Molecular Variability of Mycosphaerella graminicola as Detected by RAPD Markers

A total of 90 isolates of Mycosphaerella graminicola, the cause of septoria tritici leaf blotch of wheat, were tested for DNA polymorphism using 15 decamer random primers. There was a high level of genetic variability among isolates. In 131 random amplified polymorphic DNA (RAPD) fragments, which were produced, 96% were polymorphic. Based on multilocus analysis, 40 different molecular phenotypes were detected. These molecular phenotypes were randomly distributed among sampling sites, suggesting that no clonal structure existed in the population. Cluster analysis showed that the maximum similarity value among isolates was approximately 81% and no identical isolates were detected, indicating that every isolate was a unique genotype. The high degree of DNA polymorphism, the large number of different molecular phenotypes, their random distribution and the results of the cluster analysis all suggested that sexual reproduction has a major role in the genetic structure of M. graminicola in western Canada. The presence of sexual reproduction provides the opportunity for development of new virulent genotypes in the population and suggests that the pathogen may adapt rapidly to any race‐specific sources of resistance. Therefore, when breeding for resistance to M. graminicola, emphasis should be placed on use of non‐race‐specific resistance.     

不同探测距离对光声成像的影响

为了研究不同探测距离对光声成像的影响,采用了线性扫描和滤波反投影算法,得出了吸收体间距相对探测距离越大,则成像亮度差异明显,吸收体远离探测器,则重建图像变形很明显.采用了光声信号衰减补偿的方法,对前者重建图像取域值,对后者采用旋转扫描重建,均得到了较好的图像质量.该研究结果对光声成像探测距离的合理选择和成像效果评估具有...     

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission¹ by delivering neurotransmitters and neuropeptides onto neurons reproducibly². Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation³ has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo^4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain⁶. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current^2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be ˜ 1 μm or less to minimize diffusional ''leak'', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.Fabrication of micropipettes is facilitated with sophisticated electronic pullers⁸. Micropipettes are pulled from glass capillary tubes containing a filament that ''wicks'' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). The present protocol demonstrates microiontophoresis of acetylcholine (ACh⁺ Cl^-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40^BAC-GCaMP2 transgenic mice⁹ enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.Download video file.^{(37M, mov)}     

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem^1,2. The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light³. However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope⁴. While shown to be a valuable preparation to study the physiology of the microcirculation in rats⁵ and hamsters⁶, the cremaster muscle in mice⁷ has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function^8-11 and real-time imaging of intercellular signaling¹². The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal¹³. It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule¹². Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.Download video file.^{(45M, mov)}     

White Matter Changes in Patients with Amnestic Mild Cognitive Impairment Detected by Diffusion Tensor Imaging

Compared to normal aging adults, individuals with amnestic mild cognitive impairment (aMCI) have significantly increased risk for progressing into Alzheimer’s disease (AD). Autopsy studies found that most of the brains of aMCI cases showed anatomical features associated with AD pathology. The recent development of non-invasive neuroimaging technique, such as diffusion tensor imaging (DTI), makes it possible to investigate the microstructures of the cerebral white matter in vivo. We hypothesized that disrupted white matter (WM) integrity existed in aMCI. So we used DTI technique, by measuring fractional anisotropy (FA) and mean diffusivity (MD), to test the brain structures involved in patients with aMCI. DTI scans were collected from 40 patients with aMCI, and 28 normal controls (NC). Tract-based spatial statistics (TBSS) analyses of whole-brain FA and MD images in each individual and group comparisons were carried out. Compared to NC, aMCI patients showed significant FA reduction bilaterally, in the association and projection fibers of frontal, parietal, and temporal lobes, corpus callosum, bilateral corona radiation, right posterior thalamic radiation and right sagittal stratum. aMCI patients also showed significantly increased MD widespreadly in the association and projection fibers of frontal, parietal and temporal lobes, and corpus callosum. Assessment of the WM integrity of the frontal, parietal, temporal lobes, and corpus callosum by using DTI measures may aid early diagnosis of aMCI.     

蝮蛇抗栓酶对高粘血症患者血液流变与微循环影响的观察

静注蝮蛇抗栓酶并口服青龙胶囊治疗高粘血症１４０例患者。结果显示,蝮蛇抗栓酶能明显降低血液粘度,表现为全血粘度、血浆粘度、全血还原粘度及纤维蛋白原降低,同时降低甲襞微循环形态、流态及管周状态积分。探讨其改善血液粘度机制。     

Distribution of Feline Blood Types Detected in the Copenhagen Area of Denmark

The purpose of the present study was to make the first survey of the distribution of feline AB blood types in the Copenhagen area of Denmark. A total of 244 cats (139 purebred cats and 105 Domestic Shorthair cats) were tested. 93% of all tested cats had blood type A. Neither an AB nor an O type cat was detected and thus, the frequency of blood type B among all tested cats was 7%. Most type B cats were purebred cats (Birman, British Shorthair and Persian cats). No association between sex and blood type could be demonstrated among British Shorthair and Persian cats. Thus, the present study indicates that cats in Denmark predominantly have blood type A, and that blood type B cats are rare, except for certain breeds such as Birman and British Shorthair cats.     

