Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants.

CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops. The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes. Compared with the well-developed adenine base editors (ABEs), cytosine base editors (CBEs) and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice, significantly limiting their application. Here, we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice. We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID∗Δ, and TadA-CDd outperformed TadA-E27R/N46L. A C-to-G base editor (CGBE) engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P. In addition, a dual base editor constructed with a single protein, TadDE, enabled simultaneous, highly efficient C-to-T and A-to-G editing in rice. Collectively, our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice, providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.

Activated carbon fibers functionalized with superhydrophilic coated pDA/TiO2/SiO2 with photoluminescent self-cleaning properties for efficient oil-water separation.

The adhesion of high-viscosity oil contamination poses limitations on three-dimensional (3D) materials' practical use in treating oilfield-produced water (OPW). In this study, we developed a hybrid pDA/TiO2/SiO2 coating (PTS) on the surface of hydrophilic activated carbon (ACF1) through a combination of dopamine (DA) polymerization, ethyl orthosilicate (TEOS) hydrolysis, and the condensation of TiO2 nanoparticles (NPs) with SiO2 NPs. This coating was designed for gravity-based oil-water separation. The inherent porosity and generous pore size of ACF1-PTS conferred it an ultra-high permeation flux (pure water flux of 3.72 × 105 L∙m-2∙h-1), allowing it to effectively separate simulated oil-water mixtures and oil-water emulsions while maintaining exceptional permeation flux and oil rejection efficiency. When compared to cleaning methods involving ethanol aqueous solutions and NaClO, ultraviolet (UV) illumination cleaning proved superior, enabling oil-contaminated ACF1-PTS to exhibit remarkable flux recovery efficiency and oil-removal capabilities during cyclic separation of actual OPW. Furthermore, the ACF1-PTS material demonstrated impressive stability and durability when exposed to acidic environments (acid, alkali, and salt), robust hydraulic washout conditions, and 25-cycle tests. This study offers valuable insights and research avenues for the development of highly efficient and environmentally friendly 3D oil-water separation materials for the actual treatment of OPW.

Characterization of Canine Gingival-Derived Mesenchymal Stem Cells and Their Exosomes.

Mesenchymal stem cells (MSCs) can be isolated from numerous tissues and have the potential for self-renewal and multidirectional differentiation. Evidence is accumulating which suggests that MSCs are also present in the gingival tissue. This study aimed to evaluate the feasibility of collecting, purifying, and amplifying gingival-derived MSCs (GMSCs) from canine gingiva and to obtain GMSC-derived exosomes (GMSC-exo). GMSCs were isolated and cultured; furthermore, cellular immunofluorescence demonstrated that GMSCs possess characteristic MSC markers, and in vitro differentiation was induced, indicating that GMSCs can differentiate into multiple lineages. GMSC-exo was successfully extracted from GMSCs supernatant and found that they exhibit the typical characteristics of exosomes as analyzed by transmission electron microscopy, nanoflow analysis, and western blotting. GMSC-exo promoted the proliferation and migration of Madin-Darby canine kidney cells. It was concluded that canine gingiva is a good source of MSCs. Additionally, GMSC-exo is a potentially promising cell-free therapeutic tool for the treatment of canine gingival diseases.

Genetic Screens for Floral Mutants in Arabidopsis thaliana: Enhancers and Suppressors.

The flower is a hallmark feature that has contributed to the evolutionary success of land plants. Diverse mutagenic agents have been employed as a tool to genetically perturb flower development and identify genes involved in floral patterning and morphogenesis. Since the initial studies to identify genes governing processes such as floral organ specification, mutagenesis in sensitized backgrounds has been used to isolate enhancers and suppressors to further probe the molecular basis of floral development. Here, we first describe two commonly employed methods for mutagenesis (using ethyl methanesulfonate (EMS) or T-DNAs as mutagens), and then describe three methods for identifying a mutation that leads to phenotypic alterations: traditional map-based cloning, modified high-efficiency thermal asymmetric interlaced PCR (mhiTAIL-PCR), and deep sequencing in the plant model Arabidopsis thaliana.

