A Quick Embedding Method for Light Microscopy and Image Analysis of Cotton Fibers

A quick embedding method using UV polymerization of methacrylate plastic has been devised for embedding fibers encased in a polyvinyl chloride tube. The resulting embedments are suitable for light microscopy and image analysis.     

适用于微卫星标记的湿地松、加勒比松DNA快速提取法

以湿地松(Pinuselliottii,PEE),包括古巴加勒比松(P.caribaea var.caribaea,PCC)、洪都拉斯加勒比松(P.caribaea var.hondurensis,PCH)、巴哈马加勒比松(P.caribaeavar.bahmaensis,PCB)3个变种在内的加勒比松,侧枝顶芽为试验材料,使用RETSCHMM400混合球磨仪破碎植物组织,然后利用改良CTAB法提取基因组DNA,完成48个样品仅用1h,1个工作日可提取300个样品以上,DNA分子量大于20kb,0.3g样品可提取DNA20-60μg,能够满足几百次PCR反应;利用所提DNA进行SSR分析,条带清晰,多态性好,说明该方法提取的DNA完全可以满足SSR反应的需要。本研究为SSR标记用于湿地松、加勒比松分子标记辅助选择育种提供了经济、高效、可靠的DNA提取方法。     

A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.     

Super Resolution Microscopy Reveals that Caveolin-1 Is Required for Spatial Organization of CRFB1 and Subsequent Antiviral Signaling in Zebrafish

Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. Caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. This study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. A novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. Viral infection leads to a downregulation in Caveolin-1b (Cav-1b), disrupting clusters of CRFB1, a zebrafish type I interferon receptor (–R) subunit. Super-resolution microscopy has enabled the first single-molecule imaging of CRFB1 association with cav-1b-containing membrane domains. Strikingly, downregulation of Cav-1b, the major protein component of caveolae, caused CRFB1 clusters to disperse. Dispersal of CRFB1 clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. This response strongly suggests that CRFB1 organization within cav-1b-containing membrane domains is critical for IFN-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter IFN signaling and the antiviral immune response.     

Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.     

A Method for Isolating and Preparing Silica Bodies in Grasses for Scanning Electron Microscopy

Silica bodies are discrete deposits of dehydrated silica within epidermal cells. To describe these bodies completely, surrounding organic and unsilicified material must be removed. Methods generally used for isolating and preparing silica bodies were unsuitable for most grass species. An effective method for studying grasses is described here. After ashing the plant tissue, the ash was repeatedly rinsed with HCl in a specialized multiple funnel manifold and collected on Nuclepore filters. In addition, the silica bodies were sonicated for a few minutes to remove any remaining mineral impurities. Compared to conventional procedures, this method has a number of advantages: unsilicified material and mineral impurities were removed effectively, smaller quantities of plant tissue could be used, and the loss of silica bodies was minimized.     

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes

The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no “gold standard” for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.     

病原真菌和细菌毒性基因的分离和鉴别

本文讨论最新发展起来的病原真菌和细菌毒性基因的分离和鉴别原理和方法,涉及到基因表达分析法、基因转移方法、基因组比较法、诱变方法及其诱变子的筛选鉴定。着重讨论了这些方法的优点和局限性,评估了标记诱变法（ＳＴＭ）和限制酶介导的整合法（ＲＥＭＩ）两者相互结合在病原真菌毒性基因克隆中的应用潜力。     

一种简单有效且适于土壤微生物多样性分析的DNA提取方法

参照Zhou[11]的方法进行了改进,获得了一种简单、有效的DNA提取方法.此方法操作简单、从大量样品改为小量样品的提取,利用高浓度的PEG沉淀,不作回收纯化,所提DNA片段较大,在23 kb以上,每克土的DNA提取量从3.74～15.28 μg,OD260/OD230比值在0.89～1.21范围内,用真菌和细菌核糖体特异性引物进行PCR扩增,均获得较好的结果,DGGE图谱显示丰富性较高,可用于细菌多样性和真菌多样性的分析.此方法能够从4种不同性质土壤中提取出DNA,但提取盐渍土壤和碱性土壤的效果更好一些,为土壤微生物群落结构的多样性分析奠定良好的基础.     

