Cryo-EM phase-plate images reveal unexpected levels of apparent specimen damage.

Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 Å resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected. By comparison to simulations of images, we verified that the heterogeneity is not explained by the known features of the LPP, shot noise, or differences in particle orientation. We also demonstrate that our specimens are comparable to those previously used in the literature, based on using the final-reconstruction resolution as the metric for evaluation. All of this leads us to the hypothesis that the heterogeneity is due to damage that has occurred either during purification of the specimen or during preparation of the grids. It is not, however, our goal to explain the causes of heterogeneity; rather, we report that using the LPP has made the apparent damage too obvious to be ignored. In hindsight, similar heterogeneity can be seen in images of apoF and the 20S proteasome which others had recorded with a Volta phase plate. We therefore conclude that the increased contrast of phase-plate images (at low spatial frequencies) should also make it possible to visualize, on a single-particle basis, various forms of biologically functional heterogeneity in structure that had previously gone unnoticed.

High-power near-concentric Fabry-Perot cavity for phase contrast electron microscopy.

Transmission electron microscopy (TEM) of vitrified biological macromolecules (cryo-EM) is limited by the weak phase contrast signal that is available from such samples. Using a phase plate would thus substantially improve the signal-to-noise ratio. We have previously demonstrated the use of a high-power Fabry-Perot cavity as a phase plate for TEM. We now report improvements to our laser cavity that allow us to achieve record continuous wave intensities of over 450 GW/cm2, sufficient to produce the optimal 90° phase shift for 300 keV electrons. In addition, we have performed the first cryo-EM reconstruction using a laser phase plate, demonstrating that the stability of this laser phase plate is sufficient for use during standard cryo-EM data collection.

The Epidemiology, Cost, and Occupational Context of Spinal Injuries Sustained While 'Working for Income' in NSW: A Record-Linkage Study.

This study aimed to describe the epidemiological characteristics, the occupational context, and the cost of hospitalised work-related traumatic spinal injuries, across New South Wales, Australia. A record-linkage study of hospitalised cases of work-related spinal injury (ICD10-AM code U73.0 or workers compensation) was conducted. Study period 2013⁻2016. Eight hundred and twenty-four individuals sustained work-related spinal injuries; 86.2% of whom were males and had a mean age of 46.6 years. Falls led to 50% of the injuries; predominantly falls from building/structures, ladders or between levels. Falls occurred predominantly in the construction industry (78%). Transport crashes caused 31% of injuries and 24% in heavy vehicles. Half of all the transport injuries occurred 'off road'. The external cause was coded as 'non-specific work activity' in 44.5% of cases; missing in 11.5%. Acute care bed days numbered at 13,302; total cost $19,500,000. High numbers of work-related spinal injuries occurred in the construction industry; particularly falling from a height. Off-road transport-related injuries were significant and likely unaddressed by 'on-road' prevention policies. Medical record documentation was insufficient in injury mechanism and context specificity. Workers in the construction industry or those using vehicles off-road were at high risk of spinal injury, suggesting inefficient systems approaches or ineffective prevention policies. Reducing the use of non-specific external cause codes in patients' medical records would improve the measurement of policy effectiveness.

Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3.

Defensins are natural mucosal antimicrobial peptides and their broad spectrum activity against aerobic or facultative anaerobic bacteria has been well investigated. The aim of this study was to systematically examine the antibacterial activity of the small intestinal Paneth cell derived alpha-defensin HD5 and the major colonic beta-defensins HBD-1-3 against strict anaerobic intestinal bacteria. The antibacterial activity was assessed with a flow cytometric assay employing a membrane potential sensitive dye as marker for loss of cell viability. The majority of the tested strains belonging to the dominant anaerobe genera of the gut, Bacteroides and Parabacteroides, were only minimally affected by the constitutively expressed defensins HD5 and HBD-1. The inducible defensin HBD-2 had a limited antibacterial effect, whereas the inducible HBD-3 exhibited potent activity against most strains. The effect of HBD-3 on Bacteroides sp. appeared to be dependent on the presence of oxygen. Bacteroides fragilis strains isolated from blood during bacteremia or from extraintestinal infections were more resistant to HBD-3 than strains from the physiological gut flora. Thus, defensin resistance is not only species- but also strain-specific and may be clinically relevant in the host-bacteria interaction influencing mucosal translocation and systemic infection.

A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts.

To determine the antibacterial activity of defensins and other antimicrobial peptides in biopsy extracts, we evaluated a flow cytometric method with the membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. This assay enables us to discriminate intact non-fluorescent and depolarized fluorescent bacteria after exposure to antimicrobial peptides by measurement at the direct target, the cytoplasmic membrane and the membrane potential. The feasibility of the flow cytometric assay was evaluated with recombinant human beta-defensin 3 (HBD-3) against 25 bacterial strains representing 12 species. HBD-3 showed a broad-spectrum dose dependent activity and the minimal dose to cause depolarization ranged from 1.25 to >15 microg/ml HBD-3, depending on the species tested. The antibacterial effect was diminished with sodium chloride or dithiothreitol and could be abrogated with a HBD-3 antibody. Additionally, isolated cationic extracts from human intestinal biopsies showed a strong bactericidal effect against Escherichia coli K12, E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923, which was diminished towards E. coli at 150 mM NaCl, whereas the activity towards S. aureus ATCC 25923 remained unaffected at physiological salt concentrations. DTT blocked the bactericidal effect of biopsy extracts completely.