Generating Marker-Free Transgenic Tobacco Plants by Agrobacteriummediated Transformation with Double T-DNA Binary Vector

We have developed a 'double T-DNA' binary vector system for generating selectable marker-free transgenic plants by Agrobacterium-mediated transformation. The 'double T-DNA' binary vector pDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin phosphotransferase (nptII) gene and the other a bargene, was constructed. Transgenic tobacco (Nicotiana tabacum L.) plants were then produced by Agrobacterium-mediated transformation with this vector. Frequency of the primary transformants co-integrated with npt II gene and bar gene was 59.2%. Segregation of two T-DNA regions was found in 3 out of 4 T-1 lines from co-transformed T-0 plants with nptII and bar PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T-1 plants. The result indicated that this 'double T-DNA' vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptII gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested. Frequency of co-transformed plants was 20.0%-47.7% and relatively low as compared with that of 'double T-DNA' vector system.

Stage IV malignant transformation of mature cystic teratoma palliatively treated with concurrent chemoradiotherapy:A case report

BACKGROUND Malignant transformation(MT)of mature cystic teratoma(MCT)has a poor prognosis,especially in advanced cases.Concurrent chemoradiotherapy(CCRT)has an inhibitory effect on MT.CASE SUMMARY Herein,we present a case in which CCRT had a reduction effect preoperatively.A 73-year-old woman with pyelonephritis was referred to our hospital.Computed tomography revealed right hydronephrosis and a 6-cm pelvic mass.Endoscopic ultrasound-guided fine-needle biopsy(EUS-FNB)revealed squamous cell carci-noma.The patient was diagnosed with MT of MCT.Due to her poor general con-dition and renal malfunction,we selected CCRT,expecting fewer adverse effects.After CCRT,her performance status improved,and the tumor size was reduced;surgery was performed.Five months postoperatively,the patient developed dis-semination and lymph node metastases.Palliative chemotherapy was ineffective.She died 18 months after treatment initiation.CONCLUSION EUS-FNB was useful in the diagnosis of MT of MCT;CCRT suppressed the disea-se and improved quality of life.

Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Development of Marker-Free Transgenic Cry1Ab Rice with Lepidopteran Pest Resistance by Agrobacterium Mixture-Mediated Co-transformation

CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.

Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations

Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization(FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.

Development and drought tolerance assay of marker-free transgenic rice with OsAPX2 using biolistic particle-mediated co-transformation

Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.

Efficiencies of Generating Selectable Marker-Free Transgenic Rice with Different Transformation Methods

To study the efficiency of generating selectable marker-free （SMF） transgenic rice, two transformation methods were employed for four rice varieties （Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97）. One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions （one carrying an antisense Wx gene and the other carrying a HPTgene）. The other one, named as two-strain/two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four rice varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.

Hemorrhagic transformation in patients with large-artery atherosclerotic stroke is associated with the gut microbiota and lipopolysaccharide

