in-situ-tem-of-biological-assemblies-in-liquid

<em>In situ</em> 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 ...

erratum-in-situ-tem-of-biological-assemblies-in-liquid

Erratum: <em>In situ</em> TEM of Biological Assemblies in Liquid

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Virginia Tech Carilion Research Institute

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.

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

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.

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

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.

Visualizing viral assemblies in a nanoscale biosphere.

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.

Justin R. Tanner

Virginia Tech Carilion Research Institute

In situ TEM of Biological Assemblies in Liquid

Erratum: In situ TEM of Biological Assemblies in Liquid