Characterization and study of the antibacterial mechanisms of silver nanoparticles prepared with microalgal exopolysaccharides.

The green synthesis of biomaterials is of significant interest as it enables the safe and sustainable preparation of noble metallic nanoparticles for medical applications. Microalgae polysaccharides have received attention due to their outstanding properties such as biocompatibility, biodegradability and low cost. In addition, due to their variety of remarkable biological and physicochemical properties, polysaccharide-based nanoparticles have advantageous features yet to be explored. The primary objective of the current research was to investigate exopolysaccharides isolated from green microalgae Botryococcus braunii (EPBb) and Chlorella pyrenoidosa (EPCp), as both reducing and stabilizing agents, for the green synthesis of silver nanoparticles (AgNPs). Their antibacterial activity towards Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), and antibiotic-resistant bacteria (methicillin-resistant Staphylococcus aureus) was studied, as well as their cytotoxicity to human dermal fibroblasts. The presently synthesized AgNPs were spherical in shape and exhibited characteristic surface plasmon resonance at 430 nm. The main population had a particle size which ranged between 5 and 15 nm as analyzed by transmission electron micrographs. Zeta potentials averaged -51.81 ±â€¯3.01 mV using EPBb and -12.16 ±â€¯2.41 mV using EPCp. More importantly, AgNPs possessed strong antibacterial activity in a dose-dependent manner, even against drug-resistant bacteria. The enhanced antibacterial activity of these particles is explained due to extensive reactive oxygen species generation and bacterial cell membrane damage. In contrast, such AgNPs were not cytotoxic at the same therapeutic range to fibroblasts (0.5-10.0 µg/mL). In summary, these results showed that polysaccharide-capped AgNPs have a strong potential for numerous medical applications, such as antibacterial agents in pharmaceutical and biomedical areas.

Virus-induced gene silencing of the two squalene synthase isoforms of apple tree (Malus × domestica L.) negatively impacts phytosterol biosynthesis, plastid pigmentation and leaf growth.

MAIN CONCLUSION: The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.

Characterization of an autonomously replicating sequence in Candida guilliermondii.

Candida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, biotechnological interest, and biological control potential. During a series of preliminary experiments aiming at optimizing the electroporation procedure of C. guilliermondii cells, we observed that the efficiency of transformation of an ura5 recipient strain with the corresponding dominant marker URA5 was more than a thousand fold higher as compared with the transformation of an ura3 strain with the URA3 wild type allele. This result allowed the identification of an autonomously replicating sequence (ARS) within an A/T rich region located upstream of the URA5 open reading frame (ORF). Interestingly, linear double strand DNAs (dsDNAs) containing this putative ARS are circularized and then autonomously replicated in C. guilliermondii transformed cells. We demonstrated that the C. guilliermondii Lig4p ligase, involved in the canonical non-homologous end-joining (NHEJ) pathway, was responsible for this phenomenon since a lig4 mutant was unable to circularize and to autonomously maintain transforming dsDNAs containing the putative ARS. Finally, a functional dissection of the C. guilliermondii A/T rich region located upstream of the URA5 ORF revealed the presence of a 60 bp-length sequence essential and sufficient to confer ARS properties to shuttle plasmid and linear dsDNAs.