The role of the miR-17-92 cluster in macrophage driven innate immunity

Research into miRNA, discovered in 1993, has exploded and revolutionized our understanding of molecular regulation in biological systems. MicroRNAs are now a well established as a regu- latory mechanism of many pathways and functions within cells of eukaryotic organisms, though much needs to be learnt about the intricacies of such regulation. In recent years, targeting this system of post-translational regulation has been a goal of many therapeutics, but requires much greater insight into how miRNA work, and the broadness of their activity. The innate immune system is critical for mounting an effective response against invading pathogens and protecting the host from damage. But being such a powerful system, unchecked it can wreak havoc on the host itself. While the innate immune system is tightly regulated by many mechanisms, further understanding could lead to major advances in therapeutics of autoim- mune diseases. As the miR-17-92 cluster has already been identified as a regulator of innate immune functions, and continued research in animal models is necessary for therapeutics to become a reality. This thesis focuses on the role and function of the miR-17-92 cluster within macrophages, which are a major component of the innate immune system. It highlights the complexity and often subtle nature of microRNA regulation in biological systems. It describes the generation of a mouse line with a myeloid-specific deletion of the miR-17-92 cluster is described, and shown that despite this deletion, there is no change both to the innate immune response of these mice, or to the TLR signalling cascade. It is postulated that while the miR-17-92 cluster affects innate immune signaling in some other cell types, it is unlikely to have a similar role in macrophages

Medical Practitioners Perceived Benefits and Costs of Volunteering for a Multi-Sport Major Games

Volunteering as a medical practitioner at a multi-sport major games (MSMG) is a career highlight for many, but its benefits and costs have not been thoroughly explored. Framed by Social Exchange Theory, this study aims to address this gap by examining the experiences of medical volunteers at a recent MSMG. An online anonymous survey, based on Doherty’s (2009) study of Jeux du Canada Games volunteers, was completed by 78 Canadian medical practitioners who had volunteered at a MSMG in the previous six years. The study revealed that professional identification and networking were the greatest benefits experienced by medical volunteers, while personal inconveniences to their family, work, or vacation time were the greatest costs. Interestingly, the medical volunteers valued professional gains more than social enrichment or contributing to the event. The findings offer valuable insights into the factors that drive medical practitioners to engage in future volunteerism at MSMG

Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2\u27-O-Methyl (2\u27OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2\u27OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2\u27OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application

Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~ 45.9 kDa, ~ 37 kDa, and ~ 83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50 > 100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24 h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24 h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24 h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24 h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted

Caribou, Petroleum, and the Limits of Locality in the Canada–US Borderlands

his article discusses Karsten Heuer’s 2006 book Being Caribou in light of debates in ecocriticism and border studies about how to define the local in the context of environmental problems of vast range and uncertain temporality. It explores how Heuer’s book about following the Porcupine Caribou herd’s migration engages in multiple forms of boundary crossing—between countries, between hemispheric locations, and between species—and shows how insights from Indigenous storytelling complicate the book’s appeal to environmentalist readers by asserting a prior, transnational Indigenous presence in the transboundary landscapes of present-day Alaska and the Yukon