Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

Genetic selection of Eimeria parasites in the chicken for improvement of poultry health: implications for drug resistance and live vaccine development.

Apicomplexan parasites of the genus Eimeria are widespread in poultry flocks and can cause the intestinal disease coccidiosis. Early studies, concerned with intraspecific variation in oocyst morphology, indicated that phenotypic changes may be induced by selection experiments conducted in vivo. Genetic selection driven by targeted selection for specific phenotypes has contributed to our understanding of the phenomenon of drug resistance and the development of live attenuated vaccines. Our present knowledge regarding genetics of Eimeria is largely based upon the utilization of such selected strains as genetic markers. Practical advantages of working with Eimeria spp. in the chicken are discussed. The selection of drug-resistant strains by serial propagation has provided useful information regarding the mechanisms of drug resistance and likely longevity of anticoccidial drugs when introduced in the field. Selection experiments to develop precocious strains of Eimeria and growth in chicken embryos have contributed to the development of safe and effective live attenuated vaccines for the control of coccidiosis. Establishment of protocols for genetic complementation by transient or stable transfection of Eimeria is now supporting direct manipulation of parasite genotypes, creating opportunities to expand the range and value of live parasite vaccines. Procedures for developing drug-resistant and precocious lines of Eimeria and/or genetic markers described here are likely to prove useful for researchers investigating the propensity for resistance development to novel compounds and the development of new attenuated vaccines. Such investigations can be helpful in providing a better understanding of biochemical and molecular aspects of the biology of these parasites.

Scanning Compton X-ray microscopy.

X-ray microscopy offers the opportunity to image biological and radiosensitive materials without special sample preparations, bridging optical and electron microscopy capabilities. However, the performance of such microscopes, when imaging radiosensitive samples, is not limited by their intrinsic resolution, but by the radiation damage induced on such samples. Here, we demonstrate a novel, to the best of our knowledge, radio-efficient microscope, scanning Compton X-ray microscopy (SCXM), which uses coherently and incoherently (Compton) scattered photons to minimize the deposited energy per unit of mass for a given imaging signal. We implemented SCXM, using lenses capable of efficiently focusing 60 keV X-ray photons into the sub-micrometer scale, and probe its radio-efficient capabilities. SCXM, when implemented in high-energy diffraction-limited storage rings, e.g., European Synchrotron Radiation Facility Extremely Brilliant Source and PETRA IV, will open the opportunity to explore the nanoscale of unstained, unsectioned, and undamaged radiosensitive materials.

Pathology caused by three species of Eimeria that infect the turkey with a description of a scoring system for intestinal lesions.

Three-week-old turkey poults were infected with pure lines of three species of Eimeria (E. adenoeides, E. gallopavonis, and E. meleagrimitis) recently isolated from commercial turkey farms. The lines had been propagated from a single oocyst and identified by species-specific PCR amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Five to six days after infection their intestines were removed and examined for the presence of intestinal lesions. A description and review of the pathology caused by these parasites is provided, and a scoring system developed by which the severity of the lesions can be evaluated. The system is similar to that described by Johnson, J. and Reid, W. M. [1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology, 28, 30-36] for chickens in which a score of zero to four is assigned to lesions of increasing severity. The intestinal lesions observed here, and their assigned scores, are supported by representative illustrations. It is hoped that they may prove a useful tool for evaluating the pathology caused by E. adenoeides, E. gallopavonis, and E. meleagrimitis in the turkey.RESEARCH HIGHLIGHTSA scoring system has been developed for intestinal lesions caused by three species of Eimeria that infect the turkey.The lesions attributable to these species are illustrated.

Anticoccidial drugs of the livestock industry.

Coccidiosis is a parasitic disease of a wide variety of animals caused by coccidian protozoa. The coccidia are responsible for major economic losses of the livestock industry. For example, the annual cost due to coccidiosis to the global poultry industry has been estimated to exceed US$ 3 billion annually. Currently available drugs for the control of this disease are either polyether ionophorous antibiotics that are derived from fermentation products, or synthetic compounds, produced by chemical synthesis. Unfortunately, no new drugs in either category have been approved for use for decades. Resistance has been documented for all those of the drugs currently employed and therefore the discovery of novel drugs with unique modes of action is imperative if chemotherapy is to remain the principal means to control this disease. This chapter aims to give an overview of the efficacy and mode of action of the current compounds used to control coccidiosis in livestock and provides a brief outlook of research needs for the future.

Molecular detection of field isolates of Turkey Eimeria by polymerase chain reaction amplification of the cytochrome c oxidase I gene.

Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

Restoration of sensitivity to salinomycin in Eimeria following 5 flocks of broiler chickens reared in floor-pens using drug programs and vaccination to control coccidiosis.

Five successive flocks of broilers were reared in floor-pens and given different drug programs or were vaccinated against coccidiosis. Oocysts of Eimeria were isolated from the litter of pens during the fifth flock and their sensitivity to salinomycin (Sal) investigated by measuring new oocyst production following infection of medicated and unmedicated birds. Parasites obtained following 5 flocks given Sal were not well-controlled and it was concluded that they were partially resistant to the drug. Parasites obtained following 4 unmedicated flocks and one medicated flock were better controlled by Sal and it was concluded that in the absence of continuous medication there had been an improvement in drug efficacy. Sal almost completely suppressed oocyst production of isolates from treatments in which medication was followed by vaccination, indicating that when a drug program is followed by vaccination, restoration of sensitivity to Sal had occurred.

Contact sensitizer 2,4-dinitrochlorobenzene is a highly potent human TRPA1 agonist.

2,4-Dinitrochlorobenzene (DNCB) is widely used in human clinical studies and in experimental animal studies to evoke allergic contact dermatitis. 2,4-Dinitrochlorobenzene is a potent immunogen capable of inducing contact sensitization in all humans exposed. However, the mechanism by which DNCB evokes such symptoms is presently unknown. TRPA1 is a nonselective cation channel that is expressed in peptidergic sensory neurons and fibroblasts. TRPA1 activation was recently implicated in the pathophysiology of atopic dermatitis especially in transducing cutaneous itch signals. Here, we test the hypothesis that DNCB acts as a TRPA1 agonist and thereby evokes allergic symptoms. We found that DNCB activates human TRPA1 dose dependently in FLIPR experiments with an EC50 of 167 nM, an effect that was fully blocked by selective TRPA1 antagonists Chembridge-5861528 and A-967079. Similarly, DNCB activated nonselective TRPA1 current in patch clamp studies. Neutralization of 3 critical cysteines in TRPA1 resulted in a loss of DNCB agonism.

Biological re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria meleagrimitis Tyzzer 1929.

For the purpose of re-describing the Eimeria species that infect the turkey (Meleagris gallopavo) and to establish benchmark biological information linked to genetic markers for each species, a strain of Eimeria meleagrimitis Tyzzer 1929 was obtained from a litter sample from a turkey farm in Minnesota, USA in 2008. Multiple pure lines were derived by infecting turkey poults with a single oocyst; one of these lines was then used to re-describe biological and morphological features of E. meleagrimitis in the turkey and to designate a neotype of E. meleagrimitis in the turkey. Oocyst morphometrics of this line matched those of this species as originally described by Tyzzer (Am J Hyg 10:269-383, 1929). Three asexual generations of merogony (the first generation of meronts large in size and the second and third generations small) were detected in the intestines before the onset of gametogony; no developmental stages were detected in the cecal pouches. No mortality was induced by this line of E. meleagrimitis even when turkey poults were infected with high doses of oocysts (up to 5 × 10(5) oocysts/bird) and despite the ability of E. meleagrimitis to induce severe mucosal damage in the upper and middle duodenum. Macroscopic lesions were characterized to provide a graded lesion scoring guide that should assist assessment of the severity of infections with this species in infected turkeys. The pathogenicity of the strain was investigated, and a significant reduction in weight gain and feed conversion ratio was observed with doses of 10(4) oocysts/bird or more. The maximum yield of oocysts in the feces was obtained when birds were inoculated with 5 × 10(3) oocysts.

Milestones in avian coccidiosis research: a review.

This article describes some of the milestones in research concerned with protozoan parasites of the genus Eimeria that infect birds and cause the disease coccidiosis. The time period covered is from 1891, when oocysts were first found in the ceca of diseased chickens, to the present. Progress in our understanding has lagged behind that of other protozoan parasites such as Toxoplasma and Plasmodium despite the enormous importance of Eimeria to animal livestock production. Nevertheless, applied research by universities, government agencies, and private industry has resulted in the successful development of methods of control, research that continues today. The topics covered and the references provided are selective and include life cycles and biology, pathology, ultrastructure, biochemistry, immunity, genetics, host cell invasion, species identification, taxonomy, chemotherapy, vaccination, and literature concerned with avian coccidiosis. This review is primarily concerned with the avian species of Eimeria that infect poultry, but some important advances, principally in immunology, have been made using species that infect rodents and rabbits. These are included where appropriate.

Absorption and deposition of xanthophylls in broilers challenged with three dosages of Eimeria acervulina oocysts.

