芥菜型油菜细胞质雄性不育系WJS01A的比较鉴定北大核心CSCD

芥菜型油菜细胞质雄性不育(CMS)系WJS01A是一种稳定不育系,其败育彻底,不受环境条件的影响。该研究从形态、细胞特征、遗传和分子生物学等方面对WJS01A进行鉴定,以揭示其败育机制,为该不育系在油菜育种中的应用提供理论基础。结果表明:(1)不育系WJS01A的花序结构与正常芥菜型油菜差异不大,但在花蕾饱满度、花朵张开度及花瓣长度和宽度方面略低于正常的芥菜型油菜;它的雌蕊发育正常,但花药、花丝缩短,致使雄蕊高度显著低于柱头,花药白化无花粉产生。(2)将WJS01A衍生的甘蓝型油菜背景不育系WNJ01A以及Polima(Pol)、Ogura(Ogu)和Kosena(Kos)不育系分别与其恢复系或保持系测交,结果显示来源于WJS01A的不育类型与Pol、Ogu和Kos等材料的恢保关系明显不同,仅Hui01可以恢复WNJ01A的育性。(3)不育系WJS01A属于无花粉囊型不育,败育时期为花药原基到孢原细胞时期。(4)线粒体不育基因多重PCR可以明显区分WJS01A、WNJ01A与Pol、Ogu、Kos,但是目前的引物组合不能区分WJS01A与正常的芥菜型油菜。(5)线粒体基因组的限制性片段长度多态性(RFLP)分析表明,在所检测的8个探针/酶组合中均可以将不育系WJS01A与其他4种细胞质雄性不育系区分开,说明WJS01A是一种显著不同于Pol、Ogu和Kos等的细胞质雄性不育类型。WJS01A的利用可以丰富和拓宽当前油菜杂种优势利用的遗传基础,为缓解当前油菜杂种优势利用中不育胞质单一性问题提供新的种质。     

Environment‐specific spectral modeling: A new tool for the analysis of biological specimens

The recent discovery of fluorescent dyes for improving pathologic tissues identification has highlighted the need of robust methods for performance validation especially in the field of fluorescence‐guided surgery. Optical imaging of excised tissue samples is the reference tool to validate the association between dyes localization and the underlying histology in a controlled environment. Spectral unmixing may improve the validation process discriminating dye from endogenous signal. Here, an innovative spectral modeling approach that weights the spectral shifts associated with changes in chemical environment is described. The method is robust against spectral shift variations and its application leads to unbiased spectral weights estimates as demonstrated by numerical simulations. Finally, spectral shifts values computed pixel‐wise from spectral images are used to display additional information with potential diagnostic value.      

成熟玉米秆组织结构切片方法的比较研究

于玉米成熟期选择健壮茎秆的基部第三节间为材料,采用徒手切片法、冰冻切片法、石蜡切片法、薄切片法等4种方法,比较不同方法的玉米茎秆组织结构切片质量,为研究玉米茎秆结构与其倒伏的关系奠定技术基础。结果表明:徒手切片法是获得成熟玉米秆组织结构切片较为方便、快速的方法,切片面积较大,适合大范围观察统计;冰冻切片法是获得成熟玉米秆组织结构较快的方法,切片面积较小,适合小范围观察;薄切片是获得高质量成熟玉米秆组织结构切片的最好方法,但切片面积太小,适合高倍数显微观察和小范围电镜扫描观察组织结构;石蜡切片不适合作为成熟玉米秆的组织切片方法。研究认为,徒手切片法是最适合成熟玉米秆组织结构观察研究的制片方法。     

新疆大帽藓属6种植物茎及叶的比较解剖学研究

运用石蜡切片和电镜技术,对新疆的6种大帽藓属（Encalypta Hedw.）植物的茎和叶进行解剖学观察。结果表明：高山大帽藓（E.alpina Smith.）疣多呈菱形或“柱状”密集着生在叶表面,疣上纹饰呈纵棱和小疙瘩状;裂瓣大帽藓（E.ciliata Hedw.）茎横切面为五角形, 中轴细胞角隅加厚,叶腹面的粗疣大部分集中分布在凹陷的表面细胞壁上;剑叶大帽藓（E.spathulata C.Mull.）茎横切面没出现中柱鞘,疣在基部分叉丛生,叶腹面的表皮细胞壁不凹陷;钝叶大帽藓（E.vulgaris Hedw.）没有叶尖,中肋导水细胞大,叶表面细胞壁较光滑且强烈凹陷,其壁上的乳突不明显;天山大帽藓（E.tianschanica j.c.Zhao,R.L.Hu et S.He）茎内外皮层区分明显,内皮层细胞质浓,叶背面的分叉状粗疣呈密集生长在叶腹面的星状粗疣大多集中分布在稍下陷的细胞壁上;西藏大帽藓（E.tibetana C.Mull）中肋细胞壁加厚,叶片具层层叠叠的、密集的、不规则分叉的粗疣,叶背面有些部位被带状附属物所覆盖。这6种植物叶的背、腹面均具不同程度的分叉粗疣,但粗疣的大小、着生位置、呈现的状态、疣上不同的纹饰却各不相同;中肋细胞的层数、及导水主细胞的大小、状态也大不相同,这些细微特征的观察研究可作为大帽藓属植物分类学的依据之一。     

Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin‐fixed paraffin‐embedded adenocarcinoma tissue sections

The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI‐mass spectrometry imaging (MALDI‐MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin‐fixed paraffin‐embedded breast cancer tissue sections were used. An improved protocol for on‐tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin‐fixed paraffin‐embedded tumour tissue sections. A novel approach combining MALDI‐MSI and ion mobility separation MALDI‐tandem mass spectrometry imaging for improving the detection of low‐abundance proteins that are difficult to detect by direct MALDI‐MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI‐MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.     

Quest for a reliable,valid, and sensitive in situ hybridization procedure to detect viral nucleic acids in the central nervous system

In situ hybridization (ISH) to detect and to quantitate viral nucleic acid sequences in cryopreserved central nervous system (CNS) tissue is a reliable, valid and sensitive molecular technique. On the other hand, utilization of formaldehyde fixed paraffin embedded (FFPE) tissue to improve cytomorphology requires fundamental changes in the procedure since it is necessary to cleave the elaborate protein network cross-linked by formaldehyde using elevated concentration of proteinases in order to permit diffusion of complementary DNA probes to the targets (genomic viral nucleic acid sequences and/or viral mRNA). Adversely, this procedure hydrolized the proteinaceous glues generally used to fix tissue to glass slides resulting in loss of tissue sections during the ISH protocol. This report describes the application of a novel procedure utilizing a silano-organic compound to covalently bond to glass slides FFPE sections as well as cryopreserved tissue sections and cultured cells with and without virus infections. This covalent bonding procedure has permitted optimization of the ISH procedure for virus detection and quantification, especially for exploratory studies of specificity and wash stringency in relation to the Tm of the hybridized product. Progressive multifocal leucoencephalopathy (PML) caused by an opportunistic papovavirus (JC) was chosen because of the ready availability of tissue, stability of papovavirus nucleic acids, and specificity of³H-and³⁵S-radiolabeled JC cloned DNA probes. Further, this laboratory is utilizing the optimized sensitive procedure to search for several virus etiologies in human diseases such as multiple sclerosis, temporal lobe epilepsy, Alzheimer's disease, schizophrenia, and Parkinson's disease, as well as normal aging. Fanally, the procedure permits study of 100% of thin serial sections; hence, alternate sections can be hybridized with sense and antisense riboprobes to detect viral genome and its mRNA or stained, immunocytochemically, to detect viral proteins. Accordingly, it is anticipated that the mechanism of persistent CNS viral infections will be deciphered, at least in part by advances in cytological molecular hybridization.     

Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Chemical Dehydration of Specimens with 2,2-Dimethoxypropane (DMP) for Paraffin Processing of Animal Tissues: Practical and Economic Advantages over Dehydration in Ethanol

Chemical dehydration can be accomplished using 2,2-dimethoxy-propane (DMP). In the presence of an acid catalyst, this liquid reacts with water generating methanol and acetone as products. Although DMP is more expensive per milliliter than ethanol and other solvents used for dehydration, it is an economical alternative because a much smaller volume is needed. Slow penetration of DMP was previously thought to restrict its use to tiny specimens, but we now show that pieces of tissue as thick as 2 cm are dehydrated by overnight immersion in acidified DMP. We also show that dehydration in acidified DMP does not impair the staining of UNA or other basophilic components of animal tissues. The temperature and concentrations of methanol and H⁺ in the chemical dehydrating agent are too low to produce histochemicaUy detectable methylation or nucleic acid extraction.     

石蜡切片法中细长或薄片状材料的包埋

石蜡切片法制作细长或薄片状生物材料的横切面切片时,可进行下列简便而有效的石蜡包埋方法：先在包埋纸盒底部倒上少许熔蜡,形成约1mm厚的软蜡层;待软蜡层凝固前,再向纸盒中倒满熔蜡,然后迅速将渗好蜡的材料直立地放入熔蜡中,并将材料轻压在纸盒底部的软蜡层上。