Engineer RNA-Protein Nanowires as Light-Responsive Biomaterials.

RNA molecules have emerged as increasingly attractive biomaterials with important applications such as RNA interference (RNAi) for cancer treatment and mRNA vaccines against infectious diseases. However, it remains challenging to engineer RNA biomaterials with sophisticated functions such as non-covalent light-switching ability. Herein, light-responsive RNA-protein nanowires are engineered to have such functions. It first demonstrates that the high affinity of RNA aptamer enables the formation of long RNA-protein nanowires through designing a dimeric RNA aptamer and an engineered green fluorescence protein (GFP) that contains two TAT-derived peptides at N- and C- termini. GFP is then replaced with an optogenetic protein pair system, LOV2 (light-oxygen-voltage) protein and its binding partner ZDK (Z subunit of protein A), to confer blue light-controlled photo-switching ability. The light-responsive nanowires are long (>500 nm) in the dark, but small (20-30 nm) when exposed to light. Importantly, the co-assembly of this RNA-protein hybrid biomaterial does not rely on the photochemistry commonly used for light-responsive biomaterials, such as bond formation, cleavage, and isomerization, and is thus reversible. These RNA-protein structures can serve as a new class of light-controlled biocompatible frameworks for incorporating versatile elements such as RNA, DNA, and enzymes.

Integrated Secondary Metabolomic and Antioxidant Ability Analysis Reveals the Accumulation Patterns of Metabolites in Momordica charantia L. of Different Cultivars.

Bitter gourd (Momordica charantia L.) contains rich bioactive ingredients and secondary metabolites; hence, it has been used as medicine and food product. This study systematically quantified the nutrient contents, the total content of phenolic acids (TPC), flavonoids (TFC), and triterpenoids (TTC) in seven different cultivars of bitter gourd. This study also estimated the organic acid content and antioxidative capacity of different cultivars of bitter gourd. Although the TPC, TFC, TTC, organic acid content, and antioxidative activity differed significantly among different cultivars of bitter gourd, significant correlations were also observed in the obtained data. In the metabolomics analysis, 370 secondary metabolites were identified in seven cultivars of bitter gourd; flavonoids and phenolic acids were significantly more. Differentially accumulated metabolites identified in this study were mainly associated with secondary metabolic pathways, including pathways of flavonoid, flavonol, isoflavonoid, flavone, folate, and phenylpropanoid biosyntheses. A number of metabolites (n = 27) were significantly correlated (positive or negative) with antioxidative capacity (r ≥ 0.7 and p < 0.05). The outcomes suggest that bitter gourd contains a plethora of bioactive compounds; hence, bitter gourd may potentially be applied in developing novel molecules of medicinal importance.

Artificial Light for Improving Tomato Recovery Following Grafting: Transcriptome and Physiological Analyses.

Grafting is widely used to enhance the phenotypic traits of tomatoes, alleviate biotic and abiotic stresses, and control soil-borne diseases of the scion in greenhouse production. There are many factors that affect the healing and acclimatization stages of seedlings after grafting. However, the role of light has rarely been studied. In this study, we compared the effects of artificial light and traditional shading (under shaded plastic-covered tunnels) on the recovery of grafted tomato seedlings. The results show that the grafted tomato seedlings recovered using artificial light had a higher healthy index, leaf chlorophyll content, shoot dry weight, and net photosynthetic rate (Pn) and water use efficiency (WUE) compared with grafted seedling recovered using the traditional shading method. Transcriptome analysis showed that the differentially expressed genes (DEGs) of grafted seedlings restored using artificial light were mainly enriched in the pathways corresponding to plant hormone signal transduction. In addition, we measured the endogenous hormone content of grafted tomato seedlings. The results show that the contents of salicylic acid (SA) and kinetin (Kin) were significantly increased, and the contents of indoleacetic acid (IAA) and jasmonic acid (JA) were decreased in artificial-light-restored grafted tomato seedlings compared with those under shading treatments. Therefore, we suggest that artificial light affects the morphogenesis and photosynthetic efficiency of grafted tomato seedlings, and it can improve the performance of tomato seedlings during grafting recovery by regulating endogenous hormone levels.

