Search results for: waveform separation

Authors: F. Varga, Z. Liber, J. Jakše, A. Turudić, Z. Šatović, I. Radosavljević, N. Jeran, M. Grdiša

Abstract:

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.; Asteraceae), a source of the commercially dominant plant insecticide pyrethrin, is a species endemic to the eastern Adriatic. Genetic diversity of T. cinerariifolium was previously studied using amplified fragment length polymorphism (AFLP) markers. However, microsatellite markers (single sequence repeats - SSRs) are more informative because they are codominant, highly polymorphic, locus-specific, and more reproducible, and thus are most often used to assess the genetic diversity of plant species. Dalmatian pyrethrum is an outcrossing diploid (2n = 18) whose large genome size and high repeatability have prevented the success of the traditional approach to SSR markers development. The advent of next-generation sequencing combined with the specifically developed method recently enabled the development of, to the author's best knowledge, the first set of SSRs for genomic characterization of Dalmatian pyrethrum, which is essential from the perspective of plant genetic resources conservation. To evaluate the effectiveness of the developed SSR markers in genetic differentiation of Dalmatian pyrethrum populations, a preliminary genetic diversity study was conducted on 30 individuals from three geographically distinct natural populations in Croatia (northern Adriatic island of Mali Lošinj, southern Adriatic island of Čiovo, and Mount Biokovo) based on 12 SSR loci. Analysis of molecular variance (AMOVA) by randomization test with 10,000 permutations was performed in Arlequin 3.5. The average number of alleles per locus, observed and expected heterozygosity, probability of deviations from Hardy-Weinberg equilibrium, and inbreeding coefficient was calculated using GENEPOP 4.4. Genetic distance based on the proportion of common alleles (DPSA) was calculated using MICROSAT. Cluster analysis using the neighbor-joining method with 1,000 bootstraps was performed with PHYLIP to generate a dendrogram. The results of the AMOVA analysis showed that the total SSR diversity was 23% within and 77% between the three populations. A slight deviation from Hardy-Weinberg equilibrium was observed in the Mali Lošinj population. Allele richness ranged from 2.92 to 3.92, with the highest number of private alleles observed in the Mali Lošinj population (17). The average observed DPSA between 30 individuals was 0.557. The highest DPSA (0.875) was observed between several pairs of Dalmatian pyrethrum individuals from the Mali Lošinj and Mt. Biokovo populations, and the lowest between two individuals from the Čiovo population. Neighbor-joining trees, based on DPSA, grouped individuals into clusters according to their population affiliation. The separation of Mt. Biokovo clade was supported (bootstrap value 58%), which is consistent with the previous study on AFLP markers, where isolated populations from Mt. Biokovo differed from the rest of the populations. The developed SSR markers are an effective tool for assessing the genetic diversity and structure of natural Dalmatian pyrethrum populations. These preliminary results are encouraging for a future comprehensive study with a larger sample size across the species' range. Combined with the biochemical data, these highly informative markers could help identify potential genotypes of interest for future development of breeding lines and cultivars that are both resistant to environmental stress and high in pyrethrins. Acknowledgment: This work has been supported by the Croatian Science Foundation under the project ‘Genetic background of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) insecticidal potential’- (PyrDiv) (IP-06-2016-9034) and by project KK.01.1.1.01.0005, Biodiversity and Molecular Plant Breeding, at the Centre of Excellence for Biodiversity and Molecular Plant Breeding (CoE CroP-BioDiv), Zagreb, Croatia.

Keywords: Asteraceae, genetic diversity, genomic SSRs, NGS, pyrethrum, Tanacetum cinerariifolium

Procedia PDF Downloads 114

Authors: Rowan Moore, Kai Scheffler, Eugen Damoc, Jennifer Sutton, Aaron Bailey, Stephane Houel, Simon Cubbon, Jonathan Josephs

Abstract:

Purpose: MS analysis of monoclonal antibodies (mAbs) at the protein and peptide levels is critical during development and production of biopharmaceuticals. The compositions of current generation therapeutic proteins are often complex due to various modifications which may affect efficacy. Intact proteins analyzed by MS are detected in higher charge states that also provide more complexity in mass spectra. Protein analysis in native or native-like conditions with zero or minimal organic solvent and neutral or weakly acidic pH decreases charge state value resulting in mAb detection at higher m/z ranges with more spatial resolution. Methods: Three commercially available mAbs were used for all experiments. Intact proteins were desalted online using size exclusion chromatography (SEC) or reversed phase chromatography coupled on-line with a mass spectrometer. For streamlined use of the LC- MS platform we used a single SEC column and alternately selected specific mobile phases to perform separations in either denaturing or native-like conditions: buffer A (20 % ACN, 0.1 % FA) with Buffer B (100 mM ammonium acetate). For peptide analysis mAbs were proteolytically digested with and without prior reduction and alkylation. The mass spectrometer used for all experiments was a commercially available Thermo Scientific™ hybrid Quadrupole-Orbitrap™ mass spectrometer, equipped with the new BioPharma option which includes a new High Mass Range (HMR) mode that allows for improved high mass transmission and mass detection up to 8000 m/z. Results: We have analyzed the profiles of three mAbs under reducing and native conditions by direct infusion with offline desalting and with on-line desalting via size exclusion and reversed phase type columns. The presence of high salt under denaturing conditions was found to influence the observed charge state envelope and impact mass accuracy after spectral deconvolution. The significantly lower charge states observed under native conditions improves the spatial resolution of protein signals and has significant benefits for the analysis of antibody mixtures, e.g. lysine variants, degradants or sequence variants. This type of analysis requires the detection of masses beyond the standard mass range ranging up to 6000 m/z requiring the extended capabilities available in the new HMR mode. We have compared each antibody sample that was analyzed individually with mixtures in various relative concentrations. For this type of analysis, we observed that apparent native structures persist and ESI is benefited by the addition of low amounts of acetonitrile and formic acid in combination with the ammonium acetate-buffered mobile phase. For analyses on the peptide level we analyzed reduced/alkylated, and non-reduced proteolytic digests of the individual antibodies separated via reversed phase chromatography aiming to retrieve as much information as possible regarding sequence coverage, disulfide bridges, post-translational modifications such as various glycans, sequence variants, and their relative quantification. All data acquired were submitted to a single software package for analysis aiming to obtain a complete picture of the molecules analyzed. Here we demonstrate the capabilities of the mass spectrometer to fully characterize homogeneous and heterogeneous therapeutic proteins on one single platform. Conclusion: Full characterization of heterogeneous intact protein mixtures by improved mass separation on a quadrupole-Orbitrap™ mass spectrometer with extended capabilities has been demonstrated.

