Repositioning the Early Pathology of Type 1 Diabetes to the Extraislet Vasculature.

Type 1 diabetes (T1D) is a prototypic T cell-mediated autoimmune disease. Because the islets of Langerhans are insulated from blood vessels by a double basement membrane and lack detectable lymphatic drainage, interactions between endocrine and circulating T cells are not permitted. Thus, we hypothesized that initiation and progression of anti-islet immunity required islet neolymphangiogenesis to allow T cell access to the islet. Combining microscopy and single cell approaches, the timing of this phenomenon in mice was situated between 5 and 8 wk of age when activated anti-insulin CD4 T cells became detectable in peripheral blood while peri-islet pathology developed. This "peri-insulitis," dominated by CD4 T cells, respected the islet basement membrane and was limited on the outside by lymphatic endothelial cells that gave it the attributes of a tertiary lymphoid structure. As in most tissues, lymphangiogenesis seemed to be secondary to local segmental endothelial inflammation at the collecting postcapillary venule. In addition to classic markers of inflammation such as CD29, V-CAM, and NOS, MHC class II molecules were expressed by nonhematopoietic cells in the same location both in mouse and human islets. This CD45- MHC class II+ cell population was capable of spontaneously presenting islet Ags to CD4 T cells. Altogether, these observations favor an alternative model for the initiation of T1D, outside of the islet, in which a vascular-associated cell appears to be an important MHC class II-expressing and -presenting cell.

Representation matters: responding to the current campaign against DEI efforts.

Abandonment of diversity, equity and inclusion programs undermines fairness and the productivity of research.

Solution-processable polytriazoles from spirocyclic monomers for membrane-based hydrocarbon separations.

The thermal distillation of crude oil mixtures is an energy-intensive process, accounting for nearly 1% of global energy consumption. Membrane-based separations are an appealing alternative or tandem process to distillation due to intrinsic energy efficiency advantages. We developed a family of spirocyclic polytriazoles from structurally diverse monomers for membrane applications. The resulting polymers were prepared by a convenient step-growth method using copper-catalysed azide-alkyne cycloaddition, providing very fast reaction rates, high molecular weights and solubilities in common organic solvents and non-interconnected microporosity. Fractionation of whole Arabian light crude oil and atmospheric tower bottom feeds using these materials enriched the low-boiling-point components and removed trace heteroatom and metal impurities (comparable performance with the lighter feed as the commercial polyimide, Matrimid), demonstrating opportunities to reduce the energy cost of crude oil distillation with tandem membrane processes. Membrane-based molecular separation under these demanding conditions is made possible by high thermal stability and a moderate level of dynamic chain mobility, leading to transient interconnections between micropores, as revealed by the calculations of static and swollen pore structures.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

Synthesis of propargyl glycosides of Streptococcus pneumoniae serotypes 6A and 6B for glycoconjugate vaccines.

We developed a method for making immune responses to bacterial glycans T cell-dependent, which involves attachment of short, synthetic glycans to a virus-like nanoparticle (VLP). This strategy enhances immune responses to glycans by facilitating cognate T cell help of B cells, leading to antibody class switching and affinity maturation yielding high-affinity, anti-glycan antibodies. This method requires synthesis of bacterial glycans as propargyl glycosides for covalent attachment to VLPs, and the resulting short linker between the VLP and glycan is important for optimal T cell receptor recognition. In this work, glycans that are part of the capsular polysaccharides (CPS) produced by Streptococcus pneumoniae serotypes Sp6A and Sp6B were synthesized as disaccharides and trisaccharides. The optimal glycan epitope for antibody binding to the CPS from these serotypes is unknown, and differing "frames" of disaccharides and trisaccharides were prepared to elucidate the optimal antigen for antibody binding.

Zeolite-like performance for xylene isomer purification using polymer-derived carbon membranes.

Polymers of intrinsic microporosity (PIMs) have been used as precursors for the fabrication of porous carbon molecular sieve (CMS) membranes. PIM-1, a prototypical PIM material, uses a fused-ring structure to increase chain rigidity between spirobisindane repeat units. These two factors inhibit effective chain packing, thus resulting in high free volume within the membrane. However, a decrease of pore size and porosity was observed after pyrolytic conversion of PIM-1 to CMS membranes, attributed to the destruction of the spirocenter, which results in the "flattening" of the polymer backbone and graphite-like stacking of carbonaceous strands. Here, a spirobifluorene-based polymer of intrinsic microporosity (PIM-SBF) was synthesized and used to fabricate CMS membranes that showed significant increases in p-xylene permeability (approximately four times), with little loss in p-xylene/o-xylene selectivity (13.4 versus 14.7) for equimolar xylene vapor separations when compared to PIM-1-derived CMS membranes. This work suggests that it is feasible to fabricate such highly microporous CMS membranes with performances that exceed current state-of-the-art zeolites at high xylene loadings.

