The innate antiviral effects of extracellular viral dsRNA in rainbow trout cells

Viral double-stranded RNA (dsRNA) molecules are a potent pathogen-associated molecular pattern and play a crucial role in the innate immune response. During a viral infection, extracellular and intracellular dsRNA can initiate pathways resulting in the production of type I interferons (IFNs) and interferon-stimulated genes (ISGs). The accumulation of ISGs within a cell results in a protective antiviral state. This study used both commercially available dsRNA (poly I:C) and in vitro transcribed dsRNA molecules, based on the viral hemorrhagic septicemia virus (VHSV) genome sequence, as stimuli to investigate the effects of these molecules on the innate immune response in rainbow trout cells. The goals of the present project were to elucidate the i) IFN ii) ISG and iii) antiviral responses of fish cells to both types of dsRNA molecules. Different lengths of poly I:C and in vitro transcribed dsRNA were used to determine potential length effects of dsRNA in fish cells. The aims of the project were achieved using a functional interferon assay, an ISG-promoter reporter system, an antiviral assay, and RT-PCR. It was found that extracellular dsRNA, either poly I:C or in vitro transcribed dsRNA, is able to induce innate antiviral responses in the fish cell line, RTG-2. Consistent with mammalian studies there was a greater magnitude of immune response when cells were stimulated with longer dsRNA molecules, demonstrating dsRNA length effects in fish cells

Characterizing the Effects of Vaccine Adjuvants on Skeletal Muscle Myotubes and Macrophages

Vaccines train the immune system to recognize and defend against pathogens. Currently, six types of vaccines are in use and include live-attenuated, inactivated, viral vector, protein subunit, toxoid, and messenger RNA (mRNA), the latter of which was recently approved for humans during the COVID-19 pandemic. To increase the longevity and magnitude of immune responses, some vaccines are combined with adjuvants. Mouse models have shown that adjuvants in combination with antigens can elicit a pro-inflammatory immune system response that is required for proper development of protective immunity. There has been recent appreciation for the immunomodulatory functions of skeletal muscle, yet their contribution to the immunology of vaccination remains incompletely understood. Considering most vaccines are administered intramuscularly, we utilized C2C12 mouse myotubes and J774 macrophages to explore the cytokine response that skeletal muscle cells and macrophages evoke in response to several types of vaccine adjuvants in absence of the antigen. C2C12 myotubes and J774 macrophages were treated with 7 commonly used adjuvants or appropriate controls and collected at 6 hours and 24 hours. Cytokine secretion, cytotoxicity, and effects on myotube diameter were analyzed. Most adjuvants, except for the positive controls (LPS and PAM3CSK4), CpG 1826, and Quil-A, did not induce a pro-inflammatory response in C2C12 myotubes or J774 macrophages. Interestingly, LPS, PAM3CSK4, MF59, Quil-A, AS03, and CFA, led to increases in C2C12 myotube diameter indicating an activation of hypertrophy. The lack of pro-inflammatory effects indicates that most adjuvants need antigens or additional cell-cell interactions at the injection site to produce a pro-inflammatory cytokine response

A facility for high resolution spectroscopy: Laboratory and ground based observations in support of upper atmospheric research

This research task consists of operating a facility for making spectroscopic observations in support of upper atmospheric research. The facility responds to the needs and interests of the visiting investigators. Therefore, the research objectives are not predetermined except in broad outline. The emphasis is on studies that take advantage of the particular strengths of the Fourier Transform Spectrometer on Kitt Peak: high spectral resolution combined with wide spectral range and low noise

DsRNA-mediated antiviral immunity in fish cells: visualization, sensors, and innate immune responses

