We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKLSer358 and Drp1Ser616 phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKLSer358, and coincidence of phospho-MLKLser358 and phospho-Drp1Ser616 at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling.

Uniaxial magnetic anisotropy was imposed on a CoFeB film by applying an in-plane magnetic field during growth. Electrically driven strain from a ferroelectric 0.68Pb(Mg 1/3 Nb 2/3)O 3-0.32PbTiO 3 (011) substrate resulted in giant magnetoelectric effects, whose coupling constant peaked at a record value of $8.0 Â 10 À6 s m À1. These large magnetoelectric effects arose due to non-volatile 90 rotations of the magnetic easy axis, reflecting a competition between the fixed growth anisotropy and the voltage-controlled magnetoelastic anisotropy. In contrast to previous work, our non-volatile rotations did not require the assistance of an applied magnetic field or the setting of an in-plane substrate polarization prior to deposition.

Rationally designed mariner vectors to allow functional genomic analysis of 1 Actinobacillus pleuropneumoniae and other bacteria by transposon-directed insertion-2 site sequencing (TraDIS) 3 4 Abstract 29 30Transposon Directed Insertion Sequencing (TraDIS) is a high-throughput 31 method for mapping insertion sites in large libraries of transposon mutants. The 32 Himar1 (mariner) transposon is ideal for generating near-saturating mutant 33 libraries, especially in AT-rich chromosomes, as the requirement for integration is 34 a TA dinucleotide. In this study, we generated two novel mariner vectors, 35 pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the 36 transposase gene), in order to facilitate TraDIS identification of conditionally 37 essential genes in Actinobacillus pleuropneumoniae and other bacteria. Using the 38 pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant 39 libraries in both A. pleuropneumoniae and Pasteurella multocida that showed a 40 near random distribution of insertions around the respective chromosomes. A 41 preliminary screen of 5000 mutants each identified 8 and 15 genes, respectively, 42 that are required for growth under anaerobic conditions. 43 44 Importance 45 46 Comprehensive identification of conditionally essential genes requires 47 efficient tools for generating high-density transposon libraries that, ideally, can be 48 analysed using next-generation sequencing methods. The mariner transposon 49 has been used for mutagenesis of a wide variety of bacteria, however plasmids for 50 delivery of this transposon do not necessarily work well in all bacteria. In 51 particular, there are limited tools for functional genomic analysis of 52Pasteurellaceae species of major veterinary importance, such as swine and cattle 53 pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, 54 respectively. Here, we have developed plasmids that allow delivery of mariner and 55 the production of genome saturated mutant libraries for both of these pathogens, 56 but which should also be applicable to a wider range of bacteria. High-throughput 57 screening of the generated libraries will identify mutants required for growth under 58 different conditions, including in vivo, highlighting key virulence factors and 59 pathways that can be exploited for development of novel therapeutics and 60 vaccines. 61 62