With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.N-ethyl-N-nitrosourea | genetic mapping | forward genetics | mutagenesis | massively parallel sequencing P henotypic variation in mice can be induced with N-ethyl-Nnitrosourea (ENU), which creates single base pair substitutions in germ line DNA. However, the positional cloning of ENU-induced mutations causative for phenotypes of interest has historically been a time-consuming process, beginning with generation of an outcrossed recombinant mapping population of phenotypically mutant and WT mice, genotyping individual mice at genetic markers across the genome to create a linkage map, and finally targeted sequencing to identify the causative mutation within the critical region. The advent of massively parallel sequencing techniques has given rise to more rapid "mapping-bysequencing" methods in which genome-wide marker genotyping and DNA sequencing are combined into a single step applied to either individual or pooled groups of organisms (1). For ENUmutagenized mice, early experiments used massively parallel sequencing for mutation identification within a critical region defined by traditional or bulk segregation mapping using recombinant mapping populations produced by outcrossing the mutant to another inbred laboratory strain and backcrossing or intercrossing a second time (2-4). Later reports demonstrated mapping with the identified sequence variants themselves as markers, which eliminated...

Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/ myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)-and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.neoseptins | peptidomimetic compounds | innate immunity | proinflammatory response | crystal structure

Advanced electrocatalysts with low platinum content, high activity and durability for the oxygen reduction reaction can benefit the widespread commercial use of fuel cell technology. Here, we report a platinum-trimer decorated cobalt-palladium core-shell nanocatalyst with a low platinum loading of only 2.4 wt% for the use in alkaline fuel cell cathodes. This ternary catalyst shows a mass activity that is enhanced by a factor of 30.6 relative to a commercial platinum catalyst, which is attributed to the unique charge localization induced by platinum-trimer decoration. The high stability of the decorated trimers endows the catalyst with an outstanding durability, maintaining decent electrocatalytic activity with no degradation for more than 322,000 potential cycles in alkaline electrolyte. These findings are expected to be useful for surface engineering and design of advanced fuel cell catalysts with atomic-scale platinum decoration.

Class-switch recombination (CSR) alters the Ig isotype to diversify antibody effector functions. IgD CSR is a rare event, and its regulation is poorly understood. We report that deficiency of 53BP1, a DNA damage-response protein, caused age-dependent overproduction of secreted IgD resulting from increased IgD CSR exclusively within B cells of mucosa-associated lymphoid tissues. IgD overproduction was dependent on activation-induced cytidine deaminase, hematopoietic MyD88 expression, and an intact microbiome, against which circulating IgD, but not IgM, was reactive. IgD CSR occurred via both alternative nonhomologous end-joining and homologous recombination pathways. Microbiota-dependent IgD CSR also was detected in nasal-associated lymphoid tissue of WT mice. These results identify a pathway, present in WT mice and hyperactivated in 53BP1-deficient mice, by which microbiota signal via Toll-like receptors to elicit IgD CSR.

This study presents near-infrared (NIR) light-responsive polymer-nanostructure composite microneedles used for on-demand transdermal drug delivery. Silica-coated lanthanum hexaboride (LaB6@SiO2) nanostructures were incorporated into polycaprolactone microneedles, serving as an NIR absorber. When the microneedles were irradiated with NIR light, light-to-heat transduction mediated by the LaB6@SiO2 nanostructures caused the microneedle melting at 50 °C. This increased the mobility of the polymer chains, enabling drug release from the matrix. Drug release from the microneedles was evaluated for four laser on/off cycles. In each cycle, the samples were irradiated until the temperature reached 50 °C for 3 min (laser on); the laser was then turned off for 30 min (laser off). The results showed that light-induced phase transition in the polymer triggered drug release from the melted microneedles. A stepwise drug-release behavior was observed after multiple cycles of NIR light exposure. No notable drug leakage was found in the off state. This NIR-light-triggerable device exhibits excellent reproducibility, low off-state leakage, and noninvasive triggerability and, thus, represents an advance in transdermal delivery technology.