Expression optimization of a human papillomavirus type 16 therapeutic vaccine candidate in Nicotiana benthamiana leaves

High risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. The three approved prophylactic vaccines do not benefit already infected individuals; therefore, there is still an urgent need for therapeutic vaccines. The HPV oncoproteins E6 and E7 are ideal targets for the development of such vaccines, as they are expressed throughout the viral life cycle and in tumours. They could be used to elicit strong cytotoxic lymphocyte (CTL) responses which would aid in viral clearance, and could also be effective against tumours. Granadillo et al. (2011) developed an Escherichia coli-produced therapeutic vaccine candidate, consisting of the HPV-16 E7 protein and a cell membrane- penetrating and immunomodulatory peptide (LALF), whose fusion to HPV-16 E7 aided in the immunogenicity and antigen presentation of the oncoprotein. However, such vaccines need not only to be effective, but also to have a low cost. Plant expression systems represent an attractive alternative to conventional expression systems based on bacterial, yeast, mammalian and other cell cultures, and are potentially far more cost- effective. The aim of the present project was to produce LALF-E7 in Nicotiana benthamiana leaves via Agrobacterium-mediated transient expression, and to optimize its expression, extraction and purification. This was done by expressing LALF-E7 using different expression vectors, testing different subcellular localizations, and testing the effect of gene silencing suppressors. By using our group's replicating expression vector and targeting LALF-E7 to the chloroplasts, the expression of the candidate vaccine in N. benthamiana leaves was increased 26.8 fold compared to non-replicating vectors or cytoplasmic localization. Furthermore, silencing suppressors did not significantly increase the expression of LALF-E7 when expressed by the replicating vector and targeted to the chloroplasts. I showed, by fluorescence microscopy, that LALE-E7 was indeed being targeted to the plants' chloroplasts and that it possibly forms proteins bodies (PBs) that are closely associated to the chloroplast envelope. I further hypothesized a mechanism by which the PBs-like structures form. Once the expression of LALF-E7 was optimized in plant leaves, a purification strategy was developed by testing different extraction methods and using metal ion affinity chromatography. The extraction protocol developed pre-purified LALF-E7 by removing the majority of soluble proteins from the final extract. However, LALF-E7 was not fully purified by affinity chromatography, suggesting that other purification strategies should be used. Finally, I tested the partially purified plant-produced LALF-E7 candidate, and compared it to the E. coli-produced counterpart, in tumour regression experiments using mice as animal models. Due to low antigen doses and a large number tumourigenic cells used to inoculate the mice animal models, the effect of the plant-produced LALF-E7 as a therapeutic vaccine was inconclusive. However, it was suggested that it could potentially be comparable to the E. coli-produced counterpart. In summary, I report for the first time the entire chain of research involving the expression of LALF-E7 in plants, its extraction, purification and the testing of its immunogenicity in a mouse model. This research also suggests new avenues for the use of the LALF peptide as a PB-inducer which could be useful in increasing the expression of other recombinant proteins

Fluid compressional properties sensing at microscale using a longitudinal bulk acoustic wave transducer operated in a pulse-echo scheme

Altres ajuts: Acord transformatiu CRUE-CSICAcoustic devices have been widely used as smart chemical and biochemical sensors since they are sensitive to mechanical, chemical, optical or electrical perturbations on their surfaces; making them a reliable option for noninvasive detection of changes in physical properties of liquid samples for real-time applications. Here we present a longitudinal acoustic wave device for study of compressional properties of liquids in microfluidic systems, with the particularity of pulse-echo mode of operation. We have studied at a microscale the interaction between longitudinal acoustic waves and the compressional properties of liquid samples, interrogating the fluids with short pulses of ultrasound at GHz, finding a direct relationship between the magnitude of the bulk modulus or the specific acoustic impedance of liquids and the amplitude of the output voltage produced by acoustic echoes received by the aluminum nitride transducer. Analytical expressions and FEM simulations support the detection mechanism, while applications such as classification of liquids and detection of concentration change in solutions experimentally demonstrate the method. This contribution overcomes current restrictions of film acoustic resonators such as fragility of operation in liquid environments, high manufacturing cost or limitations regarding narrow microchannels; offering an alternative to applications that demand ultra-low consumption, miniaturization, versatility (it offers multi-frequency operation in 1 - 10 GHz range) and ease of readout (peak voltage)

