Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute(Corchorus capsularis)

Jute(Corchorus capsularis L.)is the second most important natural plant fiber source after cotton.However,developing an efficient gene editing system for jute remains a challenge.In this study,the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4,an elite jute variety widely cultivated in China.The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation(BiFC)assays.The CHLOROPLASTOS ALTERADOS 1(CcCLA1)gene,which is involved in the development of chloroplasts,was targeted for editing at two sites in Meifeng 4.Based on this hairy root transformation,the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and-GhU6.9 promoters,respectively,to assess the efficiency of gene editing.Results indicated the 50.0%(GhU6.7)and 38.5%(GhU6.9)editing events in the target 2 alleles(gRNA2),but no mutation was detected in the target 1 allele(gRNA1)in transgenic-positive hairy roots.CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation.Two CcCLA1 mutants were albinic,with a gene editing efficiency of 5.3%.These findings confirm that the CRISPR/Cas9 system,incorporating promoter GhU6.7,can be used as a gene editing tool for jute.

Comparison of Biolistic and Agrobacterium-mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.

Insight Into the Separation-of-Variable Methods for the Closed-Form Solutions of Free Vibration of Rectangular Thin Plates

The separation-of-variable(SOV)methods,such as the improved SOV method,the variational SOV method,and the extended SOV method,have been proposed by the present authors and coworkers to obtain the closed-form analytical solutions for free vibration and eigenbuckling of rectangular plates and circular cylindrical shells.By taking the free vibration of rectangular thin plates as an example,this work presents the theoretical framework of the SOV methods in an instructive way,and the bisection–based solution procedures for a group of nonlinear eigenvalue equations.Besides,the explicit equations of nodal lines of the SOV methods are presented,and the relations of nodal line patterns and frequency orders are investigated.It is concluded that the highly accurate SOV methods have the same accuracy for all frequencies,the mode shapes about repeated frequencies can also be precisely captured,and the SOV methods do not have the problem of missing roots as well.

Estimation of Chloride Diffusivity in Hydrated Tricalcium Silicate Using a Hydration-Diffusion Integrated Method

This study aims to develop a chloride diffusion simulation method that considers the hydration microstructure and pore solution properties during the hydration of tricalcium silicate(C3S).The method combines the hydration simulation,thermodynamic calculation,and finite element analysis to examine the effects of pore solution,including effect of electrochemical potential,effect of chemical activity,and effect of mechanical interactions between ions,on the chloride effective diffusion coefficient of hydrated C3S paste.The results indicate that the effect of electrochemical potential on chloride diffusion becomes stronger with increasing hydration age due to the increase in the content of hydrated calcium silicate;as the hydration age increases,the effect of chemical activity on chloride diffusion weakens when the number of diffusible elements decreases;the effect of mechanical interactions between ions on chloride diffusion decreases with the increase of hydration age.

Structural Modal Parameter Recognition and Related Damage Identification Methods under Environmental Excitations: A Review

Modal parameters can accurately characterize the structural dynamic properties and assess the physical state of the structure.Therefore,it is particularly significant to identify the structural modal parameters according to the monitoring data information in the structural health monitoring(SHM)system,so as to provide a scientific basis for structural damage identification and dynamic model modification.In view of this,this paper reviews methods for identifying structural modal parameters under environmental excitation and briefly describes how to identify structural damages based on the derived modal parameters.The paper primarily introduces data-driven modal parameter recognition methods(e.g.,time-domain,frequency-domain,and time-frequency-domain methods,etc.),briefly describes damage identification methods based on the variations of modal parameters(e.g.,natural frequency,modal shapes,and curvature modal shapes,etc.)and modal validation methods(e.g.,Stability Diagram and Modal Assurance Criterion,etc.).The current status of the application of artificial intelligence(AI)methods in the direction of modal parameter recognition and damage identification is further discussed.Based on the pre-vious analysis,the main development trends of structural modal parameter recognition and damage identification methods are given to provide scientific references for the optimized design and functional upgrading of SHM systems.