Transcriptome Analyses Reveal the Role of Light in Releasing the Morphological Dormancy of Celery Seed by Integrating Plant Hormones, Sugar Metabolism and Endosperm Weakening.

Celery seed is known to be difficult to germinate due to its morphological dormancy. Light is the key signal to release morphological dormancy and promote seed germination. However, this mechanism has rarely been studied. We performed physiological, transcriptome analyses on celery seed exposed to light and dark to decipher the mechanism by which light promotes germination of celery seed. The results showed that light significantly enhanced the expression of gibberellin synthesis genes and abscisic acid degradation genes and inhibited the expression of abscisic acid synthesis genes and gibberellin degradation genes. Moreover, gibberellin synthesis inhibitor could completely inhibit the germination capacity of celery seed, indicating that gibberellin is indispensable in the process of celery seed germination. Compared with dark, light also increased the activity of α-amylase and ß-amylase and the expression of related coding genes and promoted the degradation of starch and the increase of soluble sugar content, suggesting that light enhanced the sugar metabolism of celery seed. In addition, transcriptome analysis revealed that many genes related to endosperm weakening (cell wall remodeling enzymes, extension proteins) were up-regulated under light. It was also found that light promoted the accumulation of hydrogen peroxide in the radicle, which promoted the endosperm weakening process of celery seed. Our results thus indicated that light signal may promote the release of morphological dormancy through the simultaneous action of multiple factors.

Light from below matters: Quantifying the consequences of responses to far-red light reflected upwards for plant performance in heterogeneous canopies.

In vegetation stands, plants receive red to far-red ratio (R:FR) signals of varying strength from all directions. However, plant responses to variations in R:FR reflected from below have been largely ignored despite their potential consequences for plant performance. Using a heterogeneous rose canopy, which consists of bent shoots down in the canopy and vertically growing upright shoots, we quantified upward far-red reflection by bent shoots and its consequences for upright shoot architecture. With a three-dimensional plant model, we assessed consequences of responses to R:FR from below for plant photosynthesis. Bent shoots reflected substantially more far-red than red light, causing reduced R:FR in light reflected upwards. Leaf inclination angles increased in upright shoots which received low R:FR reflected from below. The increased leaf angle led to an increase in simulated plant photosynthesis only when this low R:FR was reflected off their own bent shoots and not when it reflected off neighbour bent shoots. We conclude that plant response to R:FR from below is an under-explored phenomenon which may have contrasting consequences for plant performance depending on the type of vegetation or crop system. The responses are beneficial for performance only when R:FR is reflected by lower foliage of the same plants.

Insight into the Reactivity of Carbon Structures for Nitrogen Reduction Reaction.

Graphene-based structures have been widely reported as promising metal-free catalysts for nitrogen reduction reaction. To explain the reactivity origin, various structures have been proposed and debated, including defects, functional groups, and doped heteroatoms. This computational work demonstrates that these structures may evolve from one to another under electrochemical conditions, generating weakly coordinated carbons, which have been identified as the active sites for N2 adsorption and activation.

Ag23Au2 and Ag22Au3: A Model of Cocrystallization in Bimetal Nanoclusters.

The field of cocrystalline nanoclusters stabilized by thiolates is in a period of rapid development. However, the types of cocrystallization have been limited to a few reported until now, so it is of great importance to investigate and understand the novel cocrystallographic structures. Herein, we design and synthesize a new type of cocrystallization, [Ag23Au2(2-EBT)18Ag22Au3(2-EBT)18]2-[2(PPh4)]2+, characterized by thermogravimetric analysis, X-ray photoelectron spectroscopy, and single-crystal X-ray crystallography. Interestingly, both of the cocrystallized nanoclusters show the same outer-shell geometric structure but diffenent cores (Ag11Au2 vs Ag10Au3). The cocrystal lattice exhibits a multilayer structure in which both of the cocrystallized nanoclusters and the counterion assemble in a layer-by-layer model. Meanwhile, the counterion is found to be critical for formation and stabilization of the target cocrystal. In addition, the target cocrystal shows high thermal stability, and this result possibly originates from the electrostatic and weak interactions in the cocrystals.

