Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

Design and Research of Active Gravity Unloading Device for Large Aperture Optical Mirror

The large aperture optical mirror for space is processed and tested in the gravity environment on the ground. After entering space, gravity disappears due to the change of environment, and the mirror surface that has met the engineering requirements on the ground will change, seriously affecting the imaging quality. In order to eliminate the influence of gravity and to ensure the consistency of space and ground, gravity unloading must be performed. In order to meet the requirements of processing and testing for the large aperture space mirror in the state of vertical optical axis, a universal gravity unloading device was proposed. It was an active support and used air cylinders to provide accurate unloading force. First, the design flow of gravity unloading was introduced;then the detailed design of the mechanical structure and control system was given;then the performance parameters of the two types of cylinders were tested and compared, including the force-pressure relationship curve and the force-position relationship curve;finally, the experimental verification of the gravity unloading device was carried out;for a mirror with an aperture of ?2100 mm, the gravity unloading device was designed and a vertical detection optical path was built. The test results showed that by using this gravity unloading device, the actual processing surface accuracy of the mirror was better than 1/50λ-RMS, which met the application requirement of the optical system. Thus, it can be seen that using this gravity unloading device can effectively unload the gravity of the mirror and realize the accurate processing and measurement of the mirror surface. .

3D Grid of Carbon Tubes with Mn3O4-NPs/CNTs Filled in their Inner Cavity as Ultrahigh-Rate and Stable Lithium Anode

Transition metal oxides are regarded as promising candidates of anode for next-generation lithium-ion batteries(LIBs)due to their ultrahigh theoretical capacity and low cost,but are restricted by their low conductivity and large volume expansion during Li^(+)intercalation.Herein,we designed and constructed a structurally integrated 3D carbon tube(3D-CT)grid film with Mn_(3)O_(4)nanoparticles(Mn_(3)O_(4)-NPs)and carbon nanotubes(CNTs)filled in the inner cavity of CTs(denoted as Mn_(3)O_(4)-NPs/CNTs@3D-CT)as high-performance free-standing anode for LIBs.The Mn_(3)O_(4)-NPs/CNTs@3D-CT grid with Mn_(3)O_(4)-NPs filled in the inner cavity of 3D-CT not only afford sufficient space to overcome the damage caused by the volume expansion of Mn_(3)O_(4)-NPs during charge and discharge processes,but also achieves highly efficient channels for the fast transport of both electrons and Li+during cycling,thus offering outstanding electrochemical performance(865 mAh g^(-1)at 1 A g^(-1)after 300 cycles)and excellent rate capability(418 mAh g^(-1)at 4 A g^(-1))based on the total mass of electrode.The unique 3D-CT framework structure would open up a new route to the highly stable,high-capacity,and excellent cycle and high-rate performance free-standing electrodes for highperformance Li-ion storage.

伴有肠母细胞分化的胃癌临床病理特征

目的:伴有肠母细胞分化的胃癌(gastric cancer with enteroblastic differentiation,GCED)是一种新的胃癌类型。该肿瘤表达胚胎干细胞标志物为SALL4以及癌胚蛋白(AFP和GPC3)等。本研究旨在探讨GCED的临床病理学特征及其分子表型。方法:回顾性分析2013年3月至2017年3月浙江大学医学院附属第一医院就诊的胃部肠型腺癌337例,其中8例可诊断为GCED,患者均为老年人,其中男性6例,女性2例,年龄68～83岁,平均年龄为76.6岁。2例治疗前血清AFP≥200μg/L。收集相关临床资料,根据其组织病理学形态,采用免疫组织化学法分析免疫学表型,应用逆转录PCR(RT-PCR)方法检测SALL4基因。结果:显微镜下观察,所有病例均具有原始的肠样结构,由立方或柱状细胞组成,胞质透明、清晰,免疫组织化学法检测显示AFP和GPC3阳性,或SALL4阳性。RT-PCR检测结果显示SALL4基因mRNA的表达水平显著增高。随访1～5年,其中5例出现肝及其他脏器转移,2例死亡,1例无瘤生存。结论:GCED是一种罕见的侵袭性肠型腺癌,预后比普通肠型腺癌差,按照普通肠型腺癌的治疗方法效果甚微。其组织学上具有特征性的改变,熟悉其瘤谱及基因学特征有助于鉴别和诊断,有助于对原始标记物,制定如SALL4的靶向治疗方案。

Medical simulation-based education improves medicos' clinical skills

Clinical skill is an essential part of clinical medicine and plays quite an important role in bridging medicos and physicians. Due to the realities in China, traditional medical education is facing many challenges. There are few opportunities for students to practice their clinical skills and their dexterities are generally at a low level. Medical simulation-based education is a new teaching modality and helps to improve medicos＇ clinical skills to a large degree. Medical simulation-based education has many significant advantages and will be further developed and applied.