1. An experiment was designed to evaluate the effect of different doses of oocysts of Eimeria acervulina on intestinal absorption and skin deposition of xanthophylls (XAs) in broilers. 2. A total of 192 broiler chickens were randomly assigned to 4 groups: an uninfected control group and three groups inoculated with either 1 × 10(2), 1 × 10(4) or 1 × 10(5) sporulated oocysts of E. acervulina by gavaging at 21 d. There were 4 replicate pens (2 male and 2 female) per group. 3. Plasma xanthophyll (PX) and skin yellowness (SY) were measured in live birds weekly. At 42 d of age, SY was measured in the breast and abdomen after chilling and in the breast 24 h post-processing on refrigerated carcasses. 4. In general, in all challenged treatments, and for the duration of the study, the average PX decreased by 0.02 µg/ml (R(2) = 61.6%) for every 1000 inoculated oocysts, whereas PX increased by 1.26 µg/ml/d in uninfected birds. 5. The average SY in live birds from 21 to 42 d of age decreased by 0.019 b*/every 1000 oocysts administered, while SY of uninfected controls increased by 0.57 b*/d. It was also noted that in all treatments females had a greater SY (6.17 b*) than males for the duration of the study. The SY of the breast and abdomen was correlated (r = 0.76) in chilled carcasses. Breast SY in 24 h refrigerated carcasses was greater in the control group and for female birds. 6. Oocyst excretion was different between inoculated treatments only on 7 d post-inoculation (PI). Coccidia lesion scores in the duodenum averaged 1+ in infected birds and 2+ in birds given the highest oocyst dose.

The extraction of single-particle diffraction patterns from a multiple-particle diffraction pattern.

The structures of biological molecules may soon be determined with X-ray free-electron lasers without crystallization by recording the coherent diffraction patterns of many identical copies of a molecule. Most analysis methods require a measurement of each molecule individually. However, current injection methods deliver particles to the X-ray beam stochastically and the maximum yield of single particle measurements is 37% at optimal concentration. The remaining 63% of pulses intercept no particles or multiple particles. We demonstrate that in the latter case single particle diffraction patterns can be extracted provided the particles are sufficiently separated. The technique has the potential to greatly increase the amount of data available for three-dimensional imaging of identical particles with X-ray lasers.

Acquisition of immunity to the protozoan parasite Eimeria adenoeides in turkey poults and cellular responses to infection.

Newly hatched turkey poults were infected with 10(2) oocysts of Eimeria adenoeides and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age, respectively. Three peaks in oocyst production were observed in the feces of poults following this series of infections. A second group of poults given the same dosing regimen was challenged with 5 × 10(4) oocysts/poult at different times to evaluate the acquisition of immunity. Judging by weight gain and mortality, no protection had been acquired at 6 d of age, but partial protection was observed by 12 and 18 d of age. A third group of poults were also infected with 10(2) oocysts and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age to evaluate cellular immune responses to infection. Sections of ceca from infected poults showed a significantly higher leukocyte infiltration on d 6, 10, 12, 16, and 18 after infection than uninfected controls. The percent area occupied by CD4+ and CD8+ lymphocytes in the ceca, as assessed by immunohistochemistry, was significantly elevated in infected poults on d 12, 16, and 18. The relative expression of chemokine CXCLi2, and cytokines IL1ß, IFNγ, IL10, IL13, IL2, IL12b, and IL18 was measured by real-time reverse-transcription PCR. The expression of CXCLi2 and IL10 was found to be elevated on d 12, and IFNγ on d 10, 12, and 16. Expression of IL13 and IL18 was increased on d 10 and IL2 on d 10 and 16, and that of IL12b on d 16 in infected poults. Increase in the infiltration of leukocytes, percent area occupied by CD4+ and CD8+ lymphocytes, and changes in the relative expression of cytokines in the ceca characterize the dynamics of immune responses in turkey poults infected with E. adenoeides early in life.

Inter-Bragg crystallographic phase retrieval from shape transforms, stacking faults and substitutional disorder.

One of the brilliant ideas of John Spence when he saw the first diffraction patterns from the Linac Coherent Light Source was that one could solve the crystallographic phase problem by utilising the intensities between Bragg peaks. Because these intensities are due to the Fourier transform of the shape of the crystal, the approach came to be known as "shape-transform phasing." Shape-transform phasing was developed over the next ten years and formed the basis for many other interesting ideas and pursuits. Here we describe the current best implementation of the original idea using a lattice occupancy formalism and show that certain types of crystal defects can also be modelled via this approach, allowing the molecular structure to be recovered from the additional information offered by the inter-Bragg intensities from these crystal defects.

