Glycopeptide characterization of Sf9-derived SARS-CoV-2 spike protein recombinant vaccine candidates expedited by the use of glycopeptide libraries.

RATIONALE: We report the N-glycosylation pattern of Sf9 insect cell-derived recombinant spike proteins being developed as candidate vaccine antigens for SARS-CoV-2 (COVID-19) (Sanofi). The method has been optimised to produce peptides with single, isolated glycosylation sites using multiple protease digests. The development and use of glycopeptide libraries from previous developmental phases allowed for faster analysis than processing datasets from individual batches from first principles. METHODS: Purified spike proteins were reduced, alkylated, and digested with proteolytic enzymes. Three different protease digests were utilised to generate peptides with isolated glycosylation sites. The glycopeptides were then analysed using a Waters Q-TOF while using a data-dependent acquisition mass spectrometry experiment. Glycopeptide mapping data processing and glycan classification were performed using Genedata Expressionist via a specialised workflow that used libraries of previously detected glycopeptides to greatly reduce processing time. RESULTS: Two different spike proteins from six manufacturers were analysed. There was a strong similarity at each site across batches and manufacturers. The majority of the glycans present were of the truncated class, although at sites N61, N234, and N717/714 high mannose structures were dominant and at N1173/1170 aglycosylation was dominant for both variant proteins. A comparison was performed on a commercially available spike protein and our results were found to be similar to those of earlier reports. CONCLUSIONS: Our data clearly show that the overall glycosylation pattern of both spike protein variants was highly similar from batch to batch, and between materials produced at different manufacturing facilities. The use of our glycopeptide libraries greatly expedited the generation of site-specific glycan occupancy data for a large glycoprotein. We compared our method with previously obtained data from a commercially available insect cell-derived spike protein and the results were comparable to published findings.

Synthesis, characterization and antifungal activities of eco-friendly palladium nanoparticles.

Palladium is a versatile catalyst, but the synthesis of palladium nanoparticles (PdNPs) is usually attained at a high temperature in the range of 160 °C to 200 °C using toxic reducing agents such as sodium borohydride. We report the synthesis of PdNPs using a low-cost and environmentally-friendly route at ambient temperatures. Quercetin diphosphate (QDP), a naturally-derived flavonoid, was employed as a reducing, capping, and stabilizing agent. The effect of temperature was optimized to produce perfectly spherical PdNP nanoparticles with sizes ranging from 0.1 to 0.3 microns in diameter. At relatively higher concentration of QDP, significantly smaller particles were produced with a size distribution of 1-7 nm. Perfectly spherical PdNP nanoparticles are a rare occurrence, especially under ambient room temperature conditions with fast reaction time. The formation of the nanoparticles was confirmed using UV-vis, TEM, EDS, and XRD. HRTEM demonstrated the lattice structure of the PdNPs. The synthesized PdNPs were also tested for their antifungal properties against Colletotrichum gloeosporioides and Fusarium oxysporum. Results showed that the size of the PdNPs played a critical role in their antifungal activity. However, for F. oxysporum, other factors beyond size could affect the antifungal activity including fine-scale, nutrient composition, and target organisms.

On the Effect of Modified Carbohydrates on the Size and Shape of Gold and Silver Nanostructures.

