Multidimensional annotation of the Escherichia coli K-12 genome

The annotation of the Escherichia coli K-12 genome in the EcoCyc database is one of the most accurate, complete and multidimensional genome annotations. Of the 4460 E. coli genes, EcoCyc assigns biochemical functions to 76%, and 66% of all genes had their functions determined experimentally. EcoCyc assigns E. coli genes to Gene Ontology and to MultiFun. Seventy-five percent of gene products contain reviews authored by the EcoCyc project that summarize the experimental literature about the gene product. EcoCyc information was derived from 15 000 publications. The database contains extensive descriptions of E. coli cellular networks, describing its metabolic, transport and transcriptional regulatory processes. A comparison to genome annotations for other model organisms shows that the E. coli genome contains the most experimentally determined gene functions in both relative and absolute terms: 2941 (66%) for E. coli, 2319 (37%) for Saccharomyces cerevisiae, 1816 (5%) for Arabidopsis thaliana, 1456 (4%) for Mus musculus and 614 (4%) for Drosophila melanogaster. Database queries to EcoCyc survey the global properties of E. coli cellular networks and illuminate the extent of information gaps for E. coli, such as dead-end metabolites. EcoCyc provides a genome browser with novel properties, and a novel interactive display of transcriptional regulatory networks

Interventional molecular imaging, a hybrid approach

Accurate detection of tumor tissue is a key feature in the diagnosis of cancer, and surgery is one of the major pillars in further management of the disease. Using preoperatively obtained images, the area of interest can be linked to the anatomy of the patient, enabling planning of the surgical intervention. During surgical exploration morbidity may be caused by the unnecessary removal of healthy tissue and/or the unintentional damage to delicate anatomical structures. Furthermore, the inability to completely excise the cancerous tissue increases the need for invasive re-excisions. More accurate surgical visualization of the area__s of interest can supplement the surgeons eyes and help improve surgical outcome. Accurate preoperative identification, surgical planning, and intraoperative visualization can be integrated using hybrid imaging agents that contain both a radioand fluorescent label. This hybrid interventional molecular imaging concept was initially validated in sentinel lymph node (SLN) biopsies, but can be further expanded by the implementation of imaging agents that specifically visualize tumor cells. In this thesis both the preclinical and clinical introduction of this hybrid concept is described.J.E. Jurriaanse stichting, Intuitive surgical, Oncovision, Storz endoscopesUBL - phd migration 201

A database and tool, IM Browser, for exploring and integrating emerging gene and protein interaction data for Drosophila

BACKGROUND: Biological processes are mediated by networks of interacting genes and proteins. Efforts to map and understand these networks are resulting in the proliferation of interaction data derived from both experimental and computational techniques for a number of organisms. The volume of this data combined with the variety of specific forms it can take has created a need for comprehensive databases that include all of the available data sets, and for exploration tools to facilitate data integration and analysis. One powerful paradigm for the navigation and analysis of interaction data is an interaction graph or map that represents proteins or genes as nodes linked by interactions. Several programs have been developed for graphical representation and analysis of interaction data, yet there remains a need for alternative programs that can provide casual users with rapid easy access to many existing and emerging data sets. DESCRIPTION: Here we describe a comprehensive database of Drosophila gene and protein interactions collected from a variety of sources, including low and high throughput screens, genetic interactions, and computational predictions. We also present a program for exploring multiple interaction data sets and for combining data from different sources. The program, referred to as the Interaction Map (IM) Browser, is a web-based application for searching and visualizing interaction data stored in a relational database system. Use of the application requires no downloads and minimal user configuration or training, thereby enabling rapid initial access to interaction data. IM Browser was designed to readily accommodate and integrate new types of interaction data as it becomes available. Moreover, all information associated with interaction measurements or predictions and the genes or proteins involved are accessible to the user. This allows combined searches and analyses based on either common or technique-specific attributes. The data can be visualized as an editable graph and all or part of the data can be downloaded for further analysis with other tools for specific applications. The database is available at CONCLUSION: The Drosophila Interactions Database described here places a variety of disparate data into one easily accessible location. The database has a simple structure that maintains all relevant information about how each interaction was determined. The IM Browser provides easy, complete access to this database and could readily be used to publish other sets of interaction data. By providing access to all of the available information from a variety of data types, the program will also facilitate advanced computational analyses

