Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.     

Imaging plant cells by two-photon excitation

Summary. Along the past recent years, two-photon excitation (TPE) microscopy has moved from the realms of technical curiosity to be a standard application in many advanced cell biology laboratories. The growing body of literature covered in this review points out the obvious advantages of TPE over any other imaging method based on fluorescence, clearly improving signal-to-noise ratio and thick-tissue penetration and showing added potential for vital imaging. Like any new technology that has to gain its own space, TPE microscopy is still going through the growing pains in which reproducible protocols, probes, and applications are scarce. Yet, the published reports and unpublished results covered in this review point out that TPE can eventually accommodate most available protocols and probes, most of the times with evident advantages. Further, the potential for plant sciences is obvious, as plant cells possess many absorbing molecules and structures and are routinely more opaque than tissues of other organisms. Since prices make it one of the most expensive microscopies, TPE is coming slow to be a generalised technology, but enough data are emerging to establish it as a method with no alternative for some objectives.Correspondence and reprints: Instituto Gulbenkian de Ciência. 2780-156 Oeiras, Portugal.     

Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites

Insect neurons are individually identifiable and have been used successfully to study principles of the formation and function of neuronal circuits. In the fruitfly Drosophila, studies on identifiable neurons can be combined with efficient genetic approaches. However, to capitalise on this potential for studies of circuit formation in the CNS of Drosophila embryos or larvae, we need to identify pre- and postsynaptic elements of such circuits and describe the neuropilar territories they occupy. Here, we present a strategy for neurite mapping, using a set of evenly distributed landmarks labelled by commercially available anti-Fasciclin2 antibodies which remain comparatively constant between specimens and over developmental time. By applying this procedure to neurites labelled by three Gal4 lines, we show that neuritic territories are established in the embryo and maintained throughout larval life, although the complexity of neuritic arborisations increases during this period. Using additional immunostainings or dye fills, we can assign Gal4-targeted neurites to individual neurons and characterise them further as a reference for future experiments on circuit formation. Using the Fasciclin2-based mapping procedure as a standard (e.g., in a common database) would facilitate studies on the functional architecture of the neuropile and the identification of candiate circuit elements.     

痛觉的脑功能成像研究进展

本文综述了近年来关于痛觉功能性脑成像的研究进展,痛觉的感觉辨别成分似与外侧丘脑、初级和次级躯体感觉区及岛叶皮层有关,而伤害性信息的认知－注意过程则与顶叶后部和前额中皮层有关。扣带回的不同部分调节着痛觉认知和情感的不同方面。文章最后对临床各种疼痛特别是神经源性痛病人的成像研究进行了分析。     

口腔内金属材料对磁共振检查的影响

检测口腔内常用金属材料在磁共振检查时是否有伪影和伪影的严重程度。对２１种口腔内常用金属材料做了磁共振成像测试,磁共振仪磁场强度为１．５Ｔ,所用序列是梯度回波。铸金片、银汞合金、银尖等９种材料无伪影;钛合金和金属烤瓷成品有轻度伪影;牙用固位钉、椿钉等１０种材料有严重伪影。部分口腔内金属材料会引起严重伪影,影响图象质量,所以在做口腔颌面部和服部磁共振成像时,须引起重视。     

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.     

Functional artificial luciferases as an optical readout for bioassays

This study elucidates functional artificial luciferases (ALucs) wholly synthesized for bioassays and molecular imaging. The ALucs bearing epitopes were newly created by amending the sequences of our previously reported ALucs in light of a multi-sequence alignment and hydrophobicity search. The synthesized ALucs are survived in live cells and stable in culture media for 25 days after secretion. The epitopes in ALucs are exposed during the secretion process and indeed valid for column purification and immunological assays. The ALucs exerted a 9400-times stronger optical intensity with a coelenterazine derivative (CTZ i), when compared with Renilla reniformis luciferase 8.6–535. A supersecondary structure of ALuc30 was predicted with respect to the X-ray crystallographic information of the coelenterazine-binding protein (CBP). The structure revealed that ALuc30 has a room for accommodating the iodide of CTZ i. This study guides on how to create functional artificial luciferases and predicts the structural details with the current bioinformatics technologies.     

Using hyperspectral satellite imagery for regional inventories: a test with tropical emergent trees in the Amazon Basin

Questions: Understanding distributions of tree species at landscape scales in tropical forests is a difficult task that could benefit from the recent development of satellite imaging spectroscopy. We tested an application of the EO‐1 Hyperion satellite sensor to spectrally detect the location of five important tree taxa in the lowland humid tropical forests of southeastern Peru. Location: Peru, Departamento de Madre de Díos. Methods: We used linear discriminant analysis with a stepwise selection procedure to analyze two Hyperion datasets (July and December 2006) to choose the most informative narrow bands for classifying trees. Results: Optimal channels selected were different between the two seasons. Classification was 100% successful for the five taxa when using 25 narrow bands and pixels that represented >40% of tree crowns. We applied the discriminant functions developed separately for the two seasons to the entire study area, and found significantly nonrandom overlap in the anticipated distributions of the five taxa between seasons. Conclusions: Despite known issues, such as signal‐to‐noise ratio and spatial resolution, Hyperion imaging spectroscopy has potential for developing regional mapping of large‐crowned tropical trees.     

Medical ultrasound imaging

The paper gives an introduction to current medical ultrasound imaging systems. The basics of anatomic and blood flow imaging are described. The properties of medical ultrasound and its focusing are described, and the various methods for two- and three-dimensional imaging of the human anatomy are shown. Systems using both linear and non-linear propagation of ultrasound are described. The blood velocity can also be non-invasively visualized using ultrasound and the basic signal processing for doing this is introduced. Examples for spectral velocity estimation, color flow imaging and the new vector velocity images are presented.     

Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.