Hemorrhagic transformation is a major complication of large-artery atheroscle rotic stroke(a major ischemic stro ke subtype)that wo rsens outcomes and increases mortality.Disruption of the gut microbiota is an important feature of stroke,and some specific bacteria and bacterial metabolites may contribute to hemorrhagic transformation pathogenesis.We aimed to investigate the relationship between the gut microbiota and hemorrhagic transformation in largearte ry atheroscle rotic stro ke.An observational retrospective study was conducted.From May 2020 to September 2021,blood and fecal samples were obtained upon admission from 32 patients with first-ever acute ischemic stroke and not undergoing intravenous thrombolysis or endovascular thrombectomy,as well as 16 healthy controls.Patients with stro ke who developed hemorrhagic transfo rmation(n=15)were compared to those who did not develop hemorrhagic transformation(n=17)and with healthy controls.The gut microbiota was assessed through 16S ribosomal ribonucleic acid sequencing.We also examined key components of the lipopolysaccharide pathway:lipopolysaccharide,lipopolysaccharide-binding protein,and soluble CD14.We observed that bacterial diversity was decreased in both the hemorrhagic transformation and non-hemorrhagic transfo rmation group compared with the healthy controls.The patients with ischemic stro ke who developed hemorrhagic transfo rmation exhibited altered gut micro biota composition,in particular an increase in the relative abundance and dive rsity of members belonging to the Enterobacteriaceae family.Plasma lipopolysaccharide and lipopolysaccharide-binding protein levels were higher in the hemorrhagic transformation group compared with the non-hemorrhagic transfo rmation group.lipopolysaccharide,lipopolysaccharide-binding protein,and soluble CD14 concentrations were associated with increased abundance of Enterobacte riaceae.Next,the role of the gut microbiota in hemorrhagic transformation was evaluated using an experimental stroke rat model.In this model,transplantation of the gut microbiota from hemorrhagic transformation rats into the recipient rats triggered higher plasma levels of lipopolysaccharide,lipopolysaccharide-binding protein,and soluble CD14.Ta ken togethe r,our findings demonstrate a noticeable change in the gut microbiota and lipopolysaccharide-related inflammatory response in stroke patients with hemorrhagic transformation.This suggests that maintaining a balanced gut microbiota may be an important factor in preventing hemorrhagic transfo rmation after stro ke.

Formation and transformation of metastable LPSO building blocks clusters in Mg-Gd-Y-Zn-Zr alloys by spinodal decomposition and heterogeneous nucleation

To study the formation and transformation mechanism of long-period stacked ordered(LPSO)structures,a systematic atomic scale analysis was conducted for the structural evolution of long-period stacked ordered(LPSO)structures in the Mg-Gd-Y-Zn-Zr alloy annealed at 300℃~500℃.Various types of metastable LPSO building block clusters were found to exist in alloy structures at different temperatures,which precipitate during the solidification and homogenization process.The stability of Zn/Y clusters is explained by the first principles of density functional theory.The LPSO structure is distinguished by the arrangement of its different Zn/Y enriched LPSO structural units,which comprises local fcc stacking sequences upon a tightly packed plane.The presence of solute atoms causes local lattice distortion,thereby enabling the rearrangement of Mg atoms in the different configurations in the local lattice,and local HCP-FCC transitions occur between Mg and Zn atoms occupying the nearest neighbor positions.This finding indicates that LPSO structures can generate necessary Schockley partial dislocations on specific slip surfaces,providing direct evidence of the transition from 18R to 14H.Growth of the LPSO,devoid of any defects and non-coherent interfaces,was observed separately from other precipitated phases.As a result,the precipitation sequence of LPSO in the solidification stage was as follows:Zn/Ycluster+Mg layers→various metastable LPSO building block clusters→18R/24R LPSO;whereas the precipitation sequence of LPSO during homogenization treatment was observed to be as follows:18R LPSO→various metastable LPSO building block clusters→14H LPSO.Of these,14H LPSO was found to be the most thermodynamically stable structure.

Quantitative analysis of microstructure evolution,stress partitioning and thermodynamics in the dynamic transformation of Fe-14Ni alloy

Dynamic transformation(DT)of austenite(γ)to ferrite(α)in the hot deformation of various carbon steels was widely investigated.However,the nature of DT remains unclear due to the lack of quantitative analysis of stress partitioning between two phases and the uncertainty of local distribution of substitu-tional elements at the interface in multi-component carbon steels used in the previous studies.Therefore,in the present study,a binary Fe-Ni alloy withα+γduplex microstructure in equilibrium was prepared and isothermally compressed inα+γtwo-phase region to achieve a quantitative analysis of microstruc-ture evolution,stress partitioning,and thermodynamics during DT.γtoαDT during isothermal compres-sion andαtoγreverse transformation on isothermal annealing under unloaded condition after deforma-tion were accompanied by Ni partitioning.The lattice strains during thermomechanical processing were obtained via in-situ neutron diffraction measurement,based on which the stress partitioning behavior betweenγandαwas discussed by using the generalized Hooke’s law.A thermodynamic framework for the isothermal deformation in solids was established based on the basic laws of thermodynamics,and it was shown that the total Helmholtz free energy change in the deformable material during the isothermal process should be smaller than the work done to the deformable material.Under the present thermody-namic framework,the microstructure evolution in the isothermal compression of Fe-14Ni alloy was well explained by considering the changes in chemical free energy,plastic and elastic energies,and the work done to the material.In addition,the stabilization of the softαphase in Fe-14Ni alloy by deformation was rationalized since theγtoαtransformation decreased the total Helmholtz free energy by decreasing the elastic and dislocation energies.