A Method for Producing Dust-, Streak- and Hole-Free Formvar Films in Laboratories Having High Atmospheric Humidity

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

实例分析PBL教学法在医学分子生物学实验教学中的应用

分子生物学实验课程对培养学生科研能力至关重要,然而,目前常规教学重动手能力,而忽略科研思维的培养。本文以证明"突变β-catenin促进结肠癌细胞增殖"为例,探讨PBL教学法在分子生物学实验教学中的实际应用。与传统孤立地讲授和实施常见分子生物学实验技术不同,该PBL(problem based learning)法通过证明"突变β-catenin促进结肠癌细胞增殖"这一科学问题将DNA提取、PCR、质粒克隆和提取、核酸电泳、胶回收、蛋白提取、Western Blot、细胞转染等实验技术有机的统一起来,强化了实验技术解决实际问题的针对性和各个方法之间的内在联系,增强了学生学习的积极性,同时也大大提高学生发现问题、综合分析问题和解决问题的科研能力。     

广东省雨水花园适用乡土植物筛选及应用分析

通过野外调查与文献分析,对广东省雨水花园适用乡土植物资源进行整合筛选。筛选出63种具有较大应用价值的乡土植物,隶属39科54属;包括乔木6种、灌木5种、藤本1种、草本51种;其中,偏阴生植物21种,阳生植物42种。根据雨水花园中心积水区、斜坡区和外围缓冲区的种植区域划分,确定相关植物类群,并对其景观搭配效果进行评价,同时结合我国城市发展趋势和特色小镇建设分析其苗木市场潜力。     

A New Statistical Method for Mixed Variates Analysis in Epidemiological Research

Epidemiologically oriented research often may not do without observational or only partially controlled studies. In many such situations both qualitative characteristics and quantitative ones are observed. In literature there are different methods of handling such problems. The paper presents a method for analyzing dependencies resp. associations between random variates of any kind. The model concerned fullfills the whole field between analysis of variance, analysis of covariance and contingency table analysis. The method is named MIVA or mixed variates analysis, bases on the class of Conditional Gaussian Distributions of the exponential family and results in a unique system of mixed and unmixed measures of association–of pairwise, partial, multiple and global type. These measures are easy to be estimated and tested on significant deviation from zero. They may be used describing or analyzing dependence structures in many epidemiological studies but also in other fields.     

A Simple Procedure for in Situ Immunolabeling, Embedding and Sectioning of Layers of Cultured Endothelial and Smooth Muscle Cells for both Light and Electron Microscopy

We present a simple procedure for in situ immunolabeling, embedding and sectioning of layers of cultured endothelial and smooth muscle cells for both light and electron microscopy. Endothelial and smooth muscle cells were seeded in tissue culture chambers /slides precoated with 30% (w/v) gelatin drops fixed with 0.5% glutaraldehyde. Live endothelial cell layers were labeled with an antibody against the surface membrane protein, anti-CD 13. After labeling, the cell layers were fixed and separated from the chambers/slides by lifting all of the samples with a spatula. Sections (1-2 mm) were cut, embedded and processed further for light or electron microscopy. Because of the delicate cell layers and the importance of preserving maximum integrity, labeling was performed under standard culture conditions and treated in situ during the entire procedure. Moreover, the small chamber size of the tissue culture dishes generated the additional advantages of requiring only a limited number of cells, small volumes of media, and little antibody.     

A Simple Method for Fixing, Dehydrating and Embedding Pollen Tubes Cultivated in Vitro for Optical and Transmission Electron Microscopy

A simple method to cultivate pollen tubes in a gelatin medium is presented. After the growth of the pollen tubes in the culture medium, they are fixed, dehydrated, and embedded in resin for ultramicrotomy. The method is easy and does not require the purchase of special materials beyond those needed for the usual techniques for studying biological specimens under transmission electron microscopy.     

Pararosaniline Or Acriflavine-Schiff Staining of Epoxy Embedded Tissue After Periodic Acid Oxidation in Ethanol: A Method Suitable for Morphometric and Fluorometric Analysis of Glycogen

A method for preparing tissue sections for automatic image analysis of glyco-gen is described. Large semithin seaions of epoxy embedded tissue fixed in glutaraldehyde osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydra-tion before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.