We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

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BackgroundFutile recanalization—when patients have a successful recanalization but fail to achieve a satisfactory functional outcome— is a common phenomenon of endovascular treatment of acute ischemic stroke (AIS). The present study aimed to identify the predictors of futile recanalization in AIS patients who received endovascular treatment.MethodsThis is a post-hoc analysis of the DIRECT-MT trial. Demographics, clinical characteristics, acute stroke workflow interval times, biochemical parameters, and imaging characteristics were compared between futile and meaningful recanalization groups. Multivariate analysis was performed to identify the predictors of futile recanalization.ResultsFutile recanalization was observed in 277 patients. In multivariable logistic regression analysis, older age (p<0.001), higher baseline systolic blood pressure (SBP) (p=0.032), incomplete reperfusion defined by extended Thrombolysis In Cerebral Infarction (eTICI) grades (p=0.020), and larger final infarct volume (FIV) (p<0.001) were independent predictors of futile recanalization.ConclusionsOld age, high baseline SBP, incomplete reperfusion defined by eTICI, and large FIV were independent predictors of futile recanalization after endovascular therapy for AIS.

We have established a stable regional geodetic reference frame using long-history (13.5 years on average) observations from 55 continuously operated Global Navigation Satellite System (GNSS) stations adjacent to the Gulf of Mexico (GOM). The regional reference frame, designated as GOM20, is aligned in origin and scale with the International GNSS Reference Frame 2014 (IGS14). The primary product from this study is the seven-parameters for transforming the Earth-Centered-Earth-Fixed (ECEF) Cartesian coordinates from IGS14 to GOM20. The frame stability of GOM20 is approximately 0.3 mm/year in the horizontal directions and 0.5 mm/year in the vertical direction. The regional reference frame can be confidently used for the time window from the 1990s to 2030 without causing positional errors larger than the accuracy of 24-h static GNSS measurements. Applications of GOM20 in delineating rapid urban subsidence, coastal subsidence and faulting, and sea-level rise are demonstrated in this article. According to this study, subsidence faster than 2 cm/year is ongoing in several major cities in central Mexico, with the most rapid subsidence reaching to 27 cm/year in Mexico City; a large portion of the Texas and Louisiana coasts are subsiding at 3 to 6.5 mm/year; the average sea-level-rise rate (with respect to GOM20) along the Gulf coast is 2.6 mm/year with a 95% confidence interval of ±1 mm/year during the past five decades. GOM20 provides a consistent platform to integrate ground deformational observations from different remote sensing techniques (e.g., GPS, InSAR, LiDAR, UAV-Photogrammetry) and ground surveys (e.g., tide gauge, leveling surveying) into a unified geodetic reference frame and enables multidisciplinary and cross-disciplinary research.