In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

Nano-CeO2 activates physical and chemical defenses of garlic (Allium sativum L.) for reducing antibiotic resistance genes in plant endosphere.

The transmission of manure- and wastewater-borne antibiotic-resistant bacteria (ARB) to plants contributes to the proliferation of antimicrobial resistance in agriculture, necessitating effective strategies for preventing the spread of antibiotic resistance genes (ARGs) from ARB in the environment to humans. Nanomaterials are potential candidates for efficiently controlling the dissemination of ARGs. The present study investigated the abundance of ARGs in hydroponically grown garlic (Allium sativum L.) following nano-CeO2 (nCeO2) application. Specifically, root exposure to nCeO2 (1, 2.5, 5, 10 mg L-1, 18 days) reduced ARG abundance in the endosphere of bulbs and leaves. The accumulation of ARGs (cat, tet, and aph(3')-Ia) in garlic bulbs decreased by 24.2-32.5 % after nCeO2 exposure at 10 mg L-1. Notably, the lignification extent of garlic stem-disc was enhanced by 10 mg L-1 nCeO2, thereby accelerating the formation of an apoplastic barrier to impede the upward transfer of ARG-harboring bacteria to garlic bulbs. Besides, nCeO2 upregulated the gene expression related to alliin biosynthesis and increased allicin content by 15.9-16.2 %, promoting a potent antimicrobial defense for reducing ARG-harboring bacteria. The potential exposure risks associated with ARGs and Ce were evaluated according to the estimated daily intake (EDI). The EDI of ARGs exhibited a decrease exceeding 95 %, while the EDI of Ce remained below the estimated oral reference dose. Consequently, through stimulating physical and chemical defenses, nCeO2 contributed to a reduced EDI of ARGs and Ce, highlighting its potential for controlling ARGs in plant endosphere within the framework of nano-enabled agrotechnology.

Toxic mechanism in Daphnia magna due to phthalic acid esters and CuO nanoparticles co-exposure: The insight of physiological, microbiomic and metabolomic profiles.

Various phthalic acid esters (PAEs) such as dibutyl phthalate (DBP) and butyl benzyl phthalate (BBP) co-exist with nanopollutants in aquatic environment. In this study, Daphnia magna was exposed to nano-CuO and DBP or BBP at environmental relevant concentrations for 21-days to investigate these combined toxic effects. Acute EC50 values (48 h) of nano-CuO, DBP, and BBP were 12.572 mg/L, 8.978 mg/L, and 4.785 mg/L, respectively. Results showed that co-exposure with nano-CuO (500 µg/L) for 21 days significantly enhanced the toxicity of DBP (100 µg/L) and BBP (100 µg/L) to Daphnia magna by 18.37% and 18.11%, respectively. The activities of superoxide dismutase, catalase, and glutathione S-transferase were enhanced by 10.95% and 14.07%, 25.63% and 25.91%, and 39.93% and 35.01% in nano-CuO+DBP and nano-CuO+BBP treatments as compared to the individual exposure groups, verifying that antioxidative defense responses were activated. Furthermore, the co-exposure of nano-CuO and PAEs decreased the population richness and diversity microbiota, and changed the microbial community composition in Daphnia magna. Metabolomic analysis elucidated that nano-CuO + PAEs exposure induced stronger disturbance on metabolic network and molecular function, including amino acid, nucleotides, and lipid metabolism-related metabolic pathways, as comparison to PAEs single exposure treatments. In summary, the integration of physiological, microflora, and untargeted metabolomics analysis offers a fresh perspective into the potential ecological risk associated with nanopollutants and phthalate pollution in aquatic ecosystems.

Simple method for transformation and gene editing in medicinal plants.

A sample delivery method, modified from cut-dip-budding, uses explants with robust shoot regeneration ability, enabling transformation and gene editing in medicinal plants, bypassing tissue culture and hairy root formation. This method has potential for applications across a wide range of plant species.

Breeding exceptionally fragrant soybeans for soy milk with strong aroma.