Virtual environment for studying the docking interactions of rigid biomolecules with haptics

Haptic technology facilitates user interaction with the virtual world via the sense of touch. In molecular docking, haptics enables the user to sense the interaction forces during the docking process. Here we describe a haptics-assisted interactive software tool, called Haptimol RD, for the study of docking interactions. By utilising GPU-accelerated proximity querying methods very large systems can now be studied. Methods for force scaling, multipoint collision response and haptic navigation are described that address force stability issues that are particular to the interactive docking of large systems. Thus Haptimol RD expands, for the first time, the use of interactive biomolecular haptics to the study of protein-protein interactions. Unlike existing approaches, Haptimol RD is designed to run on relatively inexpensive consumer-level hardware and is freely available to the community

A Biocompatible Gadolinium(Ⅲ)-Poly(Aspartic Acid-Co-Phenylalanine) for Liver Magnetic Resonance Imaging Contrast Agent

一种新型的以天门冬氨酸-苯丙氨酸共聚物为载体的大分子生物相容性材料(AP-EdA-dOTA-gd)被制备出来作为磁共振成像造影剂.首先合成了天门冬氨酸-苯丙氨酸共聚物,之后利用乙二胺将1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸(dOTA)连接到共聚物上,最后将钆离子通过配位的作用方式连接到dOTA上,最终得到大分子AP-EdA-dOTA-gd.体外溶血性试验表明AP-EdA-dOTA-gd具有较好的血液相容性.在P H=5.5的组织蛋白酶b的磷酸缓冲液中,AP-EdA-dOTA-gd能够降解.APEdA-dOTA-gd的体外弛豫效率(15.95 MMOl–1·l·S–1)为目前临床应用的gd-dOTA(5.59MMOl–1·l·S–1)的2.9倍.大鼠肝脏成像实验结果表明,AP-EdA-dOTA-gd对于肝组织的成像增强对比度为63.5±6.1%远高于gd-dOTA(24.2±2.9%).A new biocompatible gadolinium(III)-macromolecule(AP-EDA-DOTA-Gd) was developed as a magnetic resonance imaging(MRI) contrast agent.Poly(aspartic acid-cophenylalanine) was synthesized, modified via ethylenediamine, conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) and finally chelated gadolinium(III), yielding gadolinium(III)-based macromolecule(AP-EDA-DOTA-Gd).The hemolytic tests showed the hemocompatibility of this gadolinium(III)-based macromolecular conjugate.In vitro, AP-EDA-DOTA-Gd could be degraded, when it was incubated with cathepsin B in phosphate buffered solution(p H = 5.5).The T1-relaxivity(15.95 mmol–1·L·s–1) of AP-EDA-DOTA-Gd was 2.9 times of that(5.59 mmol–1·L·s–1) of the clinical MRI contrast agent(Gd-DOTA) at 1.5 T and 25 ℃.The liver enhancement of AP-EDA-DOTA-Gd was 63.5±6.1% during the maximum enhancement time(50-80 min), which was much better than that of Gd-DOTA(24.2±2.9%, 10-30 min).AP-EDA-DOTA-Gd was expected to be a potential liver MRI contrast agent.TheNationalNaturalScienceFoundationofChina(20975097and21305134

Discovery of New Roles for Chondroitin Sulfate in Neurotrophin Signaling and Retinotopic Development

