The type V effectors for CRISPR/Cas-mediated genome engineering in plants.

A plethora of CRISPR effectors, such as Cas3, Cas9, and Cas12a, are commonly employed as gene editing tools. Among these, Cas12 effectors developed based on Class II type V proteins exhibit distinct characteristics compared to Class II type VI and type II effectors, such as their ability to generate non-allelic DNA double-strand breaks, their compact structures, and the presence of a single RuvC-like nuclease domain. Capitalizing on these advantages, Cas12 family proteins have been increasingly explored and utilized in recent years. However, the characteristics and applications of different subfamilies within the type V protein family have not been systematically summarized. In this review, we focus on the characteristics of type V effector (CRISPR/Cas12) proteins and the current methods used to discover new effector proteins. We also summarize recent modifications based on engineering of type V effectors. In addition, we introduce the applications of type V effectors for gene editing in animals and plants, including the development of base editors, tools for regulating gene expression, methods for gene targeting, and biosensors. We emphasize the prospects for development and application of CRISPR/Cas12 effectors with the goal of better utilizing toolkits based on this protein family for crop improvement and enhanced agricultural production.

Genome editing for plant synthetic metabolic engineering and developmental regulation.

Plant metabolism and development are a reflection of the orderly expression of genetic information intertwined with the environment interactions. Genome editing is the cornerstone for scientists to modify endogenous genes or introduce exogenous functional genes and metabolic pathways, holding immense potential applications in molecular breeding and biosynthesis. Over the course of nearly a decade of development, genome editing has advanced significantly beyond the simple cutting of double-stranded DNA, now enabling precise base and fragment replacements, regulation of gene expression and translation, as well as epigenetic modifications. However, the utilization of genome editing in plant synthetic metabolic engineering and developmental regulation remains exploratory. Here, we provide an introduction and a comprehensive overview of the editing attributes associated with various CRISPR/Cas tools, along with diverse strategies for the meticulous control of plant metabolic pathways and developments. Furthermore, we discuss the limitations of current approaches and future prospects for genome editing-driven plant breeding.

Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools.

CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5'-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.

Electrochemical Performance of Carbon-Rich Silicon Carbonitride Ceramic as Support for Sulfur Cathode in Lithium Sulfur Battery.

As a promising matrix material for anchoring sulfur in the cathode for lithium-sulfur (Li-S) batteries, porous conducting supports have gained much attention. In this work, sulfur-containing C-rich SiCN composites are processed from silicon carbonitride (SiCN) ceramics, synthesized at temperatures from 800 to 1100 °C. To embed sulfur in the porous SiCN matrix, an easy and scalable procedure, denoted as melting-diffusion method, is applied. Accordingly, sulfur is infiltrated under solvothermal conditions at 155 °C into pores of carbon-rich silicon carbonitride (C-rich SiCN). The impact of the initial porosity and microstructure of the SiCN ceramics on the electrochemical performance of the synthesized SiCN-sulfur (SiCN-S) composites is analysed and discussed. A combination of the mesoporous character of SiCN and presence of a disordered free carbon phase makes the electrochemical performance of the SiCN matrix obtained at 900 °C superior to that of SiCN synthesized at lower and higher temperatures. A capacity value of more than 195 mAh/g over 50 cycles at a high sulfur content of 66 wt.% is achieved.

Full-Length Transcriptome Characterization and Functional Analysis of Pathogenesis-Related Proteins in Lilium Oriental Hybrid 'Sorbonne' Infected with Botrytis elliptica.