Alternating Array Stacking of Ag26 Au and Ag24 Au Nanoclusters.

An assembly strategy for metal nanoclusters using electrostatic interactions with weak interactions, such as C-H⋅⋅⋅π and π⋅⋅⋅π interactions in which cationic [Ag26 Au(2-EBT)18 (PPh3 )6 ]+ and anionic [Ag24 Au(2-EBT)18 ]- nanoclusters gather and assemble in an unusual alternating array stacking structure is presented. [Ag26 Au(2-EBT)18 (PPh3 )6 ]+ [Ag24 Au(2-EBT)18 ]- is a new compound type, a double nanocluster ion compound (DNIC). A single nanocluster ion compound (SNIC) [PPh4 ]+ [Ag24 Au(2-EBT)18 ]- was also synthesized, having a k-vector-differential crystallographic arrangement. [PPh4 ]+ [Ag24 Au(2,4-DMBT)18 ]- adopts a different assembly mode from both [Ag26 Au(2-EBT)18 (PPh3 )6 ]+ [Ag24 Au(2-EBT)18 ]- and [PPh4 ]+ [Ag24 Au(2-EBT)18 ]- . Thus, the striking packing differences of [Ag26 Au(2-EBT)18 (PPh3 )6 ]+ [Ag24 Au(2-EBT)18 ]- , [PPh4 ]+ [Ag24 Au(2-EBT)18 ]- and the existing [PPh4 ]+ [Ag24 Au(2,4-DMBT)18 ]- from each other indicate the notable influence of ligands and counterions on the self-assembly of nanoclusters.

Fcc versus Non-fcc Structural Isomerism of Gold Nanoparticles with Kernel Atom Packing Dependent Photoluminescence.

Structural isomerism allows the correlation between structures and properties to be investigated. Unfortunately, the structural isomers of metal nanoparticles are rare and genuine structural isomerism with distinctly different kernel atom packing (e.g., face-centered cubic (fcc) vs. non-fcc) has not been reported until now. Herein we introduce a novel ion-induction method to synthesize a unique gold nanocluster with a twist mirror symmetry structure. The as-synthesized nanocluster has the same composition but different kernel atom packing to an existing gold nanocluster Au42 (TBBT)26 (TBBT=4-tert-butylbenzenethiolate). The fcc-structured Au42 (TBBT)26 nanocluster shows more enhanced photoluminescence than the non-fcc-structured Au42 (TBBT)26 nanocluster, indicating that the fcc-structure is more beneficial for emission than the non-fcc structure. This idea was supported by comparison of the emission intensity of another three pairs of gold nanoclusters with similar compositions and sizes but with different kernel atom packings (fcc vs. non-fcc).

Kernel Tuning and Nonuniform Influence on Optical and Electrochemical Gaps of Bimetal Nanoclusters.

Fine tuning nanoparticles with atomic precision is exciting and challenging and is critical for tuning the properties, understanding the structure-property correlation and determining the practical applications of nanoparticles. Some ultrasmall thiolated metal nanoparticles (metal nanoclusters) have been shown to be precisely doped, and even the protecting staple metal atom could be precisely reduced. However, the precise addition or reduction of the kernel atom while the other metal atoms in the nanocluster remain the same has not been successful until now, to the best of our knowledge. Here, by carefully selecting the protecting ligand with adequate steric hindrance, we synthesized a novel nanocluster in which the kernel can be regarded as that formed by the addition of two silver atoms to both ends of the Pt@Ag12 icosohedral kernel of the Ag24Pt(SR)18 (SR: thiolate) nanocluster, as revealed by single crystal X-ray crystallography. Interestingly, compared with the previously reported Ag24Pt(SR)18 nanocluster, the as-obtained novel bimetal nanocluster exhibits a similar absorption but a different electrochemical gap. One possible explanation for this result is that the kernel tuning does not essentially change the electronic structure, but obviously influences the charge on the Pt@Ag12 kernel, as demonstrated by natural population analysis, thus possibly resulting in the large electrochemical gap difference between the two nanoclusters. This work not only provides a novel strategy to tune metal nanoclusters but also reveals that the kernel change does not necessarily alter the optical and electrochemical gaps in a uniform manner, which has important implications for the structure-property correlation of nanoparticles.

