sample variability and other experimental factors.In this work, we show that the orientation and phase retrieval steps work at photon counts dilutedto the signal levels one expects from smaller molecules or with weaker pulses, using data fromexperimental measurements of 60-nm PR772 viruses. Even when the signal is reduced to afraction as little as 1/256, the virus electron density determined using ab initio phasing is ofalmost the same quality as the high-signal data. However, we are still limited by the total numberof patterns collected, which may soon be mitigated by the advent of high repetition-rate sourceslike the European XFEL and LCLS-II., Published by Soc., Washington, DC

The crystallographic phase problem is routinely solved when imaging small molecules but remains a key component and difficult step in protein crystallography. Current phasing approaches are either experimentally based or rely on the knowledge of an homologous structure. With the recent advances in phasing algorithms called iterative projections algorithms (IPAs), ab initio phasing methods, where only minimal extra information is needed are gaining interest in protein crystallography, as they potentially offer a dramatic reduction in effort over experimental methods and are not affected by model bias. Two versions of a new ab initio phase retrieval method are presented in this paper. These rely on the measurement of the diffraction from two or more crystal forms of the same molecule. The required IPA constraint projections are derived and 2D simulations using the difference map algorithm to illustrate the convergence properties of the methods in presence of object overlap. A 3D simulation illustrates potential use and effectiveness of the algorithm.

Nature photonics 12(3), 150 - 153 (2018). doi:10.1038/s41566-018-0110-y, Ultrafast X-ray imaging on individual fragile specimens such as aerosols1, metastable particles2, superfluid quantum systems3 and live biospecimens4 provides high-resolution information that is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely defined4,5. Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lateral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond timescale., Published by Nature Publ. Group, London [u.a.]

Mobile applications (apps) are becoming more commonplace as mobile phones and tablets begin to replace traditional desktop devices. Software developers are usually located in areas where good connectivity is the norm, but large numbers of users are located in other areas where 3 G connectivity is at best poor or non-existent. This results in app development which is unsuitable for many areas of the world and, as well as being a problem in rural areas of the UK, has ramifications for mobile app use in third world countries where fixed broadband services are largely absent. Drawing on experiences of users on the margins of connectivity, this paper discusses difficulties of mobile internet access in Wales and recommends changes to app development methodology to maximise bandwidth in areas of limited network coverage.

We present a deterministic approach to the ptychographic retrieval of the wave at the exit surface of a specimen of condensed matter illuminated by X-rays. The method is based on the solution of an overdetermined set of linear equations, and is robust to measurement noise. The set of linear equations is efficiently solved using the conjugate gradient least-squares method implemented using fast Fourier transforms. The method is demonstrated using a data set obtained from a gold–chromium nanostructured test object. It is shown that the transmission function retrieved by this linear method is quantitatively comparable with established methods of ptychography, with a large decrease in computational time, and is thus a good candidate for real-time reconstruction.

Energy-filtered scanning confocal electron microscopy (EF-SCEM) is a technique that uses the reduced depth of field of an aberration-corrected transmission electron microscope to provide three-dimensional (3D) compositional information. Using a silicon sample in the orientation, we show that EF-SCEM image data can be recorded that shows lattice resolution in the plane perpendicular to the incident beam direction. The confocal effect is demonstrated through the reduction of the mean intensity as the confocal plane is displaced from the sample mid-plane, unlike optical sectioning in high-angle annular dark-field scanning transmission electron microscopy (STEM). Simulations of the EF-SCEM data show agreement with the experimental data, and allow the interpretability of the data to be explored. The effects of channelling, absorption and delocalisation complicate the quantitative and qualitative interpretation of the data, highlighting the need for matching to simulations. Finally the effects of the finite detector pin-hole aperture size are explored, and we show that the EF-SCEM contrast in the plane perpendicular to the beam direction starts to resemble that of a STEM spectrum imaging experiment as the aperture size increases. © 2013 Elsevier B.V.

