An Estimation Procedure for the Joint Distribution of Spatial Direction and Thickness of Flat Bodies Using Vertical Sections Part I: Theoretical Considerations

In soil micromorphology fissures are considered in vertical sections. To get information about the properties of the soil the joint distribution of spatial direction and width is of interest. The fissures are mathematically generalized to flat bodies which form a stationary weighted surface process with the weight “thickness”. Because of stationarity a joint distribution of spatial direction and thickness exists in a “typical point” of the surface process. A suitable parametric family of distributions is assumed. The corresponding parameters can be estimated from measurements on the vertical sections. But on the sections only the visible thickness and the visible angle of a fissure can be measured. Therefore the joint distribution of these variables is expressed by the joint spatial distribution of spatial direction and thickness. This derived distribution depends on the same parameters. The Chi-Square method is proposed for the parameter estimation. The estimation procedure is demonstrated using the Bingham-Mardia distribution for the direction and the lognormal distribution for the thickness and by defining a way to correlate the mean thickness and the direction.     

Cellular Distribution of Innexin 1 and 2 Gap Junctional Channel Proteins in Epithelia of the DrosophilaEmbryo

Invertebrate gap junctions are composed of Innexin channel proteins that are structurally and functionally analogous to the connexins in vertebrates. In situhybridization experiments have shown that most of the eight known innexingenes in Drosophilaare expressed in a complex and overlapping temporal and spatial profile, with several members showing high levels of expression in developing epithelia of the embryo. To further study the cellular roles of Innexins, we have generated antibodies against Innexins 1 and 2 and studied their protein distribution in the developing embryo. We find that both Innexins are co-expressed in a number of epithelial tissues including the epidermis, the gut and the salivary glands. On the cellular level, we find both proteins localized to the membranes of epithelial cells. Immunohistochemical analysis using cell polarity markers indicates that Innexin 1 is predominantly localized to the baso-lateral domain of epithelial cells, basal to septate junctions. In contrast, we find a variable positioning of Innexin 2 along the apico-basal axis of epithelial cells depending on the type of tissue and organ. Our findings suggest that the distribution of Innexin channel proteins to specific membrane domains of epithelial cells is regulated by tissue specific factors during the development of epithelia in the fly embryo.     

An Estimation Procedure for the Joint Distribution of Spatial Direction and Thickness of Flat Bodies Using Vertical Sections. Part II: An application in soil micromorphology

In soil micromorphology fissures are considered in vertical sections. To get information about the properties of the soil the joint distribution of spatial direction and width of these fissures is of interest. The fissures are mathematically generalized to flat bodies which are defined as stationary weighted surface processes with the weight “thickness”. In a typical point of the surface process suitable, joint parametric distributions of direction and thickness are assumed. The parameters have to be estimated from measurements on vertical sections which are taken from the soil. On these sections only a visible thickness and a visible angle can be observed. The joint distribution of these variables can be expressed by the joint distribution of spatial direction and thickness with the same parameters and in this indirect way the parameters can be estimated. The paper describes how to randomize the vertical section and how to measure the visible variables on the sections. The Chi-Square method is proposed for the parameter estimation. Further it is discussed how to derive good starting values for the numerical procedure. All this is demonstrated in a simulation study using the Bingham-Mardia distribution for the direction and the lognormal distribution for the thickness including a way to correlate the mean thickness and the direction. Finally an application in soil micromorphology is demonstrated for one soil horizon.     

Cellular Distribution of Innexin 1 and 2 Gap Junctional Channel Proteins in Epithelia of the Drosophila Embryo

Invertebrate gap junctions are composed of Innexin channel proteins that are structurally and functionally analogous to the connexins in vertebrates. In situ hybridization experiments have shown that most of the eight known innexin genes in Drosophila are expressed in a complex and overlapping temporal and spatial profile, with several members showing high levels of expression in developing epithelia of the embryo. To further study the cellular roles of Innexins, we have generated antibodies against Innexins 1 and 2 and studied their protein distribution in the developing embryo. We find that both Innexins are co-expressed in a number of epithelial tissues including the epidermis, the gut and the salivary glands. On the cellular level, we find both proteins localized to the membranes of epithelial cells. Immunohistochemical analysis using cell polarity markers indicates that Innexin 1 is predominantly localized to the baso-lateral domain of epithelial cells, basal to septate junctions. In contrast, we find a variable positioning of Innexin 2 along the apico-basal axis of epithelial cells depending on the type of tissue and organ. Our findings suggest that the distribution of Innexin channel proteins to specific membrane domains of epithelial cells is regulated by tissue specific factors during the development of epithelia in the fly embryo.     