蒙古冰草肉桂醇脱氢酶基因序列鉴定及功能分析

肉桂醇脱氢酶(CAD)作为植物次生代谢尤其是木质素生物合成过程的关键酶,在调控植物生长发育和抵御生物/非生物胁迫等过程中发挥关键作用。蒙古冰草(即沙芦草(Agropyron mongolicum))耐旱耐寒,在我国北方荒漠草原区域广泛分布。为探讨CAD基因在蒙古冰草木质素合成和非生物胁迫抗性中的作用,从蒙古冰草全长转录组数据中筛选并克隆到1个CAD基因,序列长度1083 bp,命名为AmCAD。该基因编码361个氨基酸残基,同源序列比对发现蛋白质序列保守区域含有2个Zn^(2+)结合基序和NADP(H)辅因子结合基序,属于典型的CAD蛋白,且三维结构与AtCAD5相似。AmCAD在茎秆中高表达,对AmCAD重组蛋白的酶学性质分析表明,该蛋白对不同肉桂醛类底物均具有很强的催化能力,其中对松柏醛和芥子醛的底物亲和力更强。用不同浓度甘露醇模拟干旱胁迫,蒙古冰草AmCAD基因表达受到显著诱导。研究结果表明,AmCAD在蒙古冰草木质素合成和干旱胁迫抗性中发挥重要作用,可为提高蒙古冰草品质和抗逆性分子育种提供有价值的基因资源。

Correction:Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues

Correction:aBIOTECH[2023]4:352-358 https://doi.org/10.1007/s42994-023-00119-3 In the Acknowledgements section of this article the funding number incorrectly given as SQ2022YF F1000033 and should have been 2022YFF1003204.

Co-expression of GR79 EPSPS and GAT generates highglyphosate-resistant alfalfa with low glyphosate residues

Weed competition seriously threatens the yield of alfalfa, the most important forage legume worldwide,thus generating herbicide-resistant alfalfa varieties is becoming a necessary cost-effective strategy toassist farmers for weed control. Here, we report the co-expression of plant codon-optimized forms ofGR79 EPSPS (pGR79 EPSPS) and N-acetyltransferase (pGAT) genes, in alfalfa, via Agrobacterium-mediated transformation. We established that the pGR79 EPSPS-pGAT co-expression alfalfa lines were able totolerate up to tenfold higher commercial usage of glyphosate and produced approximately ten timeslower glyphosate residues than the conventional cultivar. Our findings generate an elite herbicideresistant germplasm for alfalfa breeding and provide a promising strategy for developing high-glyphosate-resistant and low-glyphosate-residue forages.

Social-epigeneticmediators for racial disparities in pulmonary impairment among childhood cancer survivors

Dear Editor Previous research found that childhood cancer survivors of African ancestry have significantly higher morbidity and mortality than those of European ancestry[1].However,after adjusting for socio-economic factors,the magnitudes of racial health disparities are either substantially decreased or become statistically non-significant[1],suggesting that social and economic determinants may contribute to racial health disparities.Recently,we conducted epigenome-wide association studies(EWAS)for three key social determinants of health(SDOHs),namely,personal educational attainment,personal income,and neighborhood deprivation among survivors of childhood cancer,where 130 epigenome-wide significant SDOH-CpG associationswere identified among European ancestry survivors,and 25 of which were also validated in African ancestry survivors[2].Notably,many SDOH-associated CpG sites are also associated with tobacco use.

A comprehensive review concerning the problem of marine crudes sources in Tarim Basin

The issue of source of oil/gas in the platform basin area in Tarim Basin has been debated for a long time, and the debate is focused on whether the marine oil/gas resources that have been discovered in the basin were originated from hydrocarbon source rocks in the Cambrian-Lower Ordovician or in the Mid-Upper Ordovician. In this paper a summary was made in regard to the major points and supporting data by the predecessors, and a discussion was conducted toward the core issues related to the study on the oil source in the Tarim Basin area, such as choice of correlation parameters, influence of maturation and physical differentiation on oil source correlation parameters, and geological and geochemical significance of these relevant correlation indices. It is quite probable that different interpretation results could arise from the oil source correlation due to choice of parameters severely affected by the thermal maturation and physical differentiation effect, and insisted that only those parameters that

Effects of riparian vegetation patterns on the distribution and potential loss of soil nutrients： a case study of the Wenyu River in Beijing

