Manipulating Replisome Dynamics to Enhance Lambda Red-Mediated Multiplex Genome Engineering

Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening

MUC1-C drives myeloid leukaemogenesis and resistance to treatment by a survivin-mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy-resistant disease. The MUC1-C oncoprotein governs critical pathways of tumorigenesis, including self-renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1-C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1-C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1-C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1-C with silencing or pharmacologic inhibition with GO-203 led to a decrease in active β-catenin levels and, in-turn, down-regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1-C was also associated with increased sensitivity of AML cells to Cytarabine (Ara-C) treatment by a survivin-dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1-C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO-203 with Ara-C for the treatment of patients with AML

Antagonistic Mixing in Micelles of Amphiphilic Polyoxometalates and Hexaethylene Glycol Monododecyl Ether

International audienceHypothesis: Polyoxometalates (POMs) are metal oxygen clusters with a range of interesting magnetic and catalytic properties. POMs with attached hydrocarbon chains show amphiphilic behaviour so we hypothesised that mixtures of a nonionic surfactant and anionic surfactants with a polyoxometalate cluster as headgroup would form mixed micelles, giving control of the POM density in the micelle, and which would differ in size and shape from micelles formed by the individual surfactants. Due to the high charge and large size of the POM, we suggested that these would be nonideal mixtures due to the complex interactions between the two types of surfactants. The nonideality and the micellar composition may be quantified using regular solution theory. With supplementary information provided by small-angle neutron scattering (SANS), an understanding of this unusual binary surfactant system can be established.Experiments: A systematic study was performed on mixed surfactant systems containing polyoxometalate-headed amphiphiles (K10[P2W17O61OSi2(CnH(2n+1))2], abbreviated as P2W17-2Cn, where n = 12, 14 or 16) and hexaethylene glycol monododecyl ether (C12EO6). Critical micelle concentrations (CMCs) of these mixtures were measured and used to calculate the interaction parameters based on regular solution theory, enabling prediction of micellar composition. Predictions were compared to micelle structures obtained from SANS. A phase diagram was also established.Findings: The CMCs of these mixtures suggest unusual unfavourable interactions between the two species despite formation of mixed micelles. Micellar compositions obtained from SANS concurred with those calculated using the averaged interaction parameters for P2W17-2Cn/C12EO6 (n = 12 and 14). We attribute the unfavourable interactions to a combination of different phenomena: counterion-mediated interactions between P2W17 units and the unfolding of the ethylene oxide headgroups of the nonionic surfactant, yet micelles still form in these systems due to the hydrophobic interactions between surfactant tails

Cross-Transfer Effects on Muscle Strength, Size, and Quality following Unilateral Blood Flow Restriction Training

Unilateral resistance training (RT) has been shown to induce contralateral strength improvements in the untrained homologous muscle. Notably, low-load blood flow restriction (BFR) RT has shown superior increases in muscle strength and hypertrophy compared to low-load non-BFR RT. Previous literature has also reported that BFR RT has systemic cross-transfer effects of strength on other active skeletal muscles compared to low-load non-BFR RT. PURPOSE: Therefore, the purpose was to examine the cross-transfer effects of 4 weeks of unilateral BFR dorsiflexion RT on muscle strength, hypertrophy, and muscle quality. METHODS: Fourteen untrained participants were randomized into two groups: (BFR; n=8) (177.6 ± 4.1 cm, 84.8 ± 15.1 kg, 21.3 ± 1 years) or control (non-BFR; n=6) (173.2 ± 7.5 cm, 77.9 ± 10.3 kg, 23 ± 2.6 years). Subjects completed 4 weeks (8 sessions) of unilateral isokinetic dorsiflexion RT at 30% of their daily peak torque at a velocity of 60°/s. Isokinetic peak torque, echo intensity (EI), and muscle cross-sectional area (mCSA) were taken bilaterally pre and post RT. Statistical analyses included 3 separate 3-way mixed factorial ANOVAs (Group [BFR, non-BFR] x Time [pre, post] x Leg [right, left]). RESULTS: For isokinetic strength, there were no significant interactions or main effects (p\u3e.05). For EI, there were no significant interactions (p\u3e.05); however, when collapsed across time and leg, there was a significant main effect for group (p=.017; BFR (91.7 ± 1.6 vs. non-BFR (84.8 ± 1.9); mean ± SE). Furthermore, there was no significant interaction for mCSA (p\u3e.05), but there was a significant main effect for time (

Mesoporous silica formation mechanisms probed using combined Spin-Echo Modulated Small Angle Neutron Scattering (SEMSANS) and Small Angle Neutron Scattering (SANS)

International audienceThe initial formation stages of surfactant-templated silica thin films which grow at the air−water interface were studied using combined spin−echo modulated small-angle neutron scattering (SEMSANS) and small-angle neutron scattering (SANS). The films are formed from either a cationic surfactant or nonionic surfactant (C16EO8) in a dilute acidic solution by the addition of tetramethoxysilane. Previous work has suggested a twostage formation mechanism with mesostructured particle formation in the bulk solution driving film formation at the solution surface. From the SEMSANS data, it is possible to pinpoint accurately the time associated with the formation of large particles in solution that go on to form the film and to show their emergence is concomitant with the appearance of Bragg peaks in the SANS pattern, associated with the two-dimensional hexagonal order. The combination of SANS and SEMSANS allows a complete depiction of the steps of the synthesis that occur in the subphase

Does 1,8-diiodooctane affect the aggregation state of PC71BM in solution?