Effects of spatial heterogeneity on pseudo-static stability of coal mine overburden dump slope,using random limit equilibrium and random finite element methods:A comparative study

Sudden and unforeseen seismic failures of coal mine overburden(OB)dump slopes interrupt mining operations,cause loss of lives and delay the production of coal.Consideration of the spatial heterogeneity of OB dump materials is imperative for an adequate evaluation of the seismic stability of OB dump slopes.In this study,pseudo-static seismic stability analyses are carried out for an OB dump slope by considering the material parameters obtained from an insitu field investigation.Spatial heterogeneity is simulated through use of the random finite element method(RFEM)and the random limit equilibrium method(RLEM)and a comparative study is presented.Combinations of horizontal and vertical spatial correlation lengths were considered for simulating isotropic and anisotropic random fields within the OB dump slope.Seismic performances of the slope have been reported through the probability of failure and reliability index.It was observed that the RLEM approach overestimates failure probability(P_(f))by considering seismic stability with spatial heterogeneity.The P_(f)was observed to increase with an increase in the coefficient of variation of friction angle of the dump materials.Further,it was inferred that the RLEM approach may not be adequately applicable for assessing the seismic stability of an OB dump slope for a horizontal seismic coefficient that is more than or equal to 0.1.

Design of Voltage Equalization Circuit and Control Method for Lithium-ion Battery Packs

The active equalization of lithium-ion batteries involves transferring energy from high-voltage cells to low-voltage cells,ensuring consistent voltage levels across the battery pack and maintaining safety.This paper presents a voltage balancing circuit and control method.First,a single capacitor method is used to design the circuit topology for energy transfer.Next,real-time voltage detection and control are employed to balance energy between cells.Finally,simulation and experimental results demonstrate the effectiveness of the proposed method,achieving balanced voltages of 3.97 V from initial voltages of 4.10,3.97,and 3.90 V.The proposed circuit is simple,reliable,and effectively prevents overcharge and overdischarge.

An Improved Local RBF Collocation Method for 3D Excavation Deformation Based on Direct Method and Mapping Technique

Since the plasticity of soil and the irregular shape of the excavation,the efficiency and stability of the traditional local radial basis function(RBF)collocation method(LRBFCM)are inadequate for analyzing three-dimensional(3D)deformation of deep excavation.In this work,the technique known as the direct method,where the local influence nodes are collocated on a straight line,is introduced to optimize the LRBFCM.The direct method can improve the accuracy of the partial derivative,reduce the size effect caused by the large length-width ratio,and weaken the influence of the shape parameters on the LRBFCM.The mapping technique is adopted to transform the physical coordinates of a quadratic-type block to normalized coordinates,in which the deformation problem can easily be solved using the direct method.The stability of the LRBFCM is further modified by considering the irregular shape of 3D excavation,which is divided into several quadratic-type blocks.The soil’s plasticity is described by the Drucker-Prager(D-P)model.The improved LRBFCM is integrated with the incremental method to analyze the plasticity.Five different examples,including strip excavations and circular excavations,are presented to validate the proposed approach’s efficiency.

Assessment of leachate-contaminated clays using experimental and artificial methods

The investigation of leachate-contaminated clay(LCC)is essential for landfill engineering assessment and achievement of sustainable development goals.Several static and dynamic laboratory tests,including unconfined compressive strength(UCS),California bearing ratio(CBR),and cyclic simple shear,are conducted.Cyclic simple shear experiments on LCCs were performed to evaluate the damping and shear modulus.The investigated factors are vertical load(VL),leachate content(LC),frequency(F),and shear strain(ShS)for LCC.Forensic-based investigation optimization(FBIO)and equilibrium optimizer algorithm(EOA)were utilized in addition to multiple types of ensemble models,including adaptive boosting(ADB),gradient boosting regression tree(GBRT),extreme gradient boosting(XGB) and random forest(RF).The comparison of the methods showed that GBRT-FBIO and XGB-EOA models outperformed other models for shear modulus and damping of LCC.The p-value less than 0.0001 shows the significance of the used models in the response surface methodology(RSM)method.

