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

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