A riparian ecosystem is an ecological transition zone between a river channel and terrestrial ecosystems. Riparian ecosystems play a vital role in maintaining stream health and bank stabilization. The types of riparian vegetation have changed greatly because of human activities along the Wenyu River. This study examines the impact of riparian vegetation patterns on water pollution due to soil nutrient loss. Four riparian vegetation patterns from the river channel to the upland were chosen as the focus of this study： grassland, cropland, grassland- cropland, and grassland-manrnade lawn. The different distributions of soil nutrients along vegetation patterns and the potential risk of nutrient loss were observed and compared. The results showed that riparian cropland has the lowest value of total nitrogen （TN）, total phosphorus （TP）, available nitrogen （AN）, available phosphorus （AP）, and organic matter （OM）, but it has the highest soil bulk density （BD）. The distributions of soil TN, TP, AN, AP, and OM exhibited a declining trend from the upland toward the river channel for riparian cropland, whereas a different trend was observed for the riparian grassland. The vegetation patterns of grassland-cropland and grassland- manmade lawn show that the grassland in the lower slope has more nutrients and OM but lower soil BD than the cropland or manmade lawn in the upper slope. So, the lower-slope grassland may intercept and infiltrate surface runoff from the upland. The lower-slope grassland has higher levels of soil TN, TP, AN, and AP, and thus it may become a new source of nutrient loss. Our results suggest that the management of the riparian vegetation should be improved, particularly in densely populated areas, to control soil erosion and river pollution.

Hexagonally arranged arrays of urchin-like Ag hemispheres decorated with Ag nanoparticles for surface-enhanced Raman scattering substrates

The surface topography of noble metal particles is a significant factor in tailoring surface-enhanced Raman scattering （SERS） properties. Here, we present a simple fabrication route to hexagonally arranged arrays of surface-roughened urchin- like Ag hemispheres （Ag-HSs） decorated with Ag nanoparticles （Ag-NPs） for highly active and reproducible SERS substrates. The urchin-like Ag-HS arrays are achieved by sputtering Ag onto the top surface of a highly ordered porous anodic aluminum oxide （AAO） template to form ordered arrays of smooth Ag-HSs and then by electrodepositing Ag-NPs onto the surface of each Ag-HS. Owing to the ordered arrangement of the Ag-HSs and the improved surface roughness, the urchin-like hierarchical Ag-HS arrays can provide sufficient and uniform ＂hot spots＂ for reproducible and highly active SERS effects. Using the urchin-like Ag-HS arrays as SERS substrates, 10-7 M dibutyl phthalate （a member of plasticizers family） and 1.5 × 10-5 M PCB-77 （one congener of polychlorinated biphenyl, a notorious class of pollutants） are identified, showing promising potential for these substrates in the rapid recognition of organic pollutants.

Analysis and assessment of heavy metal contamination in surface water and sediments： a case study from Luan River, Northern China

Concentrations of the heavy metals Cu, Ni, Pb, Zn, Cd, and Cr were examined in surface water and sediment from the Luan River inChina,. With a decline in Cu and Ni concentration found in surface water at downstream stations. This finding suggests that water currents are a major explanatory factor in heavy metal contamination. The abundance of Cr, Pb, and Cd observed in the middle reaches of the river indicates heavy metal contamination in local areas, although there was an obvious decrease in concentrations in the water downstream of the Daheiting Reservoir. The significant rising trend in Cu, Pb, and Ni seen the sediment farther away from the river also suggests that anthropogenic activities contribute to heavy metal pollution Sediments were therefore used as environmental indicators, with sediment assessment was conducted using the geo-accumulation index （Igeo） and the potential ecological risk index （R/）. The Igeo values revealed that Cd （3.13） and Cr （2.39） had accumulated significantly in the Luan River. The R/values for most （89%） of the sampling stations were higher than 300, suggesting that sediment from the Luan River poses a severe ecological risk, with the potential ecological risks downstream higher than that in the upper and middle streams. Good correlations among Pb/Ni, Pb/Cd, Cu/Pb, and Cu/Cd in the water and Cr/Ni in the sediment were observed. Cluster analysis suggested that Cd may have various origins, being derived from anthropogenic sources.

Major transgression during Late Cretaceous constrained by basin sediments in northern Africa： implication for global rise in sea level

The global rise in sea level during the Late Cretaceous has been an issue under discussion by the international geological community. Despite the signifi- cance, its impact on the deposition of continental basins is not well known. This paper presents the systematic review on stratigraphy and sedimentary facies compiled from 22 continental basins in northern Africa. The results indicate that the region was dominated by sediments of continental facies during Early Cretaceous, which were replaced by deposits of marine facies in Late Cretaceous. The spatio- temporal distribution of sedimentary facies suggests marine facies deposition reached as far south as Taou- deni-Iullemmeden-Chad-A1 Kufra-Upper Egypt basins during Turonian to Campanian. These results indicate that northern Africa underwent significant transgression during Late Cretaceous reaching its peak during Turonian to Coniacian. This significant transgression has been attributed to the global high sea-level during this time. Previous studies show that global rise in sea level in Late Cretaceous may have been driven by an increase in the volume of ocean water （attributed to high C02 concentra- tion and subsequently warm climate） and a decrease in the volume of the ocean basin （attributed to rapid production of oceanic crust and seamounts）. Tectonic mechanism of rapid production of oceanic crust and seamounts could play a fimdamental role in driving the global rise in sea level and subsequent transgression in northern Africa during Late Cretaceous.