Micelle-Enabled Palladium Catalysis for Convenient sp²‑sp³ Coupling of Nitroalkanes with Aryl Bromides in Water Under Mild Conditions

The efficacy of custom surfactant FI-750-M, designed to mimic polar solvents such as DMF and 1,4-dioxane, has been demonstrated with the palladium-catalyzed sp²-sp³ coupling of nitroalkanes to aryl bromides using a heteroleptic palladium catalyst under unprecedentedly mild conditions. Optimized reaction conditions mostly provided good yields up to gram scale, with high selectivity and functional group tolerance for a wide scope of aryl bromides. Use of surfactant FI-750-M makes water the gross reaction medium and enables in-flask recycling. The behavior of the surfactant has been elucidated with DLS and cryo-TEM measurements, and mechanistic investigations have revealed the importance of the π-allyl ligand in the catalytic cycle

Synergistic and Selective Copper/ppm Pd-Catalyzed Suzuki–Miyaura Couplings: In Water, Mild Conditions, with Recycling

Copper-catalyzed Suzuki–Miyaura couplings can be carried out exclusively on aryl iodides under very mild conditions, which is made possible by synergistic effects due to the presence of ppm levels of palladium as cocatalyst. The coupling requires a hemilabile <i>P</i>(<i>O</i>)-<i>N</i> ligand, together with the benefits of micellar catalysis using water as the recyclable reaction medium

Sustainable HandaPhos-<i>ppm</i> Palladium Technology for Copper-Free Sonogashira Couplings in Water under Mild Conditions

Complexation of ca. 1000 ppm Pd­(OAc)₂ with the ligand HandaPhos (1–1.5:1) leads to a precatalyst that efficiently mediates Sonogashira couplings in aqueous nanomicelles under very mild conditions. Neither copper nor organic solvent is required in the reaction medium, and the product can be isolated directly from the reaction flask, leaving behind a reaction mixture that can be recycled without additional additives

Effects of candidate gene deletion and over-expression.

<p><b>A.</b> The five candidate genes were deleted from <i>Sp</i>, and the effect on resistance to 300 ppm citrinin was measured. Four deletions increased sensitivity (t-test <i>p</i> < 0.005 for each), and were studied further. Error bars show 1 S.E. <b>B.</b> The effects of three different dCas9 fusion proteins targeted to four candidate genes were measured by qPCR. <b>C.</b> Illustration of our strategy to over-express four genes via dCas9 fusion protein. <b>D.</b> We used direct competition for 40 generations to measure the relative fitness of the four-gene over-expression strain (with a Gal4-dCas9-VP64 dual fusion) vs. a control strain containing the same plasmid, but lacking any gRNAs. Conditions were YPD, and YPD + 300 ppm citrinin. Error bars show 1 S.E.</p

RNA-seq reveals candidate genes.

<p><b>A.</b> Citrinin has largely similar effects on <i>Sc</i> alleles and <i>Sp</i> alleles in the <i>Sc</i>/<i>Sp</i> hybrid; i.e. ASE is similar with or without citrinin. <b>B.</b> Candidate genes were identified based on having strong <i>Sp</i>-biased ASE (in YPD; FDR < 0.05) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005751#pgen.1005751.ref016" target="_blank">16</a>], and strong up-regulation in response to citrinin (of both <i>Sc</i> and <i>Sp</i> alleles; the smaller of these two in our RNA-seq data is plotted; binomial <i>p</i> < 10⁻⁵ for each biological replicate of each allele).</p

Promoter replacement reveals contributions of individual regulatory regions.

<p><b>A.</b> Illustration of our promoter replacement strategy. Green promoters on the left are from <i>Sc</i>, blue promoters on the right are from <i>Sp</i>. <b>B.</b> RNA-seq in the <i>Sc</i>/<i>Sp</i> hybrid, which measures the overall <i>cis-</i>regulatory divergence between these strains, is in approximate agreement with the effect of the promoter replacements, measured by qPCR (<i>r</i> = 0.92). Error bars show 1 S.E. <b>C.</b> After competitive growth for 40 generations, promoter replacement strains for 3/4 candidate genes show a similar pattern of fitness advantage in the presence of 300 ppm citrinin, and disadvantage in its absence. Error bars show 1 S.E.</p

Results of the sign test for selection on <i>cis-</i>regulation.

<p>For a description of the test and its results, see the main text and Methods. Note that only two citrinin-induced genes are shown, but four were present in the top 1% of <i>Sp</i>-biased genes, and ten in the top 25%.</p

Whole-Genome Sequencing of the World’s Oldest People

<div><p>Supercentenarians (110 years or older) are the world’s oldest people. Seventy four are alive worldwide, with twenty two in the United States. We performed whole-genome sequencing on 17 supercentenarians to explore the genetic basis underlying extreme human longevity. We found no significant evidence of enrichment for a single rare protein-altering variant or for a gene harboring different rare protein altering variants in supercentenarian compared to control genomes. We followed up on the gene most enriched for rare protein-altering variants in our cohort of supercentenarians, TSHZ3, by sequencing it in a second cohort of 99 long-lived individuals but did not find a significant enrichment. The genome of one supercentenarian had a pathogenic mutation in DSC2, known to predispose to arrhythmogenic right ventricular cardiomyopathy, which is recommended to be reported to this individual as an incidental finding according to a recent position statement by the American College of Medical Genetics and Genomics. Even with this pathogenic mutation, the proband lived to over 110 years. The entire list of rare protein-altering variants and DNA sequence of all 17 supercentenarian genomes is available as a resource to assist the discovery of the genetic basis of extreme longevity in future studies.</p></div

Pipeline to test supercentenarians for enrichment of rare protein-altering variants or genes harboring them.

<p>All female Caucasian supercentenarian genomes were annotated for protein-altering variants. (A) To test for enrichment of a single variant, we filtered against dbSNP131 and compared each remaining rare protein-altering variant against 1000G EUR. No single variant was significantly enriched. (B) To test for enrichment of a gene with rare protein-altering variants, we collapsed all variants in to their respective genes and filtered against 1000G EUR (MAF<0.015). We tested for enrichment against 34 control genomes from PGP using the RVT1 burden test or a gene-based Fisher’s Exact (for recessive model). No gene was significantly enriched for rare protein-altering variants in supercentenarians. We then Sanger validated TSHZ3 as the best candidate from our burden-test for follow-up.</p

Protein-altering variants in TSHZ3 in Georgia Centenarian cohort.

<p>Position (bp) on chromosome 19 (Chr19) of variant, reference (Ref) and Variant (Var) allele, Amino Acid (AA) position, AA1 (ref), AA2 (var), Supercentenarian carriers (shown for reference), Centenarians carriers, Nonagenarians carriers, Minor allele frequency (MAF) in 1000G EUR.</p><p>Protein-altering variants in TSHZ3 in Georgia Centenarian cohort.</p