2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation

Fluorescent glucose derivatives are valuable tools as glucose analogs in plant research to explore metabolic pathways, study enzyme activity, and investigate cellular processes related to glucose metabolism and sugar transport. They allow visualization and tracking of glucose uptake, its utilization, and distribution within plant cells and tissues. This study investigates the phenotypic and metabolic impact of the exogenously fed glucose derivative, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG) on the fibers of Gossypium hirsutum (Upland cotton) ovule in vitro cultures. The presence of 2-NBDG in the culture medium did not lead to macroscopic morphological alterations in ovule and fiber development or to the acquisition of fluorescence or yellow coloration. Confocal laser scanning microscope imaging and chromatographic analysis of cotton ovules’ outer rim cross-sections showed that the 2-NBDG is transported from the extracellular space and accumulated inside some outer integument cells, epidermal cells, and fertilized epidermal cells (fibers), but is not incorporated into the cell walls. Untargeted metabolic profiling of the fibers revealed significant changes in the relative levels of metabolites involved in glycolysis and upregulation of alternative energy-related pathways. To provide biochemical and structural evidence for the observed downregulation of glycolysis pathways in the fibers containing 2-NBDG, kinetics analysis and docking simulations were performed on hexokinase from G. hirsutum (GhHxk). Notably, the catalytic activity of heterologously expressed recombinant active GhHxk exhibited a five-fold decrease in reaction rates compared to D-glucose. Furthermore, GhHxk exhibited a linear kinetic behavior in the presence of 2-NBDG instead of the Michaelis-Menten kinetics found for D-glucose. Docking simulations suggested that 2-NBDG interacts with a distinct binding site of GhHxk9, possibly inducing a conformational change. These results highlight the importance of considering fluorescent glucose derivatives as ready-to-use analogs for tracking glucose-related biological processes. However, a direct comparison between their mode of action and its extrapolation into biochemical considerations should go beyond microscopic inspection and include complementary analytical techniques

Harnessing precursor-directed biosynthesis with glucose derivatives to access cotton fibers with enhanced physical properties

Cotton ovule in vitro cultures are a promising platform for exploring biofabrication of fibers with tailored properties. When the ovules' growth medium is supplemented with chemically synthesized cellulose precursors, it results in their integration into the developing fibers, thereby tailoring their end properties. Here, we report the feeding of synthetic glucosyl phosphate derivative, 6-deoxy-6-fluoro-glucose-1-phosphate (6F-Glc-1P) to cotton ovules growing in vitro, demonstrating the metabolic incorporation of 6F-Glc into the fibers with enhanced mechanical properties and moisture-retention capacity while emphasizing the role of molecular hierarchical architecture in defining functional characteristics and mechanical properties. This incorporation strategy bypasses the early steps of conventional metabolic pathways while broadening the range of functionalities that can be employed to customize fiber end properties. Our approach combines materials science, chemistry, and plant sciences to illustrate the innovation required to find alternative solutions for sustainable production of functional cotton fibers with enhanced and emergent properties

Sponge spicules as blueprints for the biofabrication of inorganic–organic composites and biomaterials

While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. However, of all recent animals, only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e., biosilicification) hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. Spicules lend structural stability to the sponge body, deter predators, and transmit light similar to optic fibers. This peculiar phenomenon has been comprehensively studied in recent years and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) or micro-optics (in vitro synthesis of light waveguides) with promising results

Structural Evolution of Gossypium hirsutum Fibers Grown under Greenhouse and Hydroponic Conditions

Cotton is the leading fiber source in the textile industry and one of the world’s most important crops. Despite its economic interest, cotton culture exerts an enormous pressure on natural resources (land and water) and has a negative impact on the environment (abuse of pesticides). Thus, alternative cotton growing methods are urged to be implemented. Recently, we have demonstrated that Gossypium hirsutum (“Upland” cotton) can be grown in a greenhouse (controlled conditions) and hydroponically. Here we report on the elucidation of the structural changes of the Gossypium hirsutum fibers during maturation grown [10, 14, 17, 20, 36 and 51 days post anthesis (dpa)] under a greenhouse and hydroponically, by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy with attenuated total reflectance (FT-IR ATR) and thermal gravimetric analysis/differential scanning calorimetry (TGA/DSC). The transition from primary to secondary cell wall growth occurs between 17 and 20 dpa—similarly to the soil-based cultures. However, this new cotton culture offers an advantageous pesticide and soil-free all year-round closed system with efficient water use yielding standardized mature fibers with improved properties (maturity, strength, length, whiteness)