Defect Detection Model Using Time Series Data Augmentation and Transformation

Time-series data provide important information in many fields,and their processing and analysis have been the focus of much research.However,detecting anomalies is very difficult due to data imbalance,temporal dependence,and noise.Therefore,methodologies for data augmentation and conversion of time series data into images for analysis have been studied.This paper proposes a fault detection model that uses time series data augmentation and transformation to address the problems of data imbalance,temporal dependence,and robustness to noise.The method of data augmentation is set as the addition of noise.It involves adding Gaussian noise,with the noise level set to 0.002,to maximize the generalization performance of the model.In addition,we use the Markov Transition Field(MTF)method to effectively visualize the dynamic transitions of the data while converting the time series data into images.It enables the identification of patterns in time series data and assists in capturing the sequential dependencies of the data.For anomaly detection,the PatchCore model is applied to show excellent performance,and the detected anomaly areas are represented as heat maps.It allows for the detection of anomalies,and by applying an anomaly map to the original image,it is possible to capture the areas where anomalies occur.The performance evaluation shows that both F1-score and Accuracy are high when time series data is converted to images.Additionally,when processed as images rather than as time series data,there was a significant reduction in both the size of the data and the training time.The proposed method can provide an important springboard for research in the field of anomaly detection using time series data.Besides,it helps solve problems such as analyzing complex patterns in data lightweight.

A new insight into LPSO phase transformation and mechanical properties uniformity of large-scale Mg-Gd-Y-Zn-Zr alloy prepared by multi-pass friction stir processing

A large-scale fine-grained Mg-Gd-Y-Zn-Zr alloy plate with high strength and ductility was successfully prepared by multi-pass friction stir processing(MFSP)technology in this work.The structure of grains and long period stacking ordered(LPSO)phase were characterized,and the mechanical properties uniformity was investigated.Moreover,a quantitative relationship between the microstructure and tensile yield strength was established.The results showed that the grains in the processed zone(PZ)and interfacial zone(IZ)were refined from 50μm to 3μm and 4μm,respectively,and numerous original LPSO phases were broken.In IZ,some block-shaped 18R LPSO phases were transformed into needle-like 14H LPSO phases due to stacking faults and the short-range diffusion of solute atoms.The severe shear deformation in the form of kinetic energy caused profuse stacking fault to be generated and move rapidly,greatly increasing the transformation rate of LPSO phase.After MFSP,the ultimate tensile strength,yield strength and elongation to failure of the large-scale plate were 367 MPa,305 MPa and 18.0% respectively.Grain refinement and LPSO phase strengthening were the major strengthening mechanisms for the MFSP sample.In particularly,the strength of IZ was comparable to that of PZ because the strength contribution of the 14H LPSO phase offsets the lack of grain refinement strengthening in IZ.This result opposes the widely accepted notion that IZ is a weak region in MFSP-prepared large-scale fine-grained plate.