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (¹⁴N/¹⁵N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.     

活细胞内MGTR1-3＇UTR的荧光标记及应激定位分析

利用噬菌体衣壳蛋白MS2和带有序列特异性茎环结构（含有MS2蛋白结合位点）的RNA之间的高度亲和力,对外源性入血管紧张素l型受体（angiotensin II receptor type1,AGTRl）mRNA3’端非翻译区（3＇untranslated region,3’UTR）片段进行红色荧光标记,进而在活细胞（HeLa）内研究该mRNA片段的应激生物学行为。通过在pSG5空载体质粒上先后插入两个双链DNA目的片段AGTR1-3qATR和24×MS2,构建重组质粒pSG5／AGTR1—3‘UTR／24×MS2,并将该质粒与重组质粒pERFP／MS2和pEGFP／C1-G3BP共转染入Hela细胞。荧光显微成像结果显示,AGTR1-3UTR-24×MS2 mNA片段能够携带具有入核信号的MS2-RFP融合蛋白离开胞核进入胞浆,而且在亚砷酸盐刺激下,红色荧光标记的AGTR1-3＇UTR-24×MS2 mRNA;4段可在胞浆中形成与应激蛋白G3BP—GFP共定位的颗粒。该结果表明,针对AGTR1-3‘UTR片段的MS2-RFP荧光标记系统构建成功,该荧光标记系统能有效避免假阳性的荧光信号。在细胞受到氧化应激时,AGTR1-3’UTR会被招募至胞浆中的应激颗粒结构中,启示了AGTR1-3＇UTR区域对于调控AGTR1 mRNA在细胞内的应激定位具有重要作用。     

A Review of the Potential and Versatility of Colloidal Gold Cytochemical Labeling for Molecular Morphology

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5–15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

小蓬种子萌发特性和幼苗分布格局研究

对小蓬(Nanophyton erinaceum)种子萌发特性和小蓬幼苗分布格局进行了调查研究。结果表明,小蓬在20℃条件下的发芽效果最好;小蓬种子的发芽不需要光照,但是全光照情况下发芽速度比在黑暗条件下快;在变温条件下,小蓬种子的发芽率有所提高,其中以15℃和10℃变温条件下的发芽效果最佳;不同坡向种群种子发芽率和千粒重存在明显差异,表明不同种群种子是异质的;通过调查发现小蓬主要在靠近母株一定范围内依靠种子进行自然更新。     

纳米碳标记技术用于甲状腺全切术的效果及对患者血清PTH、Ca2+水平的影响

摘要 目的：探讨纳米碳标记技术用于甲状腺全切术的效果及对患者血清甲状旁腺素(parathyroid hormone,PTH)、钙离子(calcium ion,Ca²⁺)水平的影响。方法：回顾性分析2017年1月至2018年12月于本院接受甲状腺全切术治疗的120例患者的临床资料,根据术中处理方式不同将其分为观察组和对照组,每组60例。两组患者均实施甲状腺全切术+中央区淋巴结清扫手术,观察组术中使用纳米碳混悬注射液,对照组不使用。比较两组的手术情况、治疗前后血清PTH、Ca²⁺水平的变化及术后并发症的发生情况。结果：两组术中出血量、术后引流量、甲状旁腺误切率比较差异无统计学意义(P＞0.05),观察组手术时间、住院时间比对照组明显缩短(P＜0.05);两组手术后1d、3d、7d时血清PTH、Ca²⁺均明显低于治疗前,且观察组手术后1d、3d、7d时血清PTH、Ca²⁺均明显高于对照组(P＜0.05)。观察组甲状旁腺、喉返神经功能暂时性损伤及低钙血症的发生率均明显低于对照组(P＜0.05),两组甲状旁腺、低钙血症、喉返神经功能永久性损伤比较差异无统计学意义(P＞0.05)。结论：纳米碳标记技术在甲状腺全切术中效果显著,可有效降低甲状腺误切率,降低甲状旁腺、喉返神经功能暂时性损伤、低钙血症的发生率。     

