Structural Studies on an Integral Membrane Light-Harvesting Complex

The crystallographic structure of the B800-850 peripheral light harvesting complex, an integral membrane pigment-protein complex, from the non-sulphur purple bacteria Rhodopseudomonas acidophila, strain 10050, has been determined to a resolution of 2.5 A (McDermott et al., 1995). This thesis outlines the changes made to the purification and crystallisation protocols which produced crystals which diffracted reproducibly to high resolution. In addition, microspectroscopy was utilised to determine the integrity of the complex in the crystal and to determine the effect of 'heavy atom soaking' procedures on the crystal order. The latter allowed the development of a soaking solution which maintained crystal order during heavy atom derivatisation trials. The LH structure is described and some initial interpretations of the implications that the structure has for the study of bacterial photosynthesis

Bluetongue virus infection creates light averse Culicoides vectors and serious errors in transmission risk estimates.

BackgroundPathogen manipulation of host behavior can greatly impact vector-borne disease transmission, but almost no attention has been paid to how it affects disease surveillance. Bluetongue virus (BTV), transmitted by Culicoides biting midges, is a serious disease of ruminant livestock that can cause high morbidity and mortality and significant economic losses. Worldwide, the majority of surveillance for Culicoides to assess BTV transmission risk is done using UV-light traps. Here we show that field infection rates of BTV are significantly lower in midge vectors collected using traps baited with UV light versus a host cue (CO2).MethodsWe collected Culicoides sonorensis midges in suction traps baited with CO2, UV-light, or CO2 + UV on three dairies in southern California to assess differences in the resulting estimated infection rates from these collections. Pools of midges were tested for BTV by qRT-PCR, and maximum likelihood estimates of infection rate were calculated by trap. Infection rate estimates were also calculated by trapping site within a dairy. Colonized C. sonorensis were orally infected with BTV, and infection of the structures of the compound eye was examined using structured illumination microscopy.ResultsUV traps failed entirely to detect virus both early and late in the transmission season, and underestimated virus prevalence by as much as 8.5-fold. CO2 + UV traps also had significantly lower infection rates than CO2-only traps, suggesting that light may repel infected vectors. We found very high virus levels in the eyes of infected midges, possibly causing altered vision or light perception. Collecting location also greatly impacts our perception of virus activity.ConclusionsBecause the majority of global vector surveillance for bluetongue uses only light-trapping, transmission risk estimates based on these collections are likely severely understated. Where national surveillance programs exist, alternatives to light-trapping should be considered. More broadly, disseminated infections of many arboviruses include infections in vectors' eyes and nervous tissues, and this may be causing unanticipated behavioral effects. Field demonstrations of pathogen-induced changes in vector behavior are quite rare, but should be studied in more systems to accurately predict vector-borne disease transmission

Laboratory evaluation of stable isotope labeling of Culicoides (Diptera: Ceratopogonidae) for adult dispersal studies.

BackgroundStable isotope labeling is a promising method for use in insect mark-capture and dispersal studies. Culicoides biting midges, which transmit several important animal pathogens, including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), are small flies that develop in various semi-aquatic habitats. Previous Culicoides dispersal studies have suffered from the limitations of other labeling techniques, and an inability to definitively connect collected adult midges to specific immature development sites.ResultsAdult C. sonorensis were successfully labeled with 13C and 15N stable isotopes as larvae developing in a semi-aquatic mud substrate in the laboratory. High and low-dose isotope treatments for both elements significantly enriched midges above the background isotope levels of unenriched controls. Enrichment had no effect on C. sonorensis survival, though a slight (~ 5 day) delay in emergence was observed, and there was no significant effect of pool size on 13C or 15N enrichment levels.ConclusionsStable isotope labeling is life-long, and does not interfere with natural insect behaviors. Stable isotope enrichment using 13C or 15N shows promise for Culicoides dispersal studies in the field. This method can be used to identify adult dispersal from larval source habitat where a midge developed. It may be possible to detect a single enriched midge in a pool of unenriched individuals, though further testing is needed to confirm the sensitivity of this method

Understanding the delayed prescribing of antibiotics for respiratory tract infection in primary care: a qualitative analysis

OBJECTIVE: To identify general practitioner (GP) views and understanding on the use of delayed prescribing in primary care.DESIGN: Qualitative semistructured telephone interview study.SETTING: Primary care general practices in England.PARTICIPANTS: 32 GPs from identified high-prescribing and low-prescribing general practices in England.METHOD: Semistructured telephone interviews were conducted with GPs identified from practices within clinical commissioning groups with the highest and lowest prescribing rates in England. A thematic analysis of the data was conducted to generate themes.RESULTS: All GPs had a good understanding of respiratory tract infection (RTI) management and how the delayed prescribing approach could be used in primary care. However, GPs highlighted factors that were influential as to whether delayed prescribing was successfully carried out during the consultation. These included the increase in evidence of antimicrobial resistance, and GPs' prior experiences of using delayed prescribing during the consultation. The patient-practitioner relationship could also influence treatment outcomes for RTI, and a lack of an agreed prescribing strategy within and between practices was considered to be of significance to GPs. Participants expressed that a lack of feedback on prescribing data at an individual and practice level made it difficult to know if delayed prescribing strategies were successful in reducing unnecessary consumption. GPs agreed that coherent and uniform training and guidelines would be of some benefit to ensure consistent prescribing throughout the UK.CONCLUSIONS: Delayed prescribing is encouraged in primary care, but is not always implemented successfully. Greater uniformity within and between practices in the UK is needed to operationalise delayed prescribing, as well as providing feedback on the uptake of antibiotics. Finally, GPs may need further guidance on how to answer the concerns of patients without interpreting these questions as a demand for antibiotics, as well as educating the patient about antimicrobial resistance and supporting a good patient-practitioner relationship

Switchable resolution in soft x-ray tomography of single cells.

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

AbstractSoft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant

Conformational change of the AcrR regulator reveals a possible mechanism of induction

The Escherichia coli AcrR multidrug-binding protein represses transcription of acrAB and is induced by many structurally unrelated cytotoxic compounds. The crystal structure of AcrR in space group P2221 has been reported previously. This P2221 structure has provided direct information about the multidrug-binding site and important residues for drug recognition. Here, a crystal structure of this regulator in space group P31 is presented. Comparison of the two AcrR structures reveals possible mechanisms of ligand binding and AcrR regulation

Structure of an Early Intermediate in the M-State Phase of the Bacteriorhodopsin Photocycle

AbstractThe structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230K has been determined by x-ray crystallography to a resolution of 2.0Å. Three-dimensional crystals were trapped by illuminating with actinic light at 230K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13—C14 double bond appears to be complete, but a relatively large torsion angle of 26° is still seen for the C14—C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (MN) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics