Cellulose and Lignin Nano-Scale Consolidants for Waterlogged Archaeological Wood.

Waterlogged archaeological wood comes from submerged archaeological sites (in lake, sea, river, or wetland) or from land waterlogged sites. Even if the wooden object seems to have maintained the original size and shape, the wood is more or less severely decayed because of chemical and biological factors which modify the normal ratio of cellulose and lignin in the cell wall. Drying procedures are necessary for the musealization but potentially cause severe shrinkages and collapses. The conservation practices focus not only on removing water from wood but also on substituting it with materials able to consolidate the degraded wood cell walls like polymers (e.g., PEG), sugars (e.g., lactitol), or resins (e.g., Kauramin). In the present work three different nano-scale consolidants were tested: lignin nanoparticles (LNPs) obtained form beech wood via a non-solvent method involving dialysis; bacterial nanocellulose (BC) obtained from cultures fed with agro-alimentary waste; cellulose nanocrystals (CNC) chemically extracted from native cellulose. Waterlogged archaeological wood samples of different species (oak, elm, stone pine, and silver fir) characterized by different levels of degradation were impregnated with the consolidants. The treatments efficiency was evaluated in terms of macroscopic observation of treated samples, anti-shrink efficiency (ASE) and equilibrium moisture content (EMC). The results obtained for the three consolidants showed substantial differences: LNPs and CNCs penetrated only about a millimeter inside the treated wood, while BC formed a compact layer on the surface of the cell walls throughout the thickness of the samples. In spite of successful BC penetration, physical evaluation of treatment efficiency showed that BC nanoparticles did not obtain a satisfying consolidation of the material. Based on the reported results more focused test protocols are optimized for future consolidation experiments.

Green Energy Harvester from Vibrations Based on Bacterial Cellulose.

A bio-derived power harvester from mechanical vibrations is here proposed. The harvester aims at using greener fabrication technologies and reducing the dependence from carbon-based fossil energy sources. The proposed harvester consists mainly of biodegradable matters. It is based on bacterial cellulose, produced by some kind of bacteria, in a sort of bio-factory. The cellulose is further impregnated with ionic liquids and covered with conducting polymers. Due to the mechanoelectrical transduction properties of the composite, an electrical signal is produced at the electrodes, when a mechanical deformation is imposed. Experimental results show that the proposed system is capable of delivering electrical energy on a resistive load. Applications can be envisaged on autonomous or quasi-autonomous electronics, such as wireless sensor networks, distributed measurement systems, wearable, and flexible electronics. The production technology allows for fabricating the harvester with low power consumption, negligible amounts of raw materials, no rare elements, and no pollutant emissions.

Silver-Functionalized Bacterial Cellulose as Antibacterial Membrane for Wound-Healing Applications.

Bacterial cellulose (BC) functionalized with silver nanoparticles (AgNPs) is evaluated as an antimicrobial membrane for wound-healing treatment. A facile green synthesis of silver nanoparticles inside the porous three-dimensional weblike BC network has been obtained by UV light irradiation. AgNPs were photochemically deposited onto the BC gel network as well as they were chemically bonded to the cellulose fiber surfaces. AgNPs with a narrow size distribution along with some aggregates in the BC network were evidenced from the morphological analyses. A highly crystalline nature of the BC membranes was observed in X-ray diffraction measurements, and the presence of metallic silver confirmed the photochemical reduction of Ag+ → Ag0 in Ag/BC composites. Antibacterial activity of the hybrid composites, such as pellicles, performed against the Gram-negative bacteria (Escherichia coli) by disk diffusion and growth dynamics methods showed high bacteria-killing performance. No significant amount of silver release was observed from the Ag/BC pellicles even after a long soaking time. As composite pellicles are preserved in a moist environment that also favors wound recovery, by combining all of these properties the material could be useful in wound-healing treatments.

ROS Production and Scavenging under Anoxia and Re-Oxygenation in Arabidopsis Cells: A Balance between Redox Signaling and Impairment.

