Relative persistence of AAV serotype 1 vector genomes in dystrophic muscle

The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation

Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice

The AAV9 capsid displays a high natural affinity for the heart following a single intravenous (IV) administration in both newborn and adult mice. It also results in substantial albeit relatively lower expression levels in many other tissues. To increase the overall safety of this gene delivery method we sought to identify which one of a group of promoters is able to confer the highest level of cardiac specific expression and concurrently, which is able to provide a broad biodistribution of expression across both cardiac and skeletal muscle. The in vivo behavior of five different promoters was compared: CMV, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) and cardiac troponin C (cTnC). Following IV administration to newborn mice, LacZ expression was measured by enzyme activity assays. Results showed that rAAV2/9-mediated gene delivery using the α-MHC promoter is effective for focal transgene expression in the heart and the Des promoter is highly suitable for achieving gene expression in cardiac and skeletal muscle following systemic vector administration. Importantly, these promoters provide an added layer of control over transgene activity following systemic gene delivery

Automated murmurs: the social mobile tourist application

The popularity of mobile devices and their increased computing power has given rise to surge in mobile computing technologies. Users are increasingly turning to mobile devices for information relating to their activities and location while on the move. Independent of this, the world has seen a huge uptake in the social web, which has fueled the production of applications where users are the sole providers of valuable information. In this work we present a mobile platform which leverages the popularity of mobile and social computing to produce a location sensitive messaging system which delivers user generated content to the public in the context of their physical location

A novel two-section tunable discrete mode Fabry-PÉrot laser exhibiting nanosecond wavelength switching

A novel widely tunable laser diode is proposed and demonstrated. Mode selection occurs by etching perturbing slots into the laser ridge. A two-section device is realized with different slot patterns in each section allowing Vernier tuning. The laser operates at 1.3 mum and achieves a maximum output power of 10 mW. A discontinuous tuning range of 30 nm was achieved with a side mode suppression greater than 30 dB. Wavelength switching times of approximately 1.5 ns between a number of wavelength channels separated by 7 nm have been demonstrated

Oral Delivery of Acid Alpha Glucosidase Epitopes Expressed in Plant Chloroplasts Suppresses Antibody Formation in Treatment of Pompe Mice

Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation

Ionic liquids for enzymatic sensing

The key challenges currently faced in lab-on-a-chip biochemical sensor developments are device reliability and power consumption. The major issues faced in terms of device reliability are liquid handling over extended periods of time, as the micro-dimensioned fluidic channels are prone to blockage, and unreliable micro pumps/valves. The overall aim of this proposal is to develop a biocompatible molecular sensor that will address these key issues which are holding back biocompatible sensors technologies, and thus to develop an innovative class of sensing technology at the forefront of molecular sensing. [1] Over the past decade conducting polymer electrodes have played an important role in bio-sensing and actuation. [2] Recent developments in the field of organic electronics have made available a variety of devices that bring unique capabilities at the interface with biology. [3,4] One example is organic electrochemical transistors (OECTs) that are being developed for a variety of bio-sensing applications, including the detection of ions, [5] and metabolites, such as glucose [6] and lactate [7]. Room temperature ionic liquids (RTILs) are organic salts, which are liquid at ambient temperature. Their non-volatile character and thermal stability makes them an attractive alternative to conventional organic solvents. [8] To this end, we propose to exploit enzymatic doped ionogels - new materials for inherently biocompatible molecular sensors. These particular sensors are hybrid materials that consist of monomeric components polymerised within biocompatible ionic liquids, thus allowing various platforms for modification. Further investigation shows that these sensing platforms can be incorporated into flexible materials such as carbon cloth and can be utilised for bio-sensing. Furthermore, we envisage that the proposed sensing devices can be incorporated into fabrics for “wearable” health care devices thus providing clear benefits the public health service. Long term ambitions are to build a research career with a focus on academia and industrial research collaborations. The proposed time in DCU would allow myself to demonstrate adaptability and diversity and to highlight the transferable nature of my skills through the publication of peer review articles and the securing of patents. It is hoped that the research and project management experience gained through collaborations and contacts will allow me to further my career through more senior positions and grants. Through current collaborations, engineering and chemistry support from members of the Adaptive Sensor Group has already been secured for the proposed project. Expanding my research profile through further publications and both domestic and international presentations of the research. Existing collaborations held by myself (Doug MacFarlane’s / Ekaterina Izgorodina’s group, Monash University, Gloria Elliott’s group, UNCC, North Carolina and George Malliaras & Róisín Owens at the Ecole Nationale Supérieure des Mines de Saint Etienne) will be of considerable benefit to the project

Determining fluid migration and isolation times in multiphase crustal domains using noble gases