In coherent diffractive imaging (CDI), conventional image-forming optics are replaced by the solution of an inverse scattering problem which obtains the exit surface wave from the diffraction pattern. To date CDI implementations typically use nonlinear iterative solutions, often using more than one diffraction pattern, and have been mainly implemented in the optical and x-ray regimes. Here we present single-exposure CDI reconstructions of a region of a cerium dioxide nanocrystal, illuminated by a coherent electron probe, in which the positions of both cerium and the lighter oxygen atoms are recovered simultaneously. We employ a fast deterministic algorithm based on the iterative solution of a set of linear equations with input data obtained from the inverse Fourier transform of the diffraction pattern. The solution of the linear equations proceeds using a conjugate gradient least-squares method based on fast Fourier transforms. This allows regularization of the inversion problem by iteration number, making it robust to experimental noise. This approach is successful where standard nonlinear phase retrieval techniques for the same data set are not. © 2013 American Physical Society.

We present a fast deterministic approach to the ptychographic reconstruction of the transmission function of a specimen at atomic resolution. The method is demonstrated using a data set obtained from a cerium dioxide nanoparticle using an aberration-corrected electron microscope and is compared to established approaches to ptychography. The method is based on the solution of an overdetermined set of linear equations, and is computationally efficient and robust to measurement noise. The set of linear equations is efficiently solved using the conjugate gradient least squares method implemented using fast Fourier transforms. © 2014 American Physical Society.

We propose a direct, non-iterative method for the exact recovery of the complex wave in the exit-surface plane of a coherently illuminated object from a single defocused image. The method is applicable for a wide range of illumination conditions. The defocus range is subject to certain conditions, which if satisfied allow the complex exit-surface wave to be directly recovered by solving a set of linear equations. These linear equations, whose coefficients depend on the incident illumination, are obtained by analyzing the autocorrelation function of an auxiliary wave which is related to the exit-surface wave in a simple way. This autocorrelation is constructed by taking the inverse Fourier transform of the defocused image. We present an experimental proof of concept by recovering the exit-surface wave of a microfiber illuminated by a plane wave formed using a HeNe laser.

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored.

BACKGROUND.: Vaccination against Epstein-Barr virus (EBV), inducing an antibody response to the envelope glycoprotein gp350, might protect EBV-negative children with chronic kidney disease from lymphoproliferative disease after transplantation. METHODS.: A phase I trial recruited children with chronic kidney disease to two successive cohorts given three injections of 12.5 microg (n=6) and 25 microg (n=10) recombinant gp350/alhydrogel vaccine over 6 to 8 weeks. RESULTS.: One in each cohort acquired wild EBV before the week 28 evaluation. Both doses were similarly immunogenic, inducing an IgG response in all 13 evaluable patients. Neutralizing antibodies were detected in four recipients (1/4 in the 12.5 microg and 3/9 in the 25 microg cohort). Median time from first vaccination to transplantation was 24 weeks. Immune responses declined rapidly and were unlikely to affect posttransplant events. DISCUSSION.: The vaccine was immunogenic but a prolonged vaccine schedule up to time of transplantation or improved adjuvants are required in future trials to reduce posttransplant EBV load and risk of lymphoproliferative disease.

Physical review / B covering condensed matter and materials physics 93(13), 134116(2016). doi:10.1103/PhysRevB.93.134116, We introduce an algorithm for the reconstruction of the complex transmission function of a specimen using segmented detectors in scanning transmission electron microscopy geometry. The phase of the transmission function can be related to magnetic and electric fields within the specimen and is sensitive to lighter elements. The technique is demonstrated for simulated data and also using experimental datasets taken from a MoS$_2$ monolayer and a SrTiO$_3$ crystal. We present an extension to the algorithm to account for uncertainties in the illuminating probe. The algorithm can be implemented using fast Fourier transforms, and this provides the possibility of reconstructing specimen transmission functions in real time., Published by APS, Woodbury, NY