Chondroitin sulfate (CS), a member of the glycosaminoglycan family of linear polysaccharides, is involved in the formation and maintenance of neuronal networks. CS has dual roles in regulating neuronal morphology: promoting or inhibiting neuronal outgrowth, depending on the context. A single sulfated epitope, CS-E, is capable of inducing both types of activity. Members of the neurotrophin (NT) family of growth factors are required for CS- E-induced neurite outgrowth in hippocampal neurons. Here, we demonstrate that CS is capable of forming ternary complexes with NTs and their receptors. These complexes were discovered using a novel, carbohydrate microarray-based approach that allows for the rapid screening of such interactions. To support these findings, we computationally determined the CS-E-binding site of the complexes, suggesting a structural basis for the interaction. In addition, we showed that CS-E is capable of attenuating NT signaling in cells, consistent with our computational and microarray data. This is the first demonstration that CS-E is involved in NT signaling and that CS is capable of supporting multimeric signaling complexes. In addition to stimulating growth factor signaling, CS has been known to repulsively guide retinal ganglion cell (RGC) axons for over twenty years. However, its function in vivo is unknown. RGCs are the only neuron type that transmits visual information to the brain, and their guidance, which maps a topographic projection of the retina to the superior colliculus (SC), is tightly regulated. Here, we show that CS-E is required for the proper formation of this topographic order. CS-E, but not the other major sulfation patterns, is a repellent guidance cue for RGC axons, with a graded activity profile from low to high along the dorsal-ventral axis of the retina, congruent with EphB3 expression. EphB3 binds specifically to CS-E with physiologically relevant affinity, and is required for CS-E-mediated guidance. CS-E-null mice have defects in topographic mapping in which ventral axons form ectopic termina- tions medial to their correct location in the SC. These results indicate that CS is a repulsive guidance cue required to map the dorsal-ventral axis of the retina along the lateral-medial axis of the SC. This is the first report of a non-protein topographical guidance cue.</p