Knockout of the soybean (Glycine max) betaine aldehyde dehydrogenase genes GmBADH1 and GmBADH2 using CRISPR/Cas12i3 enhances the aroma of soybeans. Soy milk made from the gmbadh1/2 double mutant seeds exhibits a much stronger aroma, which consumers prefer; this mutant has potential for enhancing quality in soy-based products.

Targeted G-to-T base editing for generation of novel herbicide-resistance gene alleles in rice.

A newly developed rice guanine base editor (OsGTBE) achieves targeted and efficient G-to-T editing (C-to-A in the opposite strand) in rice. Using OsGTBE to edit endogenous herbicide-resistant loci generated several novel alleles conferring herbicide resistance, highlighting its utility in creating valuable germplasm and enhancing genetic diversity..

Knockout of miR396 genes increases seed size and yield in soybean.

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.

Clinical application value of echocardiography in evaluating left ventricular diastolic function in patients with acute pulmonary embolism.

OBJECTIVE: To explore the clinical value of echocardiography in the assessment of left ventricular diastolic function in patients with acute pulmonary embolism (APE). METHODS: APE patients in our hospital from June 2019 to June 2021 were selected as the observation group. They were divided into low-risk group (19 cases), medium-risk group (16 cases) and high-risk group (15 cases). The non-APE people in our hospital during the same period were selected as the control group. All subjects underwent echocardiography to observe the performance of APE patients under echocardiography. The peak velocity ratio S-wave/D-wave (S/D), early diastolic annular velocity/advanced diastolic annular velocity (Ea/Aa), early filling/early diastolic annular velocity (E/Ea), and early filling/early diastolic annular velocity (E/Ea) were compared with Ar and Vp, respectively; receiver operator characteristic (ROC) curve was used to evaluate the value of echocardiography in evaluating left ventricular diastolic function in patients with APE. RESULTS: Echocardiography show different manifestations of APE patients. Compared with the control group, S/D, Ea/Aa, and Vp in the observation group were significantly decreased and E/Ea and Ar in the observation group were significantly increased (p < 0.05). With the increase of risk stratification, S/D, Ea/Aa, and Vp gradually decreased, E/Ea and Ar gradually increased, and the difference was statistically significant (p < 0.05). The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, area under curve (AUC), and 95% CI were 89.52%, 65.57%, 72.86%, 80.82%, 75.38%, 0.730, and 0.629-0.831, respectively. CONCLUSION: Echocardiography can effectively evaluate left ventricular diastolic function in patients with APE, and there are significant differences in left ventricular diastolic function in different risk stratification, which has important reference value for clinical diagnosis and treatment of APE.

High-throughput genome editing in rice with a virus-based surrogate system.

With the widespread use of clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate (WDV-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two single guide RNAs (sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency (66.8% vs. 90.1%), plantlet regeneration efficiency (2.31-fold increase), and numbers of homozygous-edited plants (36.3% vs. 70.7%). Large-scale editing using pooled sgRNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDV-Gate on sequence knock-in for protein tagging. By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.

Foliar Application of Reaction Products Derived from Selenite Removal by Iron Monosulfide for Brassica rapa ssp. Chinensis L.

The extensive application of FeS in environmental remediation requires the recovery and reuse of reaction products between FeS and pollutants. Therefore, foliar application of reaction products derived from selenite [Se(IV)] removal by FeS for pak choi was performed. The removal rate of Se(IV) by 100 mg/L FeS was 0.047 h-1. 93.2% of Se(IV) was reduced to Se(0), and FeS was correspondingly oxidized to goethite (78.9%), lepidocrocite (21.1%), and S(0) (91.5%) based on the analysis of X-ray absorption fine structure. The reaction products promoted the growth of pak choi in terms of fresh biomass, vitamin C, and protein, ascribed to the key roles of Fe and S in enhancing the electron transfer rate and light conversion rate. Furthermore, the application of reaction products decreased by 64% of disease incidence as compared with the pathogen Pseudomonas syringae pv. maculicola-infected control. The total Se content in plants increased to 576 µg/kg and was composed of 11.9% of SeMeCys, 29.8% of SeMet, and 58.3% of SeCys after exposure to reaction products, which is beneficial to the human dietary intake from pak choi. This study demonstrated that the reaction products between FeS and Se(IV) could be recovered and applied as a nano-enabled strategy to prevent crop insecurity.