The effect of exogenous calcium on cucumber fruit quality, photosynthesis, chlorophyll fluorescence, and fast chlorophyll fluorescence during the fruiting period under hypoxic stress.

BACKGROUND: Plants often suffer from hypoxic stress during waterlogging and hydroponic culturing. This study investigated the response of cucumber (Cucumis sativus L.) plant growth parameters, leaf photosynthesis, chlorophyll fluorescence, fast chlorophyll a fluorescence transient (OJIP), and fruit quality parameters to hypoxic stress alleviated by exogenous calcium. During the fruiting period, cucumber plants were exposed to hypoxia and hypoxia + Ca2+ treatment (4 mM Ca2+) for 9 d. RESULT: Exogenous calcium application enhanced the biomass and fruit quality of hypoxic stressed cucumber and also increased the net photosynthesis rate, stomatal conductance, intercellular CO2 concentration, maximum quantum efficiency of photosystem II photochemistry, actual photochemical efficiency of PSII, photochemical quenching coefficient, and non-photochemical quenching coefficient. Additionally, measurement of chlorophyll a fluorescence transients showed the positive K- and L-bands were more pronounced in leaves treated with hypoxia compared with those with hypoxia + Ca2+, indicating that hypoxic treatment induced uncoupling of the oxygen-evolving complex and inhibited electron transport beyond plastoquinone pool (Qa, Qb) including possible constraints on the reduction of end electron acceptors of photosystem I. Exogenous calcium can reduce these stress-induced damages in cucumber. CONCLUSION: This research focused the effect of exogenous calcium on cucumber photosynthesis during the fruiting period under hypoxic stress. Hypoxic stress might impair the photosynthetic electron-transport chain from the donor side of PSII up to the reduction of end acceptors of PSI, and exogenous calcium enhanced electron transport capacity and reduced hypoxic damage of cucumber leaves.

Impact of H-termination on the nitrogen reduction reaction of molybdenum carbide as an electrochemical catalyst.

Transition metal molybdenum (Mo) exhibits a strong capacity to adsorb nitrogen (N2), but the Mo-N2 interaction is too strong and thus it is difficult for ammonia (NH3) to be released from the catalyst surface. Bonding with nonmetals with strong electronegativity is helpful to weaken the Mo-N2 interaction, while the effect of hydrogen termination on catalyst surfaces needs to be evaluated given that the hydrogen evolution reaction (HER) is a key side reaction. This computational work aims to explore α-molybdenum carbide (Mo2C, orthorhombic phase) as an electrochemical catalyst for the full nitrogen reduction reaction (NRR). Our density functional theory (DFT) calculations focus on a (100) surface and demonstrate that (i) surface molybdenum and carbon can be terminated by hydrogen via the Volmer step and (ii) the NRR can occur on H-terminated Mo2C(100) with an energy requirement of 1.0-1.4 eV, depending on H-coverage. Although C-Mo bonding can remarkably reduce difficulty in NH3 release from a Mo-site, H-terminals result in performance deterioration. These results provide new insights into the development of NRR catalysts.

Metabolic engineering of lipid pathways in Saccharomyces cerevisiae and staged bioprocess for enhanced lipid production and cellular physiology.

Microbially produced lipids have attracted attention for their environmental benefits and commercial value. We have combined lipid pathway engineering in Saccharomyces cerevisiae yeast with bioprocess design to improve productivity and explore barriers to enhanced lipid production. Initially, individual gene expression was tested for impact on yeast growth and lipid production. Then, two base strains were prepared for enhanced lipid accumulation and stabilization steps by combining DGAT1, ΔTgl3 with or without Atclo1, which increased lipid content ~ 1.8-fold but reduced cell viability. Next, fatty acid (FA) biosynthesis genes Ald6-SEACSL641P alone or with ACC1** were co-expressed in base strains, which significantly improved lipid content (8.0% DCW, 2.6-fold than control), but severely reduced yeast growth and cell viability. Finally, a designed two-stage process convincingly ameliorated the negative effects, resulting in normal cell growth, very high lipid productivity (307 mg/L, 4.6-fold above control) and improved cell viability.