Plants can frequently experience low oxygen concentrations due to environmental factors such as flooding or waterlogging. It has been reported that both anoxia and the transition from anoxia to re-oxygenation determine a strong imbalance in the cellular redox state involving the production of reactive oxygen species (ROS) and nitric oxide (NO). Plant cell cultures can be a suitable system to study the response to oxygen deprivation stress since a close control of physicochemical parameters is available when using bioreactors. For this purpose, Arabidopsis cell suspension cultures grown in a stirred bioreactor were subjected to a severe anoxic stress and analyzed during anoxia and re-oxygenation for alteration in ROS and NO as well as in antioxidant enzymes and metabolites. The results obtained by confocal microscopy showed the dramatic increase of ROS, H2O2, and NO during the anoxic shock. All the ascorbate-glutathione related parameters were altered during anoxia but restored during re-oxygenation. Anoxia also induced a slight but significant increase of α-tocopherol levels measured at the end of the treatment. Overall, the evaluation of cell defenses during anoxia and re-oxygenation in Arabidopsis cell cultures revealed that the immediate response involving the overproduction of reactive species activated the antioxidant machinery including ascorbate-glutathione system, α-tocopherol and the ROS-scavenging enzymes ascorbate peroxidase, catalase, and peroxidase making cells able to counteract the stress toward cell survival.

Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

ß-Cyclodextrins enhance artemisinin production in Artemisia annua suspension cell cultures.

Artemisinin is a sesquiterpene antimalarial compound produced, though at low levels (0.1-1% dry weight), in Artemisia annua in which it accumulates in the glandular trichomes of the plant. Due to its antimalarial properties and short supply, efforts are being made to improve our understanding of artemisinin biosynthesis and its production. Native ß-cyclodextrins, as well as the chemically modified heptakis(2,6-di-O-methyl)-ß-cyclodextrin (DIMEB) and 2-hydroxypropyl-ß-cyclodextrins, were added to the culture medium of A. annua suspension cultures, and their effects on artemisinin production were analysed. The effects of a joint cyclodextrin and methyl jasmonate treatment were also investigated. Fifty millimolar DIMEB, as well as a combination of 50 mM DIMEB and 100 µM methyl jasmonate, was highly effective in increasing the artemisinin levels in the culture medium. The observed artemisinin level (27 µmol g(-1) dry weight) was about 300-fold higher than that observed in untreated suspensions. The influence of ß-cyclodextrins and methyl jasmonate on the expression of artemisinin biosynthetic genes was also investigated.

Tocopherol production in plant cell cultures.

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (alpha, beta, gamma and delta), alpha-tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic alpha-tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) alpha-tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural alpha-tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

Tocopherol biosynthesis is enhanced in photomixotrophic sunflower cell cultures.

Alpha-tocopherol is the most biologically active component of vitamin E and is synthesized only by photosynthetic organisms. Two heterotrophic cell lines of sunflower (Helianthus annuus L.) of differing alpha-tocopherol biosynthetic capability, three-fold higher in the high synthesizing cell line, HT, than in the low synthesizing one, LT, were previously identified. To investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, a new photomixotrophic sunflower cell line HS3 was established by selecting HT cells able to grow in the presence of a ten-fold reduced sucrose concentration in the culture medium. The photosynthetic properties of HS3 cells were characterized in comparison with HT and LT cells, revealing an increase in chlorophyll content, chloroplast number, and level of the photosynthesis related enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Furthermore, an enhanced expression of the gene encoding for the tocopherol biosynthetic enzyme geranyl-geranylpyrophosphate synthase (GGPPS) was observed in HS3 cells. HS3 cells also revealed a 25% and a more than three-fold higher tocopherol level than HT and LT, respectively, indicating a positive correlation between alpha-tocopherol biosynthesis of sunflower cell cultures and their photosynthetic properties. These findings can be useful for improving the tocopherol yields of the sunflower in vitro production system.