Protection efficacy of the H1 and H3 bivalent virus-like particle vaccine against swine influenza virus infection.

Swine influenza (SI) is widely prevalent in pig herds worldwide, causing huge economic losses to the pig industry and public health risks. The traditional inactivated swine influenza virus (SIV) vaccines are produced in chicken embryos, and egg-adaptive substitutions that occur during production process can impact vaccine effectiveness. Thus, developing an SI vaccine that can decrease the dependence on chicken embryos with a high immunogenicity is urgently needed. In this study, the utility of insect cell-derived SIV H1 and H3 bivalent virus-like particle (VLP) vaccines containing HA and M1 proteins of Eurasian avian-like (EA) H1N1 SIV and recent human-like H3N2 SIV were assessed in piglets. Antibody levels were monitored, and the protection efficacy of the vaccine after viral challenge was evaluated and compared with the inactivated vaccine. Results show that piglets produced high hemagglutination inhibition (HI) titers of antibodies against H1 and H3 SIV after immunization with SIV VLP vaccine. The neutralizing antibody level was significantly higher in SIV VLP vaccine than in the inactivated vaccine at 6 weeks post vaccination (p < 0.05). Furthermore, piglets immunized with the SIV VLP vaccine were protected against the challenge of H1 and H3 SIV, displaying inhibition of viral replication in piglets, and reduced lung damage. These results show that SIV VLP vaccine has good application prospects, thus laying the foundation for further research and commercialization of SIV VLP vaccine.

SUMOylation-modified Pelota-Hbs1 RNA surveillance complex restricts the infection of potyvirids in plants.

RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals. However, it is not known whether two other RNA quality control pathways, nonstop decay and no-go decay, are capable of restricting viruses in plants. Here, we show that the evolutionarily conserved Pelota-Hbs1 complex negatively regulates infection of plant viruses in the family Potyviridae (termed potyvirids), the largest group of plant RNA viruses that accounts for more than half of the viral crop damage worldwide. Pelota enables the recognition of the functional G1-2A6-7 motif in the P3 cistron, which is conserved in almost all potyvirids. This allows Pelota to target the virus and act as a viral restriction factor. Furthermore, Pelota interacts with the SUMO E2-conjugating enzyme SCE1 and is SUMOylated in planta. Blocking Pelota SUMOylation disrupts the ability to recruit Hbs1 and inhibits viral RNA degradation. These findings reveal the functional importance of Pelota SUMOylation during the infection of potyvirids in plants.

CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates.

CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

Eliciting national and subnational sets of disability weights in mainland China: Findings from the Chinese disability weight measurement study.

Background: The disability weight (DW) quantifies the severity of health states from disease sequela and is a pivotal parameter for disease burden calculation. We conducted a national and subnational DW measurement in China. Methods: In 2020-2021, we conducted a web-based survey to assess DWs for 206 health states in 31 Chinese provinces targeting health workers via professional networks. We fielded questions of paired comparison (PC) and population health equivalence (PHE). The PC data were analysed by probit regression analysis, and the regression results were anchored by results from the PHE responses on the DW scale between 0 (no loss of health) and 1 (health loss equivalent to death). Findings: We used PC responses from 468,541 respondents to estimate DWs of health states. Eight of 11 domains of health had significantly negative coefficients in the regression of the difference between Chinese and Global Burden of Disease (GBD) DWs, suggesting lower DW values for health states with mention of these domains in their lay description. We noted considerable heterogeneity within domains, however. After applying these Chinese DWs to the 2019 GBD estimates for China, total years lived with disability (YLDs) increased by 14·9% to 177 million despite lower estimates for musculoskeletal disorders, cardiovascular diseases, mental disorders, diabetes and chronic kidney disease. The lower estimates of YLDs for these conditions were more than offset by higher estimates of common, low-severity conditions. Interpretation: The differences between the GBD and Chinese DWs suggest that there might be some contextual factors influencing the valuation of health states. While the reduced estimates for mental disorders, alcohol use disorder, and dementia could hint at a culturally different valuation of these conditions in China, the much greater shifts in YLDs from low-severity conditions more likely reflects methodological difficulty to distinguish between health states that vary a little in absolute DW value but a lot in relative terms. Funding: This work was supported by the National Natural Science Foundation of China [grant number 82173626], the National Key Research and Development Program of China [grant numbers 2018YFC1315302], Wuhan Medical Research Program of Joint Fund of Hubei Health Committee [grant number WJ2019H304], and Ningxia Natural Science Foundation Project [grant number 2020AAC03436].