Hollow cerium nanoparticles synthesized by one-step method for multienzyme activity to reduce colitis in mice

BACKGROUND Inflammatory bowel disease(IBD)is a common chronic intestinal inflammatory disease.High oxidative stress is a treatment target for IBD.Cerium oxide(CeO2)nanomaterials as nanozymes with antioxidant activity are potential drugs for the treatment of colitis.AIM To synthesize hollow cerium(H-CeO2)nanoparticles by one-step method and to validate the therapeutic efficacy of H-CeO2 in IBD.METHODS H-CeO2 was synthesized by one-step method and examined its characterization and nanoenzymatic activity.Subsequently,we constructed dextran sulfate so-dium(DSS)-induced colitis in mice to observe the effects of H-CeO2 on colonic inflammation.The effects of H-CeO2 on colon inflammation and reactive oxygen species(ROS)levels in IBD mice were detected by hematoxylin and eosin staining and dichlorofluorescein diacetate staining,respectively.Finally,the biological sa-fety of H-CeO2 on mice was evaluated by hematoxylin and eosin staining,blood routine,and blood biochemistry.RESULTS H-CeO2 nanoparticles prepared by the one-step method were uniform,monodi-sperse and hollow.H-CeO2 had a good ability to scavenge ROS,∙OH and∙OOH.H-CeO2 reduced DSS-induced decreases in body weight and colon length,colonic epithelial damage,inflammatory infiltration,and ROS accumulation.H-CeO2 administration reduced the disease activity index of DSS-induced animals from about 8 to 5.H-CeO2 had no significant effect on body weight,total platelet count,hemoglobin,white blood cell,and red blood cell counts in healthy mice.No significant damage to major organs was observed in healthy mice following H-CeO2 administration.CONCLUSION The one-step synthesis of H-CeO2 nanomaterials had good antioxidant activity,biosafety,and inhibited deve-lopment of DSS-induced IBD in mice by scavenging ROS.

Retrospective comparative study of different surgical methods for gastric ulcer perforation:Efficacy and postoperative complications

BACKGROUND Gastric ulcer perforation is a critical condition that can lead to significant morbidity and mortality if not promptly addressed.It is often the result of chronic peptic ulcer disease,which is characterized by a breach in the gastric wall due to ulceration.Surgical intervention is essential for managing this life-threatening complication.However,the optimal surgical technique remains debatable among clinicians.Various methods have been employed,including simple closure,omental patch repair,and partial gastrectomy,each with distinct advantages and disadvantages.Understanding the comparative efficacy and postoperative outcomes of these techniques is crucial for improving patient care and surgical decision-making.This study addresses the need for a comprehensive analysis in this area.AIM To compare the efficacy and postoperative complications of different surgical methods for the treatment of gastric ulcer perforation.METHODS A retrospective analysis was conducted on 120 patients who underwent surgery for gastric ulcer perforation between September 2020 and June 2023.The patients were divided into three groups based on the surgical method:Simple closure,omental patch repair,and partial gastrectomy.The primary outcomes were the operative success rate and incidence of postoperative complications.Secondary outcomes included the length of hospital stay,recovery time,and long-term quality of life.RESULTS The operative success rates for simple closure,omental patch repair,and partial gastrectomy were 92.5%,95%,and 97.5%,respectively.Postoperative complications occurred in 20%,15%,and 17.5%of patients in each group,respectively.The partial gastrectomy group showed a significantly longer operative time(P<0.001)but the lowest rate of ulcer recurrence(2.5%,P<0.05).The omental patch repair group demonstrated the shortest hospital stay(mean 7.2 days,P<0.05)and fastest recovery time.CONCLUSION While all three surgical methods showed high success rates,omental patch repair demonstrated the best overall outcomes,with a balance of high efficacy,low complication rates,and shorter recovery time.However,the choice of the surgical method should be tailored to individual patient factors and the surgeon’s expertise.

