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64, P=0.003), but not in those without. The association of pEndMT with worse renal graft outcome was confirmed on 34 other early biopsies with ATN from a second transplant center. Our results suggest endothelial cell activation at the early phase of renal transplantation plays a detrimental role. This article is protected by copyright. All rights reserved.OBJECTIVES Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN-16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. MATERIALS AND METHODS The effect of TN-16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti-tumour efficacy of TN-16 was investigated in syngeneic mouse model of breast cancer. RESULTS TN-16 inhibited cancer cell proliferation by impairing late-stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy-specific substrate p62 and lack of additional LC3-II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome-lysosome fusion. Further studies revealed that TN-16-mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN-16-mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. CONCLUSIONS Our data signify that TN-16 is a potent autophagy flux inhibitor and might be suitable for (pre-) clinical use as standard inhibitor of autophagy with anti-cancer activity. © 2020 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.AIMS This study assessed nurses' perceptions of systems thinking, safe nursing care and the correlation between them. BACKGROUND Systems thinking and safe nursing care are the key elements of quality improvement approaches such as accreditation and patient safety programs. However, these two variables have not been well studied in different healthcare settings. METHODS In this cross-sectional study 300 nurses were selected using random stratified sampling method. The data were collected using a demographic data form, Systems thinking scale, and Assessment of safe nursing care questionnaire. RESULTS The scores of nurses' perceptions of systems thinking (63.25±9.20) and safe nursing care (4.13±0.60) were above average. A positive correlation was found between systems thinking, and safe nursing care (r=0.66, P less then 0.001) and its dimensions nursing skills (r=0.61, P less then 0.001), psychological needs (r=0.56, P less then 0.001), physical needs (r=0.51, P less then 0.001), teamwork (r=0.56, P less then 0.001). learn more CONCLUSION Regarding the correlation between systems thinking and safe nursing care, nurses and other medical professionals, especially novices are recommended to strengthen their systems thinking skills to improve the safe nursing care. IMPLICATIONS FOR NURSING MANAGEMENT Nurse managers should deal with organizational condition and factors affecting some poor aspects of systems thinking and safe nursing care. They must lead, support, and allocate resources to the foundations of systems thinking to achieve safe nursing care. This article is protected by copyright. All rights reserved.Site-directed mutagenesis allows the generation of novel DNA sequences that can be used for a variety of important applications such as the functional analysis of genetic variants. To overcome the limitations of existing site-directed mutagenesis approaches, we explored in vivo DNA gap repair. We found that site-specific mutations in plasmid DNA can be generated in Escherichia coli using mutant single-stranded oligonucleotides to target PCR-derived linear double-stranded plasmid DNA. We called this method DeGeRing, and we characterized its advantages, including non-biased multiplex mutagenesis, over existing site-directed mutagenesis methods such as recombineering (recombination-mediated genetic engineering), single DNA break repair (SDBR, introduced by W. Mandecki), and QuikChange (Agilent Technologies, La Jolla, CA). We determined the efficiency of DeGeRing to induce site-directed mutations with and without a phenotype in three K-12 E coli strains using multiple single-stranded oligonucleotides containing homological and heterological parts of various sizes. Virtual lack of background made the isolation of mutants with frequencies up to 10-6 unnecessary. Our data show that endogenous DNA gap repair in E coli supports efficient multiplex site-directed mutagenesis. DeGeRing might facilitate the generation of mutant DNA sequences for protein engineering and the functional analysis of genetic variants in reverse genetics. © 2020 Federation of American Societies for Experimental Biology.Cancer metastasis and secondary tumor initiation largely depend on circulating tumor cell (CTC) and vascular endothelial cell (EC) interactions by incompletely understood mechanisms. Endothelial glycocalyx (GCX) dysfunction may play a significant role in this process. GCX structure depends on vascular flow patterns, which are irregular in tumor environments. This work presents evidence that disturbed flow (DF) induces GCX degradation, leading to CTC homing to the endothelium, a first step in secondary tumor formation. A 2-fold greater attachment of CTCs to human ECs was found to occur under DF conditions, compared to uniform flow (UF) conditions. These results corresponded to an approximately 50% decrease in wheat germ agglutinin (WGA)-labeled components of the GCX under DF conditions, vs UF conditions, with undifferentiated levels of CTC-recruiting E-selectin under DF vs UF conditions. Confirming the role of the GCX, neuraminidase induced the degradation of WGA-labeled GCX under UF cell culture conditions or in Balb/C mice and led to an over 2-fold increase in CTC attachment to ECs or Balb/C mouse lungs, respectively, compared to untreated conditions.