A simple and efficient method is described for introduction of non-canonical amino acids at multiple, structurally defined sites within recombinant polypeptide sequences. E. coli MRA30, a bacterial host strain with attenuated activity for release factor 1 (RF1), is assessed for its ability to support the incorporation of a diverse range of non-canonical amino acids in response to multiple encoded amber (TAG) codons within genetic templates derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and isolated protein yield were observed to depend on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNACUA pair and the non-canonical amino acid substrate. This approach afforded elastin-mimetic protein polymers containing non-canonical amino acid derivatives at up to twenty-two positions within the repeat sequence with high levels of substitution. The identity and position of the variant residues was confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments resulting from thermolysin digestion. The accumulated data suggest that this multi-site suppression approach permits the preparation of protein-based materials in which novel chemical functionality can be introduced at precisely defined positions within the polypeptide sequence.

In palliative care, we strive to provide care to the whole patient. When we think about the whole patient, we include the people who are important in our patients' lives. Our New York City-based palliative care team has found that caring for patients' loved ones has proven to be an even more important aspect of the care we have provided during the COVID epidemic. In this article, we describe the multicomponenet interdisciplinary interventions we have implemented to enhance our ability to create a therapeutic alliance with family members and facilitate the provision of goal concordant care to patients with COVID during this extremely difficult time.

Gangliosides have diverse immunoregulatory properties. The gangliosides endogenous to macrophages may have immunoregulatory properties that distinguish them from other gangliosides. Gangliosides have been indirectly implicated in macrophage migration as putative cell surface receptors for migration inhibitory factor (MIF). In this study, a monoclonal antibody to human macrophage gangliosides (antibody 25F4) was developed and characterized. This is the first report of the development of monoclonal antibodies to gangliosides of macrophages of any species. Thin-layer chromatographic immunostaining indicated that antibody 25F4 recognized major gangliosides of human macrophages but did not recognized those previously identified as containing fucose. Immunofluorescent surface labeling of viable human macrophages indicated that antibody 25F4 recognized a surface-accessible epitope, present on all cells, and that this was abolished with lipid depletion of macrophage membranes. This epitope was not present on several human nonmacrophage cells. Finally, human macrophages pretreated with antibody 25F4 demonstrated striking inhibition of migration of an agarose droplet assay, whereas an irrelevant monoclonal antibody or monoclonal antibodies to nonganglioside surface epitopes of human macrophages had no effect on migration. Migration inhibition occurred even though antibody 25F4 was removed from the extracellular milieu and was not due to formation of cellular aggregates. These studies support a role for human macrophage gangliosides in macrophage migration.

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