T-LOC: A comprehensive tool to localize and characterize T-DNA integration sites.

Scientists have developed many approaches based on PCR or next-generation sequencing to localize and characterize integrated T-DNAs in transgenic plants generated by Agrobacterium tumefaciens-mediated T-DNA transfer. However, none of these methods has the robust ability to handle all transgenic plants with diversified T-DNA patterns. Utilizing the valuable information in the whole-genome sequencing data of transgenic plants, we have developed a comprehensive approach (T-LOC) to localize and characterize T-DNA integration sites (TISs). We evaluated the performance of T-LOC on genome sequencing data from 48 transgenic rice (Oryza sativa) plants that provide real and unbiased resources of T-DNA integration patterns. T-LOC discovered 75 full TISs and reported a diversified pattern of T-DNA integration: the ideal single-copy T-DNA between two borders, multiple-copy of T-DNAs in tandem or inverted repeats, truncated partial T-DNAs with or without the selection hygromycin gene, the inclusion of T-DNA backbone, the integration at the genome repeat region, and the concatenation of multiple ideal or partial T-DNAs. In addition, we reported that DNA fragments from the two A. tumefaciens plasmids can be fused with T-DNA and integrated into the plant genome. Besides, T-LOC characterizes the genomic changes at TISs, including deletion, duplication, accurate repair, and chromosomal rearrangement. Moreover, we validated the robustness of T-LOC using PCR, Sanger sequencing, and Nanopore sequencing. In summary, T-LOC is a robust approach to studying the TISs independent of the integration pattern and can recover all types of TISs in transgenic plants.

Arabidopsis RBV is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGONAUTE1 loading.

MicroRNAs (miRNAs) play crucial roles in gene expression regulation through RNA cleavage or translation repression. Here, we report the identification of an evolutionarily conserved WD40 domain protein as a player in miRNA biogenesis in Arabidopsis thaliana. A mutation in the REDUCTION IN BLEACHED VEIN AREA (RBV) gene encoding a WD40 domain protein led to the suppression of leaf bleaching caused by an artificial miRNA; the mutation also led to a global reduction in the accumulation of endogenous miRNAs. The nuclear protein RBV promotes the transcription of MIR genes into pri-miRNAs by enhancing the occupancy of RNA polymerase II (Pol II) at MIR gene promoters. RBV also promotes the loading of miRNAs into AGO1. In addition, RNA-seq revealed a global splicing defect in the mutant. Thus, this evolutionarily conserved, nuclear WD40 domain protein acts in miRNA biogenesis and RNA splicing.

Geminiviruses boost active DNA demethylation for counter-defense.

DNA methylation regulates gene expression under abiotic and biotic stresses. Recently, Gui et al. discovered that geminiviruses subverted DNA methylation-mediated defense through boosting the active DNA demethylation mediated by host DNA glycosylases to promote viral virulence. Their findings reveal a distinctive counter-defense strategy exploited by invading pathogens to achieve successful infection.

Protective effect of bivalent H1N1 and H3N2 VLP vaccines against Eurasian avian-like H1N1 and recent human-like H3N2 influenza viruses in a mouse model.