Nano-QSAR modeling for predicting the cytotoxicity of metallic and metal oxide nanoparticles: A review.

Given the rapid development of nanotechnology, it is crucial to understand the effects of nanoparticles on living organisms. However, it is laborious to perform toxicological tests on a case-by-case basis. Quantitative structure-activity relationship (QSAR) is an effective computational technique because it saves time, costs, and animal sacrifice. Therefore, this review presents general procedures for the construction and application of nano-QSAR models of metal-based and metal-oxide nanoparticles (MBNPs and MONPs). We also provide an overview of available databases and common algorithms. The molecular descriptors and their roles in the toxicological interpretation of MBNPs and MONPs are systematically reviewed and the future of nano-QSAR is discussed. Finally, we address the growing demand for novel nano-specific descriptors, new computational strategies to address the data shortage, in situ data for regulatory concerns, a better understanding of the physicochemical properties of NPs with bioactivity, and, most importantly, the design of nano-QSAR for real-life environmental predictions rather than laboratory simulations.

Photochemical Transformation and Catalytic Activity of Dissolved Black Nitrogen Released from Environmental Black Carbon.

Biomass combustion results in the formation and wide distribution of black carbon (BC) in soils, wherein the dissolved fractions are among the most active components. Although the presence of dissolved black nitrogen (DBN) in BC has been identified, its environmental behavior and implication are not understood. This study investigated the photochemical transformation and catalytic activity of DBN under simulated solar irradiation. DBN is more easily transformed than dissolved BC due to its photoactive heteroaromatic N structure, and the half-life of DBN produced at 500 °C (8.6 h) is two times shorter than that of the dissolved BC counterpart (23 h). Meanwhile, solar irradiation is favorable for the homoaggregation of DBN. During irradiation, DBN generates not only reactive oxygen species (e.g., 1O2, O2-, and •OH) but also reactive nitrogen species (mainly •ON), which account for its higher photocatalytic degradation of bisphenol A than dissolved BC. These findings shed new light on the impact of heteroatoms on the phototransformation and activity of BC as well as cycling of N in terrestrial systems.

Catalytic Reaction Mechanism of NO-CO on the ZrO2 (110) and (111) Surfaces.

Due to the large population of vehicles, significant amounts of carbon monoxide (CO), nitrogen oxides (NOx), and unburned hydrocarbons (HC) are emitted into the atmosphere, causing serious pollution to the environment. The use of catalysis prevents the exhaust from entering the atmosphere. To better understand the catalytic mechanism, it is necessary to establish a detailed chemical reaction mechanism. In this study, the adsorption behaviors of CO and NO, the reaction of NO reduction with CO on the ZrO2 (110) and (111) surfaces was performed through periodic density functional theory (DFT) calculations. The detailed mechanism for CO2 and N2 formation mainly involved two intermediates N2O complexes and NCO species. Moreover, the existence of oxygen vacancies was crucial for NO reduction reactions. From the calculated energy, it was found that the pathway involving NCO intermediate interaction occurring on the ZrO2 (110) surface was most favorable. Gas phase N2O formation and dissociation were also considered in this study. The results indicated the role of reaction intermediates NCO and N2O in catalytic reactions, which could solve the key scientific problems and disputes existing in the current experiments.

Toxicity of GO to Freshwater Algae in the Presence of Al2O3 Particles with Different Morphologies: Importance of Heteroaggregation.