The Fourth Alloying Mode by Way of Anti-Galvanic Reaction.

Anti-galvanic reaction (AGR) not only defies classic galvanic theory but is a promising method for tuning the compositions, structures, and properties of noble-metal nanoparticles. Employing AGR for the preparation of alloy nanoparticles has recently received great interest. Herein, we report an unprecedented alloying mode by way of AGR, in which foreign atoms induce structural transformation of the mother nanoparticles and enter the nanoparticles in a non-replacement fashion. A novel, active-metal-doped, gold nanoparticle was synthesized by this alloying mode, and its structure resolved. A CdSH motif was found in the protecting staples of the bimetal nanoparticle. DFT calculations revealed that the Au20 Cd4 (SH)(SR)19 nanoparticle is a 8e superatom cluster. Furthermore, although the Cd-doping does not essentially alter the absorption spectrum of the mother nanocluster, it distinctly enhances the stability and catalytic selectivity of the mother nanoclusters.

[Study on the 3,4-Dihydroxyphenylalanine Redox State Characterization Method of Mussel Adhesive Protein].

OBJECTIVE: To investigate the feasibility of using liquid chromatography (HPLC) to characterize the 3, 4-Dihydroxyphenylalanine (DOPA) redox state of mussel adhesive protein (MAP). METHODS: The DOPA and protein contents of MAP were determined by HPLC, Arnow and Bradford methods respectively. RESULTS: With extended oxidation time, the protein contents of MAP samples remained unchanged whereas the DOPA contents declined. The retention times of main peaks in HPLC for both the accelerated oxidation and retained samples shifted as the storage time extended, which could be related to the changes of sample redox state. CONCLUSIONS: The redox state of MAP can be characterized by the change of HPLC peak retention time. HPLC can be used in the research on the MAP redox state, which is beneficial to the product development and quality control.

Insights into the interfacial structure-function of poly(ethylene glycol)-decorated peptide-stabilised nanoscale emulsions.

The interfacial properties of nanoscale materials have profound influence on biodistribution and stability as well as the effectiveness of sophisticated surface-encoded properties such as active targeting to cell surface receptors. Tailorable nanocarrier emulsions (TNEs) are a novel class of oil-in-water emulsions stabilised by molecularly-engineered biosurfactants that permit single-pot stepwise surface modification with related polypeptides that may be chemically conjugated or genetically fused to biofunctional moieties. We have probed the structure and function of poly(ethylene glycol) (PEG) used to decorate TNEs in this way. The molecular weight of PEG decorating TNEs has considerable impact on the ζ-potential of the emulsion particles, related to differential interfacial thickness of the PEG layer as determined by X-ray reflectometry. By co-modifying TNEs with an antibody fragment, we show that the molecular weight and density of PEG governs the competing parameters of accessibility of the targeting moiety and of shielding the interface from non-specific interactions with the environment. The fundamental understanding of the molecular details of the PEG layer that we present provides valuable insights into the structure-function relationship for soft nanomaterial interfaces. This work therefore paves the way for further rational design of TNEs and other nanocarriers that must interact with their environment in controlled and predictable ways.

Self-Assembled Enzyme Nanoparticles for Carbon Dioxide Capture.

Enzyme-based processes have shown promise as a sustainable alternative to amine-based processes for carbon dioxide capture. In this work, we have engineered carbonic anhydrase nanoparticles that retain 98% of hydratase activity in comparison to their free counterparts. Carbonic anhydrase was fused with a self-assembling peptide that facilitates the noncovalent assembly of the particle and together were recombinantly expressed from a single gene construct in Escherichia coli. The purified enzymes, when subjected to a reduced pH, form 50-200 nm nanoparticles. The CO2 capture capability of enzyme nanoparticles was demonstrated at ambient (22 ± 2 °C) and higher (50 °C) temperatures, under which the nanoparticles maintain their assembled state. The carrier-free enzymatic nanoparticles demonstrated here offer a new approach to stabilize and reuse enzymes in a simple and cost-effective manner.