Proceedings of the 12th International Conference on X-Ray Microscopy, XRM 2014, Melbourne, Australia, 26 Oct 2014 - 31 Oct 2014 ; AIP conference proceedings 1696, 020049 (2016). doi:10.1063/1.4937543, Multilayer Laue lenses (MLLs) capitalize on the developments in multilayer deposition technologies for fabricating reflective coatings, specifically undertaken for EUV lithography, where layer thicknesses of several nanometers can be achieved. MLLs are deposited layer by layer, with their thicknesses following the zone plate law, and then pieces are sliced and extracted for use in focusing. Rays are reflected in the Laue geometry. The efficiency of a MLL can be very high, and is maximized by making the slice equal to about a half Pendellosung period so that most energy is transferred from the undiffracted to the diffracted beam, and by ensuring that the Bragg condition is met at each point in the zone plate. This latter condition requires that the layers are tilted to the beam by an amount that varies with layer position; e.g. for focusing a collimated beam, the layers should be normal to a cylinder of radius of twice the focal length. We have fabricated such tilted-zone MLLs and find that they exhibit improved efficiency across their entire pupil as compared with parallel-zone MLLs. This leads to a higher effective NA of the optic and hence higher resolution., Published by Inst., Melville, NY

Summary Edward Jenner's discovery of vaccination must rank as one of the most important medical advances of all time and is a prominent example of the power of rational enquiry being brought to bear during the Age of Enlightenment in 18th century Europe. In the modern era many millions of lives are saved each year by vaccines that work essentially on the same principles that were established by Edward Jenner more than 200 years ago. His country home in Berkeley, Gloucestershire, is where he carried out his work and where he spent most of his life. The building is now a museum in which the life and times of Jenner are commemorated including not only the discovery of smallpox vaccination but also his other important scientific contributions to natural history and medicine. The trustees of the Edward Jenner museum are committed to promoting the museum as a real and “virtual” educational centre that is both entertaining and informative.

Serial femtosecond nanocrystallography (SFX) is a form of x-ray coherent diffraction imaging that utilises a stream of tiny nanocrystals of the biological assembly under study, in contrast to the larger crystals used in conventional x-ray crystallography using conventional x-ray synchrotron x-ray sources. Nanocrystallography utilises the extremely brief and intense x-ray pulses that are obtained from an x-ray free-electron laser (XFEL). A key advantage is that some biological macromolecules, such as membrane proteins for example, do not easily form large crystals, but spontaneously form nanocrystals. There is therefore an opportunity for structure determination for biological molecules that are inaccessible using conventional x-ray crystallography. Nanocrystallography introduces a number of interesting image reconstruction problems. Weak diffraction patterns are recorded from hundreds of thousands of nancocrystals in unknown orientations, and these data have to be assembled and merged into a 3D intensity dataset. The diffracted intensities can also be affected by the surface structure of the crystals that can contain incomplete unit cells. Furthermore, the small crystal size means that there is potentially access to diffraction information between the crystalline Bragg peaks. With this information, phase retrieval is possible without resorting to the collection of additional experimental data as is necessary in conventional protein crystallography. We report recent work on the diffraction characteristics of nanocrystals and the resulting reconstruction algorithms.

A major challenge in high-resolution x-ray free-electron laser-based coherent diffractive imaging is the development of aerosol injectors that can efficiently deliver particles to the peak intensity of the focused X-ray beam. Here, we consider the use of a simple convergent-orifice nozzle for producing tightly focused beams of particles. Through optical imaging we show that 0.5 μm particles can be focused to a full-width at half maximum diameter of 4.2 μm, and we demonstrate the use of such a nozzle for injecting viruses into a micro-focused soft-X-ray FEL beam.