比较两种测量方法获得的血压波动对老年颈动脉斑块的影响

目的:比较两种不同血压波动测定方法测定的血压波动与颈动脉斑块发生的关系。方法:以1456名患有动脉硬化老年男性患者为研究对象,监测患者24 h动态血压,根据有无颈动脉斑块将入选患者分为2组:颈动脉斑块组(n=1012)和无颈动脉斑块组(n=444),分别采用经典的标准差方法(SD法)和个体血压波动测定方法(个体法)分别测定每位患者的血压波动,回顾性分析这两种方法测定的血压波动与颈动脉斑块形成的关联性。结果:SD法测定颈动脉斑块组24 h收缩期血压波动(SBPV)、白天SBPV、夜间SBPV以及24 h舒张期血压波动(DBPV)水平均明显高于无颈动脉斑块组(P0.05);而白天和夜间DBPV差异无统计学意义。个体法测定颈动脉斑块组24 h SBPV、白天SBPV、24 h DBPV以及白天DBPV水平较无颈动脉斑块组均明显升高(P0.05);夜间SBPV和夜间DBPV差异无统计学意义(P0.05)。比较颈动脉斑块组SBPV值出现次数,SD法测定SBPV最多的是10-15 mmHg(n=541),其次是大于15 mmHg(n=399);个体法测定颈动脉斑块组SBPV值出现次数最多的是0-8 mmHg(n=490),其次是8-10 mmHg(n=350)。结论:在老年男性动脉硬化相关疾病患者中,血压波动与颈动脉斑块的形成有着密切的关系,两种方法均可测定血压波动,但以个体血压波动测定方法更加敏感。     

Evaluation of the Microcirculation in a Rabbit Hemorrhagic Shock Model Using Laser Doppler Imaging

The aim of this study is to evaluate the feasibility of Laser Doppler imaging (LDI) for noninvasive and dynamic assessment of hemorrhagic shock in a rabbit model. A rabbit model of hemorrhagic shock was generated and LDI of the microcirculation in the rabbit ears was performed before and at 0, 30, 60, and 90 min after hemorrhage. The CCD (Charge Coupled Device) image of the ears, the mean arterial pressure (MAP) and the heart rate (HR) were monitored. The mean LDI flux was calculated. The HR of rabbits was significantly (p < 0.05) elevated and the MAP was decreased after hemorrhage, compared to the pre-hemorrhage level. Within the initial 30 min after hemorrhage, the perfusion flux lineally dropped down. In contrast, the MAP values did not differ significantly between the time points of 0 and 30 after hemorrhage (p > 0.05). Both the flux numbers and the red-to-blue color changes on LDI imaging showed the reduction of the microcirculation. LDI imaging is a noninvasive and non-contact approach to evaluate the microcirculation and may offer benefits in the diagnosis and treatment of hemorrhage shock. Further studies are needed to confirm its effectiveness in clinical practice.     

Inter Subject Variability and Reproducibility of Diffusion Tensor Imaging within and between Different Imaging Sessions

The aim of these studies was to provide reference data on intersubject variability and reproducibility of diffusion tensor imaging. Healthy volunteers underwent imaging on two occasions using the same 3T Siemens Verio magnetic resonance scanner. At each session two identical diffusion tensor sequences were obtained along with standard structural imaging. Fractional anisotropy, apparent diffusion coefficient, axial and radial diffusivity maps were created and regions of interest applied in normalised space. The baseline data from all 26 volunteers were used to calculate the intersubject variability, while within session and between session reproducibility were calculated from all the available data. The reproducibility of measurements were used to calculate the overall and within session 95% prediction interval for zero change. The within and between session reproducibility data were lower than the values for intersubject variability, and were different across the brain. The regional mean (range) coefficient of variation figures for within session reproducibility were 2.1 (0.9–5.5%), 1.2 (0.4–3.9%), 1.2 (0.4–3.8%) and 1.8 (0.4–4.3%) for fractional anisotropy, apparent diffusion coefficient, axial and radial diffusivity, and were lower than between session reproducibility measurements (2.4 (1.1–5.9%), 1.9 (0.7–5.7%), 1.7 (0.7–4.7%) and 2.4 (0.9–5.8%); p<0.001). The calculated overall and within session 95% prediction intervals for zero change were similar. This study provides additional reference data concerning intersubject variability and reproducibility of diffusion tensor imaging conducted within the same imaging session and different imaging sessions. These data can be utilised in interventional studies to quantify change within a single imaging session, or to assess the significance of change in longitudinal studies of brain injury and disease.     