Geochemical characteristics in subsurface fluid systems provide a wealth of information about fluid sources, migration, and storage conditions. Determining the extent of fluid interaction (aquifer-hydrocarbon connectivity) is important for oil and gas production and waste storage applications, but is not tractable using traditional seismic methods. Furthermore, the residence time of fluids is critical in such systems and can vary from tens of thousands to billions of years. Our understanding of the transport length scales in multiphase systems, while equally important, is more limited. Noble gas data from the Rotliegend natural gas field, northern Germany, are used here to determine the length scale and isolation age of the combined water-gas system. We show that geologically bound volume estimates (i.e., gas to water volume ratios) match closed-system noble gas model predictions, suggesting that the Rotliegend system has remained isolated as a closed system since hydrocarbon formation. Radiogenic helium data show that fluid isolation occurred 63–129 m.y. after rock and/or groundwater deposition (ca. 300 Ma), which is consistent with known hydrocarbon generation from 250 to 140 Ma, thus corroborating long-term geologic isolation. It is critical that we have the ability to distinguish between fluid systems that, despite phase separation, have remained closed to fluid loss from those that have lost oil or gas phases. These findings are the first to demonstrate that such systems remain isolated and fully gas retentive on time scales >100 m.y. over >10 km length scales, and have broad implications for saline aquifer CO2 disposal site viability and hydrocarbon resource prediction, which both require an understanding of the length and time scales of crustal fluid transport pathways

Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies

Adeno-associated viruses (AAVs) are promising viral vectors for therapeutic gene delivery, and the approval of an AAV1 vector for the treatment of lipoprotein lipase deficiency has heralded a new and exciting era for this system. However, preclinical and clinical studies show that neutralization from pre-existing antibodies is detrimental for medical application and this hurdle must be overcome before full clinical realization can be achieved. Thus the binding sites for capsid antibodies must be identified and eliminated through capsid engineering. Towards this goal and to recapitulate patient polyclonal responses, a panel of eight new mouse monoclonal antibodies (MAbs) has been generated against AAV8 and AAV9 capsids, two vectors in development for therapeutic application. Native (capsid) dot blot assays confirmed the specificity of these antibodies for their parental serotypes, with the exception of one MAb, HL2372, selected to cross-react against both capsids. Furthermore, in vitro assays showed that these MAbs are capable of neutralizing virus infection. These MAbs will be utilized for structural mapping of antigenic footprints on their respective capsids to inform development of the next generation of rAAV vectors capable of evading antibody neutralization while retaining parental tropism

An evaluation of the C/N ratio of the mantle from natural CO2-rich gas analysis: Geochemical and cosmochemical implications

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Marty, B., Almayrac, M., Barry, P. H., Bekaert, D., V., Broadley, M. W., Byrne, D. J., Ballentine, C. J., & Caracausi, A. An evaluation of the C/N ratio of the mantle from natural CO2-rich gas analysis: Geochemical and cosmochemical implications. Earth and Planetary Science Letters, 551, (2020): 116574, doi:10.1016/j.epsl.2020.116574.The terrestrial carbon to nitrogen ratio is a key geochemical parameter that can provide information on the nature of Earth's precursors, accretion/differentiation processes of our planet, as well as on the volatile budget of Earth. In principle, this ratio can be determined from the analysis of volatile elements trapped in mantle-derived rocks like mid-ocean ridge basalts (MORB), corrected for fractional degassing during eruption. However, this correction is critical and previous attempts have adopted different approaches which led to contrasting C/N estimates for the bulk silicate Earth (BSE) (Marty and Zimmermann, 1999; Bergin et al., 2015). Here we consider the analysis of CO2-rich gases worldwide for which a mantle origin has been determined using noble gas isotopes in order to evaluate the C/N ratio of the mantle source regions. These gases experienced little fractionation due to degassing, as indicated by radiogenic 4He / 40Ar* values (where 4He and 40Ar* are produced by the decay of U+Th, and 40K isotopes, respectively) close to the mantle production/accumulation values. The C/N and C/3 He ratios of gases investigated here are within the range of values previously observed in oceanic basalts. They point to an elevated mantle C/N ratio (∼350-470, molar) higher than those of potential cosmochemical accretionary endmembers. For example, the BSE C/N and 36 Ar / N ratios (160-220 and 75 x 10-7, respectively) are higher than those of CM-CI chondrites but within the range of CV-CO groups. This similarity suggests that the Earth accreted from evolved planetary precursors depleted in volatile and moderately volatile elements. Hence the high C / N composition of the BSE may be an inherited feature rather than the result of terrestrial differentiation. The C / N and 36 Ar / N ratios of the surface (atmosphere plus crust) and of the mantle cannot be easily linked to any known chondritic composition. However, these compositions are consistent with early sequestration of carbon into the mantle (but not N and noble gases), permitting the establishment of clement temperatures at the surface of our planet.M.A, D.V.B, M.W.B, D.J.B and B.M were supported by the European Research Council (PHOTONIS project, grant agreement No. 695618 to B.M.). Samples were collected as part of Study # YELL-08056 - Xenon anomalies in the Yellowstone Hotspot. We would like to thank Annie Carlson and all of the rangers at the Yellowstone National Park for providing invaluable advice and help when collecting the samples. This work was partially supported by a grant (G-2016-7206) from the Alfred P. Sloan Foundation and the Deep Carbon Observatory to P.H.B as well as NSF award 2015789 to P.H.B.. Sampling at Mt. Etna and gas analysis was supported by Instituto Nazionale di Geofisica e Vulcanologia Palermo. Fruitful discussions with Marc Hirschmann helped us to shape the ideas presented in this work. We acknowledge detailed and insightful reviews by Sami Mikhail and an anonymous reviewer, and efficient editing by Frederic Moynier. This is CRPG contribution 2741