Use of nanotechnology products is increasing; with silver (Ag) nanoparticles particularly widely used. A key uncertainty surrounding the risk assessment of AgNPs is whether their effects are driven through the same mechanism of action that underlies the toxic effects of Ag ions. We present the first full transcriptome study of the effects of Ag ions and NPs in an ecotoxicological model soil invertebrate, the earthworm Eisenia fetida. Gene expression analyses indicated similar mechanisms for both silver forms with toxicity being exerted through pathways related to ribosome function, sugar and protein metabolism, molecular stress, disruption of energy production and histones. The main difference seen between Ag ions and NPs was associated with potential toxicokinetic effects related to cellular internalisation and communication, with pathways related to endocytosis and cilia being significantly enriched. These results point to a common final toxicodynamic response, but initial internalisation driven by different exposure routes and toxicokinetic mechanisms.

Female southern flounder ( Paralichthys lethostigma ) grow 2–3 times larger than males. Therefore, all-female production will maximize profit potential for the culture of this species. We have developed protocols to produce all-female southern flounder through induction of meiotic gynogenesis with heterologous sperm of black sea bass ( Centropristis striata ). Experiments were conducted to establish these practical methods using a total of 40 spawns from 32 broodstock. The first set of experiments determined the UV dose that genetically inactivated black sea bass sperm, yet retained adequate motility for activation of flounder eggs. Milt from several black sea bass was diluted 1:10 with Ringer's solution and UV irradiated with doses ranging from 0–130 J/cm 2 . Two criteria were utilized to evaluate the UV irradiation effects: percentage of motile sperm and duration of sperm activity. Motility and duration of activity generally decreased with increases in UV dosage. At UV doses greater than or equal to 90 J/cm 2 , motility was 2 but were not different for the other treatments. Hatch rate was highest at 70 J/cm 2 . A second set of experiments examined appropriate pressure shock protocols for retention of the 2nd polar body in southern flounder eggs after activation with black sea bass sperm. A pressure shock of 8500 psi was initiated at varying times of 1, 2, and 3 min post-fertilization and maintained for 6 min. Eggs that were handled similarly, but not pressure shocked, served as negative controls. Pressure shock applied at either 1 or 2 min post-fertilization resulted in higher rates of hatch and survival. Using these methods, six separate spawns produced offspring that survived through and beyond metamorphosis. The average fertility (± SEM) was 70.9 + 12.8%. Of the fertilized eggs, percentage hatch varied with pressure shock initiation times and ranged from 1.48 + 0.52% (1 min) to 0.61 + 0.11% (3 min). Gynogenetic flounder were sex-reversed to males by high temperature and, upon reaching maturity, expressed motile sperm that resulted in successful fertilization of flounder eggs. These results indicate that the use of UV irradiated sperm from black sea bass for activation of flounder eggs and pressure shock for polar body retention is an effective method to produce gynogenetic offspring.

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the pathogenesis of a wide spectrum of malignant and non-malignant diseases. Strong evidence implicates T lymphocytes in the control of EBV replication and tumorigenesis, but cellular components of the innate immune system are poorly characterized in terms of their function in the development of EBV-specific immunity or interaction with the virus. This study demonstrates that EBV virions produced in epithelial cells surpass their B cell-derived counterparts in the capacity to enter monocytes and inhibit their development into dendritic cells (DCs). Different ratios of the gp42 and gH glycoproteins in the envelope of virions that were derived from major histocompatibility complex class II-positive or -negative cells accounted primarily for the differences in EBV tropism. EBV is shown to enter both monocytes and DCs, although the cells are susceptible to virus-induced apoptosis only if infected at early stages of DC differentiation. The purified gH/gL heterodimer binds efficiently to monocytes and DCs, but not to B cells, suggesting that high expression levels of a putative binding partner for gH contribute to virus entry. This entry takes place despite very low or undetectable expression of CD21, the canonical EBV receptor. These results indicate that the site of virus replication, either in B cells or epithelial cells, alters EBV tropism for monocytes and DCs. This results in a change in the virus's immunomodulating capacity and may have important implications for the regulation of virus–host interactions during primary and chronic EBV infection.