The roles of Al2O3 particles with different morphologies in altering graphene oxide (GO) toxicity to Chlorella pyrenoidosa were investigated. Algal growth inhibition by GO with coexisting Al2O3 particles was much lower than the sum of inhibitions from the individual materials for all the three Al2O3, showing the toxicity mitigation by Al2O3. The lowest GO toxicity was observed at the concentrations of 300, 150, and 100 mg/L for Al2O3 nanoparticles (NPs, 8-10 nm), bulk particles (BPs, 100-300 nm), and fibers (diameter: 10 nm; length: 400 nm), respectively. GO-Al2O3 heteroaggregation was responsible for the observed toxicity reduction. GO-induced algal membrane damage was suppressed by the three types of Al2O3 due to GO-Al2O3 heteroaggregation, and the reduction in intracellular reactive oxygen species generation and physical contact were confirmed as two main mechanisms. Moreover, the exposure sequence of GO and Al2O3 could highly influence the toxicity, and the simultaneous exposure of individual GO and Al2O3 showed the lowest toxicity due to minimum direct contact with algal cells. Humic acid further decreased GO-Al2O3 toxicity due to enhanced steric hindrance through surface coating of GO-Al2O3 heteroaggregates. This work provides new insights into the role of natural mineral particles in altering the environmental risk of GO.

Heteroaggregation of graphene oxide with minerals in aqueous phase.

Upon release into waters, sediments, and soils, graphene oxide (GO) may interact with fine mineral particles. We investigated the heteroaggregation of GO with different minerals, including montmorillonite, kaolinite, and goethite, in aqueous phase. GO significantly enhanced the dispersion of positively charged goethite (>50%) via heteroaggregation, while there was no interaction between GO and negatively charged montmorillonite or kaolinite. Electrostatic attraction was the dominant force in the GO-goethite heteroaggregation (pH 4.0-8.5), and the dissolved Fe ions (<0.16 mg/L) from goethite were unable to destabilize GO suspension. The GO-goethite heteroaggregation was further quantitatively investigated through GO adsorption study. All adsorption isotherms of GO at different solution pH (4.0 and 6.5) followed the Linear model. The apparent intercept (1.0-6.9 mg/g) was observed for all the adsorption isotherms, indicating that this fraction of adsorbed GO was difficult to desorb from goethite (defined here as irreversible adsorption) under the tested conditions. Desorption hysteresis was observed, which could be explained by the formation of multilayered GO-goethite complex with high configurational stability. These findings are useful for understanding the interaction of GO with mineral surfaces, and potential fate and toxicity of GO under natural conditions in aquatic environments, as well as in soils and sediments.

Mechanistic study on the increase of Microcystin-LR synthesis and release in Microcystis aeruginosa by amino-modified nano-plastics.

Ecological risk of micro/nano-plastics (MPs/NPs) has become an important environmental issue. Microcystin-leucine-arginine (MC-LR) produced by Microcystis aeruginosa (M. aeruginosa) is the most common and toxic secondary metabolites (SM). However, the influencing mechanism of MPs and NPs exposure on MC-LR synthesis and release have still not been clearly evaluated. In this work, under both acute (4d) and long-term exposure (10d), only high-concentration (10 mg/L) exposure of amino-modified polystyrene NPs (PS-NH2-NPs) promoted MC-LR synthesis (32.94 % and 42.42 %) and release (27.35 % and 31.52 %), respectively. Mechanistically, PS-NH2-NPs inhibited algae cell density, interrupted pigment synthesis, weakened photosynthesis efficiency, and induced oxidative stress, with subsequent enhancing the MC-LR synthesis. Additionally, PS-NH2-NPs exposure up-regulated MC-LR synthesis pathway genes (mcyA, mcyB, mcyD, and mcyG) combined with significantly increased metabolomics (Leucine and Arginine), thereby enhancing MC-LR synthesis. PS-NH2-NPs exposure enhanced the MC-LR release from M. aeruginosa via up-regulated MC-LR transport pathway genes (mcyH) and the shrinkage of plasma membrane. Our results provide new insights into the long-time coexistence of NPs with algae in freshwater systems might pose a potential threat to aquatic environments and human health.

Both nanoplastic and iron mineral types determine their heteroaggregation: Aggregation kinetics and interface process.