Gray mold (Botrytis elliptica) causes a deleterious fungal disease that decreases the ornamental value and yield of lilies. Lilium oriental hybrid 'Sorbonne' is a variety that is resistant to gray mold. Understanding the mechanism of resistance against B. elliptica infection in 'Sorbonne' can provide a basis for the genetic improvement in lily plants. In this study, a PacBio Sequel II system was used to sequence the full-length transcriptome of Lilium 'Sorbonne' after inoculation with B. elliptica. A total of 46.64 Gb subreads and 19,102 isoforms with an average length of 1598 bp were obtained. A prediction analysis revealed 263 lncRNAs, and 805 transcription factors, 4478 simple sequence repeats, and 17,752 coding sequences were identified. Pathogenesis-related proteins (PR), which may play important roles in resistance against B. elliptica infection, were identified based on the full-length transcriptome data and previously obtained second-generation transcriptome data. Nine non-redundant potential LhSorPR proteins were identified and assigned to two groups that were composed of two LhSorPR4 and seven LhSorPR10 proteins based on their genetic relatedness. The real-time quantitative reverse transcription PCR (qRT-PCR) results showed that the patterns of expression of nine differentially expressed PR genes under B. elliptica stress were basically consistent with the results of transcriptome sequencing. The pattern of expression of LhSorPR4s and LhSorPR10s genes in different tissues was analyzed, and the expression of each gene varied. Furthermore, we verified the function of LhSorPR4-2 gene in Lilium. The expression of LhSorPR4-2 was induced by phytohormones such as methyl jasmonate, salicylic acid, and ethephon. Moreover, the promoter region of LhSorPR4-2 was characterized by several functional domains associated with phytohormones and stress response. The overexpression of LhSorPR4-2 gene in 'Sorbonne' increased the resistance of the lily plant to B. elliptica and correlated with high chitinase activity. This study provides a full-length transcript database and functionally analyzed the resistance of PR gene to B. elliptica in Lilium, thereby introducing the candidate gene LhSorPR4-2 to breed resistance in Lilium.

Metabolic and Transcriptomic Profiling of Lilium Leaves Infected With Botrytis elliptica Reveals Different Stages of Plant Defense Mechanisms.

Botrytis elliptica, the causal agent of gray mold disease, poses a major threat to commercial Lilium production, limiting its ornamental value and yield. The molecular and metabolic regulation mechanisms of Lilium's defense response to B. elliptica infection have not been completely elucidated. Here, we performed transcriptomic and metabolomic analyses of B. elliptica resistant Lilium oriental hybrid "Sorbonne" to understand the molecular basis of gray mold disease resistance in gray mold disease. A total of 115 differentially accumulated metabolites (DAMs) were detected by comparing the different temporal stages of pathogen infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed the differentially expressed genes (DEGs) and DAMs were enriched in the phenylpropanoid and flavonoid pathways at all stages of infection, demonstrating the prominence of these pathways in the defense response of "Sorbonne" to B. elliptica. Network analysis revealed high interconnectivity of the induced defense response. Furthermore, time-course analysis of the transcriptome and a weighted gene coexpression network analysis (WGCNA) led to the identification of a number of hub genes at different stages, revealing that jasmonic acid (JA), salicylic acid (SA), brassinolide (BR), and calcium ions (Ca2+) play a crucial role in the response of "Sorbonne" to fungal infection. Our work provides a comprehensive perspective on the defense response of Lilium to B. elliptica infection, along with a potential transcriptional regulatory network underlying the defense response, thereby offering gene candidates for resistance breeding and metabolic engineering of Lilium.

Prediction of temperature tolerance in Lilium based on distribution and climate data.

There are plenty publications providing guidance for resistant taxa selection by experimental researches while the number of experimental taxa is often restricted. In this study, we presented a concise method to predict the temperature tolerance of wild Lilium in China based on open access botanical and associated environmental datasets. We divided all taxa into five groups to present an overview of Lilium's adaptability to temperature stress. Furthermore, according to the environmental conditions, the prediction of heat and cold tolerance in Lilium was made based on the combined multi-sources data at taxon level. Thirteen taxa with potential temperature tolerance were predicted of 42 taxa. The results showed that not only is tolerance prediction created by large-scale data analysis possible, but that it may supplement traditional laboratory researches with a comprehensive list of taxa.