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus. However, access to B lymphocytes during primary infection may be facilitated by replication in mucosal epithelial cells. Attachment and penetration of EBV into these two cell types are fundamentally different. Both the distribution of receptors and the cellular origin of the virus impact the efficiency of infection. Epithelial cells potentially offer a wide range of receptors with which virus can interact. We report here on analyses of epithelial cells expressing different combinations of receptors. We find that the stoichiometry of the virus glycoprotein complex that includes gHgL and gp42 affects the use of gHgL not just for entry into epithelial cells but also for attachment. Penetration can be mediated efficiently with either a coreceptor for gp42 or gHgL, but the use of gHgL for attachment as well as penetration greatly compromises its ability to mediate entry.

Epstein-Barr virus (EBV) is associated with a number of important human cancers, including nasopharyngeal carcinoma, gastric carcinoma, and Hodgkin's lymphoma. These tumors express a viral nuclear antigen, EBV nuclear antigen 1 (EBNA1), which cannot be presented to T cells in a major histocompatibility complex class I context, and the viral latent membrane proteins (LMPs). Although the LMPs are expressed in these tumors, no effective immune response is made. We report here that exposure to the cholera-like enterotoxin B subunit (EtxB) in EBV-infected lymphoblastoid cell lines (LCLs) enhances their susceptibility to killing by LMP-specific CD8 + cytotoxic T lymphocytes (CTLs) in a HLA class I-restricted manner. CTL killing of LCLs is dramatically increased through both transporter-associated protein-dependent and -independent epitopes after EtxB treatment. The use of mutant B subunits revealed that the enhanced susceptibility of LCLs to CTL killing is dependent on the B subunit's interaction with GM 1 but not its signaling properties. These important findings could underpin the development of novel approaches to treating EBV-associated malignancies and may offer a general approach to increasing the presentation of other tumor and viral antigens.

The ever-increasing brightness of synchrotron radiation sources demands improved X-ray optics to utilise their capability for imaging and probing biological cells, nanodevices and functional matter on the nanometer scale with chemical sensitivity. Here we demonstrate focusing a hard X-ray beam to an 8 nm focus using a volume zone plate (also referred to as a wedged multilayer Laue lens). This lens was constructed using a new deposition technique that enabled the independent control of the angle and thickness of diffracting layers to microradian and nanometer precision, respectively. This ensured that the Bragg condition is satisfied at each point along the lens, leading to a high numerical aperture that is limited only by its extent. We developed a phase-shifting interferometric method based on ptychography to characterise the lens focus. The precision of the fabrication and characterisation demonstrated here provides the path to efficient X-ray optics for imaging at 1 nm resolution.

