Chemical Modulation of Endocytic Sorting Augments Adeno-associated Viral Transduction

Intracellular trafficking of viruses can be influenced by a variety of inter-connected cellular sorting and degradation pathways involving endo-lysosomal vesicles, the ubiquitin-proteasome system, and autophagy-based or endoplasmic reticulum-associated machinery. In the case of recombinant adeno-associated viruses (AAV), proteasome inhibitors are known to prevent degradation of ubiquitinated AAV capsids, thereby leading to increased nuclear accumulation and transduction. However, the impact of other cellular degradation pathways on AAV trafficking is not well understood. In the current study, we screened a panel of small molecules focused on modulating different cellular degradation pathways and identified eeyarestatin I (EerI) as a novel reagent that enhances AAV transduction. EerI improved AAV transduction by an order of magnitude regardless of vector dose, genome architecture, cell type, or serotype. This effect was preceded by sequestration of AAV within enlarged vesicles that were dispersed throughout the cytoplasm. Specifically, EerI treatment redirected AAV particles toward large vesicles positive for late endosomal (Rab7) and lysosomal (LAMP1) markers. Notably, MG132 and EerI (proteasomal and endoplasmic reticulum-associated degradation inhibitors, respectively) appear to enhance AAV transduction by increasing the intracellular accumulation of viral particles in a mutually exclusive fashion. Taken together, our results expand on potential strategies to redirect recombinant AAV vectors toward more productive trafficking pathways by deregulating cellular degradation mechanisms

Cellular transduction mechanisms of adeno-associated viral vectors

Recombinant adeno-associated viral vectors (rAAV) are regarded as promising vehicles for therapeutic gene delivery. Continued development and new strategies are essential to improve the potency of AAV vectors and reduce the effective dose needed for clinical efficacy. In this regard, many studies have focused on understanding the cellular transduction mechanisms of rAAV, often with the goal of exploiting this knowledge to increase gene transfer efficiency. Here, we provide an overview of our evolving understanding of rAAV cellular trafficking pathways through the host cell, beginning with cellular entry and ending with transcription of the vector genome. Strategies to exploit this information for improving rAAV transduction are discussed

Chemical Modulation of AAV Trafficking

Adeno-associated virus is widely studied due to the promise it holds as a gene therapy vector. Gene therapy broadly describes strategies in which genetic material is introduced into a target cell in an effort to treat or cure disease. However, even with AAV being used as a gene delivery vector in over 100 clinical trials to date, there is still much unknown about the biology of the vector. Further understanding of the trafficking of the vector through the host cell will contribute to the safety and efficacy of the inevitable clinical trials and therapies that are to come. In this dissertation, we utilized small molecules to dissect and modulate the trafficking of AAV vectors. Firstly, we utilized numerous small molecules to dissect the potential role of several cellular degradation mechanisms in the AAV infectious pathway. We identified the ERAD inhibitor Eeyarestatin I (EerI) as a molecule that augments AAV transduction. EerI increased transduction by approximately 10-fold in a serotype, cell type, and genome type independent manner. Additionally, EerI and the proteasome inhibitor MG132 acted in distinct ways to augment AAV transduction. Further, EerI modulated the intracellular trafficking of AAV by redirecting AAV to enlarged Rab7/LAMP1 positive vesicles. This EerI-mediated redirection of AAV protected capsids from proteasomal degradation, thereby increasing the nuclear accumulation of AAV capsids. Next, we utilized ionomycin and BAPTA-AM to modulate the intracellular calcium environment and determined that intracellular calcium concentration influences AAV transduction. Ionomycin increases intracellular calcium concentration, and decreases transduction by approximately 10-fold. Ionomycin acts to block transduction at or before AAV nuclear entry. BAPTA-AM decreases intracellular calcium concentration, and increases transduction by approximately 10 to 100-fold in vitro and in vivo. BAPTA-AM likely acts at multiple steps in the AAV pathway to increase transduction. However, we identified that BAPTA-AM increased RNA transcription from the AAV vector genome, thereby increasing transgene protein levels. Taken together, we demonstrate multiple methods to modulate AAV trafficking using small molecules. Furthermore, we demonstrate that use of two of these molecules, EerI and BAPTA-AM, can augment AAV transduction, providing additional strategies for increasing AAV transduction in the clinic.Doctor of Philosoph

Utilization of Additive Manufacturing for the Rapid Prototyping of C-Band RF Loads