Nanoplastics (NPs) inevitably interact with iron minerals (IMs) after being released into aquatic environments, changing their transport and fate. In this study, batch heteroaggregation kinetics of four types of NPs, i.e., polymethyl methacrylate (PMMA), polystyrene (PS-Bare), amino-polystyrene (PS-NH2), and carboxyl-polystyrene (PS-COOH), with two different IMs (hematite and magnetite) were conducted. We found that the heteroaggregation of NPs and IMs and the associated interfacial interaction mechanisms are both NPs-dependent and IMs-dependent. Specifically, the NPs had stronger heteroaggregation with hematite than magnetite; the heteroaggregation order of two IMs with NPs was PMMA > PS-NH2 > PS-Bare > PS-COOH. Moreover, hydrogen bond, complexation, hydrophobic, cation-π, and electrostatic interaction were involved in the interfacial reaction between NPs and hematite, and electrons were transferred from the NPs to the hematite, causing the reduction of Fe3+ into Fe2+. Furthermore, we first revealed that both pre-homoaggregation of NPs and IMs could affect their subsequent heteroaggregation, and the homoaggregates of IMs could be interrupted by PMMA or PS-COOH NPs introduction. Therefore, the emerging NPs pollution is likely to generate an ecological effect in terms of elemental cycles such as iron cycle. This work provides new insights into assessing the environmental transfer and ecological effects of NPs in aquatic environments.

Different functional groups of carbon dots influence the formation of protein crowns and pepsin characteristic in vitro digestion.

Application of nanomaterials (NMs) in agriculture poses an ingestion risk to humans and may affect the digestive process. Different fates of NMs with differential charges in the gastrointestinal tract should be considered. In this study, the interaction between three carbon dots (CDs) carried with different functional groups (-NH2, -OH, and -COOH) and pepsin was analyzed through an in vitro digestion model. The results showed that CDs significantly reduced pepsin activity. Among them, CDs-NH2 had the greatest effect, following by CDs-OH, and CDs-COOH. Besides, molecular docking demonstrated the specific binding site of CDs to pepsin, while the most stable binding energy (-8.10 kcal/mol) was formed between CDs-NH2 and pepsin. Further, CDs formed a nanomaterial-protein crown structure with pepsin. The present study enriches the functional group properties of CDs in the digestion and provides new ideas for the potential human health of NMs.

Co-photoaging inhibited the heteroaggregation between polystyrene nanoplastics and different titanium dioxide nanoparticles.

Heteroaggregation between nanoplastics (NPs) and titanium dioxide nanoparticles (TiO2NPs) determines their environmental fates and ecological risks in aquatic environments. However, the co-photoaging scenario of NPs and TiO2NPs, interaction mechanisms of TiO2NPs with (aged) NPs, as well as the dependence of their heteroaggregation on TiO2NPs facets remain elusive. We found the critical coagulation concentration (CCC) of polystyrene nanoplastics (PSNPs) with coexisting RTiO2NPs was 1.9 - 2.2 times larger than that with coexisting ATiO2NPs, suggesting a better suspension stability of PSNPs+RTiO2NPs. In addition, CCC of TiO2NPs with coexisting photoaged PSNPs (APSNPs) was larger 1.7 - 2.2 times than that with PSNPs coexisting, indicating photoaging inhibited their heteroaggregation due to increasing electrostatic repulsion derived from increased negative charges on APSNPs and the polymer-derived dissolved organic carbon. Coexisted TiO2NPs promoted oxidation of PSNPs with the action of HO· and O2·- under UV light, leading to inhibited heteroaggregation. Moreover, Van der Waals and Lewis-acid interaction dominated the formation of primary heteroaggregates of PSNPs-TiO2NPs (ESE = ‒2.20 ∼ ‒2.78 eV) and APSNPs-TiO2NPs (ESE = ‒3.29 ∼ ‒3.67 eV), respectively. The findings provide a mechanistic insight into the environmental process of NPs and TiO2NPs, and are significant for better understanding their environmental risks in aquatic environments.