PURPOSE: The Dynamic Observing Tonometer (SmartLens, Ophthalmic Development Company AG, Zurich, Switzerland) is a diagnostic contact lens that allows continuous measurement of intraocular pressure, in addition to providing the investigator with a view of the posterior pole and anterior chamber angle. The purpose of this study was to determine the accuracy of this tonometer and the repeatability of the intraocular pressure measurements. PATIENTS AND METHODS: The intraocular pressure was measured by Goldmann applanation tonometry in one randomly chosen eye of 40 subjects (median age 66 years, range 21-77 years). The intraocular pressure, pulse amplitude and 10-second continuous tonometric recordings were then taken using the Dynamic Observing Tonometer and a pneumatonometer. Accuracy was determined by calculating the mean bias and 95% limits of agreement of measurements made with the Dynamic Observing Tonometer against measurements made with the Goldmann and pneumatonometer. Repeatability was evaluated by calculating the differences between pairs of repeated measurements against the mean value and by calculating reliability coefficients. RESULTS: Intraocular pressure measurements made with the Dynamic Observing Tonometer had a mean bias of +2.1 mm Hg (95% limits of agreement: -4.0 to +8.2 mm Hg) compared with Goldmann tonometry. There was a reasonable correlation between Goldmann and Dynamic Observing Tonometer intraocular pressure readings (r = 0.78, P < 0.01). In measuring pulse amplitude, the Dynamic Observing Tonometer was found to have a mean bias of +0.4 mm Hg (95% limits of agreement: -1.6 to +2.3 mm Hg) compared with the pneumatonometer (r = 0.78, P < 0.01). In assessing the repeatability of intraocular pressure measurements, the first Dynamic Observing Tonometer reading was on average 0.4 mm Hg higher than the second (95% limits of agreement: -3.8 to +4.6 mm Hg) with a coefficient of reliability of 0.91. For pulse amplitude readings, the first reading was on average 0.1 mm Hg lower than the second (95% limits of agreement: -1.4 to +1.2 mm Hg) with a coefficient of reliability of 0.90. CONCLUSION: Intraocular pressure measurements taken with the Dynamic Observing Tonometer had a small positive bias compared with Goldmann tonometry. The pulse amplitude values correlated well with those obtained with a pneumatonometer and the repeatability of intraocular pressure measurements was similar to that found in other commercially available tonometers.

Many studies have found that the intraocular pressure (IOP) is lower in glaucomatous populations than in normal groups. An alternative method of analysis, commonly used in the investigation of arterial blood pressure, is to reduce the pulse waveform to its component Fourier parts. The purpose of this study, therefore, was to determine whether such a technique is applicable to the IOP pulse and to investigate whether it was superior at differentiating diseased from healthy eyes.Using a pneumatonometer, continuous 10-s IOP recordings were taken from 10 glaucoma patients and 10 normal subjects. The IOP recordings were then analysed using the fast Fourier transform (FFT) to determine their spectral components. In addition, standard IOP parameters (pulse amplitude, pulse volume, and pulsatile ocular blood flow) were measured to allow comparison with the waveform analysis technique.Analysis by FFT successfully determined the IOP pulse's higher spectral components up to the fourth harmonic. In addition, although standard measurements of the IOP pulse (such as pulse amplitude) were insignificant, the second ( P=0.034), third ( P=0.015) and fourth ( P=0.013) harmonics of the waveform successfully differentiated between the glaucoma and normal groups.Spectral analysis of the IOP pulse appears to be a promising technique in the investigation of ocular vascular disease.

Epstein-Barr virus (EBV) is a tumorigenic human herpesvirus that persists for life in healthy immunocompetent carriers. The viral strategies that prevent its clearance and allow reactivation in the face of persistent immunity are not well understood. Here we demonstrate that EBV infection of monocytes inhibits their development into dendritic cells (DCs), leading to an abnormal cellular response to granulocyte macrophage–colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) and to apoptotic death. This proapoptotic activity was not affected by UV inactivation and was neutralized by EBV antibody-positive human sera, indicating that binding of the virus to monocytes is sufficient to alter their response to the cytokines. Experiments with the relevant blocking antibodies or with mutated EBV strains lacking either the EBV envelope glycoprotein gp42 or gp85 demonstrated that interaction of the trimolecular gp25–gp42–gp85 complex with the monocyte membrane is required for the effect. Our data provide the first evidence that EBV can prevent the development of DCs through a mechanism that appears to bypass the requirement for viral gene expression, and they suggest a new strategy for interference with the function of DCs during the initiation and maintenance of virus-specific immune responses.

