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Optimized protoplast isolation and transfection with a breakpoint:accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

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aBIOTECH 2024年第2期5卷 151-168页

作者： Debasmita Panda Subhasis Karmakar Manaswini Dash Swagat Kumar Tripathy Priya Das Sagar Banerjee Yiping Qi Sanghamitra Samantaray Pradipta Kumar Mohapatra Mirza J.Baig Kutubuddin A.Molla ICAR National Rice Research Institute CuttackOdisha 753006India Department of Botany Ravenshaw UniversityCuttackOdisha 753003India Department of plant science and Landscape Architecture University of MarylandCollege ParkMD 20742USA

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic ***-predicted guide RNAs(gRNAs)often fail to induce efficient cleavage at target *** target loci are inaccessible due to complex chromatin ***,there is no suitable tool availab.e to predict the architecture of genomic target sites and their ***,significant time and resources are spent on performing editing experiments with inefficient *** in vitro-cleavage assay could provide a rough assessment of gRNA efficiency,it largely excludes the interference of native genomic *** in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic ***,we developed a modified protocol that offers highly efficient protoplast isolation from rice,Arabidopsis,and chickpea,using a sucrose gradient,transfection using PEG(polyethylene glycol),and validation of single guide RNAs(sgRNAs)cleavage efficiency of *** have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and *** introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice,Arabidopsis,and chickpea *** dual sgRNAs,our CRISPRdeletion strategy offers straightforward detection of genome editing success by simple agarose gel *** sequencing of PCR products confirmed the editing efficiency of specific ***,we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability,allowing a pause between isolation and *** high-efficiency protocol for protoplast isolation and transfection enables rapid(less than 7 days)validation of sgRNA cleavage efficiency before proceeding with stable *** isolation and transfection method can als

关键词： Protoplast Genome editing Transgene-free edited plants Rice Chickpea

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Boosting genome editing in plants with single transcript unit surrogate reporter systems

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plant Communications 2024年第6期5卷 32-43页

作者： Xu Tang Qiurong Ren Xiaodan Yan Rui Zhang Li Liu Qinqin Han Xuelian Zheng Yiping Qi Hongyuan Song Yong Zhang Chongqing Key lab.ratory of plant Resource Conservation and Germplasm Innovation Integrative Science Center of Germplasm Creation in Western China(Chongqing)Science CitySchool of Life SciencesSouthwest UniversityChongqing 400715China Key lab.ratory of Agricultural Biosafety and Green Production of Upper Yangtze River Ministry of EducationChongqing 400715China College of Horticulture and Landscape Architecture Southwest UniversityChongqing 400715China Department of Biotechnology School of Life Sciences and TechnologyCenter for Informational BiologyUniversity of Electronic Science and Technology of ChinaChengdu 610054China School of Synbiology School of Life ScienceShanxi UniversityTaiyuan 030006China Department of plant science and Landscape Architecture University of MarylandCollege ParkMD 20742USA Institute for Bioscience and Biotechnology Research University of MarylandRockvilleMD 20850USA

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop ***,the challenge of low editing activity complicates the identification of editing *** this study,we introduce multiple single transcript unit surrogate reporter(STU-SR)systems to enhance the selection of genome-edited *** systems use the same single guide RNAs designed for endogenous genes to edit reporter genes,establishing a direct link between reporter gene editing activity and that of endogenous *** strategies are used to restore functional reporter genes after genome editing,including efficient single-strand annealing(SSA)for homologous recombination in STUSR-SSA ***-SR-base editor systems leverage base editing to reinstate the start codon,enriching C-to-T and A-to-G base editing *** results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice,encompassing Cas9 nuclease-based targeted mutagenesis,cytosine base editing,and adenine base *** systems exhibit compatibility with Cas9 variants,such as the PAM-less SpRY,and are shown to boost genome editing in Brassica oleracea,a dicot vegetable *** summary,we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.

关键词： CRISPR-Cas9 single transcript unit STU surrogate reporter SR single strand annealing SSA cyto-sine base editing adenine base editing

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DMFVAE: miRNA-disease associations prediction based on deep matrix factorization method with variational autoencoder

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Frontiers of Computer science 2024年第6期18卷 186912-186912页

作者： WEI Pijing WANG Qianqian GAO Zhen CAO Ruifen ZHENG Chunhou Information Materials and Intelligent Sensing lab.ratory of Anhui Province Institutes of Physical science and Information Technology Anhui University Key lab.of Intelligent Computing and Signal Processing of Ministry of Education School of Computer science and Technology Anhui University Key lab.of Intelligent Computing and Signal Processing of Ministry of Education School of Artificial Intelligence Anhui University

MicroRNAs (miRNAs) are closely related to numerous complex human diseases, therefore, exploring miRNA-disease associations (MDAs) can help people gain a better understanding of complex disease mechanism. An increasing number of computational methods have been developed to predict MDAs. However, the sparsity of the MDAs may hinder the performance of many methods. In addition, many methods fail to capture the nonlinear relationships of miRNA-disease network and inadequately leverage the features of network and neighbor nodes. In this study, we propose a deep matrix factorization model with variational autoencoder (DMFVAE) to predict potential MDAs. DMFVAE first decomposes the original association matrix and the enhanced association matrix, in which the enhanced association matrix is enhanced by self-adjusting the nearest neighbor method, to obtain sparse vectors and dense vectors, respectively. Then, the variational encoder is employed to obtain the nonlinear latent vectors of miRNA and disease for the sparse vectors, and meanwhile, node2vec is used to obtain the network structure embedding vectors of miRNA and disease for the dense vectors. Finally, sample features are acquired by combining the latent vectors and network structure embedding vectors, and the final prediction is implemented by convolutional neural network with channel attention. To evaluate the performance of DMFVAE, we conduct five-fold cross validation on the HMDD v2.0 and HMDD v3.2 datasets and the results show that DMFVAE performs well. Furthermore, case studies on lung neoplasms, colon neoplasms, and esophageal neoplasms confirm the ability of DMFVAE in identifying potential miRNAs for human diseases.

关键词： miRNA-disease association deep matrix factorization self-adjusted nearest neighbor variational encoder network structure

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Knockout of a rice K5.2 gene increases Ca accumulation in the grain

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Journal of Integrative plant Biology 2024年第2期66卷 252-264页

作者： Peitong Wang Naoki Yamaji Namiki Mitani-Ueno Jun Ge Jian Feng Ma National Key lab.ratory of Crop Genetics&Germplasm Enhancement and Utilization College of Resources and Environmental SciencesNanjing Agricultural UniversityNanjing 210095China Institute of plant science and Resources Okayama UniversityKurashiki 710-0046Japan

Rice is a staple food for half of the world’s population,but it is a poor dietary source of calcium(Ca)due to the low *** is an important issue to boost Ca concentration in this grain to improve Ca deficiency risk,but the mechanisms underlying Ca accumulation are poorly ***,we obtained a rice(Oryza sativa)mutant with high shoot Ca *** mutant exhibited 26%-53% higher Ca in shoots than did wild-type rice(WT)at different Ca *** concentration in the xylem sap was 36% higher in the mutant than in the *** was no difference in agronomic traits between the WT and mutant,but the mutant showed 25% higher Ca in the polished grain compared with the ***-based cloning combined with a complementation test revealed that the mutant phenotype was caused by an 18-bp deletion of a gene,OsK5.2,belonging to the Shaker-like K+channel ***5.2 was highly expressed in the mature region of the roots and its expression in the roots was not affected by Ca levels,but upregulated by low *** showed that OsK5.2 was mainly expressed in the pericycle of the *** together,our results revealed a novel role for OsK5.2 in Ca translocation in rice,and will be a good target for Ca biofortification in rice.

关键词： accumulation biofortification calcium OsK5.2 pericycle rice translocation

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Dissolved organic matter accelerates microbial degradation of 17 alpha-ethinylestradiol in the presence of iron mineral

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Journal of Environmental sciences 2024年第5期139卷 364-376页

作者： Huan He Min Shi Xiaoxia Yang Juhong Zhan Yanting Lin Ziwei Guo Zhicheng Liao Chaochao Lai Xiaomin Ren Bin Huang Xuejun Pan Faculty of Environmental science and Engineering Kunming University of Science and TechnologyKunming 650500China Yunnan Provincial Key lab.of Soil Carbon Sequestration and Pollution Control Kunming 650500China Research Institute for Environmental Innovation(Suzhou)Tsinghua Suzhou 215163China

Dissolved organic matter(DOM)and iron minerals widely existing in the natural aquatic environment can mediate the migration and transformation of organic ***,the mechanism of interaction between DOM and iron minerals in the microbial degradation of pollutants deserves further *** this study,the mechanism of 17 alphaethinylestradiol(EE2)biodegradation mediated by humic acid(HA)and three kinds of iron minerals(goethite,magnetite,and pyrite)was *** results found that HA and iron minerals significantly accelerated the biodegradation process of EE2,and the highest degradation efficiency of EE2(48%)was observed in the HA-mediated microbial system with pyrite under aerobic ***,it had been demonstrated that hydroxyl radicals(HO·)was the main active substance responsible for the microbial degradation of ***·is primarily generated through the reaction between hydrogen peroxide secreted by microorganisms and Fe(II),with aerobic conditions being more *** presence of iron minerals and HA could change the microbial communities in the EE2 biodegradation *** findings provide new information for exploring the migration and transformation of pollutants by microorganisms in iron-rich environments.