Ex ovo RNAi for functional gene analysis during neural development

To study gene function in vivo during development of the central nervous system (CNS) efficient model systems that allow for temporal and spatial control of gene expression are required. In our lab, we have used the chicken embryo in combination with in ovo electroporation and RNA interference (RNAi) for gene silencing during early stages of nervous system development. In particular, we used dorsal commissural neurons to study axonal pathfinding. These neurons extend their axons toward the floor plate, the ventral midline of the spinal cord. Guidance cues derived from the floor plate, the intermediate target of these axons, are important for axon growth toward and across the midline. Using in ovo RNAi, we could show that interference with the function of the transmembrane glycoprotein Endoglycan (PODXL2) resulted in the failure to turn or in erroneous caudal turns after midline crossing. Furthermore, the morphology of the floor plate was severely disturbed in the absence of Endoglycan. During later stages of neural development, Endoglycan is expressed by Purkinje cells in the cerebellum. For the functional characterization of Endoglycan during cerebellar development, we extended the experimental accessibility of chicken embryos to much older stages. We established the procedure of ex ovo electroporation and RNAi to manipulate gene expression in the developing cerebellum. The cerebellum represents a well characterized neuronal structure, and therefore, an ideal system to study CNS development, including neurogenesis, differentiation, migration, axon guidance, as well as synapse formation. To demonstrate the applicability and the efficiency of ex ovo RNAi, we analyzed the function of the cell adhesion molecule Axonin-1 in cerebellar development. Axonin-1 is expressed by postmitotic granule cells at the time when they extend their axons, the parallel fibers. In the absence of Axonin-1 the arrangement of granule cell axons within the molecular layer was aberrant and fibers no longer extended parallel but towards the cerebellar surface. The effect of Axonin-1 was not on parallel fiber elongation but affected specifically parallel fiber navigation. The same effects on parallel fiber development observed after ex ovo RNAi were reproduced in embryos treated with function-blocking anti-Axonin-1 antibodies, indicating that ex ovo RNAi efficiently and reproducibly silenced axonin-1 in the developing cerebellum. Thus, we used ex ovo RNAi to study the function of Endoglycan during cerebellar development. Endoglycan is expressed by Purkinje cells at the time when they migrate towards the pial surface to establish the Purkinje cell monolayer. Interference with Endoglycan function caused a severe migration defect of Purkinje cells. Moreover, due to the aberrant formation of the Purkinje cell monolayer, the thickness of the external germinal layer and the proliferation rate of granule cells were significantly reduced causing disturbed foliation and reduced size of the cerebellum. Für die Analyse von Genfunktionen während der Entwicklung des zentralen Nervensystems (ZNS) werden effiziente Systeme benötigt, welche es ermöglichen, die Genexpression zeitlich und räumlich zu kontrollieren. In unserem Labor benutzen wir in ovo RNAi (RNA interference), um im Hühnerembryo während früher neuraler Entwicklungsstadien Gene gezielt auszuschalten. Anhand dorsaler Kommissuralneurone studieren wir Wegweisermoleküle, welche Axone zu ihren Zielzellen dirigieren. Axone der Kommissuralneurone wachsen gegen die ventrale Mittelline des Rückenmarks, die Bodenplatte, überqueren diese und wachsen anschliessend in rostraler Richtung weiter. Wegweisermoleküle, welche direkt auf der Bodenplatte lokalisiert sind oder von dieser sezerniert werden, sind wichtig für die korrekte Wegfindung dieser Axone. Mittels in ovo RNAi konnten wir zeigen, dass das Transmembran-Glykoprotein Endoglycan (PODXL2) nach dem Überqueren der Bodenplatte für die anschliessende rostrale Drehung von Kommissuralaxonen wichtig ist. Der Verlust von Endoglycan-Aktivität führte zu Stillstehen am Ausgang der Bodenplatte und fehlerhaftem caudalen Wachsen von Kommissuralaxonen. Das Blockieren von Endoglycan führte ausserdem zu einer Veränderung der Morphologie der Bodenplatte. Während späterer Entwicklungsstadien wird Endoglycan von migrierenden Purkinje- Zellen des Kleinhirns (Cerebellum) exprimiert. Um die Funktion von Endoglycan im Kleinhirn zu untersuchen, haben wir mit ex ovo RNAi eine Methode entwickelt, welche das gezielte Hemmen einzelner Gene während fortgeschrittener Stadien der Entwicklung des Nervensystems ermöglicht. Die neuronale Struktur des Kleinhirns ist gut charakterisiert und daher ein ideales System um verschiedene Entwicklungsschritte des Nervensystems zu studieren. Um die Anwendbarkeit und Effizienz von ex ovo RNAi auf unsere Fragestellung zu testen, haben wir zunächst die Funktion des Zelladhäsionsmoleküls Axonin-1 im Kleinhirn untersucht. Während des Auswachsens der Axone, den so genannten Parallelfasern, exprimieren Körnerzellen Axonin-1. Der Verlust von Axonin-1-Aktivität hat zur Folge, dass die Organisation der Parallelfasern in der Molekularschicht des Kleinhirns defekt ist. Dieser Defekt beruht auf der fehleraften Navigation von Parallelfasern. Den gleichen Effekt auf die Entwicklung der Parallelfasern konnten wir mittels Injektionen von funktionsblockierenden Antikörpern erzielen. Dies deutet darauf hin, dass ex ovo RNAi effizient und reproduzierbar Axonin-1-Aktivität im Kleinhirn hemmen kann. Daher verwendeten wir diese Methode für die Untersuchung von Endoglycan im Kleinhirn. Dieses Gen wird von Purkinje-Zellen während ihrer radialen Migration in Richtung Oberfläche des Kleinhirns exprimiert. Das Fehlen von Endoglycan verursachte einen Migrationsdefekt der Purkinje-Zellen und eine anormale Ausbildung der Purkinje-Zellschicht. Dies wiederum führte zu einer verminderten Dicke der Körnerzellschicht bedingt durch eine Reduktion der Zellteilungsrate