X-ray lasers for structural and dynamic biology.

Research opportunities and techniques are reviewed for the application of hard x-ray pulsed free-electron lasers (XFEL) to structural biology. These include the imaging of protein nanocrystals, single particles such as viruses, pump--probe experiments for time-resolved nanocrystallography, and snapshot wide-angle x-ray scattering (WAXS) from molecules in solution. The use of femtosecond exposure times, rather than freezing of samples, as a means of minimizing radiation damage is shown to open up new opportunities for the molecular imaging of biochemical reactions at room temperature in solution. This is possible using a 'diffract-and-destroy' mode in which the incident pulse terminates before radiation damage begins. Methods for delivering hundreds of hydrated bioparticles per second (in random orientations) to a pulsed x-ray beam are described. New data analysis approaches are outlined for the correlated fluctuations in fast WAXS, for protein nanocrystals just a few molecules on a side, and for the continuous x-ray scattering from a single virus. Methods for determining the orientation of a molecule from its diffraction pattern are reviewed. Methods for the preparation of protein nanocrystals are also reviewed. New opportunities for solving the phase problem for XFEL data are outlined. A summary of the latest results is given, which now extend to atomic resolution for nanocrystals. Possibilities for time-resolved chemistry using fast WAXS (solution scattering) from mixtures is reviewed, toward the general goal of making molecular movies of biochemical processes.

A scheme for lensless X-ray microscopy combining coherent diffraction imaging and differential corner holography.

We successfully use the corners of a common silicon nitride supporting window in lensless X-ray microscopy as extended references in differential holography to obtain a real space hologram of the illuminated object. Moreover, we combine this method with the iterative phasing techniques of coherent diffraction imaging to enhance the spatial resolution on the reconstructed object, and overcome the problem of missing areas in the collected data due to the presence of a beam stop, achieving a resolution close to 85 nm.

Femtosecond dark-field imaging with an X-ray free electron laser.

The emergence of femtosecond diffractive imaging with X-ray lasers has enabled pioneering structural studies of isolated particles, such as viruses, at nanometer length scales. However, the issue of missing low frequency data significantly limits the potential of X-ray lasers to reveal sub-nanometer details of micrometer-sized samples. We have developed a new technique of dark-field coherent diffractive imaging to simultaneously overcome the missing data issue and enable us to harness the unique contrast mechanisms available in dark-field microscopy. Images of airborne particulate matter (soot) up to two microns in length were obtained using single-shot diffraction patterns obtained at the Linac Coherent Light Source, four times the size of objects previously imaged in similar experiments. This technique opens the door to femtosecond diffractive imaging of a wide range of micrometer-sized materials that exhibit irreproducible complexity down to the nanoscale, including airborne particulate matter, small cells, bacteria and gold-labeled biological samples.

Thomas K. Jeffers: pioneer of coccidiosis research.

Thomas K. Jeffers has made many significant contributions to our understanding of the biology of the parasite Eimeria, the cause of coccidiosis in poultry. His work has had direct practical application for the control of this widespread disease. Topics discussed include Jeffers' pioneering work concerned with genetics of the host response to infection, the nature of biological and immunological intraspecific variation, drug resistance and discovery, field surveys of resistance, and his most recognized achievement-the demonstration that the lifecycle of coccidia may be altered by artificial selection. Parasites so modified are attenuated but retain their immunogenicity, a discovery that has led to the development of live vaccines that are inherently non-pathogenic. This article provides a brief biography and describes the contributions that Jeffers has made to our knowledge of coccidiosis.

Focused review: The role of drug combinations for the control of coccidiosis in commercially reared chickens.

A survey of drug combinations employed by the poultry industry indicates that they have played an important role in the control of coccidiosis in chickens. The mode of action of their component drugs is described. Advantages that accrue from their use may include a reduction in potential toxicity, a broadening of their spectrum of activity against different species of Eimeria, activity against different stages of the life cycle, and improved efficacy due to synergism between component drugs. Integration of management procedures involving rotation of drug combinations with vaccination is desirable because this has been shown to result in a restoration of drug sensitivity where drug resistance is present and could contribute to the sustainable control of coccidiosis. Threats to the future use of the most widely used combinations, those that include ionophores, stem from the recent desire to eliminate antibiotics from poultry feeds.

Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration.

Cellular migration is critically dependent on an interplay between forces of attachment and detachment. Recent studies show that the serine protease urokinase and its major inhibitor and receptor regulate the adhesive properties of integrins, at least in part through initiation of cellular signals. These new functions for an old protease system imply intricate connections between proteolysis and adhesion that operate at the cell surface to regulate migration.