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AbstractLeukopoiesis is lethally arrested in mice lacking the master transcriptional regulator PU.1. Depending on the animal model, subtotal PU.1 loss either induces acute myeloid leukemia or arrests early B-cell and dendritic-cell development. Although humans with absolute PU.1 deficiency have not been reported, a small cadre of congenital agammaglobulinemia patients with sporadic, inborn PU.1 haploinsufficiency was recently described. To better estimate the penetrance, clinical complications, immunophenotypic features, and malignancy risks of PU.1-mutated agammaglobulinemia (PU.MA), a collection of 134 novel or rare PU.1 variants from publicly available databases, institutional cohorts, previously published reports, and unsolved agammaglobulinemia cases were functionally analyzed. In total, 25 loss-of-function (LOF) variants were identified in 33 heterozygous carriers from 21 kindreds across 13 nations. Of individuals harboring LOF PU.1 variants, 22 were agammaglobulinemic, 5 displayed antibody deficiencies, and 6 were unaffected, indicating an estimated disease penetrance of 81.8% with variable expressivity. In a cluster of patients, disease onset was delayed, sometimes into adulthood. All LOF variants conveyed effects via haploinsufficiency, either by destabilizing PU.1, impeding nuclear localization, or directly interfering with transcription. PU.MA patient immunophenotypes consistently demonstrated B-cell, conventional dendritic-cell, and plasmacytoid dendritic-cell deficiencies. Associated infectious and noninfectious symptoms hewed closely to X-linked agammaglobulinemia and not monogenic dendritic-cell deficiencies. No carriers of LOF PU.1 variants experienced hematologic malignancies. Collectively, in vitro and clinical data indicate heterozygous LOF PU.1 variants undermine humoral immunity but do not convey strong leukemic risks. 

Abstract Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genusOrthoflaviviruscontains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target inOrthoflavivirusgenomes. 

Voronoi diagrams are widely used to model disperse systems such as foams, powders, polycrystals and atoms in the classical limit. Voronoi tessellations partition the continuous phase into compartments, or cells, that encompass all space closer to the assigning dispersed object than any other in the system. To account for heterogeneity in object size, weights are applied to avoid unphysical partitioning across non-contacting objects. Power and additive weighting are the most common weighting schemes, wherein power is more computationally tractable but additive weighting correlates more directly with size. In general, the two schemes produce distinct spatial decompositions for any non-monodisperse system. To calibrate the divergent volumetric metrics from the two schemes, and to gain physical insight into their divergence, we compared power and additively weighted Voronoi diagrams of polydisperse ensembles representing physically relevant ranges of polydispersity, density, and overlap. When tested against experimental distributions of gas foams, the results related their divergent power and additively weighted decompositions to the polydispersity of their particle size distributions. Geometric analysis of the Voronoi cells implicated the subpopulation of small objects as the primary contributors to the divergence through their preferential assignment of larger, aspherical power cells relative to their additively weighted counterparts. 

Abstract Sequencing of melanomas has identified hundreds of recurrent mutations in both coding and non-coding DNA. These include a number of well-characterized oncogenic driver mutations, such as coding mutations in the BRAF and NRAS oncogenes, and non-coding mutations in the promoter of telomerase reverse transcriptase ( TERT ). However, the molecular etiology and significance of most of these mutations is unknown. Here, we use a new method known as CPD-capture-seq to map UV-induced cyclobutane pyrimidine dimers (CPDs) with high sequencing depth and single nucleotide resolution at sites of recurrent mutations in melanoma. Our data reveal that many previously identified drivers and other recurrent mutations in melanoma occur at CPD hotspots in UV-irradiated melanocytes, often associated with an overlapping binding site of an E26 transformation-specific (ETS) transcription factor. In contrast, recurrent mutations in the promoters of a number of known or suspected cancer genes are not associated with elevated CPD levels. Our data indicate that a subset of recurrent protein-coding mutations are also likely caused by ETS-induced CPD hotspots. This analysis indicates that ETS proteins profoundly shape the mutation landscape of melanoma and reveals a method for distinguishing potential driver mutations from passenger mutations whose recurrence is due to elevated UV damage. 

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.