Tunable substrates improve imaging of viruses and cancer proteins.

Recent breakthroughs in cryo-electron microscopy imaging technology provide an unprecedented view of biology at the nanoscale. To complement these technical advances, here we demonstrate the use of tunable substrates to streamline the isolation of biological entities from human cells. We have tested the capacity of tunable microchip devices using a variety of samples including virus assemblies and the breast cancer susceptibility protein (BRCA1) produced in cancer cells. Overall, microchip applications may shed light on ill-defined clinical issues related to molecular disease mechanisms.

A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease.

We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time.

Rotavirus core shell subdomains involved in polymerase encapsidation into virus-like particles.

The triple-layered rotavirus virion encases an 11-segmented, dsRNA genome and 11-12 copies of the viral polymerase (VP1). VP1 transcribes and replicates the genome while tethered beneath the VP2 core shell. Genome replication (i.e. minus-strand RNA synthesis) by VP1 occurs in association with core assembly. During this process, VP2 directly engages VP1, thereby (i) packaging the polymerase into a nascent core and (ii) triggering the enzyme to initiate minus-strand RNA synthesis on bound plus-strand RNA templates. Recent work has shed light on VP2 regions important for VP1 enzymic activity. In the current study, we sought to investigate VP2 subdomains involved in the encapsidation of VP1 into recombinant virus-like particles (VLPs), which are formed of VP2 and the middle layer virion protein (VP6). We showed that strain SA11 VLPs efficiently encapsidated SA11 VP1, but not the genetically divergent Bristol VP1. VLPs made with an SA11 VP2 mutant lacking residues 1-10 of the amino-terminal domain (NTD) were still able to encapsidate VP1; however, removal of the entire NTD (residues 1-102) completely abolished polymerase packaging. We also showed that a chimeric VP2 protein containing the NTD and dimer-forming subdomain of strain Bristol VP2 can efficiently encapsidate SA11 VP1. These results suggest that the VP2 NTD and dimer-forming subdomain play important, albeit non-specific, roles in both VP1 packaging and activation. When combined with previous work, the results of this study support the notion that the same VP2 regions that engage VP1 during activation are also involved in packaging the enzyme into the core.

In situ TEM of biological assemblies in liquid.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

Visualizing viral assemblies in a nanoscale biosphere.

We present a novel microfluidic platform to examine biological assemblies at high-resolution. We have engineered a functionalized chamber that serves as a "nanoscale biosphere" to capture and maintain rotavirus double-layered particles (DLPs) in a liquid environment. The chamber can be inserted into the column of a transmission electron microscope while being completely isolated from the vacuum system. This configuration allowed us to determine the structure of biological complexes at nanometer-resolution within a self-contained vessel. Images of DLPs were used to calculate the first 3D view of macromolecules in solution. We refer to this new fluidic visualization technology as in situ molecular microscopy.

Capturing RNA-dependent pathways for cryo-EM analysis.

Cryo-Electron Microscopy (EM) is a powerful technique to visualize biological processes at nanometer resolution. Structural studies of macromolecular assemblies are typically performed on individual complexes that are biochemically isolated from their cellular context. Here we present a molecular imaging platform to capture and view multiple components of cellular pathways within a functionally relevant framework. We utilized the bacterial protein synthesis machinery as a model system to develop our approach. By using modified Affinity Grid surfaces, we were able to recruit multiple protein assemblies bound to nascent strands of mRNA. The combined use of Affinity Capture technology and single particle electron microscopy provide the basis for visualizing RNA-dependent pathways in a remarkable new way.

Proliferation of antibiotic-producing bacteria and concomitant antibiotic production as the basis for the antibiotic activity of Jordan's red soils.

Anecdotes, both historical and recent, recount the curing of skin infections, including diaper rash, by using red soils from the Hashemite Kingdom of Jordan. Following inoculation of red soils isolated from geographically separate areas of Jordan, Micrococcus luteus and Staphylococcus aureus were rapidly killed. Over the 3-week incubation period, the number of specific types of antibiotic-producing bacteria increased, and high antimicrobial activity (MIC, approximately 10 microg/ml) was observed in methanol extracts of the inoculated red soils. Antibiotic-producing microorganisms whose numbers increased during incubation included actinomycetes, Lysobacter spp., and Bacillus spp. The actinomycetes produced actinomycin C(2) and actinomycin C(3). No myxobacteria or lytic bacteriophages with activity against either M. luteus or S. aureus were detected in either soil before or after inoculation and incubation. Although protozoa and amoebae were detected in the soils, the numbers were low and did not increase over the incubation period. These results suggest that the antibiotic activity of Jordan's red soils is due to the proliferation of antibiotic-producing bacteria.

Prospective randomized trial of wavefront-guided laser in situ keratomileusis with the CustomCornea and CustomVue laser systems.

PURPOSE: To compare visual function, safety, and higher-order aberrations (HOAs) after wavefront-guided laser in situ keratomileusis (LASIK) with the LadarVision CustomCornea (Alcon Laboratories, Inc.) and Star S4 CustomVue (Visx) laser systems. SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. METHODS: Seventy-eight eyes of 39 patients with myopia with or without astigmatism were randomized for LASIK treatment in 1 eye with the CustomCornea laser; the other eye was treated with the CustomVue laser. Patients were followed for 6 months after surgery. The primary outcome measures were uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), manifest refraction, and changes in HOAs. RESULTS: At 6 months, the mean logarithm of the minimum angle of resolution (logMAR) UCVA was -0.0135 +/- 0.07 (SD) in the CustomVue group and 0.0417 +/- 0.12 in the CustomCornea group (P = .023). Eighty-eight percent of eyes in the CustomVue group had 20/20 or better UCVA compared with 67% in the CustomCornea group (P<.02). At 6 months, 91% of eyes in the CustomVue group and 79% in the CustomCornea group were within +/-0.50 diopter (D) of emmetropia (P<.1); 88% and 50%, respectively, were within +/-0.25 D (P<.001). Both platforms led to a small increase in total HOAs. The CustomVue system reduced trefoil and induced less of an increase in total HOAs, whereas the CustomCornea platform increased trefoil but induced less of an increase in spherical aberrations and coma. CONCLUSIONS: Both laser systems were effective, safe, and predictable. Wavefront-guided LASIK with the CustomVue system resulted in better visual acuity, with more eyes having 20/20 acuity than in the CustomCornea group.