关键词： Dissolved organic matter Iron mineral 17 alpha-ethinylestradiol Biodegradation

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Polarizable Additive with Intermediate Chelation Strength for Stable Aqueous Zinc‑Ion Batteries

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Nano-Micro Letters 2024年第5期16卷 41-55页

作者： Yuting Xia Rongao Tong Jingxi Zhang Mingjie Xu Gang Shao Hailong Wang Yanhao Dong Chang‑An Wang State Key lab.of New Ceramics and Fine Processing School of Materials Science and EngineeringTsinghua UniversityBeijing 100084People’s Republic of China School of Materials science and Engineering Zhengzhou UniversityZhengzhou 450001HenanPeople’s Republic of China

Aqueous zinc-ion batteries are promising due to inherent safety,low cost,low toxicity,and high volumetric ***,issues of dendrites and side reactions between zinc metal anode and the electrolyte need to be solved for extended storage and cycle ***,we proposed that an electrolyte additive with an intermediate chelation strength of zinc ion—strong enough to exclude water molecules from the zinc metal-electrolyte interface and not too strong to cause a significant energy barrier for zinc ion dissociation—can benefit the electrochemical stability by suppressing hydrogen evolution reaction,overpotential growth,and den-drite ***-sodium diethylene-triaminepentaacetic acid salt was selected for such a *** has a suitable chelating ability in aqueous solutions to adjust solvation sheath and can be readily polarized under electrical loading conditions to further improve the ***||Zn symmetric cells can be stably operated over 3500 h at 1 mA cm^(-2).Zn||NH4V4O10 full cells with the additive show great cycling stability with 84.6%capacity retention after 500 cycles at 1 A g^(-1).Since the additive not only reduces H2 evolution and corrosion but also modifies Zn2+diffusion and deposition,highlyreversible Zn electrodes can be achieved as verified by the experimental *** work offers a practical approach to the logical design of reliable electrolytes for high-performance aqueous batteries.

关键词： Aqueous zinc-ion batteries Electrolyte additives DTPA-Na Chelation strength

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Mitophagy in intracerebral hemorrhage:a new target for therapeutic intervention

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Neural Regeneration Research 2024年第2期19卷 316-323页

作者： Yiyang Chen Wenxuan Tang Xinqi Huang Yumei An Jiawen Li Shengye Yuan Haiyan Shan Mingyang Zhang Institute of Forensic sciences Suzhou Medical CollegeSoochow UniversitySuzhouJiangsu ProvinceChina Shanghai Key lab.of Forensic Medicine Key Lab of Forensic ScienceMinistry of Justice(Academy of Forensic Science)ShanghaiChina Department of Obstetrics and Gynecology The Affiliated Suzhou Hospital of Nanjing Medical UniversitySuzhouJiangsu ProvinceChina

Intracerebral hemorrhage is a life-threatening condition with a high fatality rate and severe ***,there is currently no treatment availab.e for intracerebral hemorrhage,unlike for other stroke *** studies have indicated that mitochondrial dysfunction and mitophagy likely relate to the pathophysiology of intracerebral ***,or selective autophagy of mitochondria,is an essential pathway to preserve mitochondrial homeostasis by clearing up damaged *** markedly contributes to the reduction of secondary brain injury caused by mitochondrial dysfunction after intracerebral *** review provides an overview of the mitochondrial dysfunction that occurs after intracerebral hemorrhage and the underlying mechanisms regarding how mitophagy regulates it,and discusses the new direction of therapeutic strategies targeting mitophagy for intracerebral hemorrhage,aiming to determine the close connection between mitophagy and intracerebral hemorrhage and identify new therapies to modulate mitophagy after intracerebral *** conclusion,although only a small number of drugs modulating mitophagy in intracerebral hemorrhage have been found thus far,most of which are in the preclinical stage and require further investigation,mitophagy is still a very valid and promising therapeutic target for intracerebral hemorrhage in the long run.

关键词： intracerebral hemorrhage mitochondrial dysfunction mitophagy neuroinflammation neuroprotection reactive oxygen species secondary brain injury therapeutic target

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Induced CTL-S15 gene expression by Bacillus thuringiensis declines susceptibility in Spodoptera exigua

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Journal of Integrative Agriculture 2024年第9期23卷 3078-3088页

作者： Jianqiang Bao Yuxuan Chen Suwan Jiang Rui Liu Xi Zhang Fangzheng Zhang Zhiwei Chen Chen Luo Hailong Kong College of plant Protection Yangzhou UniversityYangzhou 225009China