Purpose. The purpose of this study was to investigate the influence of corneal topography and thickness on intraocular pressure (IOP) and pulse amplitude (PA) as measured using the Ocular Blood Flow Analyzer (OBFA) pneumatonometer (Paradigm Medical Industries, Utah, USA). Methods. 47 university students volunteered for this cross-sectional study: mean age 20.4 yrs, range 18 to 28 yrs; 23 male, 24 female. Only the measurements from the right eye of each participant were used. Central corneal thickness and mean corneal radius were measured using Scheimpflug biometry and corneal topographic imaging respectively. IOP and PA measurements were made with the OBFA pneumatonometer. Axial length was measured using A-scan ultrasound, due to its known correlation with these corneal parameters. Stepwise multiple regression analysis was used to identify those components that contributed significant variance to the independent variables of IOP and PA. Results. The mean IOP and PA measurements were 13.1 (SD 3.3) mmHg and 3.0 (SD 1.2) mmHg respectively. IOP measurements made with the OBFA pneumatonometer correlated significantly with central corneal thickness (r = +0.374, p = 0.010), such that a 10 mm change in CCT was equivalent to a 0.30 mmHg change in measured IOP. PA measurements correlated significantly with axial length (part correlate = -0.651, p < 0.001) and mean corneal radius (part correlate = +0.459, p < 0.001) but not corneal thickness. Conclusions. IOP measurements taken with the OBFA pneumatonometer are correlated with corneal thickness, but not axial length or corneal curvature. Conversely, PA measurements are unaffected by corneal thickness, but correlated with axial length and corneal radius. These parameters should be taken into consideration when interpreting IOP and PA measurements made with the OBFA pneumatonometer.

Dr. Edward Jenner's discovery and application of vaccination against smallpox was one of the most important medical advances of all time. In the modern era many millions of lives are saved each year by vaccines that work essentially on the same scientific principles established by Jenner more than 200 years ago. Jenner's country home in Berkeley, Gloucestershire, UK, where he carried out his work and where he spent most of his life, is now a museum and something of a shrine for vaccinologists. Jenner's house is also now the focal point of a new international learned society dedicated to advancing modern vaccinology. The aims of the new Jenner Society are to engage, support, and sustain the professional goals of vaccinologists, and to perpetuate the memory of Dr. Edward Jenner. Ultimately it is hoped that the Jenner Society will be recognized as one of the leading academic societies representing and promoting vaccine science around the world. We invite readers to consider joining the society (http://www.edwardjennersociety.org/).

Latent membrane protein 2A (LMP2A) is expressed on almost all Epstein-Barr virus (EBV)-associated tumors and is a potential target for immunotherapeutic intervention and vaccination. However, LMP2A is not efficiently processed and presented on major histocompatibility antigens class I molecules to generate potent cytotoxic T-lymphocytes (CTL) responses capable of killing these tumors. The B subunit of Escherichia coli enterotoxin (EtxB), causes rapid internalization and processing of membrane-bound LMP2A on EBV-infected B cells, and facilitates loading of processed-LMP2A peptides onto MHC class I. This re-directed trafficking/delivery of LMP2A to the MHC class I machinery enhances recognition and killing by LMP2A-specific CTL in vitro. To test the potential of EtxB to enhance immune targeting of LMP2A expressed in solid tumors, we generated a murine tumor model (Renca-LMP2A), in which LMP2A is expressed as a transgenic neoantigen on a renal carcinoma (Renca) cell line and forms solid tumors when injected subcutaneously into BALB/c mice. The data show that in BALB/c mice which have only low levels of peripheral K(d)-LMP2A-specific CD8(+) T cells, merely a transient inhibition of tumor growth is achieved compared with naïve mice; suggesting that there is suboptimal LMP2A-specifc CTL recognition and poorly targeted tumor killing. However, importantly, treatment of these mice with EtxB led to a significant delay in the onset of tumor growth and significantly lower tumor volumes compared with similar mice that did not receive EtxB. Moreover, this remarkable effect of EtxB was achieved despite progressive reduction in tumor expression of LMP2A and MHC class I molecules. These data clearly demonstrate the potential efficacy of EtxB as a novel therapeutic agent that could render EBV-associated tumors susceptible to immune control.