利用CRISPR-Cas9技术失活黑曲霉中果胶酶基因及突变株性能评价*

我国果胶酶制剂使用广泛但专一性不高,高效、专一的果胶酶制剂在市场上仍然匮乏。利用基因工程技术改造果胶酶生产菌株——黑曲霉来生产单一成分的果胶酶成为解决果胶酶应用需求的一种有效方案。构建一种高效的CRISPR-Cas9基因编辑技术,可为构建高产单一性果胶酶的黑曲霉底盘菌株提供有效的基因编辑工具。首先敲除产果胶酶黑曲霉基因组上的pyrG基因构建尿嘧啶营养缺陷型菌株AnΔpyrG,并在AnΔpyrG菌株的pyrG基因位点定点整合Cas9基因表达盒和pyrG基因表达盒,构建组成型表达Cas9基因的黑曲霉菌株AnCas9,再构建含有gpdA启动子、锤头结构核酶、HDV核酶的稳定性表达sgRNA的pLM2-sgRNA质粒,建立CRISPR-Cas9基因编辑体系。利用该技术失活AnCas9菌株中的2个聚半乳糖醛酸酶基因4978020和4983861来检测构建的CRISPR-Cas9基因编辑效率并检测4978020基因功能缺失菌株的表型变化和产酶变化,结果表明果胶酶基因编辑效率大于50%,AnΔ4978020的表型和果胶酶酶活性与出发菌株均无明显变化。在黑曲霉中成功构建了高效的Cas9基因编辑技术,4978020基因功能缺失也不影响菌株表型,为构建高产单一性果胶酶黑曲霉底盘菌株奠定基础。     

我国医疗技术管理中引入医疗技术评估的路径探讨

2015年以来,在简政放权的大环境下,我国医疗技术管理面临着从重审批的分级分类管理向重监管转型的挑战和机遇,亟需探索建立更加科学合理的医疗技术管理体系。使用医疗技术评估的科学工具和手段是在医疗技术管理领域国际通行的理念和做法。本文梳理了我国医疗技术管理政策沿革,介绍了国际典型代表英国医疗技术管理的过程和方法,并为完善我国医疗技术管理提供了几条政策建议。     

王府井东方广场遗址骨制品研究

王府井东方广场遗址不仅出土了丰富的石制品,而且也含有较为丰富的骨制品。骨制品可分为骨核、人工骨块、骨片、骨屑和骨器五大类;骨片约占骨制品总数一半;骨器包括刮削器、尖头器、雕刻器和骨铲四类。观察发现：79件骨制品和骨骼可以拼合成33组,45件上有古人类砍砸、切割或刻划的痕迹。丰富的骨制品进一步反映了王府井古人类文化的进步性;同华北地区类似制品比较,它们之间存在着密切的渊源关系。遗物分布特征表明,东方广场遗址是一处古人类的临时居住地。出土的石器、骨器遗物以及大量的动物骨骼化石表明,王府井古人类不仅能够制作石器与骨器用来宰杀与支解猎物,而且具有用火、控火进行烹饪的能力。     

虎头梁遗址中的细石器技术

本文从应用目的的角度出发,对虎头梁遗址中的细石器技术进行了界定。通过数量统计、模拟计算等方法论证了虎头梁遗址中共存在着2种细石器技术,它们分别以Ⅰ型细石核和Ⅱ型细石核为代表。这两种细石器技术体现出了不同的剥片效率以及古人类在对外部环境适应方面的不同需求。Ⅰ型细石核因其相对较高的剥片效率以及构思巧妙的外部形态、加工精细的刃缘具有石核和工具的双重功能,常被古人外出时携带使用。Ⅱ型细石核则是古人类仅用来在固定地点剥制细石叶而使用的。     

Semen cryopreservation in golden‐headed lion tamarin,Leontopithecus chrysomelas

Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden‐headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden‐headed lion tamarins using two different exenders: BotuBOV® (BB) and Test Yolk Buffer® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, susceptibility to oxidative stress, and sperm–egg‐binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short‐term measure for the conservation of golden‐headed lion tamarins.     

基于CRISPR/Cas系统的多重基因编辑与调控技术

规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及其相关Cas蛋白所构建的CRISPR/Cas系统是古细菌或细菌中特有的一种获得性免疫系统。研究人员将其开发成基因编辑工具之后,凭借其高效、精准和通用性强等优点迅速成为合成生物学领域的热门研究方向,在生命科学、生物工程技术、食品科学及农作物育种等多个领域引发了革命性的影响。目前基于CRISPR/Cas系统单基因编辑与调控技术日益完善,但在多重基因编辑和调控方面仍存在挑战。本文聚焦基于CRISPR/Cas系统的多重基因编辑与调控技术开发及应用,针对单个细胞内实现多位点基因编辑或调控和细胞群体内实现多位点基因编辑或调控技术,依据作用原理对其进行了系统总结和阐述,包括基于CRISPR/Cas系统的双链断裂、单链断裂以及多重基因调控技术等。这些工作丰富了多重基因编辑与调控的工具,为CRISPR/Cas系统在多领域的应用作出了贡献。     

Manufacturing and characterization of Pd nanoparticles formed on immobilized bacterial cells

AIMS: To fabricate and analyse Pd nanoparticles on immobilized bacterial cells. METHODS AND RESULTS: Biological ceramic composites (biocers) were used as a template to produce Pd(0) nanoparticles. The metal-binding cells of the uranium mining waste pile isolate, Bacillus sphaericus JG-A12 were used as a biological component of the biocers and immobilized by using sol-gel technology. Vegetative cells and surface-layer proteins of this strain are known to bind high amounts of Pd(II) that can be reduced to Pd(0) particles by the addition of a reducing agent. Sorption of Pd(II) by the biocers from a metal complex solution was studied by inductively coupled plasma mass spectroscopy analyses. After embedding into sol-gel ceramics, the cells retained their Pd(II)-binding capability. Pd(0) nanoclusters were produced by the addition of hydrogen as reducing agent after the sorption of Pd(II). The interactions of Pd(0) with the biocers and the formed Pd(0) nanoparticles were investigated by extended X-ray absorption fine structure spectroscopy. The particles had a size of 0.6-0.8 nm. CONCLUSIONS: Bacterial cells that were immobilized by embedding into sol-gel ceramics were used as a template to produce Pd nanoclusters of a size smaller than 1 nm. These particles possess interesting physical and chemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of embedded bacterial cells as template enabled the fabrication of immobilized Pd(0) nanoparticles. These particles are highly interesting for technical applications, such as the development of novel catalysts.     

肉类及肉制品中动物源性成分鉴别方法研究进展

肉类掺假问题直接影响着人类健康、公共卫生安全以及社会稳定等方面,成为当今食品安全热点话题之一,因此,高效、精确的肉类及肉制品中动物源性成分的检测鉴定势在必行。基于此,主要介绍了对于动物源性成分检测及鉴别的不同研究方法,分析了利弊,并对后续肉类及肉制品中动物源性成分的鉴别方法的研发方向进行了展望,以期为此领域提供资料性参考。     

肖黄栌红色素的理化性质研究

本文研究了肖黄栌红色素的提取 ,并研究了不同糖浓度、温度、光照、紫外线、Na2 HPO4 -KH2 PO4 缓冲液、维生素C、氧化剂H2 O2 、还原剂Na2 SO3 等对肖黄栌红色素稳定性的影响。结果表明 ,肖黄栌红色素适应的pH范围较广、抗热、抗氧化、抗还原、耐糖、耐维生素C。因而是一种有重要开发价值的天然红色素