AdNAC20 Regulates Lignin and Coumarin Biosynthesis in the Roots of Angelica dahurica var. Formosana.

Angelica dahurica var. formosana (ADF), which belongs to the Umbelliferae family, is one of the original plants of herbal raw material Angelicae Dahuricae Radix. ADF roots represent an enormous biomass resource convertible for disease treatment and bioproducts. But, early bolting of ADF resulted in lignification and a decrease in the coumarin content in the root, and roots lignification restricts its coumarin for commercial utility. Although there have been attempts to regulate the synthesis ratio of lignin and coumarin through biotechnology to increase the coumarin content in ADF and further enhance its commercial value, optimizing the biosynthesis of lignin and coumarin remains challenging. Based on gene expression analysis and phylogenetic tree profiling, AdNAC20 as the target for genetic engineering of lignin and coumarin biosynthesis in ADF was selected in this study. Early-bolting ADF had significantly greater degrees of root lignification and lower coumarin contents than that of the normal plants. In this study, overexpression of AdNAC20 gene plants were created using transgenic technology, while independent homozygous transgenic lines with precise site mutation of AdNAC20 were created using CRISPR/Cas9 technology. The overexpressing transgenic ADF plants showed a 9.28% decrease in total coumarin content and a significant 12.28% increase in lignin content, while knockout mutant plants showed a 16.3% increase in total coumarin content and a 33.48% decrease in lignin content. Furthermore, 29,671 differentially expressed genes (DEGs) were obtained by comparative transcriptomics of OE-NAC20, KO-NAC20, and WT of ADF. A schematic diagram of the gene network interacting with AdNAC20 during the early-bolting process of ADF was constructed by DEG analysis. AdNAC20 was predicted to directly regulate the transcription of several genes with SNBE-like motifs in their promoter, such as MYB46, C3H, and CCoAOMT. In this study, AdNAC20 was shown to play a dual pathway function that positively enhanced lignin formation but negatively controlled coumarin formation. And the heterologous expression of the AdNAC20 gene at Arabidopsis thaliana proved that the AdNAC20 gene also plays an important role in the process of bolting and flowering.

Resolving the influence of lignin on soil organic matter decomposition with mechanistic models and continental-scale data.

Confidence in model estimates of soil CO2 flux depends on assumptions regarding fundamental mechanisms that control the decomposition of litter and soil organic carbon (SOC). Multiple hypotheses have been proposed to explain the role of lignin, an abundant and complex biopolymer that may limit decomposition. We tested competing mechanisms using data-model fusion with modified versions of the CN-SIM model and a 571-day laboratory incubation dataset where decomposition of litter, lignin, and SOC was measured across 80 soil samples from the National Ecological Observatory Network. We found that lignin decomposition consistently decreased over time in 65 samples, whereas in the other 15 samples, lignin decomposition subsequently increased. These "lagged-peak" samples can be predicted by low soil pH, high extractable Mn, and fungal community composition as measured by ITS PC2 (the second principal component of an ordination of fungal ITS amplicon sequences). The highest-performing model incorporated soil biogeochemical factors and daily dynamics of substrate availability (labile bulk litter:lignin) that jointly represented two hypotheses (C substrate limitation and co-metabolism) previously thought to influence lignin decomposition. In contrast, models representing either hypothesis alone were biased and underestimated cumulative decomposition. Our findings reconcile competing hypotheses of lignin decomposition and suggest the need to precisely represent the role of lignin and consider soil metal and fungal characteristics to accurately estimate decomposition in Earth-system models.

Trade-offs in soil carbon protection mechanisms under aerobic and anaerobic conditions.