Bioinspired fabrication of biosilica-based bone substitution materials

Until today, autogenic bone grafts from various donor regions represent the gold standard in the field of bone reconstruction, providing both osteoinductive and osteoconductive characteristics. However, due to low availability and a disequilibrium between supply and demand, the risk of disease transfer and morbidity, usually associated with autogeneic bone grafts, the development of biomimic materials with structural and chemical properties similar to those of natural bone have been extensively studied. So far,rnonly a few synthetic materials, so far, have met these criteria, displaying properties that allow an optimal bone reconstitution. Biosilica is formed enzymatically under physiological-relevant conditions (temperature and pH) via silicatein (silica protein), an enzyme that was isolated from siliceous sponges, cloned, and prepared in a recombinant way, retaining its catalytic activity. It is biocompatible, has some unique mechanical characteristics, and comprises significant osteoinductive activity.rnTo explore the application of biosilica in the fields of regenerative medicine,rnsilicatein was encapsulated, together with its substrate sodium metasilicate, into poly(D,L-lactide)/polyvinylpyrrolidone(PVP)-based microspheres, using w/o/wrnmethodology with solvent casting and termed Poly(D,L-lactide)-silicatein silicacontaining-microspheres [PLASSM]. Both silicatein encapsulation efficiency (40%) and catalytic activity retention upon polymer encapsulation were enhanced by addition of an essential pre-emulsifying step using PVP. Furthermore, the metabolic stability, cytoxicity as well as the kinetics of silicatein release from the PLASSM were studied under biomimetic conditions, using simulated body fluid. As a solid support for PLASSM, a polyvinylpyrrolidone/starch/Na2HPO4-based matrix (termed plastic-like filler matrix containing silicic acid [PMSA]) was developed and its chemical and physical properties determined. Moreover, due to the non-toxicity and bioinactivity of the PMSA, it is suggested that PMSA acts as osteoconductive material. Both components, PLASSM and PMSA, when added together, form arnbifunctional 2-component implant material, that is (i)non-toxic(biocompatible), (ii)moldable, (iii) self-hardening at a controlled and clinically suitable rate to allows a tight insertion into any bone defect (iv) biodegradable, (v)forms a porous material upon exposure to body biomimetic conditions, and (vi)displays both osteoinductive (silicatein)and osteoconductive (PMSA) properties.rnPreliminary in vivo experiments were carried out with rabbit femurs, by creatingrnartificial bone defects that were subsequently treated with the bifunctional 2-component implant material. After 9 weeks of implantation, both computed tomography (CT) and morphological analyses showed complete resorption of the implanted material, concurrent with complete bone regeneration. The given data can be considered as a significant contribution to the successful introduction of biosilica-based implants into the field of bone substitution surgery.Autogenetische Knochentransplantate von verschiedenen Spenderregionen stellen bis heute den höchsten Stand auf dem Gebiet der Knochenrekonstruktion dar, indem sie sowohl osteoinduktive als auch osteokonduktive Charakteristika aufweisen. Deren geringe Verfügbarkeit, die Unausgewogenheit zwischen Angebot und Nachfrage, und die normalerweise von autogenetischen Knochentransplantaten ausgehende Ansteckungsund Erkrankungsgefahr haben jedoch zur Entwicklung von biomimetischen Materialien geführt, die aufgrund ihrer strukturellen und chemischen Eigenschaften stark denen natürlicher Knochen ähneln. Soweit konnten allerdings nur einige wenige synthetische Materialien diese Kriterien erfüllen, da deren Eigenschaften eine optimale Knochenrekonstruktion erlauben. Biosilica wird unter physiologisch-relevanten Bedingungen (Temperatur und pH-Wert) mit Hilfe von Silicatein (Silica Protein)enzymatisch gebildet. Bei Letzterem handelt es sich um ein aus Silikat-Schwämmen isoliertes, kloniertes und rekombinant aufbereitetes Enzym, das katalytische Aktivität aufweist. Biosilica ist biologisch verträglich, hat einzigartige mechanische Eigenschaften und besitzt eine bedeutende osteoinduktive Aktivität.rnUm die Anwendung von Biosilica auf dem Gebiet der regenerativen Medizin zu erforschen, wurde Silicatein zusammen mit seinem Substrat Sodium-Metasilikat inrnMikrosphären eingekapselt, die auf Poly(D,L-Lactid)/Polyvinylpyrrolidon (PVP) basieren. Dies erfolgte unter Einsatz der w/o/w Methode durch Gießen von Lösungsmittel und führte zur Bezeichnung Poly(D,L-Lactid)-Silikatein-Silica-enthaltende Mikrosphären[PLASSM]. Durch einen weiteren essentiellen Schritt vor der Emulgierung mittels PVP wurde sowohl die Effizienz der Silicatein Einkapselung (40%) als auch die metabolische Stabilität, die Zytotoxizität als auch die Kinetik der Silicatein-Freisetzung aus den PLASSM unter biomimetischen Bedingungen untersucht. Als Stützpunkt für die PLASSM wurde eine auf Polyvinylpyrrolidon/Stärke/Na2HPO-basierende Matrix entwickelt, deren chemische sowie auch physikalische Eigenschaften daraufhin bestimmt wurden. Diese Plastik-ähnelnde Füllmatrix enthält Kieselsäure und wird somit kurz PMSA genannt. PMSA scheint sogar durch seine fehlende Toxizität und seine biologische Trägheit als osteokonduktives Material zu agieren. Wenn beide Komponenten, PLASSM und PMSA, gemeinsam hinzugefügt werden, bilden sie ein bifunktionelles, aus Zwei-Komponenten bestehendes Material für Implantate, das (i) nichttoxisch (biokompatibel), (ii) formbar und (iii) zu einem kontrollierbarem sowie klinisch-angemessenem Grad selbst-härtend ist, um es auf stabile Weise bei Knochendefekten jeder Art einzusetzen. Darüber hinaus ist es (iv) biologisch abbaubar, (v) wandelt sich nach Exposition zu Bedingungen, wie sie im Körper vorkommen, in ein poröses Material um und (vi) weist sowohl osteoinduktive (Silicatein)als auch osteokonduktive (PMSA) Eigenschaften auf.rnVorläufige in vivo Experimente wurden an Hasen-Femora ausgeführt, indemrnkünstliche Knochenschäden hervorgerufen wurden, die anschließend mit dem neuenrnImplantat-Material behandelt wurden. Neun Wochen nach der Implantation zeigtenrnsowohl Computertomographie (CT) als auch morphologische Analysen eine vollständige Resorption des eingepflanzten Materials sowie gleichzeitig eine komplette Regeneration des Knochens. Die in dieser Arbeit erhaltenen Daten können als ein bedeutender Beitrag zur erfolgreichen Einführung von Biosilica-enthaltenden Implantaten in das Gebiet der Knochenersatz-Chirurgie betrachtet werden