A Model for Adjusting for Nonignorable Verification Bias in Estimation of the ROC Curve and Its Area with Likelihood‐Based Approach

Summary In estimation of the ROC curve, when the true disease status is subject to nonignorable missingness, the observed likelihood involves the missing mechanism given by a selection model. In this article, we proposed a likelihood‐based approach to estimate the ROC curve and the area under the ROC curve when the verification bias is nonignorable. We specified a parametric disease model in order to make the nonignorable selection model identifiable. With the estimated verification and disease probabilities, we constructed four types of empirical estimates of the ROC curve and its area based on imputation and reweighting methods. In practice, a reasonably large sample size is required to estimate the nonignorable selection model in our settings. Simulation studies showed that all four estimators of ROC area performed well, and imputation estimators were generally more efficient than the other estimators proposed. We applied the proposed method to a data set from research in Alzheimer's disease.     

Modified Behavior in Baculovirus-Infected Lepidopteran Larvae and Its Impact on the Spatial Distribution of Inoculum

Quantifying the rate of dispersal of target insects when infected with a disease agent will aid the development of biorational pest control programs. The effect of nucleopolyhedrovirus (NPV) infection on the mobility of second and fourth instarMamestra brassicaelarvae was investigated in the laboratory and field. NPV infection altered larval mobility, with the changes in behavior varying with the timecourse of infection. Diseased larvae moved three to five times further than healthy ones during the middle stages of infection. By the 7th day postinfection diseased larvae were less mobile than healthy counterparts. The same pattern of modified behavior was observed in both instars. Fourth instar larvae moved further than second instars under laboratory and field conditions. In the field, infected larvae tended to die on the apex of the cabbage leaves. Bioassay of the leaves showed a linear decrease in inoculum from central to peripheral plants within the plots, which occurred to the same extent for second and fourth instars. Leaves from plots where infected fourth instar larvae had been introduced had higher inoculum density than those from plots with second instars.     

双单抗的免疫层析一步法用于早妊诊断的研究

在试管式、微孔式和斑点式的酶免测定法测定人绒毛膜促性腺激素（ＨＣＧ）的基础上,发展了应用双单克隆抗体的免疫层析一步法测定ＨＣＧ。此法用胶体金标记抗βＨＣＧ单克隆抗体,将抗αＨＣＧ单克隆抗体包被在硝酸纤维素膜上。无需分离步骤,特别是在进行测定时除加入样品外无需再加任何试剂,此方法特别迅速、简便,２～５ｍｉｎ即可得结果。凡ＨＣＧ浓度＞２５ＩＵ／Ｌ的样品可得到阳性结果。在人体血或尿中可能出现的高浓度的干扰物质,如抗坏血酸、乙酰水杨酸、雌二醇、蛋白质、胆红素、甘油三酯等对本测定均无干扰作用,在促黄体激素（ＬＨ）浓度高达５００ＩＵ／Ｌ时仍与ＨＣＧ没有交叉反应。能进行测定的最高值大于３００ＩＵ／ｍｌ,这表示,当ＨＣＧ浓度达到妊娠期的最高值时仍不会有假阴性结果。     

The Cellular and Mechanical Basis for Response Characteristics of Identified Primary Afferents in the Rat Vibrissal System

1. Download : Download high-res image (188KB)
2. Download : Download full-size image

     

Cellular and Subcellular Distribution of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Its mRNA in the Rat Central Nervous System