人宫颈癌细胞Hela移植模型肿瘤生长的荧光分析和卡尺测量的比较

目的建立稳定表达绿色荧光蛋白的人宫颈癌细胞系,建立移植瘤模型并比较移植模型肿瘤生长的荧光分析和卡尺测量的优缺点。方法以Lipofectamine 2000介导chickenβ-actin-GFP-NEO转染人宫颈癌细胞Hela,经梯度浓度G418筛选获得稳定表达绿色荧光蛋白的细胞克隆并扩大培养。BALB/cA-nu裸鼠皮下接种1×10^6个发光细胞使其成瘤,利用活体荧光成像系统和游标卡尺观察肿瘤的生长情况。结果获得了稳定表达GFP的人宫颈癌细胞株,将其接种到裸鼠体内可成瘤。活体荧光成像观察发现,1至3周随着肿瘤体积逐渐增大,平均荧光光子数逐渐增加;4周时随着肿瘤出现明显坏死,平均荧光光子数呈现下降趋势,而游标卡尺测量结果显示肿瘤在4至5周仍然不断的增大。结论绿色荧光蛋白能够在人宫颈癌细胞Hela中长期稳定表达,用绿色荧光蛋白标记的人宫颈癌细胞Hela建立的裸鼠肿瘤模型可以为人宫颈癌研究提供理想的实验材料,应用小动物活体成像系统能够客观定量评价活的肿瘤细胞在动物体内的生长情况,而不是肿瘤体积的变化。     

Vessel Wall Inflammation of Takayasu Arteritis Detected by Contrast-Enhanced Magnetic Resonance Imaging: Association with Disease Distribution and Activity

Aims

The assessment of the distribution and activity of vessel wall inflammation is clinically important in patients with Takayasu arteritis. Magnetic resonance imaging (MRI) is a useful tool, but the clinical utility of late gadolinium enhancement (LGE) in Takayasu arteritis has yet to be determined. The aim of the present study was to evaluate the utility of LGE in assessing vessel wall inflammation and disease activity in Takayasu arteritis.

Methods and Results

We enrolled 49 patients with Takayasu arteritis who had undergone 1.5 T MRI. Patients were divided into Active (n = 19) and Inactive disease (n = 30) groups. The distribution of vessel wall inflammation using angiography and LGE was assessed by qualitative analysis. In 79% and 63% of patients in Active and Inactive groups, respectively, greater distribution of vessel wall inflammation was observed with LGE than with conventional angiography. MRI values of pre- and post-contrast signal-to-noise ratios (SNR), SNR increment (post-SNR minus pre-SNR), pre- and post-contrast contrast-to-noise ratios (CNR), and CNR increment (post-CNR minus pre-CNR) were evaluated at arterial wall sites with the highest signal intensity using quantitative analysis of post-contrast LGE images. No statistically significant differences in MRI parameters were observed between Active and Inactive groups. Contrast-enhanced MRI was unable to accurately detect active disease.

Conclusion

Contrast-enhanced MRI has utility in detecting the distribution of vessel wall inflammation but has less utility in assessing disease activity in Takayasu arteritis.     

Study of Microcirculation and Blood Rheology in Persons with Different Levels of Maximum Oxygen Consumption

Human Physiology - Oxygen delivery to cellular microdistricts is ensured by the coordinated work of systemic hemodynamics, microcirculation, and effective blood flow. However, the contribution of...     

Control of Blood Microcirculation Parameters in Therapy with Alpha-Lipoic Acid in Patients with Diabetes Mellitus

Human Physiology - In this study, we assessed the possibility of using portable laser Doppler flowmeters for determining the state of the blood microcirculatory system in upper and lower limbs of...     

Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy

The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.     

利用激光扫描共聚焦显微镜的肿瘤血管在体成像研究

传统的观察血管的方法需将组织制成切片,然后通过光学显微镜进行观察。显示的只是血管的某一片段而无法观察到血管的全貌。应用激光扫描共聚焦显微镜,可对活体动物血管进行断层成像,从而再现血管的结构。本方法为对肿瘤等病变组织血管进行研究提供了一种新的检测手段。     

PCR技术检测食源性致病菌的研究进展

食源性致病菌的检测技术是食源性疾病预防与控制的关键环节。PCR是近年来广泛应用于食源性致病菌快速检测的方法之一。在食源性致病菌中,用于PCR检测的靶基因包括各种毒力基因、酶基因及特异性鉴别基因。这些靶基因的发现推动了食源性致病菌PCR快速检测的发展。     

IP-FCM: Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.Download video file.^{(71M, mov)}     

用RAPD分析多年生黑麦草品种间遗传多样性

多年生黑麦草是世界上应用最广泛的冷季型草坪草兼牧草之一,具有建植快速,分蘖能力强,适口性良好等特点。为了评价其品种间的遗传多样性,选取了多年生黑麦草的14个草坪型品种和2个意大利黑麦草品种用RAPD进行遗传多样性分析,并且对每个品种的几项坪用特性进行了观察。61个引物共扩增出408条带,多态条带比率(PPB)为89．95％。聚类分析结果表明,多年生黑麦草品种之间的遗传多样性较低,相似系数分布于44．55％～95．69％之间。一年生黑麦草的2个品种TopOne和A,vance没有单独聚成一类,而是与14个多年生黑麦草品种混合聚在一起,而且Avance与其他多年生黑麦草之间的相似系数较高。聚类结果与形态特征没有明确的对应关系。