1,8-Diiodooctane (DIO) is an additive used in the processing of organic photovoltaics and has previously been reported, on the basis of small-angle X-ray scattering (SAXS) measurements, to deflocculate nano-aggregates of [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM) in chlorobenzene. We have critically re-examined this finding in a series of scattering measurements using both X-rays and neutrons. With SAXS, we find that the form of the background solvent scattering is influenced by the presence of DIO, that there is substantial attenuation of the X-rays by the background solvent and that there appears to be beam-induced aggregation. All three factors call into question the suitability of SAXS for measurements on these samples. By contrast, small-angle neutron scattering (SANS) measurements, performed at concentrations of 15 mg ml−1 up to and including 40 mg ml−1, show no difference in the aggregation state for PC71BM in chlorobenzene with and without 3% DIO; we find PC71BM to be molecularly dissolved in all solvent cases. In situ film thinning measurements of spin-coated PC71BM solution with the DIO additive dry much slower. Optical imaging shows that the fullerene films possess enhanced molecular mobility in the presence of DIO and it is this which, we conclude, improves the nanomorphology and consequently solar cell performance. We propose that any compatible high boiling solvent would be expected to show the same behaviour

Influence of viral infection on the relationships between airway cytokines and lung function in asthmatic children

Abstract Background Few longitudinal studies examine inflammation and lung function in asthma. We sought to determine the cytokines that reduce airflow, and the influence of respiratory viral infections on these relationships. Methods Children underwent home collections of nasal lavage during scheduled surveillance periods and self-reported respiratory illnesses. We studied 53 children for one year, analyzing 392 surveillance samples and 203 samples from 85 respiratory illnesses. Generalized estimated equations were used to evaluate associations between nasal lavage biomarkers (7 mRNAs, 10 proteins), lung function and viral infection. Results As anticipated, viral infection was associated with increased cytokines and reduced FVC and FEV1. However, we found frequent and strong interactions between biomarkers and virus on lung function. For example, in the absence of viral infection, CXCL10 mRNA, MDA5 mRNA, CXCL10, IL-4, IL-13, CCL4, CCL5, CCL20 and CCL24 were negatively associated with FVC. In contrast, during infection, the opposite relationship was frequently found, with IL-4, IL-13, CCL5, CCL20 and CCL24 levels associated with less severe reductions in both FVC and FEV1. Conclusions In asthmatic children, airflow obstruction is driven by specific pro-inflammatory cytokines. In the absence of viral infection, higher cytokine levels are associated with decreasing lung function. However, with infection, there is a reversal in this relationship, with cytokine abundance associated with reduced lung function decline. While nasal samples may not reflect lower airway responses, these data suggest that some aspects of the inflammatory response may be protective against viral infection. This study may have ramifications for the treatment of viral-induced asthma exacerbations.https://deepblue.lib.umich.edu/bitstream/2027.42/146519/1/12931_2018_Article_922.pd

Phase 1 Integrated Complete Trip Deployment Plan - University of Washington ITS4US Deployment Project

693JJ321C000004This document presents the Integrated Complete Trip Deployment Plan (ICTDP) for the University of Washington (UW) ITS4US Deployment Project, which is developing and deploying the Transportation Data Equity Initiative (TDEI). This ITS4US Deployment Project currently is in Phase 1, planning and systems engineering development, in which the preliminary idea is developed into a structured concept that is suitable for further design, building, testing, and operation. This ICTDP summarizes the refined deployment concept developed in Phase 1 and sets forth the high-level Phase 2 (Design/Build/Test) and Phase 3 (Operate/Maintain/Evaluate) Schedule. It provides a refined Phase 1 deployment concept and the technical approach the UW ITS4US team will use for phases 2 and 3. It also provides the current deployment schedule for phases 2 and 3, along with a cost estimate for those phase

Fiber Reinforced Polymer (FRP) Seismic Retrofit of Reinforced Concrete Bridge Columns Vulnerable to Long-Duration Subduction Zone Earthquakes

69A3551947137This research was focused on characterization of the behavior of FRP jacketed bridge columns under long-duration earthquakes. Six tests were conducted on cantilever bridge columns with FRP jackets at the base. The FRP jackets were 0.40\u201d and extended over a height of one column diameter. The FRP had specified strength of 128-ksi and elastic modulus of 14.2ksi in the circumferential direction. Test variables for the columns included longitudinal reinforcement ratio, longitudinal bar diameter, axial load ratio, and loading protocol. All tested columns had lateral deformation capacity of at least 6% drift, with lateral deformation capacity considered to occur at 20% strength loss. Axial failure was not achieved in any of the test columns, and the tests were stopped after multiple cycles at 10% drift. Five of the six test columns were nominally identical to a set of previously tested columns with FRP jackets