RNA localization during early development of the axolotl.

The asymmetric localization of biomolecules is critical for body plan development. One of the most popular model organisms for early embryogenesis studies is Xenopus laevis but there is a lack of information in other animal species. Here, we compared the early development of two amphibian species-the frog X. laevis and the axolotl Ambystoma mexicanum. This study aimed to identify asymmetrically localized RNAs along the animal-vegetal axis during the early development of A. mexicanum. For that purpose, we performed spatial transcriptome-wide analysis at low resolution, which revealed dynamic changes along the animal-vegetal axis classified into the following categories: profile alteration, de novo synthesis and degradation. Surprisingly, our results showed that many of the vegetally localized genes, which are important for germ cell development, are degraded during early development. Furthermore, we assessed the motif presence in UTRs of degraded mRNAs and revealed the enrichment of several motifs in RNAs of germ cell markers. Our results suggest novel reorganization of the transcriptome during embryogenesis of A. mexicanum to converge to the similar developmental pattern as the X. laevis.

Investigation of drug dissolution and uptake from low-density DPI formulations in an impactor-integrated cell culture model.

Besides deposition, pulmonary bioavailability is determined by dissolution of particles in the scarce epithelial fluid and by cellular API uptake. In the present work, we have developed an experimental in vitro model, which is combining the state-of-the-art next generation impactor (NGI), used for aerodynamic performance assessment of inhalation products, with a culture of human alveolar A549 epithelial cells to study the fate of inhaled drugs following lung deposition. The goal was to investigate five previously developed nano-milled and spray-dried budesonide formulations and to examine the suitability of the in vitro test model. The NGI dissolution cups of stages 3, 4, and 5 were transformed to accommodate cell culture inserts while assuring minimal interference with the air flow. A549 cells were cultivated at the air-liquid interface on Corning® Matrigel® -coated inserts. After deposition of aerodynamically classified powders on the cell cultures, budesonide amount was determined on the cell surface, in the interior of the cell monolayer, and in the basal solution for four to eight hours. Significant differences in the total deposited drug amount and the amount remaining on the cell surface at the end of the experiment were found between different formulations and NGI stages. Roughly 50% of budesonide was taken up by the cells and converted to a large extent to its metabolic conjugate with oleic acid for all formulations and stages. Prolonged time required for complete drug dissolution and cell uptake in case of large deposited powder amounts suggested initial drug saturation of the surfactant layer of the cell surface. Discrimination between formulations with respect to time scale of dissolution and cell uptake was possible with the present test model providing useful insights into the biopharmaceutical performance of developed formulations that may be relevant for predicting local bioavailability. The absolute quantitative result of cell uptake and permeation into the systemic compartment is unreliable, though, because of partly compromised cell membrane integrity due to particle impaction and professed leakiness of A549 monolayer tight junctions, respectively.

Production of fast-dissolving low-density powders for improved lung deposition by spray drying of a nanosuspension.

We combined high-energy wet media milling and spray drying to engineer dry powders for inhalation consisting of geometrically large, low-density particles with superior aerodynamic properties and fast dissolution. Peclet number proved to be a useful instrument to guide choice of the additives and process conditions for generating low-density powders by spray drying. Composite dry powders consisted of milled and stabilized budesonide nanoparticles, leucine or albumin as matrix formers, and ammonium carbonate as a pore former. Powders of different composition showed fairly large and comparable geometric particle sizes (de,50 > 4.4 µm) with effective densities strongly depending on the present matrix former. Powders with lowest density reached an aerosol performance of up to 60%, which is well above most commercial, carrier-based products. It was also demonstrated that the nanomilling step was indispensable to yield such good aerosol performance. Dissolution of aerodynamically classified particle fractions showed a very fast onset and was largely completed within 30 min irrespective of the formulation and the impactor stage. Mathematical kinetic modeling was used to deduce the API dissolution rate coefficient from the results obtained using a modified USP 2 apparatus. Dissolution rate was found to be determined by the properties of the API nanoparticles rather than those of the composite particles. The employment of industrially established, solely water-based processes allows introducing the presented approach as a platform technology for the development of well-performing pulmonary formulations.

Immunomagnetic molecular probe with UHPLC-MS/MS: a promising way for reliable bronchial asthma diagnostics based on quantification of cysteinyl leukotrienes.

A sensitive and precise method for simultaneous quantification of cysteinyl leukotrienes (=cys LTs) - leukotriene C4 (=LTC4), leukotriene D4 (=LTD4) and leukotriene E4 (=LTE4) - essential biomarkers of bronchial asthma present in exhaled breath condensate (=EBC) was developed. An immunomagnetic molecular probe was prepared by anchoring cysteinyl leukotrienes antibody on the surface of functionalized monodispersed magnetic particles and used to selectively isolate cys LTs from biological matrices - EBC, plasma and urine. Immobilization and the immunoaffinity capture procedures were optimized to maximize the amount of separated cys LTs, which were detected "off-beads" after acidic elution by UHPLC-ESI-MS/MS operated in a multiple reaction monitoring mode. The developed method was characterized with high precision ≤13.6% (intra-day precision determined as RSD) and ≤14.5% (inter-day precision determined as RSD), acceptable accuracy ≤18.5% (determined as RE), and high recovery of immunoseparation (≥93.1%) in aforementioned biological matrices. The applicability of the method was demonstrated on EBC, plasma and urine clinical samples of patients with various subtypes of bronchial asthma (occupational, steroid-resistant, moderate with and without corticosteroids therapy) and healthy subjects where reasonable differences in cys LTs concentration levels were found. Combining extremely selective immunomagnetic separation with highly sensitive and precise detection step, the developed method was used to aid diagnosis, predict the most effective therapy, and monitor the response to treatment. The detection of elevated inflammatory mediators (cys LTs) in EBC of subjects with relatively asymptomatic asthma and normal pulmonary function tests could offer a novel way for monitoring the lung inflammation and perhaps initiating treatment in an earlier stage.