The Effect of a Low Degree of Fluorine Substitution on Cotton Fiber Properties

Abstract Cellulose modification often employs chemical processes to tailor its properties and functionalities to fit the demands of a wide range of applications, maximizing its potential as a versatile and sustainable material. From both synthetic and environmental standpoints, one of the ultimate goals is to achieve significant modifications to enhance the end properties of the cellulose while minimizing the number of modified building blocks. The current study demonstrates that a synthetic glucose derivative, 6‐deoxy‐6‐fluoro‐glucose (6F‐Glc), fed into the fertilized cotton ovules, resulted in the accumulation of fluorine inside the cotton fibers with no apparent alteration to their morphology or development. These fibers exhibited a degree of substitution of 0.006, which is 170 times lower than that reported for chemical methods for cellulose modification. However, the physical characterization of the modified fibers showed a surprisingly large impact of this low‐level modification on the cellulose structure (e.g., hydrogen bonding network rearrangement) and a modest increase in the mechanical properties of the fibers. The obtained results exemplify the use of biological systems to introduce low quantities of new functionalities while maximizing the impact on fiber properties

Molybdenum trioxide nanoparticles with intrinsic sulfite oxidase activity

Sulfite oxidase is a mitochondria-located molybdenum-containing enzyme catalyzing the oxidation of sulfite to sulfate in the amino acid and lipid metabolism. Therefore, it plays a major role in detoxification processes, where defects in the enzyme cause a severe infant disease leading to early death with no efficient or cost-effective therapy in sight. Here we report that molybdenum trioxide (MoO3) nanoparticles display an intrinsic biomimetic sulfite oxidase activity under physiological conditions, and, functionalized with a customized bifunctional ligand containing dopamine as anchor group and triphenylphosphonium ion as targeting agent, they selectively target the mitochondria while being highly dispersible in aqueous solutions. Chemically induced sulfite oxidase knockdown cells treated with MoO3 nanoparticles recovered their sulfite oxidase activity in vitro, which makes MoO3 nanoparticles a potential therapeutic for sulfite oxidase deficiency and opens new avenues for cost-effective therapies for gene-induced deficiencies

Impact of the mesoporous silica SBA-15 functionalization on the mode of action of Ph3Sn(CH2)6OH