Common but different : the expanding realm of Cladosporium

The genus Cladosporium (Cladosporiaceae, Dothideomycetes), which represents one of the largest genera of dematiaceous hyphomycetes, has been intensively investigated during the past decade. In the process, three major species complexes (C. cladosporioides, C. herbarum and C. sphaerospermum) were resolved based on morphology and DNA phylogeny, and a monographic revision of the genus (s. lat.) published reflecting the current taxonomic status quo. In the present study a further 19 new species are described based on phylogenetic characters (nuclear ribosomal RNA gene operon, including the internal transcribed spacer regions ITS1 and ITS2, as well as partial actin and translation elongation factor 1-α gene sequences) and morphological differences. For a selection of the species with ornamented conidia, scanning electron microscopic photos were prepared to illustrate the different types of surface ornamentation. Surprisingly, during this study Cladosporium ramotenellum was found to be a quite common saprobic species, being widely distributed and occurring on various substrates. Therefore, an emended species description is provided. Furthermore, the host range and distribution data for several previously described species are also expanded.http://www.studiesinmycology.orgam2016Forestry and Agricultural Biotechnology Institute (FABI

Multi-messenger searches via IceCube’s high-energy neutrinos and gravitational-wave detections of LIGO/Virgo

We summarize initial results for high-energy neutrino counterpart searches coinciding with gravitational-wave events in LIGO/Virgo\u27s GWTC-2 catalog using IceCube\u27s neutrino triggers. We did not find any statistically significant high-energy neutrino counterpart and derived upper limits on the time-integrated neutrino emission on Earth as well as the isotropic equivalent energy emitted in high-energy neutrinos for each event

In-situ estimation of ice crystal properties at the South Pole using LED calibration data from the IceCube Neutrino Observatory

The IceCube Neutrino Observatory instruments about 1 km3 of deep, glacial ice at the geographic South Pole using 5160 photomultipliers to detect Cherenkov light emitted by charged relativistic particles. A unexpected light propagation effect observed by the experiment is an anisotropic attenuation, which is aligned with the local flow direction of the ice. Birefringent light propagation has been examined as a possible explanation for this effect. The predictions of a first-principles birefringence model developed for this purpose, in particular curved light trajectories resulting from asymmetric diffusion, provide a qualitatively good match to the main features of the data. This in turn allows us to deduce ice crystal properties. Since the wavelength of the detected light is short compared to the crystal size, these crystal properties do not only include the crystal orientation fabric, but also the average crystal size and shape, as a function of depth. By adding small empirical corrections to this first-principles model, a quantitatively accurate description of the optical properties of the IceCube glacial ice is obtained. In this paper, we present the experimental signature of ice optical anisotropy observed in IceCube LED calibration data, the theory and parametrization of the birefringence effect, the fitting procedures of these parameterizations to experimental data as well as the inferred crystal properties.</p

Non-standard neutrino interactions in IceCube

Non-standard neutrino interactions (NSI) may arise in various types of new physics. Their existence would change the potential that atmospheric neutrinos encounter when traversing Earth matter and hence alter their oscillation behavior. This imprint on coherent neutrino forward scattering can be probed using high-statistics neutrino experiments such as IceCube and its low-energy extension, DeepCore. Both provide extensive data samples that include all neutrino flavors, with oscillation baselines between tens of kilometers and the diameter of the Earth. DeepCore event energies reach from a few GeV up to the order of 100 GeV - which marks the lower threshold for higher energy IceCube atmospheric samples, ranging up to 10 TeV. In DeepCore data, the large sample size and energy range allow us to consider not only flavor-violating and flavor-nonuniversal NSI in the μ−τ sector, but also those involving electron flavor. The effective parameterization used in our analyses is independent of the underlying model and the new physics mass scale. In this way, competitive limits on several NSI parameters have been set in the past. The 8 years of data available now result in significantly improved sensitivities. This improvement stems not only from the increase in statistics but also from substantial improvement in the treatment of systematic uncertainties, background rejection and event reconstruction