Intrahepatic portal venous systems in adult patients with cavernous transformation of portal vein: Imaging features and a new classification

Background: Cavernous transformation of the portal vein(CTPV) due to portal vein obstruction is a rare vascular anomaly defined as the formation of multiple collateral vessels in the hepatic hilum. This study aimed to investigate the imaging features of intrahepatic portal vein in adult patients with CTPV and establish the relationship between the manifestations of intrahepatic portal vein and the progression of CTPV. Methods: We retrospectively analyzed 14 CTPV patients in Beijing Tsinghua Changgung Hospital. All patients underwent both direct portal venography(DPV) and computed tomography angiography(CTA) to reveal the manifestations of the portal venous system. The vessels measured included the left portal vein(LPV), right portal vein(RPV), main portal vein(MPV) and the portal vein bifurcation(PVB). Results: Nine males and 5 females, with a median age of 40.5 years, were included in the study. No significant difference was found in the diameters of the LPV or RPV measured by DPV and CTA. The visualization in terms of LPV, RPV and PVB measured by DPV was higher than that by CTA. There was a significant association between LPV/RPV and PVB/MPV in term of visibility revealed with DPV( P = 0.01), while this association was not observed with CTA. According to the imaging features of the portal vein measured by DPV, CTPV was classified into three categories to facilitate the diagnosis and treatment. Conclusions: DPV was more accurate than CTA for revealing the course of the intrahepatic portal vein in patients with CTPV. The classification of CTPV, that originated from the imaging features of the portal vein revealed by DPV, may provide a new perspective for the diagnosis and treatment of CTPV.

ViTAU:基于Vision transformer和面部动作单元的面瘫识别与分析

面部神经麻痹(Facial nerve paralysis,FNP),通常称为贝尔氏麻痹或面瘫,对患者的日常生活和心理健康产生显著影响,面瘫的及时识别和诊断对于患者的早期治疗和康复至关重要.随着深度学习和计算机视觉技术的快速发展,面瘫的自动识别变得可行,为诊断提供了一种更准确和客观的方式.目前的研究主要集中关注面部的整体变化,而忽略了面部细节的重要性.面部不同部位对识别结果的影响力并不相同,这些研究尚未对面部各个区域进行细致区分和分析.本项研究引入结合Vision transformer(ViT)模型和动作单元(Action unit,AU)区域检测网络的创新性方法用于面瘫的自动识别及区域分析.ViT模型通过自注意力机制精准识别是否面瘫,同时,基于AU的策略从StyleGAN2模型提取的特征图中,利用金字塔卷积神经网络分析受影响区域.这一综合方法在YouTube Facial Palsy(YFP)和经过扩展的Cohn Kanade(CK+)数据集上的实验中分别达到99.4%的面瘫识别准确率和81.36%的面瘫区域识别准确率.通过与最新方法的对比,实验结果展示了所提的自动面瘫识别方法的有效性.

基于贝叶斯图注意力Transformer的航空发动机剩余使用寿命概率预测

航空发动机作为飞机的心脏,其健康状态对飞机的安全飞行至关重要.深度学习强大的数据挖掘能力,为通过海量历史数据预测航空发动机的剩余使用寿命提供了新方法.然而,传统基于深度学习的方法大都关注于挖掘数据在时间上的关联,而忽略了多个传感器监测数据之间复杂的非欧氏空间关系.此外,少有研究考虑数据或者预测过程本身具有的不确定性,缺乏对预测结果可靠性的评估.为解决上述问题,本文提出了一种基于贝叶斯网络和图注意力Transformer的航空发动机剩余使用寿命概率预测方法.将图注意力机制融入Transformer的时间多头注意力模块,结合图注意力网络在空间特征提取上的优势和Transformer模型在时间特征提取的优势,实现数据特征时空关系的联合提取.同时,利用改进的贝叶斯网络度量预测不确定性,在得到剩余使用寿命预测点值的同时给出相应的置信区间.最后,通过在公开航空发动机数据集上的实验,证明了所提模型的有效性和先进性.

Unraveling the nexus between cellular senescence and malignant transformation:a paradigm shift in cancer research

Cellular senescence, a natural process wherein cells cease division and undergo irreversible growth arrest, has long captivated the curiosity of scientists because of its many implications in aging and disease. Recent research has shed light on the nexus between cellular senescence and malignant transformation, thus leading to a paradigm shift in understanding cancer development and progression.

