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Background Neutrophils are thought to be short‐lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. Methods and Results We permanently ligated the left anterior descending coronary arteries of mice and performed single‐cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady‐state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late‐emerging granulocyte expressing SiglecF, a marker classically used to define eosinophils. SiglecFHI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post‐MI. Validation studies using quantitative polymerase chain reaction of fluorescent‐activated cell sorter sorted Ly6G+SiglecFHI and Ly6G+SiglecFLO neutrophils confirmed the distinct nature of these populations. To confirm that the cells were neutrophils rather than eosinophils, we infarcted GATA‐deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G+SiglecFHI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G+SiglecFHI neutrophils expressed high levels of Myc‐regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. Conclusions Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and reveal a dynamic proinflammatory cardiac Ly6G+SigF+(Myc+NFϰB+) neutrophil that has been overlooked because of negative selection.

Fixed-charge (non-polarizable) forcefields are accurate and computationally efficient tools for modeling the molecular dynamics of nucleic acid polymers, particularly DNA, well into the µs timescale. The continued utility of these forcefields depends in part on expanding the residue set in step with advancing nucleic acid chemistry and biology. A key step in parameterizing new residues is charge derivation which is self-consistent with the existing residues. As atomic charges are derived by fitting against molecular electrostatic potentials, appropriate structural models are critical. Benchmarking against the existing charge set used in current AMBER nucleic acid forcefields, we report that quantum mechanical models of deoxynucleosides, even at a high level of theory, are not optimal structures for charge derivation. Instead, structures from molecular mechanics minimization yield charges with up to 6-fold lower RMS deviation from the published values, due to the choice of such an approach in the derivation of the original charge set. We present a contemporary protocol for rendering self-consistent charges as well as optimized charges for a panel of nine non-canonical residues that will permit comparison with literature as well as studying the dynamics of novel DNA polymers.

Ca2+-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca2+]o) homeostasis and many (patho)physiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT) domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR’s cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs) in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.