Oxygen (O2 ) limitation is generally understood to suppress oil carbon (C) decomposition and is a key mechanism impacting terrestrial C stocks under global change. Yet, O2 limitation may differentially impact kinetic or thermodynamic versus physicochemical C protection mechanisms, challenging our understanding of how soil C may respond to climate-mediated changes in O2 dynamics. Although O2 limitation may suppress decomposition of new litter C inputs, release of physicochemically protected C due to iron (Fe) reduction could potentially sustain soil C losses. To test this trade-off, we incubated two disparate upland soils that experience periodic O2 limitation-a tropical rainforest Oxisol and a temperate cropland Mollisol-with added litter under either aerobic (control) or anaerobic conditions for 1 year. Anoxia suppressed total C loss by 27% in the Oxisol and by 41% in the Mollisol relative to the control, mainly due to the decrease in litter-C decomposition. However, anoxia sustained or even increased decomposition of native soil-C (11.0% vs. 12.4% in the control for the Oxisol and 12.5% vs. 5.3% in the control for the Mollisol, in terms of initial soil C mass), and it stimulated losses of metal- or mineral-associated C. Solid-state 13 C nuclear magnetic resonance spectroscopy demonstrated that anaerobic conditions decreased protein-derived C but increased lignin- and carbohydrate-C relative to the control. Our results indicate a trade-off between physicochemical and kinetic/thermodynamic C protection mechanisms under anaerobic conditions, whereby decreased decomposition of litter C was compensated by more extensive loss of mineral-associated soil C in both soils. This challenges the common assumption that anoxia inherently protects soil C and illustrates the vulnerability of mineral-associated C under anaerobic events characteristic of a warmer and wetter future climate.

Shape Effect of Nanoparticles on Tumor Penetration in Monolayers Versus Spheroids.

The physical properties of nanoparticles (NPs), such as size, surface chemistry, elasticity, and shape, have exerted a profound influence on tumor penetration. However, the effect of shape on cellular uptake and tumor penetration is still unclear because of the different chemical compositions and shapes of tested particles and the use of inapposite cellular models. To discover the effect of NP shapes on cellular uptake and tumor penetration and bridge the gap between models in vivo and in vitro, elongated polystyrene (PS) NPs with a fixed volume, an identical chemical composition, and the same zeta potential, but with different aspect ratios (ARs), were generated. The physical properties, cellular uptake, tumor penetration, and corresponding mechanisms of these NPs were thoroughly investigated. We discovered that the elongated PS particles with higher ARs had lower uptake rates in the 2-dimensional cell monolayer culture model in vitro, but they showed optimal ARs in the evaluated three-dimensional spheroid model. Although the elongated PS particles had a similar tumor penetration mechanism (mainly through extracellular pathways), the percentage of penetration using these mechanisms was strongly dependent on the ARs. As an alternative model for studies in vivo, spheroids were used instead of the cell monolayer for the development of drug delivery systems. In addition, the physicochemical properties of NPs must be delicately balanced and adjusted to achieve the best therapeutic outcomes.

Enrichment of Lignin-Derived Carbon in Mineral-Associated Soil Organic Matter.

A modern paradigm of soil organic matter proposes that persistent carbon (C) derives primarily from microbial residues interacting with minerals, challenging older ideas that lignin moieties contribute to soil C because of inherent recalcitrance. We proposed that aspects of these old and new paradigms can be partially reconciled by considering interactions between lignin decomposition products and redox-sensitive iron (Fe) minerals. An Fe-rich tropical soil (with C4 litter and either 13C-labeled or unlabeled lignin) was pretreated with different durations of anaerobiosis (0-12 days) and incubated aerobically for 317 days. Only 5.7 ± 0.2% of lignin 13C was mineralized to CO2 versus 51.2 ± 0.4% of litter C. More added lignin-derived C (48.2 ± 0.9%) than bulk litter-derived C (30.6 ± 0.7%) was retained in mineral-associated organic matter (MAOM; density >1.8 g cm-3), and 12.2 ± 0.3% of lignin-derived C vs 6.4 ± 0.1% of litter C accrued in clay-sized (<2 µm) MAOM. Longer anaerobic pretreatments increased added lignin-derived C associated with Fe, according to extractions and nanoscale secondary ion mass spectrometry (NanoSIMS). Microbial residues are important, but lignin-derived C may also contribute disproportionately to MAOM relative to bulk litter-derived C, especially following redox-sensitive biogeochemical interactions.