Effects of Biochar Inoculation with Bacillus megaterium on Rice Soil Phosphorus Fraction Transformation and Bacterial Community Dynamics

Rice husk biochar inoculated with Bacillus megaterium(BM)(referred to as BM-inoculated biochar, BMB) and uninoculated rice husk biochar(RHB) were added to soil at two rates(0.5%, as BMB1 and RHB1, respectively, and 1.0%, as BMB2 and RHB2, respectively) in an incubation experiment to comprehensively evaluate their effects on basic soil properties, phosphorus(P) fractions, bacterial community composition, and P-cycling genes.

Hydrogen-based mineral phase transformation mechanism investigation of pyrolusite ore

Pyrolusite comprises the foremost manganese oxides and is a major source of manganese production.An innovative hydrogenbased mineral phase transformation technology to pyrolusite was proposed,where a 96.44%distribution rate of divalent manganese(Mn^(2+))was observed at an optimal roasting temperature of 650℃,a roasting time of 25 min,and an H2 concentration of 20vol%;under these conditions.The manganese predominantly existed in the form of manganosite.This study investigated the generation mechanism of manganosite based on the reduction kinetics,phase transformation,and structural evolution of pyrolusite and revealed that high temperature improved the distribution rate,and the optimal kinetic model for the reaction was the random nucleation and growth model(reaction order,n=3/2)with an activation energy(E_(a))of 24.119 kJ·mol^(−1).Throughout the mineral phase transformation,manganese oxide from the outer layer of particles moves inward to the core.In addition,pyrolusite follows the reduction sequence of MnO_(2)→Mn_(2)O_(3)→Mn_(3)O_(4)→MnO,and the reduction of manganese oxides in each valence state simultaneously proceeds.These findings provide significant insight into the efficient and clean utilization of pyrolusite.

The nitrogen transformation behavior based on the pyrolysis products of wheat straw

In order to provide basic design parameters for the industrial pyrolysis process,the transformation behavior of nitrogen was investigated using wheat straw as raw material.The distributions of nitrogen in pyrolysis char,oil,and gas were obtained and the nitrogenous components in the products were analyzed systematically by X-ray photoelectron spectroscopy(XPS),pyrolysis-gas chromatography/mass spectrometry(Py-GC/MS)and thermogravimetric-Fourier transform infrared spectrometry(TG-FTIR).The nitrogen distribution ranges of the pyrolysis char,oil,and gas were 37.34%–54.82%,32.87%–40.94%and 10.20%–28.83%,respectively.More nitrogen was retained in char at lower pyrolysis temperature and the nitrogen distribution of oil was from rise to decline with increasing temperature.The most abundant N-containing compounds in three-phase products were pyrrole-N,amines,and HCN,respectively.In addition,the transformation mechanism of nitrogen from wheat straw to pyrolysis products was concluded.

Critical approaches in the catalytic transformation of sugar isomerization and epimerization after Fischer-History,challenges,and prospects

The transformation of aldose to ketose or common sugars into rare saccharides,including rare ketoses and aldoses,is of great value and interest to the food industry and for saccharidic biomass utilization,medicine,and the synthesis of drugs.Nowadays,high-fructose corn syrup(HFCS)is industrially produced in more than 10 million tons annually using immobilized glucose isomerase.Some low-calorie saccharides such as tagatose and psicose,which are becoming popular sweeteners,have also been produced on a pilot scale in order to replace sucrose and HFCS.However,current catalysts and catalytic processes are still difficult to utilize in biomass conversion and also have strong substrate dependence in producing high-value,rare sugars.Considering the specific reaction properties of saccharides and catalysts,since the pioneering discovery by Fischer,various catalysts and catalytic systems have been discovered or developed in attempts to extend the reaction pathways,improve the reaction efficiency,and to potentially produce commercial products.In this review,we trace the history of sugar isomerization/epimerization reactions and summarize the important breakthroughs for each reaction as well as the difficulties that remain unresolved to date.