No abstract

5Aflatoxins, a group of polyketide-derived furanocoumarins (Fig. 1), are the most toxic and carcinogenic compounds among the known mycotoxins. Among the at least 16 structurally related aflatoxins characterized, however, there are only four major aflatoxins,

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.

Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cellbased assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1β that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions. H uman epidermal growth factor receptor (EGFR) and its homologs, known as ErbBs or HERs, are essential receptor tyrosine kinases that mediate cell proliferation and differentiation during animal development and are the targets of multiple cancer therapies (1). EGFR is the archetype of single-pass membrane-spanning receptors thought to transmit signals by ligandinduced dimerization (2, 3), and structural studies show that ligand binding to human EGFR promotes rearrangement of its four extracellular domains from a tethered to an extended conformation in which a loop, termed the dimerization arm, becomes exposed and mediates formation of symmetric receptor dimers (4) (Fig. 1A). At odds with a ligand-induced dimerization model of EGFR signaling, however, are recent studies showing dimers of human EGFR in the absence of ligand (5-8) as well as negative cooperativity when epidermal growth factor (EGF) binds to EGFR (9). Curiously, the single Drosophila EGFR homolog adopts an extended conformation in the absence of ligand and forms asymmetric receptor dimers with a single high-affinity ligand bound (10, 11), suggesting different mechanisms may regulate EGFR activation in Drosophila and humans.We report here evidence for active, singly ligated homodimers of human EGFR and its homolog, ErbB4. We also report the crystal structure of the ErbB4 extracellular region bound to its ligand Neuregulin-1β, which allows resolution of two types of human EGFR/ErbB dimers, one of which resembles the asymmetric Drosophila EGFR dimer and appears to reflect a singly ligated ErbB dimer state. These results compel reappraisal of canonical views of ligand-induced dimerization and show that several previously anomalous properties of human EGFR and its homologs represent vertebrate innovations on a core signaling mechanism present in invertebrates. Results a...