Herein appropriateness of nonfunctionalized mesoporous silica nanoparticles SBA-15 and functionalized with (3-chloropropyl)triethoxysilane (→ SBA-15~Cl) and (3-aminopropyl)triethoxysilane (→ SBA-15~NH2) on delivery of physically adsorbed Ph3Sn(CH2)6OH (Sn6) is evaluated. Fluorescent nanomaterial, bearing isatoic moiety, loaded with Sn6 (→ SBA-15~NF|Sn6) was used for cellular uptake study. The fluorescent nanomaterial is efficiently acquired and distributed into the cytoplasm of the cells even after 2 h of cultivation. According to the attained data, all SBA-15 materials loaded with Sn6 diminished cellular viability in dose dependent manner while carriers alone (SBA-15, SBA-15~Cl, SBA-15~NH2) did not show cytotoxicity against B16 cells. According to the MC50 values structural modification of SBA-15 did not improve the efficacy of tested drug. While progressive apoptosis was detected upon the treatment with SBA-15|Sn6, exposure of cells to SBA-15~NH2|Sn6 revealed extinguished apoptosis in time, accompanied with lower caspase activity. This effect is probably due to triggered autophagic process under the treatment with the SBA-15~NH2|Sn6, thus opposed to apoptosis. Presented results suggested that functionalization of SBA-15 was not beneficial for the efficacy of loaded drug, thus, all of them are almost equally efficient considering loaded Sn6 content. Importantly, functionalization of SBA-15 does have an influence on the mode of action and differentiation inducing properties

DataSheet_1_2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation.pdf

Fluorescent glucose derivatives are valuable tools as glucose analogs in plant research to explore metabolic pathways, study enzyme activity, and investigate cellular processes related to glucose metabolism and sugar transport. They allow visualization and tracking of glucose uptake, its utilization, and distribution within plant cells and tissues. This study investigates the phenotypic and metabolic impact of the exogenously fed glucose derivative, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG) on the fibers of Gossypium hirsutum (Upland cotton) ovule in vitro cultures. The presence of 2-NBDG in the culture medium did not lead to macroscopic morphological alterations in ovule and fiber development or to the acquisition of fluorescence or yellow coloration. Confocal laser scanning microscope imaging and chromatographic analysis of cotton ovules’ outer rim cross-sections showed that the 2-NBDG is transported from the extracellular space and accumulated inside some outer integument cells, epidermal cells, and fertilized epidermal cells (fibers), but is not incorporated into the cell walls. Untargeted metabolic profiling of the fibers revealed significant changes in the relative levels of metabolites involved in glycolysis and upregulation of alternative energy-related pathways. To provide biochemical and structural evidence for the observed downregulation of glycolysis pathways in the fibers containing 2-NBDG, kinetics analysis and docking simulations were performed on hexokinase from G. hirsutum (GhHxk). Notably, the catalytic activity of heterologously expressed recombinant active GhHxk exhibited a five-fold decrease in reaction rates compared to D-glucose. Furthermore, GhHxk exhibited a linear kinetic behavior in the presence of 2-NBDG instead of the Michaelis-Menten kinetics found for D-glucose. Docking simulations suggested that 2-NBDG interacts with a distinct binding site of GhHxk9, possibly inducing a conformational change. These results highlight the importance of considering fluorescent glucose derivatives as ready-to-use analogs for tracking glucose-related biological processes. However, a direct comparison between their mode of action and its extrapolation into biochemical considerations should go beyond microscopic inspection and include complementary analytical techniques.</p

Merging models of biomineralisation with concepts of nonclassical crystallisation: is a liquid amorphous precursor involved in the formation of the prismatic layer of the Mediterranean Fan Mussel Pinna nobilis?

16 pagesInternational audienceThe calcitic prisms of Pinna nobilis (Pinnidae, Linnaeus 1758) are shown to be perfect examples of a mesocrystalline material. Based on their ultrastructure and on the occurrence of an amorphous transient precursor during the early stages of prism formation, we provide evidence for the pathway of mesocrystallisation proposed by Seto et al. (2012), which proceeds not by self-organized oriented attachment of crystalline nano-bricks but by aggregation of initially amorphous nanogranules which later transform by epitaxial nucleation to a threedimensional array of well aligned nanocrystals. We further fathom the role of a liquid amorphous calcium carbonate in biomineralisation processes and provide strong evidence for the occurrence of PILP-like intermediates during prism formation. We develop a new scenario of prism formation based on the presented findings presented findings and discuss the implications of a speculative liquid amorphous calcium carbonate (LACC) intermediate in vivo