Towards a blank design method for manufacturing big-tapered profiled ring disk by spinning-rolling process

The big-tapered profiled ring disk is a key component of engines for rockets and missiles.A new forming technology,as called spinning-rolling process,has been proposed previously for the high performance,high efficiency and low-cost manufacturing of the component.Blank design is the key part of plastic forming process design.For spinning-rolling process,the shape and size of the blank play a crucial role in process stability,deformation behavior and dimensional accuracy.So this work proposes a blank design method to determine the geometry structure and sizes of the blank.The mathematical model for calculating the blank size has been deduced based on volume conservation and neutral layer length invariance principle.The FE simulation and corresponding trial production of an actual big-tapered profiled ring disk show that the proposed blank design method is applicative.In order to obtain a preferred blank,the influence rules of blank size determined by different deformation degrees(rolling ratio k)on the spinning-rolling process are revealed by comprehensive FE simulations.Overall considering the process stability,circularity of the deformed ring disk and forming forces,a reasonable range of deformation degree(rolling ratio k)is recommended for the blank design of the new spinning-rolling process.

Sensitivity Analysis of Structural Dynamic Behavior Based on the Sparse Polynomial Chaos Expansion and Material Point Method

This paper presents a framework for constructing surrogate models for sensitivity analysis of structural dynamics behavior.Physical models involving deformation,such as collisions,vibrations,and penetration,are devel-oped using the material point method.To reduce the computational cost of Monte Carlo simulations,response surface models are created as surrogate models for the material point system to approximate its dynamic behavior.An adaptive randomized greedy algorithm is employed to construct a sparse polynomial chaos expansion model with a fixed order,effectively balancing the accuracy and computational efficiency of the surrogate model.Based on the sparse polynomial chaos expansion,sensitivity analysis is conducted using the global finite difference and Sobol methods.Several examples of structural dynamics are provided to demonstrate the effectiveness of the proposed method in addressing structural dynamics problems.

Agrobacterium-mediated Transformation of Kentucky Bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

Agrobacterium-Mediated Transformation of Rice: Constraints and Possible Solutions

Genetic transformation of rice(Oryza sativa L.) by introducing beneficial traits is now a central research instrument in plant physiology and a practical tool for plant improvement. Many approaches are verified for stable introduction of foreign genes into the plant genome. The review examined the different constraints that limit the success of rice genetic transformation via Agrobacterium-mediated approach and suggested possible solutions. Explant identification, gene transfer technique and construct to tailor the integration, transgene expression without collateral to genetic damage and transformant selection are among the technical challenges affecting the rice transformation. Despite the contests, Agrobacteriummediated transformation system has been a better option for producing transgenic rice varieties because of its exact T-DNA processing and simple integration of low copy-number transgene. This information is necessary for improving the transformation system for recalcitrant rice varieties.

Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon (Citrus Limon (L.) Burm. F. cv. Eureka)

The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.

In planta Agrobacterium-Mediated Transformation of Rice

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog(MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD_(600) = 0.8–1.0 and the co-cultivation at 25 ℃ for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gus A gene by PCR using the gus A specific primers in the T_0 lines, there were 4 transgenic lines from 286 T_0 lines(1.4% transformation efficiency). However, histochemical glucuronidase(GUS) assay demonstrated that only three of four transgenic lines exhibited gus A expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

Effect of Phytosulfokine-α on Agrobacterium-Mediated Transformation in Rice

Phytosulfokine- α （PSK- α ）, a biologically active peptide acting as a growth factor, plays a key role in cellular differentiation and proliferation. To test if PSK- α has some influence on agrobacterium-mediated transformation in rice, PSK-α at a series of concentrations was added into co-culture medium respectively. The results showed that PSK- α indeed affected the recovery of resistant calli and the transformation frequency of rice varieties Taipei 309 and Lijiangxintuanheigu, PSK- α at the concentration of 10 nmol/L could increase induction of resistant callus and efficiency of transformation, with a 11% and 4.9% top increase, respectively than the control. However, PSK- αat 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effect of PSK-α on agrobacterium-mediated transformation is related with the concentration of 2, 4-D in selection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSK- α plus 2 mg/L 2, 4-D.

Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea(Pisum sativum L.)

Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.