Quantitation of DNA double-strand break resection intermediates in human cells

5′ strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5′ strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle–dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.     

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer

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Bioproduction of benzaldehyde in a solid–liquid two-phase partitioning bioreactor using <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The bioproduction of benzaldehyde from benzyl alcohol using Pichia pastoris was examined in a solid–liquid two-phase partitioning bioreactor (TPPB) to reduce substrate and product inhibition. Rational polymer selection identified Elvax 40W as an effective sequestering phase, possessing partition coefficients for benzyl alcohol and benzaldehyde of 3.5 and 35.4, respectively. The use of Elvax 40W increased the overall mass of benzaldehyde produced by approx. 300% in a 5 l bioreactor, relative to a single phase biotransformation. The two-phase system had a molar yield of 0.99, indicating that only minor losses occurred. These results provide a promising starting point for solid–liquid TPPBs to enhance benzaldehyde production, and suggest that multiple, targeted polymers may provide relief for transformations characterized by multiple inhibitory substrates/product/by-products.     

Strike Fast,Strike Hard: The Red-Throated Caracara Exploits Absconding Behavior of Social Wasps during Nest Predation

Red-throated Caracaras Ibycter americanus (Falconidae) are specialist predators of social wasps in the Neotropics. It had been proposed that these caracaras possess chemical repellents that allow them to take the brood of wasp nests without being attacked by worker wasps. To determine how caracaras exploit nests of social wasps and whether chemical repellents facilitate predation, we: (1) video recorded the birds attacking wasp nests; (2) analyzed surface extracts of the birds'' faces, feet, and feathers for potential chemical repellents; and (3) inflicted mechanical damage on wasp nests to determine the defensive behavior of wasps in response to varying levels of disturbance. During caracara predation events, two species of large-bodied wasps mounted stinging attacks on caracaras, whereas three smaller-bodied wasp species did not. The “hit-and-run” predation tactic of caracaras when they attacked nests of large and aggressive wasps reduced the risk of getting stung. Our data reveal that the predation strategy of caracaras is based on mechanical disturbance of, and damage to, target wasp nests. Caracara attacks and severe experimental disturbance of nests invariably caused wasps to abscond (abandon their nests). Two compounds in caracara foot extracts [sulcatone and iridodial] elicited electrophysiological responses from wasp antennae, and were also present in defensive secretions of sympatric arboreal-nesting Azteca ants. These compounds appear not to be wasp repellents but to be acquired coincidentally by caracaras when they perch on trees inhabited with Azteca ants. We conclude that caracara predation success does not depend on wasp repellents but relies on the absconding response that is typical of swarm-founding polistine wasps. Our study highlights the potential importance of vertebrate predators in the ecology and evolution of social wasps.     

Monitoring insecticide resistance in Australian Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) detects fipronil and spinosad resistance

Abstract  Insecticide resistance monitoring using a Potter precision spray tower with discriminating concentration and log dose probability techniques underpins the Australian insecticide management strategy for Frankliniella occidentalis Pergande. Abamectin, acephate, chlorpyrifos, dichlorvos, dimethoate, endosulfan, fipronil, malathion, methamidophos methidathion, methiocarb, methomyl, pyrazophos and spinosad are recommended for use against F. occidentalis but abamectin, methiocarb and pyrazophos are the only chemicals where insecticide resistance has not been detected. Although not registered, chlorfenapyr was effective against F. occidentalis and should be pursued for that purpose. In contrast, chlorpyrifos, dichlorvos and malathion resistance were detected at low to moderate levels throughout the study period putting their sustainable use for F. occidentalis control in doubt . Although it appears that acephate, dimethoate, endosulfan, fipronil, methamidophos, methidathion and spinosad remain effective, some populations contained a small percentage of thrips that survived exposure to a concentration that killed 100% of the susceptible strain. Subsequent laboratory selection of one such population separately with fipronil and spinosad caused an increase in resistance to these insecticides. These products must now be considered at risk. This is the first report of fipronil or spinosad resistance in populations of F. occidentalis.     

EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation

Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.