Sleep disturbance and quality of life in clinically stable inpatients with schizophrenia in rural China.

BACKGROUND: Studies have identified numerous factors that may affect the sleep quality and quality of life (QOL) in outpatients with schizophrenia. However, the clinically stable inpatients who represent a large proportion of the population with schizophrenia in China have not received enough attention. The present study was performed to explore the sociodemographic and clinical correlates of sleep disturbance and QOL in clinically stable inpatients with schizophrenia in rural China. METHODS: A cross-sectional study was designed, and 207 clinically stable inpatients with schizophrenia were selected from Chifeng Anding Hospital, located in Inner Mongolia Autonomous Region, in northern China. All subjects were interviewed by the same investigator using standardized assessment instruments. QOL and sleep disturbance were measured using the Schizophrenia Quality of Life Scale (SQLS) and Pittsburgh Sleep Quality Index (PSQI), respectively. Univariate and multiple regression analyses were used to identify the factors influencing sleep disturbance and QOL. Antipsychotics taken by individuals were converted into olanzapine equivalent doses as the main confounding factor to be controlled. RESULTS: The prevalence of sleep disturbance was 58%, and sleep disturbance was significantly associated with depression (OR 1.33, 95% CI 1.17-1.52) and coping mechanisms (OR 0.95, 95% CI 0.91-0.98). We observed large differences between the sexes: the QOL of male inpatients with schizophrenia was substantially better than that of female inpatients, with a standard coefficient of 0.19 ± 1.62. Other factors related to QOL were depression (0.42 ± 0.30), hope (- 0.21 ± 0.19), general psychopathology symptoms (0.21 ± 0.24) and personal and social performance (- 0.12 ± 0.07). CONCLUSIONS: The depressive symptoms of inpatients with schizophrenia should receive more attention. More targeted interventions, such as the early identification and treatment of depression, should be promptly administered to improve the patient's hospitalization experience.

Mediation and moderation analyses: exploring the complex pathways between hope and quality of life among patients with schizophrenia.

BACKGROUND: The underlying mechanism between hope and quality of life is as yet unknown. We aim to examine the potential mediating effect of depression and resilience and the moderated effect of sex in this well-established association. METHODS: Two hundred seven patients diagnosed with schizophrenia were administered a questionnaire battery that measured hope, depression, resilience and QOL. A multiple mediation model was used to examine the mediating effect of resilience and depression on the association between hope and QOL. A subgroup analysis was performed and a moderated mediation model was examined to find and test the moderated effect of sex on the mediation model. We used Mplus to perform moderation and mediation analyses so that the mediators and moderator could function together in the same model. RESULT: Sex was the moderator on the direct path between hope and QOL. The relationship between hope and QOL was mediated by resilience and depression in both sexes. When compared with female patients, the effect of hope on QOL was completely mediated by resilience and depression in males. In female patients, the model was partially mediated, and the direct effect of hope on QOL was significantly negatively correlated with the level of hope. CONCLUSION: We present a conceptual model containing the mediated effects of resilience and depression and the moderated effect of sex between hope and QOL, which we believe facilitates the understanding of these associations. This model should be useful in the formulation of strategies to improve QOL.

Microstructure and Dielectric Properties of LPCVD/CVI-SiBCN Ceramics Annealed at Different Temperatures.

SiBCN ceramics were introduced into porous Si3N4 ceramics via a low-pressure chemical vapor deposition and infiltration (LPCVD/CVI) technique, and then the composite ceramics were heat-treated from 1400 °C to 1700 °C in a N2 atmosphere. The effects of annealing temperatures on microstructure, phase evolution, dielectric properties of SiBCN ceramics were investigated. The results revealed that α-Si3N4 and free carbon were separated below 1700 °C, and then SiC grains formed in the SiBCN ceramic matrix after annealing at 1700 °C through a phase-reaction between free carbon and α-Si3N4. The average dielectric loss of composites increased from 0 to 0.03 due to the formation of dispersive SiC grains and the increase of grain boundaries.