The 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs) are closely related oligodendrocyte proteins whose in vivo function is unknown. To identify subcellular sites of CNP function, the distribution of CNP and CNP mRNA was determined in tissue sections from rats of various developmental ages. Our results indicate that CNP gene products were expressed exclusively by oligodendrocytes in the CNS. CNP mRNA was concentrated around oligodendrocyte perinuclear regions during all stages of myelination. Developmentally, initial detection of CNP mRNA closely paralleled initial detection of its translation products. In electron micrographs of immunostained ultrathin cryosections, CNP was associated with oligodendrocyte membranes during the earliest phase of axonal ensheathment. In more mature fibers, immunocytochemistry established that the CNPs are not major components of compact myelin but are concentrated within specific regions of the oligodendrocyte and myelin internode. These include (a) the plasma membrane of oligodendrocytes and their processes, (b) the periaxonal membrane and inner mesaxon, (c) the outer tongue process, (d) the paranodal myelin loops, and (e) the "incisure-like" membranes found in many larger CNS myelin sheaths. A cytoplasmic pool of CNP was also detected in oligodendrocyte perikarya and larger oligodendrocyte processes. CNP was also enriched in similar locations in myelinated fibers of the PNS.     

Evaluation of a Push-Pull Approach for Aedes aegypti (L.) Using a Novel Dispensing System for Spatial Repellents in the Laboratory and in a Semi-Field Environment

The increase in insecticide resistant mosquito populations necessitates the exploration of novel vector control intervention measures. Push-pull strategies for insect control have been successful when used in integrated crop pest management. Through the combinatory use of deterring and attracting stimuli, the abundance of insect pests can be changed in a given area. A push-pull strategy might also significantly reduce human-vector contacts and augment existing mosquito control strategies, e.g. through the combination of an attractive trapping system and a potent spatial repellent. Our approach includes the BG-Sentinel (BGS) trap in combination with catnip oil (Nepeta cataria), a known spatial repellent for Aedes aegypti. To impart a deterrent effect on mosquitoes at a distance, a homogenous and continuous dispersal of volatile repellent compounds is crucial. We have developed a repellent dispensing system that is easy to use and provides a homogenous dispersal of repellent in an air curtain. The use of five 9 V fans and custom-made repellent sachets containing 10% catnip essential oil created a repellent loaded air curtain that provided coverage of an area of 2 m² (1.2 x 1.65 m). Air was sampled at four different heights in the curtain and analysed via thermal desorption (TD) and consecutive gas chromatography—mass spectrometry (GC-MS). Nepetalactone, the main constituent of the oil, was detected in air at a concentration range of 80 to 100 μg/m³ and the amounts were comparable at all four sampling positions. When a human volunteer was sitting behind the repellent curtain and a BGS trap was installed in front of the curtain in laboratory push-pull trials, Ae. aegypti landing collections decreased significantly by 50% compared to repellent-free controls. However, in a semi-field environment, comparable protective effects could not be achieved and further research on suitable repellent concentrations for outdoor implementation will be required.     

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling,Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available¹. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment². Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice³. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown⁴, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)⁵ and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate^5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for ¹⁵N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein⁷ (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state⁸. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE1⁹.     

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.     

戊型肝炎病毒细胞吸附模型的建立及病毒吸附区域初步研究

E.coli中表达的HEV衣壳蛋白片段P239(aa368~606)形成的类病毒颗粒与戊肝患者恢复期血清及中和单抗具有良好的反应性,较好地模拟了天然HEV病毒颗粒的表面空间结构。利用P239吸附HepG2细胞的模型来模拟HEV对宿主细胞的吸附,多株中和单抗对吸附的阻断验证了吸附的特异性。P239与多株传代细胞系的吸附结果则表明了这种特异性吸附的细胞选择性。对阻断P239吸附的线性单抗进行定位,初步确定了P239与细胞相互作用的区域:ORF2上的aa423~443很可能和病毒上的细胞膜受体结合部位非常靠近,或可能直接参与构成了病毒与细胞特异性识别的表位。此本研究为进一步研究HEV与宿主细胞的相互作用提供一定的线索。