It has been reported that C-type lectins(CTLs),which are pattern recognition receptors of the insect innate immunity response,may compete with Cry toxin for the receptor alkaline phosphatase to decrease its toxicity in ***,to date,which CTLs affect larval susceptibility to Bt in Spodoptera exigua is not *** this study,33 CTL genes were identified from *** on the number of carbohydrate-recognition domains(CRDs)and the domain architectures,they were classified into three groups:(1)nineteen CTL-S(single-CRD),(2)eight immulectin(dual-CRD)and(3)six CTL-X(CRD with other domains).RT-qPCR analysis revealed that expression levels of SeCTL-S15,IML-4 and CTL-X6 were upregulated after challenge with Bt and *** and developmental stage expression analysis showed that only SeCTL-S15 was mainly expressed in the midgut and larva,*** of SeCTL-S15 significantly increased Bt susceptibility,as indicated by reduced survival and larval *** results suggest that CTL-S15 might play a vital role in the low susceptibility of larvae to Bt in *** results provide new insights into CTL function in insects.

关键词： Spodoptera exigua Bacillus thuringiensis susceptibility C-type lectins

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Community structure and carbon metabolism functions of bacterioplankton in the Guangdong coastal zone

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Marine Life science & Technology 2024年第3期6卷 547-561页

作者： Ziqi Peng Pandeng Wang Xiaoqing Luo Qiqi Deng Ziwen Yang Jiaxue Wu Wendong Xian Weicong Yan Xiaozhen Mou Yang Yuan Wenjun Li Jialing Li State Key lab.ratory of Biocontrol Guangdong Provincial Key Laboratory of Plant Stress BiologySchool of Marine SciencesSchool of EcologySchool of Life SciencesSun Yat-Sen UniversityGuangzhou 510275China Southern Marine science and Engineering Guangdong lab.ratory(Guangzhou) Guangzhou 511458China Department of Biological sciences Kent State UniversityKentOH 44242USA Marine Microorganism Ecological&Application lab Marine Science and Technology CollegeZhejiang Ocean UniversityZhoushan 316000China

Coastal ecosystems are an important region for biogeochemical cycling,are a hotspot of anthropogenic disturbance and play a crucial role in global carbon cycling through the metabolic activities of *** can be broadly classified into two lifestyles:free-living(FL)and particle-attached(PA).However,how coastal bacterioplankton the community structure,co-occurrence networks and carbon metabolic functions with different lifestyles are differentiated is still largely *** these processes is necessary to better determine the contributions of coastal bacterioplankton to carbon ***,the characteristics of community structure and carbon metabolism function of bacterioplankton with two lifestyles in the coastal areas of Guangdong Province were investigated using amplicon sequencing,metagenomic,and metatranscriptomic *** results show that the main bacterioplankton responsible for carbon metabolism were the Pseudomonadota,Bacteroidota,and *** microbial community structure,carbon metabolic function,and environmental preferences differ between different *** and PA bacteria exhibited higher carbon fixation and degradation potentials,respectively.A range of environmental factors,such as dissolved oxygen,pH,and temperature,were associated with the community structure and carbon metabolic functions of the *** activities,such as nutrient discharge,may affect the distribution of functional genes and enhance the carbon degradation functions of *** conclusion,this study increased the understanding of the role of microorganisms in regulating carbon export in coastal ecosystems with intense human activity.

关键词： Particle-attached and free-living bacterioplankton Community structure Carbon metabolism Coastal ecosystem

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The NAC056 transcription factor confers freezing tolerance by positively regulating expression of CBFs and NIA1 in Arabidopsis

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plant Communications 2024年第7期5卷 251-260页

作者： Peipei Xu Wei Ma Huafeng Feng Weiming Cai lab.ratory of Photosynthesis and Environment CAS Center for Excellence in Molecular Plant SciencesShanghai Institute of Plant Physiology and EcologyChinese Academy of SciencesNo.300 Fenglin RoadShanghai 200032China State Key lab.ratory of NBC Protection for Civilian Beijing 102205China Department of Food science College of Hospitality ManagementShanghai Business SchoolShanghai 200235China These authors contributed equally to this article

Freezing stress can seriously affect plant growth and development,but the mechanisms of these effects and plant responses to freezing stress require further ***,we identified a NAM,ATAF1/2,and CUC2(NAC)-family transcription factor(TF),NAC056,that can promote freezing tolerance in ***056 mRNA levels are strongly induced by freezing stress in roots,and the nac056 mutant exhibits compromised freezing ***056 acts positively in response to freezing by directly promoting key C-repeat-binding factor(CBF)pathway ***,we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants;therefore,NAC056–CBF1–NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing *** addition,35S::NAC056 transgenic plants show enhanced freezing tolerance,which is partially reversed in the cbfs triple ***,NAC056 confers freezing tolerance through the CBF pathway,mediating plant responses to balance growth and freezing stress tolerance.

关键词： CBFs freezing tolerance NAC transcription factor nitrate assimilation root growth

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