Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction.

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism

Structure-based vaccine design by electron microscopy

Modern vaccine design relies on multiscale, interdisciplinary efforts that take advantage of innovative technologies such as in silico identification of antigens, high throughput screening of antigen immunogenicity, and gene expression profiling to predict host immune responses. In recent years, structural analysis has played an increasingly important role in vaccine development as a means to improve antigen stability, immunogenicity and large scale production. Transmission electron microscopy (TEM), and in particular cryo-TEM, is an established and powerful imaging technique applicable to many specimens, including the three-dimensional (3D) reconstruction of macromolecules and their associated complexes to high resolution. The technique is parsimonious in its material requirements and captures the specimens in their fully hydrated state, close to their native environment. The resolution of cryo-TEM reconstructions was limited to the subnanometer range until the recent development of direct electron detectors and improvements in image processing software, which has led to a so-called “resolution revolution” in the cryo-TEM field. Several protein structures have now been solved at near atomic resolution, establishing the technique as a viable alternative to X-ray analysis for high resolution structure determination. We have determined several structures with and without bound compounds at 2.9 Å – 3.6 Å resolution, which are being integrated into drug discovery and development workflows by our clients. Here we present the 2.4Å resolution structure of apoferritin determined with our Titan Krios electron microscope as an example of the cryo-TEM services available at NIS. These services are significantly enhanced with unique access by NIS to a new instrument, Spotiton, a robotic device that dispenses picoliter-volumes of sample onto a self-blotting nanowire grid as it flies past en route to vitrification. This provides several advantages over standard vitrification methods, including more automated and reproducible preparation of specimens and reducing the deleterious effects of particles interacting with the air-water interface. While high resolution 3D structure determination by cryo-TEM is at the forefront of structural biology, averages of 2D projection images at moderate resolution in negative stain or vitreous ice can also provide a wealth of information that may be difficult to obtain using other methods. This is illustrated in a number of case studies, including (1) mapping of neutralizing epitopes on the CMV pentameric glycoprotein complex; (2) mapping of neutralizing epitopes on the HIV-1 envelope glycoprotein trimer; (3) assessment of structure and conformational stability of pre- and post-fusion RSV-F protein; (4) characterization of novel adjuvants and protein delivery systems. In summary, both the moderate resolution TEM and high resolution cryo-TEM methods are well suited to extensively characterize antigen structure-function relationships, some of which may be refractory to other experimental methods

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation.

While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity

Developing a toolkit for the assessment and monitoring of musculoskeletal ageing

The complexities and heterogeneity of the ageing process have slowed the development of consensus on appropriate biomarkers of healthy ageing. The Medical Research Council–Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) is a collaboration between researchers and clinicians at the Universities of Liverpool, Sheffield and Newcastle. One of CIMA’s objectives is to ‘Identify and share optimal techniques and approaches to monitor age-related changes in all musculoskeletal tissues, and to provide an integrated assessment of musculoskeletal function’—in other words to develop a toolkit for assessing musculoskeletal ageing. This toolkit is envisaged as an instrument that can be used to characterise and quantify musculoskeletal function during ‘normal’ ageing, lend itself to use in large-scale, internationally important cohorts, and provide a set of biomarker outcome measures for epidemiological and intervention studies designed to enhance healthy musculoskeletal ageing. Such potential biomarkers include: biochemical measurements in biofluids or tissue samples, in vivo measurements of body composition, imaging of structural and physical properties, and functional tests. This review assesses candidate biomarkers of musculoskeletal ageing under these four headings, details their biological bases, strengths and limitations, and makes practical recommendations for their use. In addition, we identify gaps in the evidence base and priorities for further research on biomarkers of musculoskeletal ageing

An economical and highly adaptable optogenetics system for individual and population-level manipulation of Caenorhabditis elegans

This dataset contains the raw imaging files of the following study. Background: Optogenetics allows the experimental manipulation of excitable cells by a light stimulus without the need for technically challenging and invasive procedures. The high degree of spatial, temporal and intensity control that can be achieved with a light stimulus, combined with cell type-specific expression of light-sensitive ion channels, enables highly specific and precise stimulation of excitable cells. Optogenetic tools have therefore revolutionized the study of neuronal circuits in a number of models, including Caenorhabditis elegans. Despite the existence of several optogenetic systems that allow spatial and temporal photoactivation of light-sensitive actuators in C. elegans, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, modular and adjustable optogenetics device for use on different microscopes and worm trackers, which we called the OptoArm. Results: The OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles and light-color. We demonstrate the OptoArm′s power in a population-based multi-parameter study on the contributions of motor circuit cells to age-related motility decline. We found that individual components of the neuromuscular system display different rates of age-dependent deterioration. The functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit aging. Conclusion: We have assembled an economical, reliable and highly adaptable optogenetics system which can be deployed to address diverse biological questions. We provide a detailed description of the construction as well as technical and biological validation of our set-up. Importantly, use of the OptoArm is not limited to C. elegans and may benefit studies in multiple model organisms, making optogenetics more accessible to the broader research communit

Interprofessional Management of Median Arcuate Ligament Syndrome (Dunbar Syndrome) Related to Lumbar Lordosis and Hip Dysplasia: A Patient’s Perspective

We present a 53-year-old female patient with median arcuate ligament syndrome (MALS), also known as Dunbar syndrome or celiac artery compression syndrome, related to lumbar lordosis and hip dysplasia. She utilized interprofessional management strategies, which were beneficial in reducing lumbar lordosis and MALS-related symptoms. This finding is important because there are no other reports in the literature describing interprofessional strategies to manage symptoms for patients who are waiting for surgery or are not candidates for surgery

Experimental Correlation between Nonlinear Optical and Magnetotransport Properties Observed in Au-Co Thin Films

Magnetic materials where at least one dimension is in the nanometer scale typically exhibit different magnetic, magnetotransport, and magnetooptical properties compared to bulk materials. Composite magnetic thin films where the matrix composition, magnetic cluster size, and overall composite film thickness can be experimentally tailored via adequate processing or growth parameters offer a viable nanoscale platform to investigate possible correlations between nonlinear magnetooptical and magnetotransport properties, since both types of properties are sensitive to the local magnetization landscape. It has been shown that the local magnetization contrast affects the nonlinear magnetooptical properties as well as the magnetotransport properties in magnetic-metal/nonmagnetic metal multilayers; thus, nanocomposite films showcase another path to investigate possible correlations between these distinct properties which may prove useful for sensing applications