Determining the structure of interfacial peptide films: comparing neutron reflectometry and molecular dynamics simulations.

The peptides AM1 and Lac21E self-organize into switchable films at an air-water interface. In an earlier study, it was proposed that both AM1 and Lac21E formed monolayers of α-helical peptides based on consistency with neutron reflectivity data. In this article, molecular dynamics simulations of assemblies of helical and nonhelical AM1 and Lac21E at an air-water interface suggest some tendency for the peptides to spontaneously adopt an α-helical conformation. However, irrespective of the structure of the peptides, the simulations reproduced not only the structural properties of the films (thickness and distribution of the hydrophobic and hydrophilic amino acids) but also the experimental neutron reflectivity measurements at different contrast variations. This suggests that neutron reflectometry alone cannot be used to determine the structure of the peptides in this case. However, together with molecular dynamics simulations, it is possible to obtain a detailed understanding of peptide films at an atomic level.

Exploring engineering strategies that enhance de novo production of exotic cyclopropane fatty acids in Saccharomyces cerevisiae.

Cycloalkanes have broad applications as specialty fuels, lubricants, and pharmaceuticals but are not currently available from renewable sources, whereas, production of microbial cycloalkanes such as cyclopropane fatty acids (CFA) has bottlenecks. Here, a systematic investigation was undertaken into the biosynthesis of CFA in Saccharomyces cerevisiae heterologously expressing bacterial CFA synthase. The enzyme catalyzes formation of a 3-membered ring in unsaturated fatty acids. Monounsaturated fatty acids in phospholipids (PL) are the site of CFA synthesis; precursor cis-Δ9 C16 and C18 fatty acids were enhanced through OLE1 and SAM2 overexpression which enhanced CFA in PL. CFA turnover from PL to storage in triacylglycerols (TAG) was achieved by phospholipase PBL2 overexpression and acyl-CoA synthase to increase flux to TAG. Consequently, CFA storage as TAG reached 12 mg g-1 DCW, improved 3-fold over the base strain and >22% of TAG was CFA. Our research improves understanding of cycloalkane biosynthesis in yeast and offers insights into processing of other exotic fatty acids.

Coassembled Multicomponent Protein Nanoparticles Elicit Enhanced Antibacterial Activity.

The waning pipeline of the useful antibacterial arsenal has necessitated the urgent development of more effective antibacterial strategies with distinct mechanisms to rival the continuing emergence of resistant pathogens, particularly Gram-negative bacteria, due to their explicit drug-impermeable, two-membrane-sandwiched cell wall envelope. Herein, we have developed multicomponent coassembled nanoparticles with strong bactericidal activity and simultaneous bacterial cell envelope targeting using a peptide coassembly strategy. Compared to the single-component self-assembled nanoparticle counterparts or cocktail mixtures of these at a similar concentration, coassembled multicomponent nanoparticles showed higher bacterial killing efficiency against Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli by several orders of magnitude (about 100-1,000,000-fold increase). Comprehensive confocal and electron microscopy suggest that the superior antibacterial activity of the coassembled nanoparticles proceeds via multiple complementary mechanisms of action, including membrane destabilization, disruption, and cell wall hydrolysis, actions that were not observed with the single nanoparticle counterparts. To understand the fundamental working mechanisms behind the improved performance of coassembled nanoparticles, we utilized a "dilution effect" system where the antibacterial components are intermolecularly mixed and coassembled with a non-antibacterial protein in the nanoparticles. We suggest that coassembled nanoparticles mediate enhanced bacterial killing activity by attributes such as optimized local concentration, high avidity, cooperativity, and synergy. The nanoparticles showed no cytotoxic or hemolytic activity against tested eukaryotic cells and erythrocytes. Collectively, these findings reveal potential strategies for disrupting the impermeable barrier that Gram-negative pathogens leverage to restrict antibacterial access and may serve as a platform technology for potential nano-antibacterial design to strengthen the declining antibiotic arsenal.