Gold (Au) and silver (Ag) nanostructures have widespread utilization from biomedicine to materials science. Therefore, their synthesis with control of their morphology and surface chemistry have been among the hot topics over the last decades. Here, we introduce a new approach relying on sugar derivatives that work as reducing, stabilizing, and capping agents in the synthesis of Au and Ag nanostructures. These sugar derivatives are utilized alone and as mixture, resulting in spherical, spheroid, trigonal, polygonic, and star-like morphologies. The synthesis approach was further tested in the presence of acetate and dimethylamine as size- and shape-directing agents. With the use of transmission electron microscopy (TEM), selected area electron diffraction (SAED), x-ray diffraction (XRD), scanning electron microscopy (SEM), and ultraviolet-visible (UV-vis) absorption spectroscopy techniques, the particle size, shape, assembly, aggregation, and film formation characteristics were evaluated. NPs' attributes were shown to be tunable by manipulating the sugar ligand selection and sugar ligand/metal-ion ratio. For instance, with an imine side group and changing the sugar moiety from cellobiose to lactose, the morphology of the Ag nanoparticles (NPs) transformed from well dispersed cubic to rough and aggregated. The introduction of acetate and dimethylamine further extended the growth pattern and morphological properties of these NPs. As examples, L5 AS, G5AS, and S5AS ligands formed spherical or sheet-like structures when used alone, which upon the use of these additives transformed into larger multicore and rough NPs, revealing their significant effect on the NP morphology. Selected samples were tested for their stability against protein corona formation and ionic strength, where a high chemical stability and resistance to protein coating were observed. The findings show a promising, benign approach for the synthesis of shape- and size-directed Au and Ag nanostructures, along with a selection of the chemistry of carbohydrate-derivatives that can open new windows for their applications.

Antimicrobial Activity of a New Class of Phosphorylated and Modified Flavonoids.

The surge of resistant food pathogens is a major threat worldwide. Previous research conducted on phytochemicals has shown their antibacterial activity against pathogenic bacteria. The design of antimicrobial agents to curb pathogenic disease remains a challenge demanding critical attention. Flavonoids such as apigenin and quercetin were evaluated against Gram-positive and Gram-negative bacteria. The results indicated that the antibacterial activity of each flavonoid occurred at a different minimum inhibitory concentration. However, the antimicrobial activity results of the modified flavonoids were also reported, and it was observed that the Gram-positive bacteria were more susceptible in comparison to the Gram-negative bacteria. The cell wall structure of the Gram-positive and Gram-negative bacteria could be the main reason for the bacteria susceptibility. Modified flavonoids could be used as a suitable alternative antimicrobial agent for the treatment of infectious diseases. Our results indicated 100% inhibition of Listeria monocytogenes, Pseudomonas aeruginosa, and Aeromonas hydrophila with modified flavonoids.

Photochemical Synthesis and Catalytic Applications of Gold Nanoplates Fabricated Using Quercetin Diphosphate Macromolecules.

The demand for safer design and synthesis of gold nanoparticles (AuNPs) is on the increase with the ultimate goal of producing clean nanomaterials for biological applications. We hereby present a rapid, greener, and photochemical synthesis of gold nanoplates with sizes ranging from 10 to 200 nm using water-soluble quercetin diphosphate (QDP) macromolecules. The synthesis was achieved in water without the use of surfactants, reducing agents, or polymers. The edge length of the triangular nanoplates ranged from 50 to 1200 nm. Furthermore, the reduction of methylene blue was used to investigate the catalytic activity of AuNPs. The catalytic activity of triangular AuNPs was three times higher than that of the spherical AuNPs based on kinetic rate constants (k). The rate constants were 3.44 × 10-2 and 1.11 × 10-2 s-1 for triangular and spherical AuNPs, respectively. The X-ray diffraction data of gold nanoplates synthesized by this method exhibited that the nanocrystals were mainly dominated by (111) facets which are in agreement to the nanoplates synthesized by using thermal and chemical approaches. The calculated relative diffraction peak intensity of (200), (220), and (311) in comparison with (111) was found to be 0.35, 0.17, and 0.15, respectively, which were lower than the corresponding standard values (JCPDS 04-0784). For example, (200)/(111) = 0.35 compared to 0.52 obtained from the standard (JCPDS 04-0784), indicating that the gold nanoplates are dominated by (111) facets. The calculated lattice from selected area electron diffraction data of the as-synthesized and after 1 year nanoplates was 4.060 and 4.088 Å, respectively. Our calculations were found to be in agreement with 4.078 Å for face-centered cubic gold (JCPDS 04-0784) and literature values of 4.07 Å. The computed QDP-Au complex demonstrated that the reduction process took place in the B ring of QDP. This approach contributes immensely to promoting the ideals of sustainable nanotechnology by eradicating the use of hazardous and toxic organic solvents.