The global aquaculture industry is a multibillion dollar business that is threatened by pathogens, including a wide array of aquatic viruses. Currently there are no antiviral treatments available to combat viral outbreaks, and as such viral infection can cause vast economic loss. Two important species for aquaculture include rainbow trout (Oncorhynchus mykiss), destined for human consumption, and fathead minnow (Pimephales promelas), a common species grown for bait purposes. Double-stranded (ds)RNA is a potent immunomodulating molecule produced during viral replication; dsRNA treatment induces a robust antiviral state that makes host cells refractive to viral replication. This thesis explored dsRNA-induced innate antiviral pathways from two angles. Firstly, the differences in dsRNA-induced responses between virally-produced dsRNA, synthesized dsRNA with natural sequence variation, and synthesized dsRNA with a homogenous sequence were analyzed in rainbow trout cells. The dsRNA, regardless of source, was sensed at the cell surface by a common receptor in rainbow trout cells and induced an innate immune response and antiviral state against two aquatic viruses, viral hemorrhagic septicemia virus and infectious pancreatic necrosis virus; the virally-produced and lab-synthesized dsRNA molecules with natural sequence variation produced the most similar responses. The second angle of approach was to better understand the host’s response to exogenous dsRNA treatment. To this end, novel dsRNA sensors were identified and characterized in rainbow trout. This included class A scavenger receptors, the purported surface receptors for dsRNA, including MARCO, SCARA3, SCARA4, and SCARA5, as well as two novel cytoplasmic dsRNA sensors from the DExH/D-box family, DDX3 and DHX9. The receptors found in rainbow trout contained the same conserved domains that are found in their mammalian counterparts, a first indication of conserved functionality. Two MARCO variants were identified and found to bind to two gram-negative and one gram–positive bacteria, but surprisingly not to dsRNA. Rainbow trout DDX3 and DHX9 are both functional in their ability to bind to dsRNA. The culmination of these findings was the development of a dsRNA molecule with sequence variation that can act as a potent antiviral therapy in vitro in fathead minnow cells. The findings from this thesis demonstrate the importance of ‘natural’ dsRNA as an innate immune signalling molecule and its potential to function as a prophylactic antiviral therapeutic for fish

The Regeneration Games: Commodities, Gifts and the Economics of London 2012

This paper considers contradictions between two concurrent and tacit conceptions of the Olympic ‘legacy’, setting out one conception that understands the games and their legacies as gifts alongside and as counterpoint to the prevailing discourse, which conceives Olympic assets as commodities. The paper critically examines press and governmental discussion of legacy, in order to locate these in the context of a wider perspective contrasting ‘gift’ and ‘commodity’ Olympics – setting anthropological conceptions of gift-based sociality as a necessary supplement to contractual and dis-embedded socioeconomic organizational assumptions underpinning the commodity Olympics. Costbenefit planning is central to modern city building and mega-event delivery. The paper considers the insufficiency of this approach as the exclusive paradigm within which to frame and manage a dynamic socio-economic and cultural legacy arising from the 2012 games

Inhalation exposure of animals.

Relative advantages and disadvantages and important design criteria for various exposure methods are presented. Five types of exposures are discussed: whole-body chambers, head-only exposures, nose or mouth-only methods, lung-only exposures, and partial-lung exposures. Design considerations covered include: air cleaning and conditioning; construction materials; losses of exposure materials; evenness of exposure; sampling biases; animal observation and care; noise and vibration control, safe exhausts, chamber loading, reliability, pressure fluctuations; neck seals, masks, animal restraint methods; and animal comfort. Ethical considerations in use of animals in inhalation experiments are also discussed

Outflow and dense gas emission from massive Infrared Dark Clouds

Infrared Dark Clouds are expected to harbor sources in different, very young evolutionary stages. To better characterize these differences, we observed a sample of 43 massive Infrared Dark Clouds, originally selected as candidate high-mass starless cores, with the IRAM 30m telescope covering spectral line tracers of low-density gas, high-density gas, molecular outflows/jets and temperature effects. The SiO(2-1) observations reveal detections toward 18 sources. Assuming that SiO is exclusively produced by sputtering from dust grains, this implies that at least in 40% of this sample star formation is on-going. A broad range of SiO line-widths is observed (between 2.2 and 65km/s), and we discuss potential origins for this velocity spread. While the low-density tracers 12CO(2-1) and 13CO(1-0) are detected in several velocity components, the high-density tracer H13CO+(1--0) generally shows only a single velocity component and is hence well suited for kinematic distance estimates of IRDCs. Furthermore, the H13CO+ line-width is on average 1.5 times larger than that of previously observed NH3(1,1). This is indicative of more motion at the denser core centers, either due to turbulence or beginning star formation activity. In addition, we detect CH3CN toward only six sources whereas CH3OH is observed toward approximately 40% of the sample. Estimates of the CH3CN and CH3OH abundances are low with average values of 1.2x10^{-10} and 4.3x10^{-10}, respectively. These results are consistent with chemical models at the earliest evolutionary stages of high-mass star formation. Furthermore, the CH3OH abundances compare well to recently reported values for low-mass starless cores.Comment: 22 pages (ApJ referee style), 7 figures, accepted for Ap