Fabrication of a Micellar Supramolecular Hydrogel for Ocular Drug Delivery.

In this paper, we describe a simple method for constructing a micellar supramolecular hydrogel, composed of a low-molecular-weight methoxy poly(ethylene glycol) (Mn = 2000 Da) block polymer and α-cyclodextrin (α-CD), for topical ocular drug delivery. Adding aqueous block polymer micelles into an α-CD aqueous solution resulted in the formation of a micellar supramolecular hydrogel through host-guest inclusion. The effects of the drug payload, block polymer, and α-CD concentrations as well as the block polymer structure on gelation time were investigated. The resultant micellar supramolecular hydrogels were thoroughly characterized by X-ray diffraction, rheological studies, and scanning electron microscopy. The hydrogels exhibited thixotropic properties, which are beneficial to ocular drug delivery. In vitro release studies indicated that the α-CD concentration strongly influenced the release rate of diclofenac (DIC) from supramolecular hydrogel. The hydrogels showed relatively low cytotoxicity toward L-929 and HCEC cells and did not significantly affect the migration of the latter after 24 h incubation. The hydrogel was nonirritant toward the rabbit eye, as indicated by the Draize test, fluorescein staining, and histological observation. Nile Red-labeled micellar supramolecular hydrogel showed that it could significantly extend the retention time on the corneal surface in rabbits, compared with a plain micellar formulation. In vivo pharmacokinetics indicated that the hydrogel could greatly improve ocular drug bioavailability, compared with that of micellar formulation. Our results suggest that the micellar supramolecular hydrogel is a promising system for ocular drug delivery.

Enhanced Ocular Delivery of Beva via Ultra-Small Polymeric Micelles for Noninvasive Anti-VEGF Therapy.

Pathological ocular neovascularization resulting from retinal ischemia constitutes a major cause of vision loss. Current anti-VEGF therapies rely on burdensome intravitreal injections of Bevacizumab (Beva). Herein ultrasmall polymeric micelles encapsulating Beva (P@Beva) are developed for noninvasive topical delivery to posterior eye tissues. Beva is efficiently loaded into 11 nm micelles fabricated via self-assembly of hyperbranched amphiphilic copolymers. The neutral, brush-like micelles demonstrate excellent drug encapsulation and colloidal stability. In vitro, P@Beva enhances intracellular delivery of Beva in ocular cells versus free drug. Ex vivo corneal and conjunctival-sclera-choroidal tissues transport after eye drops are improved 23-fold and 7.9-fold, respectively. Anti-angiogenic bioactivity is retained with P@Beva eliciting greater inhibition of endothelial tube formation and choroid sprouting over Beva alone. Remarkably, in an oxygen-induced retinopathy (OIR) model, topical P@Beva matching efficacy of intravitreal Beva injection, is the clinical standard. Comprehensive biocompatibility verifies safety. Overall, this pioneering protein delivery platform holds promise to shift paradigms from invasive intravitreal injections toward simplified, noninvasive administration of biotherapeutics targeting posterior eye diseases.

Contrasting geochemical and fungal controls on decomposition of lignin and soil carbon at continental scale.

