Shaping Single-Walled Metal Oxide Nanotubes from Precursors of Controlled Curvature

We demonstrate new molecular-level concepts for constructing nanoscopic metal oxide objects. First, the diameters of metal oxide nanotubes are shaped with angstrom-level precision by controlling the shape of nanometer-scale precursors. Second, we measure (at the molecular level) the subtle relationships between precursor shape and structure and final nanotube curvature. Anionic ligands are used to exert fine control over precursor shapes, allowing assembly into nanotubes whose diameters relate directly to the curvatures of the ‘shaped’ precursors

Self-Assembly of an α‑Helical Peptide into a Crystalline Two-Dimensional Nanoporous Framework

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, <b>3FD-IL</b>, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide <b>3FD-IL</b> displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonal packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the <b>3FD-IL</b> nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. The resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria

<div><p>Background</p><p>Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria.</p><p>Results</p><p>Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents.</p><p>Conclusions</p><p>These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.</p></div

Vesicles isolated from supernatant of RBC storage units are membrane-bound, intact, and contain RBC surface antigen and RBC-specific miRNA.

<p><b>A</b>, Unstained vesicles (red, bottom left) or vesicles co-stained with calcein-AM and fluorescent anti-GPA (blue, top right) were analyzed by flow cytometry. >99% of the vesicles were positive for calcein-AM and anti-GPA. <b>B</b>, RNA from vesicles was analyzed by Agilent Bioanalyzer. Peak on electropherogram at 25 nt is internal standard and small peak to the right reflects small RNA. This electropherogram is representative of Bioanlyzer data from six different RBC storage units. <b>C</b>, Levels (Ct values) of miR-451 were assessed by qRT-PCR in stored RBCs, the RMP pellet, and in human aortic endothelial cells (HAECs, negative control). <b>D</b>, Hemoglobin-alpha content of RMP pellet and stored RBCs, as assessed by Western blot. Blot is representative of six different RBC storage units.</p

Representative electron micrographs of pelleted material from supernatant of RBC storage units.

<p><b>A, B,</b> Negative-stain EM images of the unfixed material, showing pleomorphic structures. <b>C,D,</b> Thin-section TEM images, showing membrane encapsulate vesicles. <b>E,F,</b> SEM images. <b>G,H,</b> Negative-stain EM of fixed (G) versus unfixed (H) structures. Vesicles were isolated and imaged as described in Methods section.</p

Rational Design of Helical Nanotubes from Self-Assembly of Coiled-Coil Lock Washers

Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide <b>GCN4-pAA</b>. In solution and the crystalline state, <b>GCN4-pAA</b> adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of <b>GCN4-pAA</b> afforded peptide <b>7HSAP1</b>, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of <b>GCN4-pAA</b>. Fluorescence studies of the interaction of <b>7HSAP1</b> with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity

Representative 3D cryo-electron tomograpahy data and visual characterization of RBC-derived microparticles (RMPs).

<p><b>A,</b> Slices through 3D volumes of RMPs. <b>B,</b> RMPs were measured and were characterized as either round or pleiomorphic, and as either full or empty. The number of each particle type, percent of the total, mean diameter, and standard deviation are presented in the table.</p

Rational Design of Helical Nanotubes from Self-Assembly of Coiled-Coil Lock Washers

Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide <b>GCN4-pAA</b>. In solution and the crystalline state, <b>GCN4-pAA</b> adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of <b>GCN4-pAA</b> afforded peptide <b>7HSAP1</b>, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of <b>GCN4-pAA</b>. Fluorescence studies of the interaction of <b>7HSAP1</b> with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity

Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (<i>e</i>.<i>g</i>., labeling an envelope protein) and the rescue of viral infectivity (<i>e</i>.<i>g</i>., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses