Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

Site‐specific incorporation of non‐standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell‐free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA^? endA^?), we synthesized 550±40 μg mL^?1 of modified superfolder green fluorescent protein containing p‐acetyl‐L ‐phenylalanine. This yield was increased to ～1300 μg mL^?1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell‐free synthetic biology.     

Characterization of best linear unbiased estimates generated from national genetic evaluations of reproductive performance,survival, and milk yield in dairy cows

Genetic evaluations decompose an observed phenotype into its genetic and nongenetic components; the former are termed BLUP with the solutions for the systematic environmental effects in the statistical model termed best linear unbiased estimates (BLUE). Geneticists predominantly focus on the BLUP and rarely consider the BLUE. The objective of this study, however, was to define and quantify the association between 8 herd-level characteristics and BLUE for 6 traits in dairy herds, namely (1) age at first calving, (2) calving to first service interval (CFS), (3) number of services, (4) calving interval (CIV), (5) survival, and (6) milk yield. Phenotypic data along with the fixed and random effects solutions were generated from the Irish national multi-breed dairy cow fertility genetic evaluations on 3,445,557 cows; BLUE for individual contemporary groups were collapsed into mean herd-year estimates. Data from 5,707 spring-calving herds between the years 2007 and 2016 inclusive were retained; association analyses were undertaken using linear mixed multiple regression models. Pearson coefficient correlations were used to quantify the relationships among individual trait herd-year BLUE, and transition matrices were used to understand the dynamics of mean herd BLUE estimates over years. Based on the mean annual trends in raw, BLUP, and BLUE, it was estimated that BLUE were associated with at least two-thirds of the improvement in CIV and milk production over the past 10 yr. Milk recording herds calved heifers for the first time on average 15 d younger, had an almost 2 d longer CFS but 2.3 d shorter CIV than non-milk-recording herds. Larger herd sizes were associated with worse BLUE for both CFS and CIV. Expanding herds and herds that had the highest proportion of cows born on the farm itself, on average, calved heifers younger and had shorter CIV. By separating the raw performance of a selection of herds into their respective BLUE and BLUP, it was possible to identify herds with inferior management practices that were being compensated by superior genetics; similarly, herds were identified with superior BLUE, but because of their inferior genetic merit, were not reaching their full potential. This suggests that BLUE could have a pivotal role in a tailored decision support tool that would enable producers to focus on the most limiting factor hindering them from achieving their maximum performance.     

Longitudinal outcomes of young high-risk adolescents with imaginary companions.

The creation and cultivation of an imaginary companion is considered to be a healthy form of pretend play in early childhood, but there tends to be a less positive view of older children who have them. To test the extent that having an imaginary companion in middle school is associated with positive or negative outcomes, an ethnically diverse sample of 152 middle school children at high risk for developing problem behaviors were interviewed about imaginary companions, coping styles, and problem behaviors. Although having a current imaginary companion (n = 13) was associated with using more positive coping strategies, peer nomination data indicated that these children had low social preference with peers. In addition, our data indicated that these children were perceived by their parents as having more problem behaviors compared with young adolescents who never had imaginary companions (n = 108) or children who had imaginary companions in the past (n = 31). However, a longitudinal follow-up at the end of high school indicated that the children who had imaginary companions in middle school showed greater positive adjustment on a multiple-indicator adjustment construct. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 microM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of alpha-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.