Eurasian avian-like (EA) H1N1 swine influenza viruses (SIVs) are currently the most prevalent SIVs in Chinese swine populations, but recent human-like H3N2 SIV subtypes have also been frequently isolated. Hence, there is an urgent need to develop an effective vaccine against both EA H1N1 and recent human-like H3N2 infections. In this study, we utilized the baculovirus expression system to produce virus-like particles (VLPs) containing hemagglutinin protein (HA) and matrix protein (M1) based on A/Swine/Guangdong/YJ4/2014 (H1N1) and A/swine/Guangdong/L22/2010 (H3N2). An immunological experiment showed that in a mouse model, bivalent VLP vaccines against H1N1 and H3N2 can induce stronger humoral and cellular immune responses than whole influenza virus vaccines. Compared with monovalent inactivated vaccines that cannot offer protection against different SIV subtypes, monovalent H1N1 or H3N2 VLP vaccines can provide partial protection against lethal challenge by viruses of different subtypes. Meanwhile, bivalent VLP vaccines against H1N1 and H3N2 can provide full protection against lethal doses of homologous and heterologous viruses belonging to the EA H1N1 or recent human-like H3N2 lineage. These results suggest a promising approach to the development of vaccines against SIVs.

A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants.

BACKGROUND: The high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects. RESULTS: In this study, we perform a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, reveal that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicates that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the genome. Furthermore, we detect off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tend to cluster, while off-target A>G DNA mutations rarely clustered. CONCLUSION: Our findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.

High-efficiency and multiplex adenine base editing in plants using new TadA variants.

Recently reported adenine base editors (ABEs) exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding. However, editing efficiency varies significantly among different target sites. Here, we investigated the activities of three evolved E. coli adenosine deaminase TadA variants (TadA8e, TadA8.17, and TadA8.20) side-by-side in transgenic rice. We found that TadA8e outperforms TadA8.17 and TadA8.20, and induces efficient A-to-G conversion at all tested sites in the rice genome, including those that were uneditable by ABE7.10 in our previous experiments. Furthermore, V82S/Q154R mutations were incorporated into TadA8e, resulting in a new variant that we named TadA9. Our data show that TadA9 is broadly compatible with CRISPR/SpCas9, CRISPR/SpCas9-NG, and CRISPR/SpRY, as well as CRISPR/ScCas9 nickase systems, achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e. Finally, TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in weed control. Collectively, we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome. Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.

High-Rate and Long-Cycle Cathode for Sodium-Ion Batteries: Enhanced Electrode Stability and Kinetics via Binder Adjustment.

Sodium-ion batteries (SIBs) are heralded as promising candidates for grid-scale energy storage systems due to their low cost and abundant sodium resources. Excellent rate capacity and outstanding cycling stability are always the goals for SIBs. Up to now, nearly all attention has been focused on the control of morphology and structure of electrode materials, but the influence of binders on their performance is neglected, especially in cathode materials. Herein, using Na3V2(PO4)2O2F (NVPOF) as a cathode material, the influence of four different binders (sodium alginate, SA; carboxymethylcellulose sodium, CMC; poly(vinylidene fluoride), PVDF; and poly(acrylic latex), LA133) on its electrochemical performance is studied. As a result, when using SA as the binder, the electrochemical performance of the NVPOF electrode is improved significantly, which is mainly because of the high water solubility, rich carboxyl and hydroxyl groups, and high adhesive and cohesive properties of the SA binder, leading to the uniform distribution of active materials NVPOF and carbon black in electrodes, good integrity, low polarization, and superior kinetic properties of the NVPOF electrodes, as demonstrated by scanning electron microscopy, cyclic voltammetry, electrochemical impedance spectroscopy, and galvanostatic intermittent titration technique. More importantly, when coupled with a hard carbon anode, the fabricated sodium-ion full cells also exhibit excellent rate performance, thus providing a preview of their practical application. This work shows that the battery performance can be improved by matching suitable binder systems, which is believed to have great importance for the further optimization of the electrochemical performance of SIBs.