Multivalent Targeting of the Asialoglycoprotein Receptor by Virus-Like Particles.

The asialoglycoprotein receptor (ASGPR) is expressed in high density on hepatocytes. Multivalent variants of galactosyl carbohydrates bind ASGPR with high affinity, enabling hepatic delivery of ligand-bound cargo. Virus-like particle (VLP) conjugates of a relatively high-affinity ligand were efficiently endocytosed by ASGPR-expressing cells in a manner strongly dependent on the nature and density of ligand display, with the best formulation using a nanomolar-, but not a picomolar-level, binder. Optimized particles were taken up by HepG2 cells with greater efficiency than competing small molecules or the natural multigalactosylated ligand, asialoorosomucoid. Upon systemic injection in mice, these VLPs were rapidly cleared to the liver and were found in association with sinusoidal endothelial cells, Kupffer cells, hepatocytes, dendritic cells, and other immune cells. Both ASGPR-targeted and nontargeted particles were distributed similarly to endothelial and Kupffer cells, but targeted particles were distributed to a greater number and fraction of hepatocytes. Thus, selective cellular trafficking in the liver is difficult to achieve: even with the most potent ASGPR targeting available, barrier cells take up much of the injected particles and hepatocytes are accessed only approximately twice as efficiently in the best case.

Oral Delivery of Nanoparticles Carrying Ancestral Uricase Enzyme Protects against Hyperuricemia in Knockout Mice.

The therapeutic value of delivering recombinant uricase to human patients has been appreciated for decades. The development of therapeutic uricases has been hampered by the fact that humans do not encode an endogenous uricase and therefore most recombinant forms of the protein are recognized as foreign by the immune system and are therefore highly immunogenic. In order to both shield and stabilize the active enzyme, we encapsulated a functional ancestral uricase in recombinant, noninfectious Qß capsid nanoparticles and characterized its catalytic activity. Oral delivery of the nanoparticles moderated key symptoms of kidney dysfunction in uricase-knockout mice by lowering uric acid levels. Histological kidney samples of the treated mice suggest that delivery of recombinant uricase had a protective effect against the destructive effects of uric acid that lead to renal failure caused by hyperuricemia.

Modular Catalysis: Aptamer Enhancement of Enzyme Kinetics in a Nanoparticle Reactor.

Enzyme activity requires sequential binding and chemical transformation of substrates. While directed evolution and random mutagenesis are common methods for improving catalytic activity, these methods do not allow for independent control of KM and kcat. To achieve such control, we envisioned that the colocalization of aptamers and enzymes that act on the same molecule could increase catalytic efficiency through preconcentration of substrate. We explored this concept with cocaine esterase and anticocaine aptamers having varying KD values, both encapsulated in MS2 virus-like particles. Rate enhancements were observed with magnitudes dependent on both aptamer:enzyme stoichiometry and aptamer KD, peaking when aptamer KD and enzyme KM were roughly equivalent. This beneficial effect was lost when either aptamer binding was too tight or the aptamers were not constrained to be close to the catalyst. This work demonstrates a modular way to enhance catalysis by independently controlling substrate capture and release to the processing enzyme.

Preparation and Biological Properties of Oligonucleotide-Functionalized Virus-like Particles.

Oligonucleotides are powerful molecules for programming function and assembly. When arrayed on nanoparticle scaffolds in high density, the resulting molecules, spherical nucleic acids (SNAs), become imbued with unique properties. We used the copper-catalyzed azide-alkyne cycloaddition to graft oligonucleotides on Qß virus-like particles to see if such structures also gain SNA-like behavior. Copper-binding ligands were shown to promote the click reaction without degrading oligonucleotide substrates. Reactions were first optimized with a small-molecule fluorogenic reporter and were then applied to the more challenging synthesis of polyvalent protein nanoparticle-oligonucleotide conjugates. The resulting particles exhibited the enhanced cellular uptake and protection from nuclease-mediated oligonucleotide cleavage characteristic of SNAs, had similar residence time in the liver relative to unmodified particles, and were somewhat shielded from immune recognition, resulting in nearly 10-fold lower antibody titers relative to unmodified particles. Oligonucleotide-functionalized virus-like particles thus provide an interesting option for protein nanoparticle-mediated delivery of functional molecules.