Keywords: disulfide bond analysis, intact analysis, native analysis, mass spectrometry, monoclonal antibodies, peptide mapping, post-translational modifications, sequence variants, size exclusion chromatography, therapeutic protein analysis, UHPLC

Procedia PDF Downloads 362

Authors: Jocelyn Doucet, Jean-Philippe Laviolette, Ali Eslami

Abstract:

The end-of-life management and recycling of polymer wastes remains a key environment issue in on-going efforts to increase resource efficiency and attaining GHG emission reduction targets. Half of all the plastics ever produced were made in the last 13 years, and only about 16% of that plastic waste is collected for recycling, while 25% is incinerated, 40% is landfilled, and 19% is unmanaged and leaks in the environment and waterways. In addition to the plastic collection issue, the UN recently published a report on chemicals in plastics, which adds another layer of challenge when integrating recycled content containing toxic products into new products. To tackle these important issues, innovative solutions are required. Chemical recycling of plastics provides new complementary alternatives to the current recycled plastic market by converting waste material into a high value chemical commodity that can be reintegrated in a variety of applications, making the total market size of the output – virgin-like, high value products - larger than the market size of the input – plastic waste. Access to high-quality feedstock also remains a major obstacle, primarily due to material contamination issues. Pyrowave approaches this challenge with its innovative nano-recycling technology, which purifies polymers at the molecular level, removing undesirable contaminants and restoring the resin to its virgin state without having to depolymerise it. This breakthrough approach expands the range of plastics that can be effectively recycled, including mixed plastics with various contaminants such as lead, inorganic pigments, and flame retardants. The technology allows yields below 100ppm, and purity can be adjusted to an infinitesimal level depending on the customer's specifications. The separation of the polymer and contaminants in Pyrowave's nano-recycling process offers the unique ability to customize the solution on targeted additives and contaminants to be removed based on the difference in molecular size. This precise control enables the attainment of a final polymer purity equivalent to virgin resin. The patented process involves dissolving the contaminated material using a specially formulated solvent, purifying the mixture at the molecular level, and subsequently extracting the solvent to yield a purified polymer resin that can directly be reintegrated in new products without further treatment. Notably, this technology offers simplicity, effectiveness, and flexibility while minimizing environmental impact and preserving valuable resources in the manufacturing circuit. Pyrowave has successfully applied this nano-recycling technology to decontaminate polymers and supply purified, high-quality recycled plastics to critical industries, including food-contact compliance. The technology is low-carbon, electrified, and provides 100% traceable resins with properties identical to those of virgin resins. Additionally, the issue of low recycling rates and the limited market for traditionally hard-to-recycle plastic waste has fueled the need for new complementary alternatives. Chemical recycling, such as Pyrowave's microwave depolymerization, presents a sustainable and efficient solution by converting plastic waste into high-value commodities. By employing microwave catalytic depolymerization, Pyrowave enables a truly circular economy of plastics, particularly in treating polystyrene waste to produce virgin-like styrene monomers. This revolutionary approach boasts low energy consumption, high yields, and a reduced carbon footprint. Pyrowave offers a portfolio of sustainable, low-carbon, electric solutions to give plastic waste a second life and paves the way to the new circular economy of plastics. Here, particularly for polystyrene, we show that styrene monomer yields from Pyrowave’s polystyrene microwave depolymerization reactor is 2,2 to 1,5 times higher than that of the thermal conventional pyrolysis. In addition, we provide a detailed understanding of the microwave assisted depolymerization via analyzing the effects of microwave power, pyrolysis time, microwave receptor and temperature on the styrene product yields. Furthermore, we investigate life cycle environmental impact assessment of microwave assisted pyrolysis of polystyrene in commercial-scale production. Finally, it is worth pointing out that Pyrowave is able to treat several tons of polystyrene to produce virgin styrene monomers and manage waste/contaminated polymeric materials as well in a truly circular economy.

Keywords: nanorecycling, nanomaterials, plastic recycling, depolymerization

Procedia PDF Downloads 66