Structural basis for the Smad5 MH1 domain to recognize different DNA sequences.

Smad proteins are important intracellular mediators of TGF-ß signalling, which transmit signals directly from cell surface receptors to the nucleus. The MH1 domain of Smad plays a key role in DNA recognition. Two types of DNA sequence were identified as Smad binding motifs: the Smad binding element (SBE) and the GC-rich sequence. Here we report the first crystal structure of the Smad5 MH1 domain in complex with the GC-rich sequence. Compared with the Smad5-MH1/SBE complex structure, the Smad5 MH1 domain contacts the GC-rich site with the same ß-hairpin, but the detailed interaction modes are different. Conserved ß-hairpin residues make base specific contacts with the minimal GC-rich site, 5'-GGC-3'. The assembly of Smad5-MH1 on the GC-rich DNA also results in distinct DNA conformational changes. Moreover, the crystal structure of Smad5-MH1 in complex with a composite DNA sequence demonstrates that the MH1 domain is targeted to each binding site (GC-rich or SBE) with modular binding modes, and the length of the DNA spacer affects the MH1 assembly. In conclusion, our work provides the structural basis for the recognition and binding specificity of the Smad MH1 domain with the DNA targets.

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis.

Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family regulates their ubiquitylation activity. However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that the C2-WW-HECT ligase Smurf1 is activated by neddylation. Smurf1 physically interacts with Nedd8 and Ubc12, forms a Nedd8-thioester intermediate, and then catalyses its own neddylation on multiple lysine residues. Intriguingly, this autoneddylation needs an active site at C426 in the HECT N-lobe. Neddylation of Smurf1 potently enhances ubiquitin E2 recruitment and augments the ubiquitin ligase activity of Smurf1. The regulatory role of neddylation is conserved in human Smurf1 and yeast Rsp5. Furthermore, in human colorectal cancers, the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12 correlates with cancer progression and poor prognosis. These findings provide evidence that neddylation is important in HECT ubiquitin ligase activation and shed new light on the tumour-promoting role of Smurf1.

Permeabilization of Microbacterium oxylans shifts the conversion of puerarin from puerarin-7-O-glucoside to puerarin-7-O-fructoside.

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 +/- 31.9 microM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 +/- 48.0 microM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, (13)C NMR, (1)H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 +/- 5.4 microM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 +/- 27.7 microM puerarin-7-O-glucoside and 187.8 +/- 29.5 microM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 +/- 24.0 microM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product's composition.

Preparation of a super-long two column chromatography system and its application in separating glycosylated puerarin.

Separation of Puerarin-7-O-glucoside from its precursor, puerarin, using a common chromatography column packed with AB-8 macroporous resin was unsuccessful. Therefore, in this study a 8 m super-long flexible reinforced PVC column was externally added to the common column in order to improve the chromatography efficiency by increasing the number of theoretical plates. Both the PVC and common columns were separately packed with AB-8 macroporous resin slurry. The packed PVC column was coiled after washing and stored until use. The microbial transformation mixture with puerarin-7-O-glucoside and puerarin (250 mL) was loaded onto the common column, followed by washing with 2000 mL H(2)O. After attaching the coiled external PVC column to the common column, a linear gradient of 10-30% ethanol was applied to elute the target compound. Two peaks appeared: peak I contained puerarin-7-O-glucoside at 97.9% purity and 88.1% recovery rate, and peak II was puerarin at 98.7% purity and 87.0% recovery rate. The use of the coiled external flexible reinforced PVC column avoided spatial restriction for long columns, which made it much more convenient for column packing and chromatography operations. Furthermore, this method eliminated the resin blockage problem caused by stationary water pressure in a rigid vertical long column. Using an external super-long column, the PVC tube was connected with the common column only during elution, which avoided delay in time period during sample loading and column washes associated with the use of long external columns.