Additive manufacturing is a versatile technique that shows promise in providing quick and dynamic manufacturing for complex engineering problems. Research has been ongoing into the use of additive manufacturing for potential applications in radiofrequency (RF) component technologies. Here we present a method for developing an effective prototype load produced out of 316L stainless steel on a direct metal laser sintering machine. The model was tested within simulation software to verify the validity of the design. The load structure was manufactured utilizing an online digital manufacturing company, showing the viability of using easily accessible tools to manufacture RF structures. The produced load was able to produce an S$_{11}$ value of -22.8 dB at the C-band frequency of 5.712 GHz while under vacuum. In a high power test, the load was able to terminate a peak power of 8.1 MW. Discussion includes future applications of the present research and how it will help to improve the implementation of future accelerator concepts

From Regular to Chaotic States in Atomic Nuclei

An interesting aspect of nuclear dynamics is the co--existence, in atomic nuclei, of regular and chaotic states. In the first part of the present work, we review the state of the art of nuclear dynamics and use a schematic shell model to show how a very simple and schematic nucleon--nucleon interaction can produce an order$\to$chaos transition. The second part is devoted to a discussion of the wave function behaviour and decay of chaotic states using some simple models (to be published in Rivista Nuovo Cimento).Comment: 65 pages, LaTex (the figures are not included), Preprint DFPD/94/TH/26, University of Padov

‘Swallow your pride and fear’: the educational strategies of high-achieving non-traditional university students

With more graduates, degree outcomes have a renewed significance for high-achieving students to stand out in a graduate crowd. In the UK, over a quarter of undergraduates now leave university with the highest grade – a ‘first-class’ degree – although students from non-traditional and underprivileged backgrounds are the least likely. This paper explores the experiences of high-achieving non-traditional (HANT) university students. Drawing on in-depth interviews with 30 final-year students who are on course to achieve a first-class degree from working-class, minority ethnic and/or mature backgrounds, we examine their pathways to academic success through identity works and negotiations. We argue that early successes are crucial for students to re-evaluate their self-expectations as students who can achieve in higher education, while self-esteem, pride or fear can prevent students from maximising their available resources and opportunities. Implications for practice and policy are discussed, including the reflective advice from HANT students toward academic success

Evaluating the Effects of Rumenocentesis on Health and Performance in Dairy Cows

The objective of this study was to evaluate the effects of the rumenocentesis procedure on dairy cows by determining selected metabolic and physiological variables representing the health status. Two groups of 6 cows either underwent rumenocentesis (GA) or sham (GB) procedures. Superficial skin temperature of the rumenocentesis area was measured using infrared thermography before the procedure (-1 h), immediately after (0 h), and at 48 h, 96 h, and 20 days following rumenocentesis. Blood samples were collected at all times, except for immediately after the procedure (0 h), and selected immunologic response variables were determined. Milk yield and rectal temperatures were measured daily. Rumenocentesis did not influence the white blood cell count, haptoglobin or total protein concentrations. Electrophoretic protein fractions were not different between GA and GB treatments. Milk yield and rectal temperature were not affected by rumenocentesis. Skin temperature of the rumenocentesis region in GA group cows increased by 1.0 °C immediately after rumenocentesis and returned to baseline after 48 h where it remained constantly until the end of the study; similar to observation in GB group cows. Results of this study would suggest minimal adverse effects on cow health and production when performing rumenocentesis for the diagnosis of subacute ruminal acidosis. Additional more intensive studies addressing animal welfare issues relative to the diagnostic techniques are needed

Prevalence of filarioid nematodes and trypanosomes in American robins and house sparrows, Chicago USA

AbstractHosts are commonly infected with a suite of parasites, and interactions among these parasites can affect the size, structure, and behavior of host–parasite communities. As an important step to understanding the significance of co-circulating parasites, we describe prevalence of co-circulating hemoparasites in two important avian amplification hosts for West Nile virus (WNV), the American robin (Turdus migratorius) and house sparrow (Passer domesticus), during the 2010–2011 in Chicago, Illinois, USA. Rates of nematode microfilariemia were 1.5% of the robins (n=70) and 4.2% of the house sparrows (n=72) collected during the day and 11.1% of the roosting robins (n=63) and 0% of the house sparrows (n=11) collected at night. Phylogenetic analysis of nucleotide sequences of the 18S rRNA and cytochrome oxidase subunit I (COI) genes from these parasites resolved two clades of filarioid nematodes. Microscopy revealed that 18.0% of American robins (n=133) and 16.9% of house sparrows (n=83) hosted trypanosomes in the blood. Phylogenetic analysis of nucleotide sequences from the 18s rRNA gene revealed that the trypanosomes fall within previously described avian trypanosome clades. These results document hemoparasites in the blood of WNV hosts in a center of endemic WNV transmission, suggesting a potential for direct or indirect interactions with the virus

Functional Analysis of the Putative Integrin Recognition Motif on Adeno-associated Virus 9

Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system