Lignin is an abundant and complex plant polymer that may limit litter decomposition, yet lignin is sometimes a minor constituent of soil organic carbon (SOC). Accounting for diversity in soil characteristics might reconcile this apparent contradiction. Tracking decomposition of a lignin/litter mixture and SOC across different North American mineral soils using lab and field incubations, here we show that cumulative lignin decomposition varies 18-fold among soils and is strongly correlated with bulk litter decomposition, but not SOC decomposition. Climate legacy predicts decomposition in the lab, and impacts of nitrogen availability are minor compared with geochemical and microbial properties. Lignin decomposition increases with some metals and fungal taxa, whereas SOC decomposition decreases with metals and is weakly related with fungi. Decoupling of lignin and SOC decomposition and their contrasting biogeochemical drivers indicate that lignin is not necessarily a bottleneck for SOC decomposition and can explain variable contributions of lignin to SOC among ecosystems.

Lignin lags, leads, or limits the decomposition of litter and soil organic carbon.

Lignin's role in litter and soil organic carbon (SOC) decomposition remains contentious. Lignin decomposition was traditionally thought to increase during midstage litter decomposition, when cellulose occlusion by lignin began to limit mass loss. Alternatively, lignin decomposition could be greatest in fresh litter as a consequence of co-metabolism, and lignin might decompose faster than bulk SOC. To test these competing hypotheses, we incubated 10 forest soils with C4 grass litter (amended with 13 C-labeled or unlabeled lignin) over 2 yr and measured soil respiration and its isotope composition. Early lignin decomposition was greatest in 5 of 10 soils, consistent with the co-metabolism hypothesis. However, lignin decomposition peaked 6-24 months later in the other five soils, consistent with the substrate-limitation hypothesis; these soils were highly acidic. Rates of lignin, litter, and SOC decomposition tended to converge over time. Cumulative lignin decomposition was never greater than SOC decomposition; lignin decomposition was significantly lower than SOC decomposition in six soils. Net nitrogen mineralization predicted lignin decomposition ratios relative to litter and SOC. Although the onset of lignin decomposition can indeed be rapid, lignin still presents a likely bottleneck in litter and SOC decomposition, meriting a reconsideration of lignin's role in modern decomposition paradigms.

An insight into the influence of hydrogen bond acceptors on cellulose/1-allyl-3-methyl imidazolium chloride solution.

Although ionic liquids have been well established as effective solvents for the dissolution and processing of natural cellulose fibers, the detailed dissolution mechanism at the molecular level still remains unclear. Herein, the turbidimetric measurement showed that the solubility of cellulose in 1-allyl-3-methyl imidazolium chloride (AmimCl) decreased with increasing temperature. The temperature dependence of the OH stretching vibration band of cellulose in AmimCl was investigated by infrared spectroscopy. The interaction between AmimCl and different hydrogen bond acceptors were investigated by turbidimetry and NMR spectroscopy, which indicated that the excellent compatibility of the hydrogen bond acceptors with AmimCl provides more interaction sites for the hydroxyl groups of the cellulose. In addition, ionic liquids with a similar anionic structure of hydrogen bond acceptors have been synthesized. This study provides a green and safe guide for the preparation of ionic liquids with excellent solubility of cellulose.

Controlled synthesis of NaYF4 nanoparticles and upconversion properties of NaYF4:Yb, Er (Tm)/FC transparent nanocomposite thin films.

Monodisperse oleic acid stabilized pure NaYF(4) nanoparticles with controlled size and shape have been successfully synthesized by changing the initial reaction temperature. Transparent nanocomposite thin films consisting of NaYF(4):Yb, Er (Tm) upconverting nanoparticles (UCNPs) and fluorocarbon resin (FC) are deposited on the slide glass by dip-coating method. The results show that these nanocomposite thin films exhibit intense green and blue upconversion photoluminescence under 980 nm laser excitation and higher transparency than blank substrate. The NaYF(4):Yb,Er (Tm) nanoparticles and NaYF(4):Yb,Er (Tm)/FC nanocomposite thin films have been characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), scanning electron microscopy (SEM), SEM/back-scattered electron (BSE), atomic force microscopy (AFM), UV-Vis spectrophotometer (UVPC), and photoluminescence (PL) spectra. These nanocomposite thin films can be potentially used in solar cells field.